Sperm kinematic subpopulations of the American crocodile (Crocodylus acutus).

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.

Análisis de subpoblaciones cinéticas y morfométricas de espermatozoides con sistemas CASA: revisión

La valoración subjetiva de la calidad seminal ha dado paso al uso de las técnicas objetivas de valoración por medio de la tecnología CASA (computer-assisted semen analysis). Se pueden aplicar una serie de pruebas cinéticas, morfométricas, morfológicas o de integridad del ADN para caracterizar y entender la biología reproductiva del espermatozoide. En los últimos años, se ha dado un cambio significativo de paradigma conceptual respecto de qué es un eyaculado. Cabe decir que, hasta tiempos recientes, se consideró que una población representativa (miles de millones) de espermatozoides estaba formada por células "equivalentes" con un objetivo común: ser el que finalmente pudiese fecundar el ovocito. La comprobación de que el conjunto de espermatozoides se agrupa en subpoblaciones bien definidas de acuerdo con sus características cinéticas y/o morfométricas, ha abierto el camino hacia una visión más cooperativa. Además, se ha visto que la distribución subpoblacional es diferente entre individuos, lo que parece indicar diferentes estrategias que se pueden entender dentro de otro paradigma: el de la competencia espermática entre diferentes eyaculados. A pesar de que aún no se conoce el papel de las diferentes subpoblaciones, se deben continuar los trabajos en esa dirección. En esta revisión, se caracterizó la evaluación de la fertilidad con la tecnología CASA y se examinaron los principales métodos multivariados en la evaluación de subpoblaciones de espermatozoides analizados mediante un sistema CASA.

In recent years, there has been a substantive change in the conceptual paradigm regarding what an ejaculate is. Until recently, it was considered that a 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. New data suggesting that a set of spermatozoa is grouped in distinct subpopulations, according to their kinematic and morphometric characteristics, has opened the way towards a more cooperative vision. In addition, recently, it has been established that the subpopulation distribution is different among males, which seems to indicate that there are different strategies that can be understood within another paradigm: sperm competition between different ejaculates. These heterogeneous subpopulations of spermatozoa in the ejaculate, that show kinematics and morphometric patterns, has been widely known for a while, but the biological meaning of these different sperm subpopulations is still not clear. For this reason, the subjective evaluation of seminal quality is being displaced by objective assessment techniques using CASA technology (computer-assisted semen analysis). Also, the application of principal components (PC) and clustering methods to reveal subpopulations of spermatozoa are complementary tools used to characterize raw semen and processed cell suspensions. Despite the advances in such powerful tools, researchers are not well versed on such techniques and its advantages. For instance, PC analysis is a multivariate statistical method that reduces the number of variables used in subsequent calculations for describing the data. By integrating the original data into new complex mathematical variables, homogenous subpopulations of spermatozoa can define clearly. Furthermore, kinematic, morphometric, morphological or DNA integrity tests can be also applied to characterize and understand the reproductive biology of the spermatozoon. Although the role of the different subpopulations of spermatozoa remains unknown, the work should continue by implementing novel technologies and coordinated tools. In this review, we characterize the fertility evaluation with CASA technology and examine the main multivariate methods in the assessment of sperm subpopulations analyzed by CASA systems.

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

The development of analytical methods for the evaluation of crocodilian semen is an important component for the assessment of male breeding soundness and the development of assisted breeding technology in this taxon. Computer-Assisted Semen Analysis (CASA) technology is becoming an increasingly common technique in seminal evaluations for animals but there has been no application of this technique for reptilian spermatozoa. The aim of this study was to analyse sperm kinematic and morphometric variables in Caiman crocodilus fuscus semen samples and to determine whether there were sperm subpopulations. Four ejaculates from four sexually mature captive caimans were used for this study. A CASA-Mot and CASA-Morph system was used with an image acquisition rate of 50 Hz for 2 s of capture. The ISAS®D4C20 counting chambers were used and spermatozoa incubated at 25 °C. Total and progressive motilities did not differ among animals (P > 0.05). There was a significant animal effect in the model with respect to sperm morphometry, and kinematic indices including linearity (LIN) and straightness (STR) (P < 0.05). Results for principal component (PC) analysis indicated variables were grouped into four components: PC1 related to velocity, PC2 to progressivity, PC3 to oscillation and PC4 to sperm path cross-linking. Subpopulation (SP) structure analysis indicated there were four groups, namely, rapid non-progressive (SP1), slow non-progressive (SP2), rapid progressive (SP3) and medium progressive (SP4), representing 14.5%, 45.4%, 18.7%, and 21.4% respectively. Findings in the present study indicate the importance of continuing development of reliable protocols regarding the standardisation of computer-based semen analyses in reptilian species.