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APost graduate Credit Seminar on
Equine Embryo Transfer
PARMAR VASANT R.
04 -0318-2007
VOBG-900
Minor AdvisorDR. P. B. Patel
Major AdvisorDR. B. N. SUTHAR
Department of ARGO College of Veterinary Science and Animal Husbandry,
SardarKrushinagar Dantiwada Agriculture University, S.K.NAGAR2008
Introduction
Introduction
The horse was possibly the first of the domestic large animal species to be subjected to AI and last to undergo embryo recovery and transfer.
Compared with cattle, ET was relatively slow to develop commercially in equids during 1970s and 1980s.
(Allen and Rowson, 1975; Squires et al., 1985).
Equine Embryo Transfer increase steadily because of the recent (2002) legal removal of limit on the number of foals registerable to one mare in same year.
(Hudson and Mc Cue, 2004)
1st Successful Embryo Transfer was done in Rabbit by Walter Heape in 1890.
Surgical Embryo Transfer by Allen and Rowson in 1972.
Non-Surgical Embryo Recovery by Oguri and Tsutsumi in 1972.
Successful Non-Surgical Embryo Transferby Oguri and Tsutsumi in 1974.
In India
1st Embryo Transferred FoalBy Lt Gen S Pattabhiraman
ARMY PRIDE (GAURAV)
February 10, 2006 at Equine Breeding Stud,
Babugarh
Applications
1. To Obtain foals from Performer older mares.
2. To provide a Genetically promising foals.
3. To increase the No. of foals/mare/lifetime
4. To obtain foals from physically disable mares
5. Use as a Research tool
6. To allow Freezing, Cloning, Embryo Sexing etc
7. To obtain the foals during non-breeding season
(Squires and Seidel, 1995; Aguilar and Woods, 1997;Vanderwall, 2000)
Selection of Donor
Selection of Donor
1. Genetic superiority
2. Ability to produce large number of usable embryos
3. Size and tone of Uterus and Cervix
4. Normal fertility and genitalia
5. Regular estrus cycles(Squires et al., 1999)
Selection of Recipient
Selection of Recipient
1. Body weight 400 to 550kg.
2. Age 3 to 10 yrs.
3. Normal Estrus cycle.
4. Free of Ovarian and Uterine abnormalities.
(Squires et al., 1999)
Ovariectomised Recipient
Progesterone (100-300mg)
5 to 7 days before transfer
Day 100 to 140 of pregnancy
(Hinrichs et al.,1985; McKinnon et al.,1988; Anderson, 1992)
Advantage. – Eliminate the need for synchronization
Disadvantage. – Need for daily administration of Progestin
Group Preg. 12d
%
Preg. 50d
%
ED
%
Cycling 75 61.8 17.5
P4 treated 75.9 58.9 22.3
Total 75.4 60.6 19.6
Comparison of Cycling and Ovariectomised mares As Recipient
(Rocha filho et al., 2004)
Estrus Synchronization
Estrus Estrus SynchronizationSynchronization
The degree of synchrony between recipient and donor appears to be The degree of synchrony between recipient and donor appears to be not criticalnot critical
Synchrony between ovulation in recipient and donor mares is +1 to -3 Synchrony between ovulation in recipient and donor mares is +1 to -3 days.days.
(Allen et al.,1976; Imel et al.1981; Douglas, 1982; McLinnon and (Allen et al.,1976; Imel et al.1981; Douglas, 1982; McLinnon and Squires,1988; Squires and Seidel, 1995)Squires,1988; Squires and Seidel, 1995)
Method of Synchrony:
Single I/M injection of PGF2α Donor mare 1 or 2 days ahead of similar therapy applied to recipient mare.
Both are between 6 – 14 days of diestrus.
(1) Use of PGF2α:
(Holtan et al.,1977; Yardaydin et al., 1993; Allen, 2001)
(2) Use of Progesterone:
Both donor and mare given 9-10 day course of
progesterone inj.
Donor mare 1 or 2 days ahead of similar therapy applied
to recipient mare.
