Inter and intra stallion analysis of the kinematic sperm subpopulations in fresh ejaculates of Pura Raza Española horses

PIROSANTO YAMILA; TERAN ESTER; MOLINA ANTONIO; DEMYDA-PEYRÁS, SEBASTIÁN

Lima

Congreso; 49th international Embryo Tecnology Society; 2023

Artificial insemination is one of the most used reproductive assisted technologies employed in the Pura Raza Española breeding system. But also, stallions are known for an extensive variability in semen quality, being some individuals less suitable for the gamete cryopreservation. This fact limits their use as breeders, particularly in foreign countries. Several studies have demonstrated that using CASA systems, it is possible to identify and quantify different sperm subpopulations within an ejaculate with specific patterns of movement due to its objectivity and repeatability. Some of those subpopulations were linked positively with the fertility of stallions in field conditions. But also, the existence of specific subpopulation patterns at the individual level was also previously described. In this study, we aim to determine the existence of subpopulations in Pura Raza Española stallions based on sperm kinetic data obtained from CASA. 141 ejaculates from 24 Pura Raza Española stallions (ranging from 6 to 14 years of age) were collected between February 2021 and 2022. Collections were made by natural mating using a Missouri AV. Samples were first adjusted to a standard concentration of 30 x 106 spz/mL and characterized kinematically using a CASA Androvision™ system. In each ejaculate, we determined eight different parameters, including curved line velocity (VCL, um/s), velocity average path (VAP, um/s), velocity straight line (VSL, um/s), amplitude of lateral head displacement (ALH, um), beat-cross frequency (BCF, Hz), linearity (LIN, VSL/VCLx100), righteousness (STR, VSL/VAPx100) and oscillation (WOB, VAP/VCLx100). In total, in 239,546 spermatozoa were analyzed. Thereafter, we performed a principal component analysis (PCA) followed by a hierarchical and non-hierarchical clustering procedure to assign each spermatozoon to a given kinematic subpopulation. Finally, differences among individuals and ejaculates were analyzed using a GLM statistical test, where the dependent variable is the proportion of the individual corresponding to each subpopulation and the individual and ejaculates were used as a fixed factors.Results showed that PCA clustering based on velocities (VAP and VSL) explained 46% of the variation, whereas PCA analysis based on linearity and head oscillation (LIN and ALH) accounted for 34%. These results allowed to cluster of the spermatozoa in 3 kinematic subpopulations: fast and progressive (SP1), fast but non-linear (SP2), and slow (SP3). Statistical differences were observed among individuals (p