To begin cytological evaluation for confirming estra cycle synchronization, gently collect the vaginal cells from the CD1 mouse by rolling the swab against the vaginal wall using a small pre moisten swab smear the collected vaginal cells into a 20 microliter drop of sterile water on a microscope slide and air dry. Under a microscope, use 100X magnification with bright field illumination to evaluate for the presence of cornified epithelial cells. For cervical manipulation, place a recipient female on the top of a cage with a wire rack approximately 0.5 hours before insemination allowing the mouse to grab the cage bar surface grasp near the base of the mouse's tail using the thumb and forefinger and angle the tail upward while stabilizing the animal.
Insert the blunt end of a small plastic rod vaginally to contact the cervix and vibrate for 30 seconds by contact with the trimmer. To perform a non-surgical sperm transfer for artificial insemination, place the insemination device on a P200 pipette set to 40 microliters and remove the protective cover. Remove an aliquot of the capacitated sperm sample from the dish placed at 37 degrees Celsius and 5%carbon dioxide and transfer it to a 35 millimeter tissue culture dish without oil at 37 degrees Celsius, press the plunger of the pipette to the first stop.
Lower the tip of the catheter into the sperm sample and slowly load 40 microliters of the sample into the transfer device. Remove residual oil from the exterior of the insemination device using an absorbent tissue before setting the pipette aside. Next place the recipient female on the wire rack cage top.
Hold the female mouse in position by grasping near the base of the tail, using the thumb and forefinger and angle the tail upward while stabilizing the animal. Place the small speculum into the vagina and insert the insemination device catheter into the speculum through the cervix and into the uterus. Once the device hub contacts the speculum dispense the sperm by pressing the pipette plunger to the first stop.
Once done, remove the device and speculum.