On last day of progesterone therapy Inj. Prostaglandin
analogues given
(Driancourt and Palmer, 1982; Yardaydin et al., 1993 )(Driancourt and Palmer, 1982; Yardaydin et al., 1993 )
Superovulation
Superovulation
Equine chorionic gonadotropin
GnRH
Porcine FSH
Immunization against inhibin
Equine chorionic gonadotropin
GnRH
Porcine FSH
Immunization against inhibin
()()
(Day,1940)
(Ginther and Bergfelt, 1990)
(McCue, 1996)
(Fortune and
Kimmich, 1993)
ECGECG
Superovulation in ruminant
Even in large doses, less effective
(Allen,1982)GnRHGnRH
Multiple follicle in seasonally anoestrus mare
(Johnson, 1987)
Ineffective to cyclic mare
Porcine FSHPorcine FSH
High levels of porcine FSHtwice daily
1.2 – 1.7 ovulations
(Fortune and Kimmich, 1983; Squires et al.,1986)
Inhibin
Immunization against inhibin
Suppression of FSH
Suppression of FSH
(Ginther et al., 2001)
Inhibin α subunit doubling of ovulation rate
(McCue et al., 1993)
On day 0 and day 35
Ovulations 1.86
Then goes to 2.29
On day 0 and day 35
Ovulations 1.86
Then goes to 2.29
(Mc Kinnon et al., 1992; McCue et al., 1993)
Equine Pituitary Extract
Prepared according to Braseton & McShan (1970).
Latter by Gullou & Combarnous (1983).
6gm of crude pituitary extract /kg of pituitaries.
Appro. 6-10% LH & 2-4% FSH
Multiple ovulation in both seasonally anovulatory and cycling mares.
(Douglas, 1979; Woods, 1982)
D-0 D-5 D-7
hCG
(3000IU)
Ov
D-0D-7
Ovulation
Start EPE tre
atment
PGF2α EPE discontinue
Embryo collection
EPE dose rate of 25mg twice /day
(Dippert, et al.,1992; Scoggin et al., 2002)
AI 1 day after hCG
No of treatment days – 6.6 days
Ovulations
Embryo
Alvarenga et al., 2001
Once daily
2.4 1.6
Twice Daily
7.1 3.5
Ovulation Embryo
Douglas, 1979 2.3 ----
Woods and Ginther, 1983
3.0 ----
Squires et al., 1987
3.8 2.0
Squires et al., 2003
1.7 – 3.8 -----
Ov
Day-5 6 7
Start eFSH
PGF2α
(2nd day of treatment)
Stop treatment
(Follicle >32-35mm)
hCG
(30-40hrs after end of treatment)
AI
1day after hCG
Ov Embryo collection
(Day 7 - 8)
Equine FSH
Purified Equine Pituitary Extract
Used at a dose rate of 12.5 mg twice per day
(Niswender et al., 2003)
Parameter Groups
G I G II G III
Ovulations/mare
3.3+1.6 5.0+2.1 4.8+1.3
Embryos
/mare
1.2+1.3 1.8+1.3 2.6+0.5
Embryos
/Ovulations
0.36+1.2 0.36+1.8 0.54+1.3
(Machado et al., 2004)G I – 25mgEPE twice a day
G II – EPE in decreasing doses
G III – 12.5mg eFSH twice daily
Comparison between EPE and eFSH
Factors affecting on Superovulation
۩ Day of initial treatment
۩ Size of largest follicle at initial treatment
۩ Frequency of injection
۩ Hormone used
(Squires and McCue, 2007)
Follicle population
Before appearance of a dominant follicle
Day of initial treatment
Treatment Day 15-19 19-23
Ov. 2.9 1.3
Day 15-19 19-23
Ov. 2.9 1.3
(Woods and Ginther, 1983)Day 5 12
Ov. 2.9 1.1
Day 5 12
Ov. 2.9 1.1 (Dippert et al., 1992)
Size of largest follicle at initial treatment
Follicles < 25mm
Progesterone Estradiol
GnRH Analogues
(Pierson and Ginther, 1990) (Dippert et al., 1992)
Greater No of larger Preovulatory follicles Follicle size of 15 or 20 mm at initiated treatment
(Pierson and Ginther, 1990)
Frequency of injection
Twice daily
Once daily
EPE (Douglas et al., 1974)
(lapin and Ginther 1977; Douglas, 1979)
OvulationOvulationss
Embryo Embryo RecoveryRecovery
OnceOnce 2.42.4 1.61.6
TwiceTwice 7.17.1 3.53.5
Compare once with twice daily inj. of EPE
