Factors influencing success of embryo transfer programs

Factors influencing success of
embryo transfer programs
John F. Hasler
Bioniche Animal Health USA, Inc.
jfhasler05@msn.com

The goal of bovine embryo transfer

Some variables in embryo transfer
Donors
Breed
Parity
Age
Genetics
Lactational status
Reproductive history
Nutrition
Climate
Season
Superovulation
gonadotropin
dose
route of injection
frequency of inj.
stage of cycle
method of synchr.
Insemination
quality
no. sperm
Management system
Stress
Embryos
Super vs. single
Donor
Breed
Age
Fertility
Sire
Size of response
Age of embryo
Stage of embryo
Quality of embryo
Storage medium
Length of storage
Conditions of storage
Fresh
Frozen
Split
IVF
Cloned
Transgenic
Recipients
Breed
Parity
Age
Lactational status
Reprod. history
Nutrition
Climate
Season
Estrus induction
Synchrony w. embryo
CL quality
Side of transfer
Method of transfer
Site of transfer
Drugs/hormones
Stress
Practitioner
Experience
Skill
‘Luck’

Gonadotrophin preparations used in commercial
embryo transfer in the past 35 years

Number of bovine flushes and embryos
recovered yearly in the USA between
2002 and 2008
Year No. Flushes No. Embryos Mean no. Emb. / Flush
2002 28,109 172,118 6.1
2003 34,896 205,441 5.9
2004 40,701 248,469 6.1
2005 48,233 305,129 6.3
2006 51,802 319,984 6.2
2007 54,808 332,486 6.1
2008 52,804 329,171 6.2
(Compliments of the American Embryo Transfer Association)

Superovulation variability among beef
cattle in Texas over time
1986 2001
No. collections 2,048 1,156
Mean total ova 11.5 12.7
Mean degenerates 2.2 2.4
Mean embryos 6.2 6.5
Mean UFO 3.1 3.8
(Looney, 2004)

Effect of cattle breed on superovulation
Beef a No. supered Total ova Good embryos % good
Beefmaster 80 13.8 7.8 57
Brangus 1290 12.0 6.4 53
Brahma 144 8.9 5.6 63
Simmental 141 10.5 6.5 62
Dairy b
Holstein 6787 11.6 6.2 53
Ayrshire 332 14.8 7.4 50
Brown Sw. 84 10.1 5.5 54
Jersey 332 14.3 7.3 51
( a Looney, 1986; b Sauvé, 2003)

Effect of age on superovulatory responses
of 633 reproductively-healthy Holsteins
Age group No. animals Mean no. ova
Mean no. good
embryos
Heifers 28 6.1 3.8
2 – 6 years 308 10.4 6.7
7 – 10 years 224 10.6 6.9
11 – 14 years 64 9.7 5.3
>15 years 9 5.6 2.6
(Hasler, et al., 1983)

Variation in superovulation among 5
individual Holstein cows
No. Mean % % Mean no.
Donor supers. total ova* unfert.* degen.* good*
A 11 18.6 45 7 8.9
B 13 12.2 26 31 5.2
C 9 10.3 7 1 9.2
D 15 8.7 10 14 6.3
E 13 5.4 36 15 2.5
*P

8
Effect of day FSH started
on superovulation
No. transferable embryos
7
6
5
4
3
2
1
Donaldson
Hasler
McDonald
Steel
0
6 7 8 9 10 11 12 13
Day of cycle FSH started
14
Donaldson, n = 933 (beef); Hasler, n = 639 (dairy); McDonald, n = 3,724 (dairy);
Steel, Donaldson, n = 2,340 n = (dairy)
(Donaldson, 1984; Hasler et al., 1983; McDonald, unpub.; Steel, unpub.)

