Sperm collection and computer-assisted sperm analysis in the teleost model Japanese medaka (Oryzias latipes)

Abstract

Japanese medaka (Oryzias latipes) are a teleost fish and an emerging vertebrate model for ecotoxicology, developmental, genetics, and physiology research. Medaka are also used extensively to investigate vertebrate reproduction, which is an essential biological function as it allows a species to perpetuate. Sperm quality is an important indicator of male fertility and thus reproduction success. Techniques for extracting sperm and sperm analysis are well documented for many species, including for teleost fish. Collecting semen is relatively simple in larger fish but can be more complicated in small model fish as they produce less sperm and are more delicate. This article therefore describes two methods of sperm collection in the small model fish Japanese medaka: testes dissection and abdominal massage. We demonstrate that both approaches are viable for medaka and show that abdominal massage can be performed a repeated number of times as the fish quickly recover from the procedure. We also describe a protocol for computer-assisted sperm analysis in medaka to objectively assess several important indicators of medaka sperm quality (motility, progressivity, duration of motility, relative concentration). The use of these procedures combined with the other advantages of using this small teleost model will greatly improve the understanding of the environmental, physiological, and genetic factors influencing fertility in vertebrate males.

Impact of light pollution at night on male reproductive success in Japanese medaka (Oryzias latipes)

“…The 10 minutes of video just before the female spawned were analyzed to measure three parameters: 1-protective (mate guarding) behavior, 2-aggressive behavior toward the other male, 3-amount of time spent spawning. For the protective behavior (supplemental video 1 (Closs et al, 2023)), we recorded the tank from above for 10 minutes with only 1-2 cm of water to prevent vertical swimming (figure 1A). We took a picture every minute from the video and measured the distance of each male to the female using ImageJ.…”

“…For the other two parameters, the video was recorded from the side of the tank. We manually counted the number of fights and "chase with hit" (respectively supplemental videos 2 and 3 (Closs et al, 2023)) as aggressive behavior during the 10 minutes prior to the female spawning. We measured the time spent spawning by each male, as defined by body contractions while alongside females (supplemental video 4 (Closs et al, 2023)), while the female was releasing eggs, as well as any premature spawning attempts during those 10 minutes.…”