Sperm quality separation through density gradient in dogs

The sperm separation by density gradient is a technique used to remove the extender from cryopreserved samples and select sperm with high motility for its use in reproductive biotechnologies. The 45% and 90% Percoll® gradient separate the semen into two distinct fractions: low sperm motility in the interface between the two Percoll® layers; and high motility sperm in the sediment. However, studies in dogs are still required to confirm the quality of spermatozoa selected by Percoll®, in both fresh and cryopreserved samples. The aim of this study was to verify the quality of sperm samples separated by Percoll® from fresh and cryopreserved-thawed canine semen. Nine dogs were used to obtain semen samples, which were divided into two aliquots: one designed for immediate evaluation and the other was cryopreserved by standard protocol and submitted to post-thaw evaluation. Semen samples were layered over two-steps pads of Percoll® (1 ml of 90% Percoll® and 1 ml 45% Percoll®). The tubes were centrifuged for 20 min at 300 xg . After separation, fresh and post-thaw samples from the interface and sediment were analyzed by computerized assessment of sperm motility; integrity of plasma and acrosomal membrane by eosin/nigrosin and Fast-Green/Rosa-Bengala stains, respectively; and mitochondrial activity by 3,3 '- diaminobenzidine (DAB) stain. We used Student's t-test and Wilcoxon test. The level of significance adopted was P<0.05. Both fresh and post-thaw semen had higher percentage of sperm with intact acrosome in the sediment (thawed: 81.8% ± 14.5; fresh: 93.4% ± 14.1) compared to the interface (thawed: 53.3% ± 7.7; fresh: 78.8% ± 19.7). We observed differences on the percentage of spermatozoa with high mitochondrial activity in the interface (thawed: 23.9% ± 18.4; fresh: 56.3% ± 29.6) and sediment (thawed: 65.8%± 26.2; fresh: 76.8% ± 17.6). High sperm motile spermatozoa was selected in the post-thaw semen sediment (interface: 16.2% ± 16.6; sediment: 31.7% ± 48.4) and fresh semen sediment (interface: 4.5% ±8.6; sediment: 35.3% ± 35.9). No difference on plasma membrane integrity among samples from sediment and interface were verified. However, post-thaw sediment samples (26.4% ± 18.2) had lower percentage of sperm with intact plasma membrane compared to fresh samples (65.2% ± 27.5). In conclusion, Percoll® gradient selects the best quality sperm in fresh and post-thaw samples. In addition, cryopreserved cells have greater sensitivity during passage through the gradient, resulting in plasma membrane injury.