Motile sperm subpopulations in bull semen using different clustering approaches - Associations with flow cytometric sperm characteristics and fertility.

There are sperm subpopulations (SPs) with different kinematic characteristics in various species, however, biological relevance of these SPs is still uncertain. The objective of the present study was to investigate associations of motile sperm SPs with sperm characteristics determined by evaluations with flow cytometry and assessment of bull fertility, using multiple approaches for sperm clustering. Semen from 24 bulls was evaluated concomitantly using computer-assisted sperm analysis (CASA) and flow cytometry before freezing and after thawing. Motile SPs were determined utilizing two acknowledged clustering methods (TwoStep and K-Means) and one customized method. With the customized method, there was utilization of mean values of sperm velocity and linearity as thresholds for direct assignment of motile spermatozoa into four SPs. Regardless of approach for identifying SPs, sperm quality, as determined using flow cytometry, was correlated particularly with the subpopulation (SP) of fast and linear spermatozoa immediately after thawing and with the SP of fast and nonlinear spermatozoa before freezing and 3 h after thawing. Furthermore, there was a positive correlation between proportion of spermatozoa with fast and nonlinear movements before freezing and bull non-return to estrous rates. These results indicate that with different sperm SPs, there are different biological implications which can be evaluated to gain useful information concerning semen quality as determined using flow cytometry and fertility. Furthermore, determining SPs by assigning motile spermatozoa into clusters based on a combination of "below and "above" threshold values for sperm velocity and linearity might be considered a practical alternative to otherwise intricate clustering procedures.