We collected mature, unfertilized eggs from our golden breeding colony, which is maintained in flow-through Plexiglas aquaria as part of a recirculating system. You can find further details on how the colony is maintained in "How We Made it" as part of "The Need for a Histology Atlas of the Medaka Post-Hatch Development".
To fertilize the eggs, we followed
in vitro fertilization techniques originally described by Yamamoto (1939). We selected mature, ovulating females and separated them from males for 48 hours. After anesthetizing the fish using tricaine methanesulfonate (MS-222), we gently stripped the eggs by applying light pressure to the abdomen with a blunt spatula with a rostral to caudal motion just above the location of the ovary. This method allowed clusters of eggs to be released from the ovarian cavity. Immediately after collection, the egg clusters were transferred into Ringer’s solution (Yamamoto 1939) and then separated into individual eggs using surgical scissors. Ringer´s medium, with an osmolality around 267 mOsmol/kg, closely matches the osmotic conditions inside the egg (260 to 320 mil mOsmol/kg). This balance helps preserve egg viability, extending the fertilizable period. Females were transferred to a recovery bath with gently aerated water. We selected only non-activated, mature eggs—identified by the absence of a cortical reaction and the even distribution of oil goblets around the periphery—and transferred them in groups of about 10 into the wells of a 24-well plate filled with Ringer’s solution.
To collect sperm, mature males were also anesthetized (MS-222) after being isolated from females for 96-120 hours. We used a Pasteur pipette (1.19-mm internal diameter) to collect milt by placing the tip at the urogenital opening and gently pressing the abdomen with blunt forceps. The collected sperm was then added directly to the wells containing the eggs. Males were then returned to freshwater to recover. Fertilization was confirmed by observing the characteristic fertilization wave (Yamamoto, 1939), and the time of fertilization was recorded. Fertilized eggs were then transferred to small glass dishes with system water and incubated under semi-static conditions (14 hours light: 10 hours dark; 25 ± 3°C) with daily water renewal.
We staged the embryos based on features visible through a dissecting stereomicroscope (70-80X). To photograph each stage, we used an Olympus SZH dissecting stereomicroscope equipped with an Olympus C-5060 digital camera. Each image shows two embryos at the same developmental stage, viewed from different angles to help highlight key structures. To hold them in place temporarily, eggs were placed in a depression plate with a 1-2% methylcellulose solution, then rinsed in dechlorinated tap water after imaging. Each egg was used only once for observations and photography for each developmental stage. Developmental times are shown in hours and minutes post-fertilization (hpf and min), and are intended as general guidelines only. Normal biological variability often leads to differences among individuals, even under controlled conditions.
To preserve authenticity, none of the images were modified or digitally altered. From the thousands of photos captured, we carefully selected the final images featured in this atlas. These were chosen for being the most representative and for clearly showing the features that define each developmental stage of the medaka embryo.