Cấy truyền phôi (Embryo transfer)

Embryo Transfer (ET)
Embryo
collection

Donor
cow

Repeat
Superovulation

AI
Fresh
embryo

Frozen
embryo

Recipient

Estrus
synchronization

Embryo
transfer

Calf


Advantage of ET

Advantage of ET
Treatment for repeat breeding cow
Efficiency of ET on repeat breeding cow
No. of ET

No. of Preg.

Preg. Rate (%)

Normal cow

28

23

82

Repeat breeding cow

23

16

70

Efficiency of ET of IVF embryos after AI on repeat breeding cow
Heifer
Cow

No. of ET

No. of Preg.

Preg. Rate (%)

AI+ET

61

30

49.2

ET

61

18

29.5

AI+ET

273

114

41.5

ET

137

28

20.4


Superovulation of the cow
Progesterone

Day 0

Estrus

AI

Luteal phase
10

11

12

13

14

15

Estrus/ovulation
FSH with decreasing manner
(2 times / day, 3 or 4 days)
PGF2α

Principle
　 Induction of growth and ovulation of multiple follicles
1. FSH treatment (decreasing manner)
Stimulate multiple follicle growth
2. Timing of FSH treatment
Stimulate 2nd follicular wave
3. PGF2α treatment
Control of CL regression and
induction of estrus/ovulation


FSH treatment (decreasing manner)
Half life of FSH: 5 hours
Twice / day, 3 to 4 days, 28-50 IU (total amount )
PGF2α treatment
Inject at 3 days after 1st FSH injection
Estrus will occur at 36-48 hours after PGF2α injection
Ovulation will occur at 24-36 hours after estrus
＊ eCG (equine chronic gonadotropin, PMSG)
Similar to FSH and LH
Half life: 40 hours
Large molecular weigh: induce antibody
and reduce response

Follicular wave occurrence
2-wave cycle: 2nd wave occur at 9-10 days after estrus
3-wave cycle: 2nd wave occur at 8-9 days after estrus,
3rd wave occur at 15-16 days after estrus
Follicle selection
FSH level decrease at 2-3 days after wave occurrence
Follicular growth depend on LH after follicle selection
Endogenous FSH
FSH peak appear at one day before wave occurrence
Treatment with exogenous FSH
Optimum timing:
from one day before to 2 days after wave occurrence



Improvement of response to hormonal treatment
　

Aspiration of large follicle or estradiol treatment
　　 Induction of new follicular wave by removing suppressive
effects of large follicle on the growth of another follicles
　　 Aspiration of large follicle
　　　　　　 New follicular wave will occur within 2 days
　　　　 Induction of atresia of large follicle by estrogen injection
　　　　　　 New follicular wave will occur at 3-4 days after injection
　

Progeaterone releasing device: CIDR, PRID
Luteal phase could be made artificially

Growth of bovine embryo
Oocyte

Ovary

fertilization 2-cell

fimbriae

4-cell

isthmus
ampulla
oviduct

Days after fertilizaion

8-cell

16-cell Morula Blastcyst

Uterotubal
junction

Uterus
horn

Haching

Embryo collection
Uterus flushing at 7 days after insemination
　（ embryos enter to uterus at 4-5 days after ovulation)
Flushing medium
Balloon catheter

Filter unit

Uterus flushing using balloon catheter


Anesthesia
to stop rectal contraction


Remove mucus of luteal phase


Balloon catheter

Injection of air or saline into balloon

Location of balloon


Location of balloon


Flushing medium

　 PBS
　　　 Glucose （ 1g/L ）
　　　 Sodium pyruvate （ 0.036g/L ）
　　　 Bovine serum albumin （ 3g/L ）
　　　 Antibiotics

3-way flushing line

Filter unit for embryo collection
　 Pore size: 70µm
　 Embryo diameter: 120-140 µm
Microscope
　 Searching embryo


How to recover flushing medium


Searching embryos


Development of bovine embryo

Immature oocyte

Unfertilized oocyte

2-cell embryo

4-cell embryo

8-cell embryo

16-cell embryo

Early blastcyst

Blastcyst

Morula embryo

Expanded blastcyst

Hatching blastcyst

Hatched blastcyst


Evaluation of the embryos
Regularity of shape (embryo and zona)
Compactness of blastmeres
Variation in cell size
Color and texture of cytoplasm
Overall diameter
Abnormal
Presence of extruded cells
Irregular shape
Presence of vesicles
Presence of vesicles

Normal
Regular shape

Presence of
extruded cells
Dark color
Presence of
degenerated cells

Clear color

A rank: Degenerated area <10%
B rank: Degenerated area 10-30
C rank: Degenerated area 30-50%
D rank: Degenerated area >50%
Evaluation of embryo quality


Pregnancy rate

Synchronization of estrus cycle
between donor and recipient

Difference in time of estrus cycle


Synchronization of estrus cycle
between donor and recipient

Synchronization of estrus cycle
Twice injection of PGF2α with 12 days apart

Hormonal treatment same as timed-AI
Ovsynch, PreSynch/Ovsynch
CIDR-Synch
PRID


Follicular growth
Oulation

Ovulation
Progesterone

FSH

0

5

10
15
Days after estrus

20

・ 2 or 3 wave of follicular gorwth
・ FSH stimulate small follicle growth (recruitment)

Hypothalamus

Hypothalamus

Pituitary

Pituitary
（−）

LH surge

（＋）

Pituitary

FSH

FSH

（＋）

LHRH

LHRH

LHRH

Hypothalamus

Ovary

Small follicle
growth by FSH

（−）

Inhibin

Inhibition of follicle
growth by dominant follicle

（＋）
Signal for ovulation
from mature follicle

Estradiol