Sperm motility with computer assisted sperm analysis (CASA)

Sperm motility was assessed with Computer Assisted Sperm Analysis (CASA) software using computer-aided video microscopy (CASA: Hamilton Thorne, Beverley, MA; microscope: Olympus BX41, Center Valley, PA). To prevent the need for multiple serial dilutions, egg-sperm bundle collection targeted the ideal sperm concentration (ca. 5 × 10⁶ to 5 × 10⁷ cells/mL) for the specialized fixed depth, 20 µm counting chambers (Leja Products BV, The Netherlands). Transfer pipettes were used to carefully collect the bundles in minimal seawater, and samples were then diluted with filtered seawater to the desired volume. Hawaiian Montipora corals have a toxin (found both in the adult tissue and eggs) that can immediately kill sperm if an egg is ruptured in a small volume of water⁹⁶. Due to concerns for the toxin and desired starting concentrations for CASA, egg-sperm bundles were collected in 15 mL tubes with 100 bundles in 10 mL of seawater for M. capitata (2018: n = 55, N = 9; 2019: n = 23, N = 16) and 100 bundles per 5 mL of seawater for Montipora spp. (2018: n = 137, N = 16; 2019: n = 61, N = 14). If fewer bundles were released (primarily for Montipora spp.), starting seawater volume was adjusted as needed to maintain the target starting sperm concentration.

The bundles were allowed to break apart with minimal agitation, and sperm was transferred to a separate 1.5 mL Eppendorf away from the eggs. The CASA slides were loaded with 4 μL of each sperm sample, and per standard CASA software recommendation, at least five videos and a minimum of 200 sperm were recorded per sample to record total motility. If needed and depending on concentration, sperm was diluted in filtered seawater to a target of 1 × 10⁷ cells/mL. Both initial and one-hour motility measurements were taken for most samples; a new slide of the existing sample was loaded for the one-hour reassessment. CASA parameters for all samples were modified from Zuchowicz et al.⁹⁷.