CIDR®
(controlled intravaginal drug releasing device)

Superovulation protocol set-ups
1
Day 0* 1 2 3 4 10
FSH FSH FSH FSH Heat Flush
(AM & PM) (AM & PM) ( AM & PM) (AM & PM) (AM)
PG
(Am & PM)
Heat
(PM or D4 )
(*Day 8-13 of cycle)
2
Day 0 4 5 6 7 9 16
CIDR FSH FSH FSH FSH Heat Flush
(AM & PM) (AM & PM) (AM & PM) (AM & PM)
EB + P4
PG
(AM & PM)
CIDR X
(PM)
3
Day 0 2 4 5 6 7 8 9 16
CIDR GnRH FSH FSH FSH FSH FSH Heat Flush
(PM) (PM) (AM & PM) (AM & PM) (AM & PM) (AM)
PG
(PM)
PG
(AM)
CIDR X
(AM)
GnRH

Superovulation Results
(6 ET practitioners in 6 different states)
Set-up No. donors Total ova Good embryos
1 16 16.0 9.1
1 8 15.3 9.0
1 12 8.2 4.1
2 7 22.4 15.3
2 10 11.0 2.9
3 10 14.8 8.1

Reducing the number of injections
involved in superovulation
8 injections versus 2 injections of FSH
Fewer trips through the chute =
• savings in labor & expense
• less stress on cattle
• reduction of injury to personnel and cattle

Superovulation of beef cattle with two
injections of Folltropin®FSH dissolved in
a slow release formula (SRF) diluent
Percent Pregnant
18
16
14
12
10
8
6
4
a
b
b
controls
SRF 0.5%
SRF 1.0%
2
0
ova fertilized embryos
ab P

More embryos per flush
Have we improved this statistic in the
past 35 years?

Yearly Mean Total Ova and Embryos from ~15,000
Collections of Dairy cattle over a 20-Year Period
Percent Pregnant
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
Total Ova
Embryos
AETA
CETA
1985 1990 1995 2000 2005
Change from FSH-P to Folltropin
Year
(Sauvé-unpublished)

Semen

Effect of AI interval post estrus on fertilization and
the number of good embryos recovered
from superovulated cows
AI Treatment a
CL (Mean)
% Ova
fertilized
% Transferable
embryos (Mean)
12 h 10.0 72 b 61 b (4.7)
24 h 12.8 79 b 66 b (6.7)
36 h 13.0 54 b 30 c (3.0)
48 h 8.1 19 c 1.7 d (0.2)
12,24,36 & 48 h 10.0 77 b 61 b (3.7)
a
20 million sperm/straw, 2 straws per AI, 12 donors per group
b,c,d P

Effect of 9 sires on fertilization rate and
embryo production in 1596
superovulated dairy cows
% of total embryos/UFO
60
50
40
30
20
10
0
% good embryos
% UFO
1 2 3 4 5 6 7 8 9
Sires used for AI
(Compliments of MacDonald, unpublished)

Effect of semen quality on fertilization rate and
embryo quality in superovulated cattle
(227 bulls used in 742 donors)
Semen % Fertilized % Excellent
Quality* (n = 9,732) Embryos (n = 4,035)
Excellent 82.1 a 61.2 a
Good 67.7 b 55.7 b
Fair 58.3 c 53.9 c
Poor 51.8 d 33.7 d
abcd P

Comparison of semen quality when shipped to ET
center directly from commercial AI unit compared
to semen provided by breeder client
Source of semen
Semen quality at thawing Direct from AI center Provided by client
No. of shipments 1,278 981
Poor quality semen (%) 22 (1.7 a ) 66 (6.7 b )
Semen with no motility (%) 0 (0 a ) 24 (2.4 b )
a,b Percentages in the same row differ significantly (P

Embryo Production of Superovulated Angus
Cows Inseminated with Sexed Semen
14
12
11.7
12.0
Conventional
Sexed
No. Embryos/Ova
10
8
6
4
2
6.5
**
4.5
3.1
6.3
**
2.0
1.0
0
Total Transferable Unfertilized Degenerate
** Treatments differ (P < 0.05) Category
(32 cows per treatment inseminated at onset of heat and 12 (2X)
& 24 h post. Control = 15M; Sexed = 2M)
(Lamb et al., 2007)

Embryo Factors

Effect of bovine embryo grade (9,021 fresh
& 5,287 frozen) on pregnancy rate
Pregnant (%)
80
70
60
50
40
30
20
a
a
b
b
Fresh
Frozen
c c c b,c
10
0
1 2 3 4
Embryo Grade
a,b,c
Values differ (P

