• In vitro fertilization (IVF):a process by which egg cells are fertilized by sperm outside the body: in vitro. The fertilized egg (zygote) is then transferred to the uterus with the intent to establish a successful pregnancy.

1. Collecting oocyte

• 1. Collected from ovary:
• 2).Living collecting:
• Transvaginal Oocyte Retrieval

2. In vitro maturation of oocyte

1. Culture medium: M199, plus serum and hormone
2. temperature: 38～39℃, 5％C02, air, 95% humility
3. Maturation time in vitro: the time for oocyte of cow and sheep is about 22-24h, horse 30-36h, pig 40-44h

3.Fertilized in vitro

1. Sperm preparation ：spin directly, swimming up; density centrifuge with percoll
2. Capacitation：heparin, Ca2+, higher osmosis solution
3. fertilization solution: Tyrode’s modified solution
4. method of fertilization：microdroping, microplate
5. Culture time and temperature: After fertilization, oocyte moved to 39℃，5％CO2 CO2 incubator for 24～44h.

4. In vitro culture

1. culture method: After fertilization, the oocyte moved to prepared granular cell using microdrop culture
2. Embry harvest : for the cow, from the maturation of oocyte to blastocyte, need 8 days

5. Embryo transfer

• Surgery implant： inject the solution with embryo to the Uterine horn， fallopian tube through the cut side of overy.