A] Collection of embryos :
1) Sacrifice the 2.5-day post coital vaginal plug positive females by cervical dislocation to collect the 8-cell to compacted morulae-stage embryos.
2) Open the abdominal cavity and excise both the oviducts along with a small piece of the adjoining uterus and place in a drop of 50 µl of F1 (K-SICS-5000) solution in 60 mm Petri dish.
3) Flush the oviducts slowly with F1 solution using a 30- gauge blunt needle by holding the infundibulum using fine forceps under a stereomicroscope to expel the embryos.
4) Siphone the embryos with mouth pipette and transfer to a fresh drop of F1 medium, and wash several times in the same medium to get rid off the debris which may contribute to low viability.
5) Criteria for selection of embryos: Eight-cells to compacted morulae-stage embryos should be selected microscopically based on round form, normal size, normal cytoplasmic granulation and intact zona pellucida.
B] Freezing of embryos [2, 10]:
1) Transfer all clean embryos to F2 (KSICS- 5000) medium for 10 min of equilibration.
2) After 10 min, transfer the embryos to F3 (KSICS- 5000) medium for another 10 min.
3) Loading of Embryos in plastic straw: On average, 20–25 embryos can be loaded into each labeled 0.25 ml capacity plastic straw using three columns of F3 medium separated by air bubbles from a central column containing the embryos as shown in Fig. 1A.
4) Seal the open ends of the straws with the help of appropriately heated plastic sealing machine.
5) Insert the straws in a cryo-chamber in the vertical position, keeping the cotton plug at the upper side.
6) Cool down the straws as follows:
• From room temperature (24°C) to -7°C at the rate of 2°C/min. Initial holding time at -7°C may be for 5 min.
• Manual seeding is performed at -7°C by touching the cotton bud dipped in liquid nitrogen at the upper end of the central column of media containing embryos as shown in Fig. 1A.
• Lower the temperatures of straws to -30°C at the rate of 0.5°C/min.
• Lower the temperatures of straws from -30°C to -120°C at a rate of 1°C/min.
• After holding them at -120°C for 5 min, directly plunge the straws into liquid nitrogen for long-term storage.
C] Thawing of embryos :
1) Thawing is performed by quickly removing each straw from the liquid nitrogen, holding them in the room air at 24°C for 40 s and then dipping them in a 37°C water bath for 30 s.
2) Immediately after thawing, expel all the embryos in a drop of T1 media onto a petri dish. Transfer all embryos serially from T1 to T4 (K-SITS-5000) medium to remove the cryoprotectant as well as sucrose from the embryos with 5 min. of equilibration in each medium.
3) Viability is assessed based on the morphology of embryos observed under microscope (Fig. 1B).
D] In vitro and in vivo survival:
1) Wash the frozen-thawed embryos in T4 medium for 5 min and culture in a 75 μl drop of the Blastocyst Medium covered with paraffin oil in a petri plate.
2) Keep these plates in a CO2 incubator at 37°C in 5% CO2 for 24 h.
3) Survival of the 8-cell to morulae-stage embryos is assessed by their ability to develop into fully expanded blastocysts with a blastocoel cavity (Fig. 1C).
E] Uterine Transfer :
1) Weigh the 3.5 day old pseudo-pregnant mouse and use those animals whose body weight is ranging between 25-30 gm for embryo transfer.
2) Anesthetize the mice by Isoflurane gas anesthesia in ventro-dorsal position.
3) Load the transfer pipette in a sequence of small amount of F1 medium, small air bubble, F1 medium and, second air bubbledraw blastocysts with minimal volume of medium.
4) Wipe the back of the recipient mouse with 70% ethanol and remove the patch of the fur.
5) Make 5-10 mm. incision along the the paralumbar fossa. Slide the skin to the left or right until the the ovary (Orange-pink) or fat pad (White) are visible through abdominal wall.
6) Pick the abdominal wall with pointed forcep and make 5 mm of incision with fine scissor.
7) Pick up the fat pad associated over the ovary by blunt forceps and clip with Bulldog clamp so that ovary, oviduct and uterus remain outside the body wall.
8) Gently place the mouse under stereomicroscope and hold the top of the uterus with blunt forceps. With the help of 26- gauge needle make a hole in the uterus a few millimeters down from the utero-tubular junction. Make sure that the needle has entered in the uterine lumen but not lodged in the wall of the uterus. Keeping the eye on the hole made by the needle, pull out the needle and insert the transfer pipette into the lumen of the uterus. Gently blow the blastocysts into the uterine lumen leaving behind the air gap.
9) Unclip the Bulldog clamp and remove the mouse from stereomicroscope. With the help of blunt forceps, place the fat pad, ovary and uterus inside the body cavity and suture the abdominal wall with surgical thread and seal the skin wound by Vetbond.
10) After surgery, if the mice is still in unconscious stage, place the mice on warm plate maintaining temperature 37 °C for quicker recovery.
F] Vasectomy for making Sterile Males :
1) Anesthetize healthy male mouse of 4-5 weeks age by using Iso-flurane gas anesthesia.
2) Keep the mouse dorso-ventrally so that the abdomen of the mouse is exposed. Shave the area above the penis carefully and wipeout with 70% Ethanol.
3) Give approx. 1 cm transverse cut to the skin 1-2 cm above the penis with fine scissor. To approach the body cavity, again give the transverse incision to abdominal wall.
4) With the help of blunt forceps, gently pull the white fat pad associated above the testis so that testis along with vas deference and epididymis comes out.
5) Vas deference can be identified as small tube like structure running along with small blood vessel. Ligate the vas deferens at two sites by keeping approx. 1 cm distance and cut the middle portion of the ligation.
6) Repeat the procedure sl. no. 4 and 5 for other vas deferens of the same animal.
7) Slowly push the fat pad in the body cavity and suture the abdominal wall.
8) Seal the skin incision with Vetbond and place the mouse on thermal plate for recovery from anesthesia.
G] Making pseudo-pregnant females:
1) Select the females which are in natural estrus preferably CFW (SW), CD1 (ICR)/Cri and B6D2F1 hybrid strains which weigh in between 25-30 gm.
2) Avoid underweight as well as over weight females for pseudo pregnancy.
3) Place the one female with one vasectomised male in cage for mating.
4) Observe the vaginal plug in next morning and consider the pseudo pregnancy of 0.5 day.
5) Transfer the cultured blastocyst in utero-tubular junction of 3.5 day old pseudo- pregnant females.