Cryopreservation
Freezing Embryos

The number of bovine embryos recovered,
transferred, and frozen in 2006 as reported by the
AETA and the CETA
AETA
CETA
No. donors collected 51,893 14,623
No. embryos
recovered
No. embryos
transferred (%)
319,703 96,285
104,722 (32.8%) 24,727 (25.7%)
No. embryos frozen 207,871 (65.0%) 65,568 (68.1%)
Glycerol
Ethylene Glycol
8,246 (4%)
199,625 (96%)
5,494 (21%)*
20,458 (79%)
(*No. thawed and transferred in 2006)
(Official statistics of the AETA and CETA)

Equipment Needed
Glycerol (10 to 20 minutes)
Warm water thaw container
Trained Embryologist
Microscope
Sucrose solutions
Holding medium
Reload straw
Ethylene Glycol (1 minute)
Warm water thaw container

Embryo Recipient Cattle in Hungary, 1980

Drugs & hormones used in attempts to
improve pregnancy rates following AI
and/or ET
• Progesterone
• LH
• hCG
• eCG (PMSG)
• GnRH
• Clenbuterol (smooth muscle relaxant)
• Ibuprofen (anti-inflammatory & anti-PG synthesis)
• Banamine (Flunixin Meglumine) (anti-inflammatory and anti-PG
release)
• PG Receptor Competitive Antagonist (AL8810 or ‘Embryo Armor’)
• Growth Hormone (Posilac)
• Estradiol

Effect of embryo-recipient estrus synchrony on pregnancy
rates in recipients of different breeds and parity (fresh &
frozen embryos)
80
75
70
% Pregnant
65
60
55
50
45
40
35
Dairy heifers: n= 10,108
Beef cows: n= 1,256
Dairy cows: n= 1,297
30
48 36 24 12 0 -12 -24 -36 -48
(Plus) Synchrony (hours) (Minus) (Hasler, 2001)

Effect of recipient-donor estrus synchrony on
pregnancy rate with fresh and frozen embryos
80
75
% Pregnant
70
65
60
55
50
45
40
35
Fresh n= 8996
Frozen n= 5271
30
48 36 24 12 0 -12 -24 -36 -48
(Plus) Synchrony (hours) (Minus) (Hasler, 2001)

HEAT DETECTION
1 Shot PG
CIDR ® -PG
MGA ® -PG
MGA
1 14
FIXED-TIME AI (TAI)*
CO-Synch + CIDR ®
Perform TAI at 54 ± 2 hr after PG with GnRH at TAI.
MGA ® -PG
GnRH
Perform TAI at 72 ± 2 hr after PG with GnRH at TAI.
MGA
1 14
CIDR ® Select
BEEF HEIFER PROTOCOLS
HEAT DETECT & TIME AI (TAI)
PG
0 5
12
treatment day
Heat detect & AI
… 19 d …
treatment day
CIDR ®
PG
0 7
treatment day
… 19 d …
treatment day
Perform TAI at 72 ± 2 hr after PG with GnRH at TAI.
CIDR ®
0 7 13
Heat detect & AI
treatment day
PG
PG
33 39
Heat detect & AI
.. 54 ± 2 hr ..
GnRH
AI
9
GnRH
PG
AI
72 ± 2 hr
33 36
GnRH
Select Synch + CIDR ® & TAI
Heat detect and AI day 7 to 10 and TAI all non-responders 72 - 84 hr after PG
with GnRH at TAI.
MGA ® -PG & TAI
MGA
1 14
GnRH
AI
CIDR ®
.. 72 - 84 hr ..
0
treatment day
7
10
Heat detect & AI
Heat detect and AI day 33 to 36 and TAI all non-responders 72 - 84 hrs after PG
with GnRH at TAI.
COMPARISON OF PROTOCOLS FOR BEEF HEIFERS
PG
GnRH
GnRH
PG
AI
.. 72 - 84 hr ..
33
36
Heat detect & AI
HEAT DETECTION COST LABOR
1 Shot PG Low High
CIDR ® -PG Medium Medium
MGA ® -PG Low Low/Medium
HEAT DETECT & TAI
Select Synch + CIDR ®
(TAI nonresponders
72-84 hr after PG)
High Medium
MGA ® -PG (TAI nonresponders
72-84 hr after PG)
Medium Medium
FIXED-TIME AI (TAI)
CO-Synch + CIDR ®
(TAI 54 ± 2 hr after PG
High Medium
with GnRH at TAI)
… 19 d …
treatment day
MGA ® -PG (TAI 72 ± 2 hr after PG
with GnRH at TAI)
Medium
Medium
CIDR
0 14
GnRH
... 9d … .. 7d ..
23
treatment day
PG
AI
GnRH CIDR ® Select
(TAI 72 ± 2 hr after
72 ± 2 hr
Cystorelin ® , Factrel ® , Fertagyl ® , OvaCyst ® High Medium/High
PG with GnRH at TAI)
30 33
Estrumate ® , In-Synch ® , Lutalyse ® , ProstaMate ® ,
PG
• The times estroPLAN listed for “Fixed-time ® AI” should be considered as the approximate average time of
insemination. This should be based on the number of heifers to inseminate, labor, and facilities.
Beef Reproduction Task Force

