This technique enables the sampling of milt from a small model fish, Japanese Madaka, and the investigation of its quality in a consistent manner. Specifically sperm collection by abdominal massage allows repeated sampling on the same fish as this approach is not invasive. Furthermore, this technique can be applied for Madaka sperm cryo preservation.
Sperm quality is an essential indicator of fertilization success, and therefore an important parameter to analyze in ecological and whole productive physiology studies. To begin, prepare a soft holding sponge, by cutting it to fit smugly in a Petri dish. Cut a straight line in the middle of the sponge, that is long enough to hold the fish on its ventral side up.
After anesthetizing the fish, dry the abdomen of the fish by gently wiping it with a paper towel. Place the fish in the trough of the damp holding sponge with the ventral side up, so its gills are exposed to the anesthetic solution in the sponge. If the area around the cloaca is wet, gently dry the underside of the abdomen with a disposable tissue wipe.
Under a dissecting microscope, place the micro pipette with an attached aspirator tube against the cloaca of the fish. Now massage the abdomen of the fish by gently squeezing with a blunt end smooth forceps in a rostral to caudal motion. For recovery of the fish, release it from the sponge into the previously prepared recovery water before returning the fish to the aquarium system.
Transfer the collected milt into a tube containing preheated activating solution and pipette solution up and down several times using the aspirator tube assembly. Homogenize the diluted sperm gently by flicking the tubes before analysis. After euthanizing the fish dry it as demonstrated previously and place the fish under a dissecting microscope with its left lateral side facing up.
Using small dissecting scissors, cut a flap dorsally from the cloaca and then across the ribs to the gills to expose the internal organs. Locate the testes and cut the attachment at both ends with fine forceps. Remove the testes and transfer them to a prepared tube containing pre-heated activating solution.
Use the forceps to crush the testes several times against the side of the tube to release the sperm, which can be visualized by the slightly cloudy appearance of the solution. To begin the analysis, open the sperm analysis software and select the motility module. Set the configurations in the software for all parameters.
Place a pre-warmed disposable 20 micrometer counting chamber slide under the microscope on a heated stage, set to 27 degrees Celsius. Pipette the sample into the chamber on the slide until it fills, but without overfilling. Carefully wipe away excess samples from the entrance of the chamber to prevent drift movement of cells in the solution.
Select analyze to look at the sample under the microscope. Ensure the microscope is focused and select analyze again to record the sperm in the field. Move the slide to a new area of the sample in the frame and repeat the procedure for three to five different fields of view, avoiding air bubbles, cell masses, or artifacts.
To view the results, select results, and double click on a field to visualize the individual field or to manually check for any mislabeled or untracked spermatozoa. If necessary, right click on individual spermatozoa to relabel the motility. In the present study, computer assisted sperm analysis was used to record various velocity and movement parameters.
The calculated values include the straightness index, linearity index, and wobble. The osmolarity of the activation solutions impacted the motility of the sperm from testees dissection. The sperm were immotile in aquarium water, but motile in HBSS with high and low osmolarity.
However, motility is significantly reduced at 36 milli osmol per kilogram, compared to higher osmolality solutions. Motility was also affected by the sperm collection method, wherein the sperm are significantly more motile in the testis dissection method compared to abdominal massage. Also, a higher percentage of the cells were progressive and more cells were either medium or rapid moving.
The percentage motility and progressivity are significantly higher in the sperm stored at four degrees Celsius or on ice, compared to 27 degrees Celsius storage. The environmental and housing conditions also slightly impacted sperm motility. No significant difference in sperm motility was seen in the sperm collected after and before spawning.
Males housed without females for a month showed high motility, but the difference was insignificant. For sperm collection by abdominal massage, training is needed to learn the proper forceps pressure and movement and to avoid feced contamination. This technique will be very useful to investigate for instance, the effects of diverse pollutants on male fertility in ecotoxicologial studies, where Medaka is a commonly used model.
