The overall goal of this procedure is to cryopreserve mouse sperm. This is accomplished by first collecting sperm from mouse EPIs using cryo preservation straws. The straws are then frozen in liquid nitrogen where they're stored until they're recovered by thawing them in warm water.
The recovered mouse sperm are ultimately used for in vitro fertilization. The main advantage of this kit over existing methods is the kit contains all the necessary materials needed to cryopreserve and recover mouse sperm Generally in the individuals new to this method may strangle with handling and loading straws. Begin by assembling 15 sperm cryo-preservation straws per line of mice to be cryopreserved.
First, connect each 0.25 milliliter straw to a one milliliter syringe with the cotton plug closest to the syringe. Second load eight centimeters of IVF medium into each straw, followed by one centimeter of air. Third, label each of the 15 straws with the name of the line to be preserved for each line to be cryopreserved.
Fill a well of a four well dish with 240 microliters of cryo protective agent. Attach a one milliliter pipette to the handle of the freezing canister to extend the handle. Lastly, fill the foam insulated box provided with the kit with 15 centimeters of liquid nitrogen and close the lid euthanize.
Two fertile adult male mice. For each line to be cryopreserved and surgically, remove their codal EPIs. Place the mouse in dorsal recu and open the ventral body wall.
Pull gently on the vast deference and remove any excess fat. Next, locate the epididymus and remove it intact with a small piece of fast deference. Place the EPIs into a well of the prepared four well dish loaded with CPA Under a dissecting scope.
Make five to seven longitudinal cuts in each epididymus. Then using forceps, gently squeeze each fast difference. To push out the sperm, incubate the CPA and epididymis mixture at room temperature for three to five minutes.
Gently agitate the dish by hand two to three times during the incubation. This will allow sperm to swim out by aspiration. Load each prepared straw with five millimeters of the sperm suspension, followed by one centimeter of air.
It is important for the freezing process that all straws are loaded equally. After loading all 15 straws, carefully heat seal both ends of each straw. To ensure sperm survival, complete the process for all 15 straws within 10 minutes.
Once all the straws are prepared, load them into the freezing canister sperm column. First, place the canister into the foam box and allow it to float vertically in the liquid nitrogen. For 10 minutes, the canister must float vertically or the sperm freeze rate will be affected.
Then immerse the canister in the liquid nitrogen. The sperm is thus cryopreserved and is ready for long-term liquid nitrogen storage. Approximately 60 hours before a planned sperm recovery.
Follow the written protocol to super ovulate females before IVF. For each straw to be thawed, prepare and equilibrate all media for at least one hour at 37 degrees Celsius and 5%carbon dioxide. These materials include a 35 millimeter Petri dish with a 90 microliter drop of sperm treatment medium covered with oil.
A 35 millimeter Petri dish with three milliliters of IVF medium to serve as a cutting dish and per every five super ovulated females prepare an IVF dish, which is one well of a four well dish loaded with 500 microliters of IVF medium. For each IVF dish, prepare a washing dish, which is a 35 millimeter Petri dish with three milliliters of KSOM and an embryo culture dish, which is a 35 millimeter Petri dish with 50 microliter drops of KSOM medium covered with oil. With all the required materials prepared quickly remove a straw from storage and place it in a 37 degree Celsius water bath for two to three minutes.
Dry off the straw. Then make an angled cut in the air column below the cotton plug. At the other end of the straw, make another angled cut over another air column.
Avoid cutting the sperm column. Now aspirate the sperm suspension from the end of the straw, using a 200 microliter pipette and deposit it in the center of the sperm treatment medium.Drop. Then incubate the dish for 45 to 60 minutes.
During the sperm incubation, isolate the ucs of the super ovulated females and transfer them into the cutting dish. Use a 30 gauge needle or forceps to tear each oviduct releasing the cumulus cyte complexes. Transfer the C Cs from five females into each IVF.
Well, with a minimum amount of media. Avoid transferring fat and blood as dirty media inhibits fertilization efficiency. After the incubation, collect 10 to 15 microliters of the sperm suspension from the periphery of the STM drop and deposit the suspension into one IVF.
Well now co-culture the cells for four to six hours for fertilization. After the fertilization, pass the embryos through at least 2K SOM washes in the washing dish. Then culture.
The embryos in KSOM until the desired developmental stage is reached. Sperm from five Charles River inbred mouse strains were frozen. The protection ratios of sperm motility and rapid progressive motility ranged from 81%to 47%across five strains from those sperm.
The IVF rates with frozen thawed sperm varied in different strains from 26 to 88%over one and a half years. A total of 49 GM mouse lines were archived by sperm cryopreservation and later recovered by IVF in four different female strains. C 57 black six BC DBA one and 1 29 SV Once mastered the sperm cryopreservation technique can be done in 30 minutes if it's performed properly.
For this procedure, it's important to flow to canister vertically and liquid nitrogen.
