Embryo of Taoyuan Pig

Sow and
 Piglets

Embryo
Location of Preservation: Department of Animal Physiology
Taiwan Livestock Research Institute
112 Farm Road
Hsinhua, Tainan 71210 Taiwan
Republic of China
Tel: 886-6-5911211
Fax: 886-6-5911210
Contributor: Mr. Ming-Che Wu, Mr. Lin-Ren Chen, Mr. Teng-Tsao Hsu

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Protocol of Embryo Cryopreservation

A. Collection of Embryo 

1. 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

  1. Take straw out from liquid nitrogen container and put directly into 37 C water bath for thawing (approximately 1~3 minutes).

  2. Find embryos in 0.5 M glycerol/PBS and stay for 10 minutes.

  3. Transfer embryos into 0.25 M glycerol/PBS and keep 10 minutes.

  4. Pipette 16% FCS/PBS medium and drop slowly into the embryo culture.

  5. Pipette out embryos and wash with 16% FCS/PBS three times.

  6. Embryo transfer can be conducted by putting embryos into uterine horns after examination.

¡@ The Appearance of Pig Hatched Blastocyst After Thawing

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