(Alvarenga et al., 2001)
Parameters
Treatment groups
25-SID 50-SID 12.5-BID 25-BID
Ovulations/mare
3.4+0.6 4.4+0.7 3.4+0.8 1.2+0.1
Embryos/
mare
1.2+0.4 1.5+0.3 2.6+0.6 2.1+0.6
Embryos/
ovulations(%)
12/34(35.5) 12/33(36.4) 23/34(67.6) 19/44(43.2)
Mares with >2 ovulations
1/10(20.0) 4/8(50.0) 6/9(66.7) 5/9(55.6)
(Scoggin et al., 2002)SID- Once a day
BID – Twice a day
Mare treated with EPE Once or Twice daily
Hormones used
Highly contaminated with LH
High LH : FSH Ratio
Luteinization of follicles without ovulation
(Hofferer et al.,1993; Rosas et al., 1998;Briant et al., 2004)
Embryo collectionEmbryo
collection
Embryo collection
Equineembryo
Day 5 ½ - 6 post ovulation
To traverse the oviduct and enter the uterus
(Freeman et al., 1991; Battut et al., 2000)
After entering the uterine lumen,
it develop in to an expanded blastocyst
Size of embryo on different Size of embryo on different collection daycollection day
Day of collection
Mean size of embryo (μm)
7 498 (120-2000)
8 873 (150-3000)
9 1835 (220-4500)
(Squires et al., 1985;Fleury and Alvarenga, 1999)
Significantly low Embryo recovery on day 6
(Battut et al., 1997)Flushing on day 6
Deep freezing
Bisection to produce identical twins
(Carnevale, 2004)
(Skidmore et al.,1989)
Flushing on day 9
Lower Embryo success rate
(Squires and Seidel,1995)
Embryos are commonly recovered on day 7 & 8
Procedure
Most common method
Non-surgical Transvaginal Embryo recovery
Extended two way Foley catheter (French size 30 or 33)
(Imel et al., 1981; Vanderwall, 2000)
(Kuzan and Seidel, 1986)
1% (v/v) fetal or newborn calf serum, Penicillin (100 units/ml) Streptomycin (100 ug/ml) Or Commercially available equine embryo flush solution
1% (v/v) fetal or newborn calf serum, Penicillin (100 units/ml) Streptomycin (100 ug/ml) Or Commercially available equine embryo flush solution
DPBS
Modified Dulbecco’s Phosphate buffered Saline
A foley catheter passed mannually through the vagina and cervix
5 cm in to the uterine body
Cuff of the catheter is inflated with 60 ml of Sterile Saline/Water or Air
Catheter is then drawn back against the internal os of cervix
1-2 liter’s of pre warmed (35-37ºC) DPBS
Infuse in to the uterus
The catheter is clamped
Inlet tubing disconnected from catheter
Out let tubing connected
The clamp is opened
Fluid is allowed to drain out of uterus by gravity flow,
1-2 liter’s of pre warmed (35-37ºC) DPBS
Infuse in to the uterus
The catheter is clamped
Inlet tubing disconnected from catheter
Out let tubing connected
The clamp is opened
Fluid is allowed to drain out of uterus by gravity flow,
75 micron filter positioned over a collecting 75 micron filter positioned over a collecting cylindercylinder
Maintain atleast 20ml of fluidMaintain atleast 20ml of fluid
Repeat procedure for 3-4 timesRepeat procedure for 3-4 times
75 micron filter positioned over a collecting 75 micron filter positioned over a collecting cylindercylinder
Maintain atleast 20ml of fluidMaintain atleast 20ml of fluid
Repeat procedure for 3-4 timesRepeat procedure for 3-4 times
Recover majority (93 – 98%) of fluid Inject PGF2α : 10 mg I/M.
(Iuliano, 1983; Vanderwall, 2000)
Grade 1 – Excellent or Good
Symmetrical and spherical embryo mass with cells of uniform size, color, and density.
Embryo mass has clear edges without indentation.
Grade 2 - Fair
Moderate irregularities in the overall shape of the embryo.
