Our primary research focus is the development of assisted reproduction techniques that provide refinements and reductions in keeping with the 3Rs of animal experimentation. We have developed non-surgical embryo transfer and non-surgical artificial insemination procedures for mice and a non-surgical embryo transfer procedure for rats. With the addition of the cervical manipulation procedure to provide pseudopregnant females, we create 3Rs improvements and assisted reproduction in rodents.
The protocols described here provides streamlined methods for recovering pups using non-surgical embryo transfer methods and artificial dissemination in mice. The cervical manipulation protocol provides a refinement of procedures producing pseudopregnant female recipients for assisted reproduction techniques. It reduces the number of females needed as pseudopregnant recipients, and it eliminates the need for vasectomized males.
The protocols outlined here can be used for strategic vivarium management. The protocols can be used with cryopreservation in vitro fertilization, rederivation, the production of age match cohorts, and the production of genetically modified animals. To begin cytological evaluation for confirming ESRA cycle synchronization, gently collect the vaginal cells from the CD1 mouse by rolling the swab against the vaginal wall using a small premoistened 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 brightfield 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 tremor. 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.
Begin non-surgical embryo transfer in the CD1 mouse on day 6 by placing a 20 microliter drop of M2 medium onto the lid of a tissue culture dish. Use reflective lighting under a microscope and load 10 to 20 blast assists into the M2 medium drop with a standard embryo handling pipette. Secure the non-surgical embryo transfer device onto a P2 pipette set to 1.8 microliters, and remove the protective catheter cover.
Press the plunger of the pipette to the first stop and lower the tip of the catheter into the medium before slowly pulling the embryos into the catheter tip of the device. Then remove the catheter from the medium. Next, set the pipette volume to 2.0 microliters to generate a small air bubble at the tip of the catheter.
Gently lay the pipette loaded with the embryos near the mouse cage. Place the recipient female on the wire rack cage top and hold the mouse in position by grasping near the base of the tail using the forefinger and the thumb, then angle the tail upward while stabilizing the animal. Place a small speculum into the vagina and insert the transfer device catheter into the speculum through the cervix and into the uterus.
Once the device hub contacts the speculum, dispense the embryos by pressing the pipette plunger to the first stop. Avoid the transfer of extra air into the uterine horn. Remove the device and speculum.
