To create first-generation (F1) hybrids of Mus subspecies, we crossed 2 M. m. castaneus males to 3 M. m. domesticus females and 2 M. m. domesticus males to 5 M. m. castaneus females in a harem-mating scheme. In total, we produced 8 male F1s in each direction of the cross. F1 males whose sire was M. m. castaneus (CAST genome) are referred to as CW, and those whose sire was M. m. domesticus (WSB genome) as WC. All males were housed individually for a minimum of two weeks prior to sacrifice between 90 and 120 days of age.
To enrich for viable sperm from each F1 male, we performed a standard swim-up assay [32 (link)]. First, immediately following sacrifice, we collected and flash-froze liver and tail control tissues (liver samples, N = 16; tail samples N = 8). Then, we removed and lacerated the epididymides of each male, placed this tissue in 1.5 ml of human tubal fluid (Embryomax HTF, Millipore), and maintained the sample at a constant 37°C for 10 minutes. Next, we isolated the supernatant, containing sperm that swam out of the epididymides, and spun this sample for 10 minutes at 250 g. We then discarded the supernatant, repeated the wash, and this time allowed sperm to swim up into the solution for an hour to select the most robust cells. Finally, we removed the solution, transferred them to new vial, pelleted these sperm by centrifugation, and froze them at -80°C.
To enrich for viable sperm from each F1 male, we performed a standard swim-up assay [32 (link)]. First, immediately following sacrifice, we collected and flash-froze liver and tail control tissues (liver samples, N = 16; tail samples N = 8). Then, we removed and lacerated the epididymides of each male, placed this tissue in 1.5 ml of human tubal fluid (Embryomax HTF, Millipore), and maintained the sample at a constant 37°C for 10 minutes. Next, we isolated the supernatant, containing sperm that swam out of the epididymides, and spun this sample for 10 minutes at 250 g. We then discarded the supernatant, repeated the wash, and this time allowed sperm to swim up into the solution for an hour to select the most robust cells. Finally, we removed the solution, transferred them to new vial, pelleted these sperm by centrifugation, and froze them at -80°C.