Bx53 differential interference contrast microscope

After the fertility test, three knockout male mice and three same-aged wild-type B6D2F1/J mice were euthanized by cervical dislocation following anesthesia to examine their testis weights, testicular and epididymal histology, sperm morphology, and sperm motility. Testes and epididymides were fixed in Bouin fluid (Polysciences, Inc., Warrington, PA), embedded in paraffin wax, sectioned at a thickness of 5 μm on a Microm HM325 microtome (Microm, Walldorf, Germany), and stained with 1% periodic acid (Nacalai Tesque, Kyoto, Japan) and Schiff's reagent (Wako, Osaka, Japan) followed by counterstaining with Mayer's hematoxylin solution (Wako, Osaka, Japan). Spermatozoa were extracted from cauda epididymis and dispersed in TYH medium for 10 min. Sperm morphology was observed under an Olympus BX53 differential interference contrast microscope equipped with an Olympus DP74 color camera (Olympus, Tokyo, Japan). Sperm motility was measured with the CEROS II sperm analysis system (Hamilton Thorne Biosciences, Beverly, MA) at 10 min and 2 h of incubation.

After euthanasia, testes and epididymides were dissected. After measuring the testicular weight, testes, caput and cauda epididymides were fixed in Bouin’s fluid (Polysciences, Inc., Warrington, PA, USA), and embedded in paraffin, sectioned rehydrated and treated with 1% periodic acid for 10 min, followed by treatment with Schiff’s reagent (FUJIFILM Wako, Osaka, Japan) for 20 min. The sections were stained with Mayer’s hematoxylin solution (FUJIFILM Wako) prior to imaging and observed using an Olympus BX53 differential interference contrast microscope equipped with an Olympus DP74 color camera (Olympus, Tokyo, Japan).