Embryo of Yellow Cattle

Taiwan Livestock Research Institute

1 Ranch Road, Kenting

Hengchun, Pintung, Taiwan

Republic of China

Tel: 886-8-8861341~4

Fax: 886-8-8861345

Equipments for Embryo Collection

　

	Flask for carrying PBS Lubricate 5 ml syringe 70% alcohol Procain anaesthetics PGF2a Penicillin antibiotics Gloves Hemostatic forcep Betadine 500 ml cylinder
	Foley Catheter : catheter steel pole for insertion into the uterine horn upper left-inlet for inflation of air bubble cuff inlet for flushing and collecting embryo inflatable cuff

　

Three-Way Bovine Collection Catheter Catheter with three inlets Stainless steel stilette with a central core and an introducer 50 ml syringe

　

French Type Catheter three-way catheter 20 ml syringe for air inflation 50 ml syringe for flushing flask for fluid collection flask for flushing medium expanding pincers for fitting the air bubble onto the catheter

　

　

Apparatus for Micromanipulation of Embryos Microscope for embryo examination Intracellular cryoprotecting medium: 10% glycerol, 20% 1, 2-propanediol Extracellular cryoprotecting medium: 25% glycerol, 25% 1, 2-propanediol Dilutent: 1 M sucrose Culture dish Pipette Straw

Programmable Freezer

Protocol of Embryo Cryopreservation

A. Collection of Embryo

1. Induction of estrus: 　Method A: 1500~3000 IU PMSG, 48 hours after, 2~3 ml PGF2a, 　　　　　 36~48 hours after, 1500~2500 IU HCG intravenously　 　Method B: 　　day 1: 1.2 ml FSH in AM; 1.2 ml FSH in PM 　　day 2: 0.8 ml FSH in AM; 0.8 ml FSH in PM 　　day 3: 0.4 ml FSH and 3 ml PGF2a in AM; 0.4 ml FSH in PM 　　day 4: 0.4 ml FSH in AM; 0.4 ml FSH in PM 　　day 5: 1500~2500 IU HCG in AM 2. Breeding with artificial insemination or nature service 3. Embryo collection on 6~8 days after mating

　

　

B. Cryopreservation of Embryo

1. Bovine embryos were collected into petri dish for examination and selection of 8-cell stage embryo.
2. Good and excellent grade embryos are ready for freezing.

Vitrification method

　

3. Feezing methods:

1. PBS wash 3 times
2. Move embryo into 1 M sucrose/PBS for 10 minutes at 20C
3. Pipette embryos into 25% glycerol-25% 1, 2-propanediol/PBS
4. Put into 0.25 or 0.5 ml straw as indicated
   
5. Seal with straw seal-powder
6. Put straw into liquid nitrogen for cryopreservation

　

Programmable Freezing Method

(1). Medium:

Solution A: PBS+10% FCS (54 ml PBS+ 6 ml FCS = 60 ml)　
Solution B: PBS+10% glycerol (27 ml Solution A+ 3 ml glycerol =30 ml)
Soultion C: PBS+5%  glycerol (5 ml Solution A+5 ml Solution B =10 ml)

(2). Freezing steps:

a. Embryos wash with Solution A three times at room temperature
b. Put embryos into Solution C for five minutes at room temperature
c. Put embryos into Solution B for 10~20 minutes at room temperature
d. Pipette embryos into the straw as indicated
e. Sealed straw were put into the programmable freezer:
              Fast cooling from 20 down to 0 C
              1 C/min from 0 C to -7 C
              Seeding for 5~10 min at -7 C
              0.3~0.6 C/min from -7 C to -35 C
              Put into liquid nitrogen (-196 C)
f. Move frozen straw into liquid nitrogen container for cryopreservation



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