Motile sperm head morphometry in relation to sperm kinematics and freezability in cats

Sperm head morphometry exhibits significant variation across species, among individuals, and even within single ejaculates, potentially influencing sperm functions such as motility and cryotolerance. The aim of this study was to evaluate whether sperm head morphometry affects sperm kinematics and freezability in domestic cats. Semen was collected from 16 male cats, once from each cat. Kinematic and morphometric characteristics were analyzed simultaneously at the single-spermatozoon level using a CASA system (MOT-morph, n = 10,400 sperm cells). Additionally, sperm head morphometry was performed on fix-stained samples from the same cats (FS-morph, n = 1788 sperm cells). Morphometric data were subjected to Principal Component Analysis and subsequent clustering analysis to identify distinct morphometric subpopulations. For each sample, 20 × 10⁶ spermatozoa were cryopreserved. After thawing, samples were classified as 'good freezing' (GF, n = 8) or 'bad freezing' (BF, n = 8) based on post-thaw membrane integrity (GF >50 %; BF <45 %), assessed via flow cytometry and fluorescent staining (SYBR-14 and propidium iodide). In MOT-morph, four morpho-kinetic subpopulations were distinguished, while in FS-morph, two morphometric subpopulations were identified. A technical bias, wherein faster-swimming cells appeared larger, precluded the establishment of a definitive relationship between morphometry and kinematics. Consequently, subpopulations established via MOT-morph represent artificial models conflating sperm size and speed rather than accurate descriptors of real sperm groups. In FS-morph, there were no significant differences between the GF and BF groups in any morphometric parameter or subpopulation. However, in MOT-morph, spermatozoa in the GF group exhibited a significantly (p < 0.05) lower probability of being classified as MOT-SP1 and a higher probability of being classified as MOT-SP3 and MOT-SP4 than those in the BF group. These results suggest that while simultaneous morphometric and kinematic analysis of feline spermatozoa presents challenges, derived subpopulations hold potential for predicting freezability in domestic cats. Notably, the software used in this study facilitates rapid analysis of nearly six times more cells than classic morphometric methods, without the need for slide preparation, fixation, or staining. Validation of results using larger number of individuals and with refined technological solutions is required.