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Multivariate cluster analysis to study motility activation of Solea senegalensis spermatozoa: a model for marine teleosts

¹ Biology of Reproduction Group, National Wildlife Research Institute (IREC) (CSIC-UCLM-JCCM), 02071 Albacete, Spain² Institute for Regional Development (IDR), UCLM, 02071 Albacete, Spain³ Center for Marine Sciences (CCMAR), University of Algarve, 8000-139 Faro, Portugal⁴ Animal Reproduction and Obstetrics, University of León, 24071 León, Spain

Correspondence should be addressed to F Martínez-Pastor who is now at Ciencia y Tecnología Agroforestal, ETSIA, University of Castilla-La Mancha, Av. España s/n, 02071 Albacete, Spain; Email: felipe.martinez{at}uclm.es

Computer-assisted sperm analysis (CASA) and clustering analysis have enabled to study sperm subpopulations in mammals, but their use in fish sperm has been limited. We have used spermatozoa from Senegalese sole (Solea senegalensis) as a model for subpopulation analysis in teleostei using two different activating solutions. Semen from six males was activated using 1100 mOsm/kg solutions: artificial seawater (ASW) or sucrose solution (SUC). Motility was acquired at 15, 30, 45, and 60 s post-activation. CASA parameters were combined into two principal components, which were used in a non-hierarchical clustering analysis, obtaining four subpopulations (CL): CL1 (slow/non-linear), CL2 (slow/linear), CL3 (fast/non-linear), and CL4 (fast/linear). We detected spermatozoa lysis, especially in ASW. Sperm motility was higher for SUC and decreased with time. The subpopulation proportions varied with time and activating treatment, showing both an increase in CL1 and CL2 and a decrease in CL3 and CL4 with time. Both CL3 and CL4 were higher in samples activated with SUC, at least in early post-activation. Proportions of CL3 and CL4 at 15 s were associated with higher quality at 60 s and with lower lysis. A second clustering analysis was conducted, classifying the males accordingly to their motility subpopulations. This analysis showed a high heterogeneity between samples. Subpopulation analysis of CASA data can be applied to Solea spermatozoa, allowing identification of potentially interesting sperm subpopulations. Future studies might benefit from these techniques to establish the relationship of these subpopulations with fish sperm quality and fertility, helping to characterize males according to their reproductive potential.