Synchronization and Management of Recipients
Heat
Detection
GnRH
C I D R
PGF 2
α
Heat
check
7 d
( ± 1.5 d)
T
R
A
N
S
F
E
R
Day 0 Day 7
Day 16
Timed
Transfers
GnRH
C I D R
PGF 2
α
No Heat
Detection
48 hr
GnRH
7 d
T
R
A
N
S
F
E
R
Courtesy of Randall Hinshaw

Conception rates following embryo transfer in
anestrus vs. cycling recipient beef cows
Cow status 1
No.
received
CIDR
No. estrus
detected (%)
No.
received
embryo (%) 2
No.
Pregnant
(%) 2
Anestrus 321 161 (50.1) a 140 (87.0) 56 (40.0) a
Cycling 376 338 (89.9) b 311 (92.0) 206 (66.2) b
1 Status determined by palpation per rectum and ultrasound at time of CIDR
insertion; 2 Percentages based on previous column.
a,b P

Beef cattle on pasture
Polled Herefords in Oklahoma, 2004

Commercial Dairy
Holsteins in New Mexico, 2004

Embryo transfer outcomes in two
different management systems
Factor Beef ranch Commercial dairy
No. donors 45 36
No. superovulated &
94 87
inseminated
Mean no. ova 14.4 9.1
Mean no. unfertilized
(% of total)
Mean no. transferable
embryos (% of total)
3.1 (21.5) 4.3 (47.6)
9.1 (63.2) 3.4 (37.7)
ET recipient pregnancy
rate
>70%

‘The practitioner effect’

ET conception rates of IVP embryos in the
Netherlands relative to the day of the week on which
transfers were conducted
Day of week No. trans. % pregnant
Tuesday 633 50.8
Friday 1103 42.2
(Van Wagtendonk-de Leeuw et al., 1997)

Effect of technician on pregnancy rate of
13,485 IVP embryos in Brazil
Percent Pregnant
50
45
40
35
30
25
20
15
10
5
0
1 3 5 7 9 11 13 15 17 19 21 23
Embryo Transfer Technician
(Minimum of 200 transfers per technician)
(Technician no. 1 = 1 pregnant of 251 transfers) Lamb, et al., 2005

Influence of technicians (n=12) on conception
rates in AI (>6,000 inseminations in Holland)
Note: There was a highly significant (P

“Luck”
Probabilities of success resulting from the transfer of
four embryos if the true conception rate is 50%
No. Pregnant Average frequency Frequency %
0/4 1/16 6
1/4 1/4 25
2/4 3/8 38
3/4 1/4 25
4/4 1/16 6
(Seidel et al., 2003)

Key to Success
If ET is conducted within reasonably
acceptable parameters involving
practitioner competence, media, embryo
quality, hygiene, etc., almost certainly the
biggest factors affecting success are
DONOR & RECIPIENT QUALITY &
MANAGEMENT