Embryo of Yellow Cattle

Cow

Embryo


Location of Preservation: Hengchun Research Station
Taiwan Livestock Research Institute
1 Ranch Road, Kenting
Hengchun, Pintung, Taiwan
Republic of China
Tel: 886-8-8861341~4
Fax: 886-8-8861345
Contributor: Mr. Shang-Hsiang Wen, Mr. Kuang-Fuh Lee

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Equipments for Embryo Collection

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  1. Flask for carrying PBS
  2. Lubricate
  3. 5 ml syringe
  4. 70% alcohol
  5. Procain anaesthetics
  6. PGF2a
  7. Penicillin antibiotics
  8. Gloves
  9. Hemostatic forcep
  10. Betadine
  11. 500 ml cylinder

Foley Catheter :

  1. catheter
  2. steel pole for insertion into the uterine horn
  3. upper left-inlet for inflation of air bubble cuff
  4. inlet for flushing and collecting embryo
  5. inflatable cuff

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Three-Way Bovine Collection Catheter

  1. Catheter with three inlets
  2. Stainless steel stilette with a central core and an introducer
  3. 50 ml syringe

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French Type Catheter

  1. three-way catheter
  2. 20 ml syringe for air inflation
  3. 50 ml syringe for flushing
  4. flask for fluid collection
  5. flask for flushing medium
  6. expanding pincers for fitting the air bubble onto the catheter
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Apparatus for Micromanipulation of Embryos

  1. Microscope for embryo examination
  2. Intracellular cryoprotecting medium: 10% glycerol, 20% 1, 2-propanediol
  3. Extracellular cryoprotecting medium: 25% glycerol, 25% 1, 2-propanediol
  4. Dilutent: 1 M sucrose
  5. Culture dish
  6. Pipette
  7. Straw

Programmable Freezer

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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

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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

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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

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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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