Kinematic and head morphometric characterisation of spermatozoa from the Brown Caiman (Caiman crocodilus fuscus)

Sperm kinematics and morphometric subpopulations analysis with CASA systems: a review. The subjective evaluation of seminal quality has given way to the use of objective assessment techniques by CASA technology (computer-assisted semen analysis). The application of principal components (PC) and clustering methods to reveal subpopulations of spermatozoa is a powerful tool to evaluate raw semen and processed cell suspensions, but not many researchers are aware of the technique. PC analysis is a multivariate statistical method that reduces the number of variables used in subsequent calculations used to describe the data. By integrating the original variables according to their coherence in a database into a new complex mathematical variable, clearly defined homogenous subpopulations of spermatozoa can be defined. Kinematic, morphometric, morphological or DNA integrity tests may apply to characterize it and understand the reproductive biology of the spermatozoon. In recent years there has been a substantive change in the conceptual paradigm regarding what an ejaculate is. It should be said that until recently it was considered that the representative population (billions) of sperm was made up of "equivalent" cells with a common goal: to be the one that could finally fertilize the oocyte. The verification that the set of spermatozoa is grouped in distinct subpopulations, according to their kinematic and morphometric characteristics, opens the way towards a more cooperative vision. Besides, we have seen that the subpopulation distribution is different among males, which seems to indicate different strategies that can be understood within another paradigm, that of sperm competition between different ejaculates. The existence of heterogeneous subpopulations of spermatozoa in the ejaculate that show kinematics and morphometric patterns is widely known but the biological meaning of these different sperm subpopulations is still not clear. Although the role of the different subpopulations remains unknown, the work should continue in that direction.

There has been very limited use of computer assisted semen analysis (CASA) to evaluate reptile sperm. The aim of this study was to examine sperm kinematic variables in American crocodile (Crocodylus acutus) semen samples and to assess whether sperm subpopulations could be characterized. Eight ejaculates (two ejaculates/male) from four sexually mature captive crocodiles were obtained. An ISAS®v1 CASA-Mot system, with an image acquisition rate of 50 Hz, and ISAS®D4C20 counting chambers were used for sperm analyses. The percentages of motile and progressively motile spermatozoa did not differ among animals (P > 0.05) but there was a significant animal effect with regards to kinematic variables (P < 0.05). Principal component (PC) analysis revealed that kinematic variables grouped into three components: PC1, related to velocity; PC2 to progressiveness and PC3 to oscillation. Subpopulation structure analysis identified four groups (P < 0.05), which represented, on average, 9.8%, 32.1%, 26.8%, and 31.3% of the total sperm population. Males differed in the proportion of sperm in each of the kinematic subpopulations. This new approach for the analysis of reptile sperm kinematic subpopulations, reflecting quantifiable parameters generated by CASA system technology, opens up possibilities for future assessments of crocodile sperm and will be useful in the future development of assisted reproduction for these species.

There has been very limited use of computer assisted semen analysis (CASA) to evaluate reptile sperm. The aim of this study was to examine sperm kinematic variables in American crocodile (Crocodylus acutus) semen samples and to assess whether sperm subpopulations could be characterized. Eight ejaculates (two ejaculates/male) from four sexually mature captive crocodiles were obtained. An ISAS®v1 CASA-Mot system, with an image acquisition rate of 50 Hz, and ISAS®D4C20 counting chambers were used for sperm analyses. The percentages of motile and progressively motile spermatozoa did not differ among animals (P > 0.05) but there was a significant animal effect with regards to kinematic variables (P < 0.05). Principal component (PC) analysis revealed that kinematic variables grouped into three components: PC1, related to velocity; PC2 to progressiveness and PC3 to oscillation. Subpopulation structure analysis identified four groups (P < 0.05), which represented, on average, 9.8%, 32.1%, 26.8%, and 31.3% of the total sperm population. Males differed in the proportion of sperm in each of the kinematic subpopulations. This new approach for the analysis of reptile sperm kinematic subpopulations, reflecting quantifiable parameters generated by CASA system technology, opens up possibilities for future assessments of crocodile sperm and will be useful in the future development of assisted reproduction for these species.