Sperm Collection and Computer-Assisted Sperm Analysis in the Teleost Model Japanese Medaka (<em>Oryzias latipes</em>)

Sperm handling and management in the teleost model fish Japanese medaka (Oryzias latipes)

“…In our previous study, we found that medaka sperm were motile in HBSS (287 mOsm/kg) 13 , in accordance with the finding from Yang et al 2 . Therefore, we chose this osmolality (~ 300 mOsm/kg hereafter as HBSS 300) for testing sperm activation in two different activation solutions (prepared in the laboratory by the authors): (1) Hanks' balanced salt solution (HBSS 300) (137 mM NaCl, 5.4 mM KCl, 1.3 mM CaCl 2 , 1.0 mM MgSO 4 , 0.25 mM Na 2 HPO 4 , 0.44 mM KH 2 PO 4 , 4.2 mM NaHCO 3 , and 5.55 mM glucose, pH 7.2, 300 mOsm/kg) and (2) Kurokura (K 180) (180 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl 2 , and 2.38 mM NaHCO 3 ).…”

“…While it is more aligned with natural conditions for sperm to become active in the water where the fish raises and acclimates 14 , only a limited number of previous studies have used aquarium water for medaka sperm activation. In a previous study, the sperm were immotile in aquarium water 13 . In the current study, we observed either a complete absence of motility or very short motility lasting less than a few seconds in aquarium water (data are not shown).…”

“…In most previous studies, medaka sperm was collected after dissection of the testes 2,8,9 , and to the best of our knowledge, there is only one study that used the sample collected by stripping not into the tubes but directly in a cavity slide containing test solution 10 . In our previous study, we demonstrated the technique of sperm collection by abdominal massage into capillary tubes 13 . Sperm collection using stripping under anesthesia has several advantages over testes dissection.…”

“…The fish were fed twice per day with Gemma micro 500 (Skretting, Norway) and once per day with baby brine shrimps (Artemia spp.). Methods for sperm collection are described in our previous methodological study 13 . We used the same methods with some adjustments where needed.…”

“…Total motility was analyzed using computer assisted sperm analysis (CASA) and IVOS II system (IMV technologies) using Leja chamber slides (Leja, Netherlands) with a depth of 20 µm (Leja20, four-chamber, LOT: 481815B1) (see Supplementary Video S1). The settings for CASA software were adapted for medaka based on our previous study 13 . The following CASA parameters were analyzed: (1) total motility (%); (2) progressive motility (%); (3) kinematics parameters: curvilinear velocity (VCL, µm/s), straight-line velocity (VSL, µm/s), average path velocity (VAP, µm/s), linearity (LIN = VSL/VCL, %), wobble (WOB = VAP/VCL, %) and straightness (STR = VSL/ VAP, %).…”