S2215

The attraction of the sperm was estimated by using manufactured analysis chambers with cross‐type columns. The cross‐type columns were constructed to be 7 mm long vertically (width: 5 mm) and 41 mm long horizontally (width: 3 mm). They were connected to the object glass (S2215; Matsunami, Kishiwada, Japan) with a 13 mm long vertical (width: 1 mm) part by using double‐sided tape that was 50 μm in thickness (No. 7046; Teraoka, Tokyo, Japan) and then adhered together with a 22 mm× 40 mm cover glass (No. 1; Matsunami) and filled with BO medium (Figure 1). Both ends of the wide column were covered with mineral oil after the sperm were introduced.

To prepare coacervates in the absence of protein, 25 μL of polyanion solution were mixed with a proper volume of PLL with the cation to anion ratio (C : A) 1 : 1 via gentle pipetting. The final volume was adjusted to 120 μL by adding a proper buffer. Salt concentration was adjusted using a 2 M NaCl buffer before mixing polymer solutions. The mixture was equilibrated at 4 °C, at least, for 3 h followed by centrifugation at 1000 G, 10 min (KUBOTA 5922, Kubota Corporation, Osaka, Japan). The supernatant (∼100 μL) was carefully aspirated and used for DLS measurement (Malvern ZETASIZER PRO, Malvern Panalytical Ltd, Malvern, UK) and TEM (JEM-2010, JEOL, Ltd, Japan) observation. The formation of coacervate was verified by visual observation of distinct macro-phase separation in the tube and microscopic observation of the distinct droplet formation by brightfield imaging using a BZ-X810 microscope (Keyence Corporation, Osaka, Japan) with 40× objective lens (PlanApoλ NA 0.95, Nikon, Tokyo, Japan). For microscope observation, first, coverslip (22 × 40 mm, 0.17 mm thickness) was attached to the pre-cleaned standard glass-slide (S2215, Matsunami) using two-sided tape to have little gap in between glass-slide and coverslip. Then, approximately 20 μL of dispersed coacervate solution was loaded into the gap and observed under the microscope.