Contributor: Mr. Ming-Che Wu, Mr. Lin-Ren Chen, Mr. Teng-Tsao HsuTaiwan Livestock Research Institute 112 Farm Road Hsinhua, Tainan 71210 Taiwan Republic of China Tel: 886-6-5911211 Fax: 886-6-5911210
A. Collection of Embryo1. Induction of estrus: Method A: s.c. 1000 IU PMSG 72 hours after, 500 IU HCG intravenously. Often for prepubertal gilts. Method B: often for sows day 1 : i.m. 500 ug PGF2a day 3 : 1000 IU PMSG intramuscularly day 6 : 500 IU HCG intravenously 2. Breeding with artificial insemination or nature service 3. Embryo collection on 6~7 days after mating
B. Cryopreservation of Embryo
1. Embryos were collected into petri dish for examination. 2. Good and excellent grade embryos are ready for freezing. 3. Freezing steps: a. 16% FCS/PBS wash 3 times b. Move embryo into 0.25 M glycerol/PBS for 10 minutes c. Pipette embryos into 0.5 M glycerol/PBS for 10 minutes d. Pipette embryos into 1.0 M glycerol/PBS for 10 minutes e. Pipette into 0.25 ml straw and seal. f. Put straw into liquid nitrogen for cryopreservation g. Sealed straw were put into the programmable freezer: Fast cooling from 20 C down to 0 C 1 C/min from 0 C to -6.8 C Seeding for 5~10 min at -6.8 C 0.3 C /min from -6.8 C to -3.5 C Put into liquid nitrogen (-196 C) 4. Move frozen straw into liquid nitogen container for cryopresservation
Thawing of Frozen Embryo
Take straw out from liquid nitrogen container and put directly into 37 C water bath for thawing (approximately 1~3 minutes).
Find embryos in 0.5 M glycerol/PBS and stay for 10 minutes.
Transfer embryos into 0.25 M glycerol/PBS and keep 10 minutes.
Pipette 16% FCS/PBS medium and drop slowly into the embryo culture.
Pipette out embryos and wash with 16% FCS/PBS three times.
Embryo transfer can be conducted by putting embryos into uterine horns after examination.
¡@ The Appearance of Pig Hatched Blastocyst After Thawing