The Effect of Density Gradient Centrifugation and Magnetic-Activated Cell Sorting with the Combination of Both on Bovine Spermatozoa Kinematics, Viability and Acrosome Status

Conventional and novel spermatozoa separation techniques have their own strength and weaknesses. Both are widely used in assisted reproductive technologies (ARTs). In this study, combinations of two separation methods against single separation methods were evaluated. Freshly ejaculated semen was collected and divided into five groups; control, density gradient centrifugation (DGC), magnetic activated cell sorting (MACS), DGC-MACS and MACS-DGC; based on the sequence of separation. Evaluation of spermatozoa kinematics, viability and acrosome status for each group were done by computer assisted semen analysis (CASA), hypo-osmotic swelling test (HOST) and chlorotetracycline (CTC) staining respectively. Results show that DGC yield the fastest spermatozoa velocity and highest percentage of viable spermatozoa fraction compared to other groups. However, they also produced a high percentage of acrosome-reacted spermatozoa. Spermatozoa isolated through DGC-MACS shows the highest percentage of non-capacitated spermatozoa. However, spermatozoa isolated through DGC-MACS yields slower spermatozoa velocity population and lower percentage of viable sperm compared to spermatozoa isolated through DGC. Isolation of spermatozoa through MACS-DGC yields slower population of spermatozoa velocity with a lower percentage of non-capacitated spermatozoa compared to spermatozoa isolated through DGC-MACS. DGC separation and its combination with MACS had yield different spermatozoa quality. This distinct characteristic is likely suitable for in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) procedure respectively.