Validation of computer-assisted sperm-motility analysis in the amphibian Silurana tropicalis

We have developed and validated a computer-assisted sperm-motility assessment (CASA) method for use with the emerging amphibian model Silurana tropicalis. The testicular sperm-activation method was validated by analysing activation replicate coefficients of variation, effects of tracking time settings on velocity distributions and the relative partitioning of differentially motile sperm subpopulations between matched right and left testes. Two major sperm subpopulations were identified using multivariate pattern analysis and their relative frequencies were consistent between samples from matched right and left testes and from randomly drawn subsets of six frogs sampled from the total set of 16 frogs. The power of this approach for detecting treatment effects targeting the hypothalamic-pituitary--gonadal axis was investigated by injecting a group of frogs with 100IU human chorionic gonadotrophin (hCG) 2h before sampling and comparing their sperm-subpopulation frequencies with non-injected controls. While parametric analysis across sperm samples failed to detect treatment effects, subpopulation analysis showed that hCG significantly increased the proportion of progressive and non-sinuous spermatozoa compared with controls (Chi square=6.40, DF=1, P=0.011). This demonstrated the potential value of analysing objectively measured sperm behaviour as an endpoint.