Few extruded blastomeres.
Grading of Embryo
Grade 3 – Poor
Major irregularities in the shape of the embryonic mass or in the size, color, or density of the constituent cells.
Presence of extruded blastomere and degenerated cells.
Grade 4 – Degenerated or Dead
Embryonic cells of irregular size and color.
Numerous extruded blastomeres, degenerated cells .
(Stringfellow and Seidel, 1998)
Embryo Transfer
Two methods
Surgical Transfer
Non-Surgical Transfer
Surgical Embryo Transfer
Via a Ventral midline laparotomy Via a Ventral midline laparotomy
performed under general anesthesia. performed under general anesthesia.
(Allen, 1982)
Via a Flank laparotomy carried out Via a Flank laparotomy carried out under local infiltration anesthesia. under local infiltration anesthesia.
(Squires (Squires et alet al., 1985)., 1985)
Procedure
Proximal 1/3 of uterine horn is exteriorized.
Inject Embryo and a minimal amount (<0.5 ml) of culture medium by fire polished glass pipette.
Proximal 1/3 of uterine horn is exteriorized.
Inject Embryo and a minimal amount (<0.5 ml) of culture medium by fire polished glass pipette.
Advantages
• Invasive • Impracticality • Increased costs
Disadvantages
High pregnancy rates.(75-90%)
(Castleberry et al., 1980; Squires et al., 1982;
Allen, 2003)
McKinnon et al., 1988)
Non Surgical Embryo Transfer
Preferable to surgical for its
1. Speed,
2. Simplicity,
3. Economy,
4. lack of post transfer complications
Transcervical Transfer
(Lascombres and Pashen, 2000)
C Standard Artificial insemination Pipette
B Disposable plastic “ Insemination gun “
A Reusable stainless steel “Insemination gun “
Gun protected with operator's hand Pass through vagina and into cervix.
Transrectal manipulation
Pregnancy rates up to 75-85% achieve. (Meadows et al., 2000; Jasko 2002).
Proce
dure
Wilsher and Allen’s method
Polansky’s duck billed vaginoscope
Modified Vulsellum forceps
Embryo loaded disposable transfer pipette
Very quick and simple to perform Success rate of >90%
Maintain minimal contamination
Expanding arms of the polansky’s spaculum restrains the vaginal mucosa
Advantages
(Wilsher and Allen, 2004)
Microsurgical transfer
Endoscopy
60 cm non-flexible, Long optical section of
Endoscope and a needle
Flank of standing mare
Abdominal cavity
Uterine lumen
(Muller, 2001)
Ultrasound guided transfer
Transvaginally Transducer-needle guide introduce by puncturing vaginal Wall
Transvaginally Transducer-needle guide introduce by puncturing vaginal Wall
Abdominal cavity. Abdominal cavity.
Lumen of UterusLumen of Uterus
(Muller, 2001)
Comparison of Microsurgical, Surgical and Comparison of Microsurgical, Surgical and Non surgical method of Embryo TransferNon surgical method of Embryo Transfer
Method No. of Transfer
No. of pregnancy (%)
Endoscopy 07 3 (43)
Ultrasonography 04 2 (50)
Total 11 5 (46)
Surgical 31 18 (72)
Non Surgical 43 14 (33)
(Muller, 2001)
Factors affecting embryo recovery
Day of recovery
No. of ovulation
Age of the donor mare
Quality of semen
Day of recovery
No. of ovulation
Age of the donor mare
Quality of semen
(Grifflth et al., 1981;
Squires et al., 1995)
(Squires et al., 1987)
(Squires et al., 1982; Vogelsang et al., 1989)
(Francl et al., 1987)
Day of recovery
Commercially Embryos flushed on day 7 or 8
Day 6 Lower (30%) recovery rate
(Iuliano et al., 1985)
Appropriate for freezing
(Slade, 1985)
Day 9 Less viable for transfer
(Squires and Seidel,1995)
Day of collection
Recovery rate%
Mean size of embryo (μm)
7 49.30 (106/215) 498 (120-2000)
8 58.0 (388/669) 873 (150-3000)
9 54.50 (18/33) 1835 (220-4500)
(Fleury and Alvarenga, 1999)
Comparison of Recovery rates
Effect of day post-ovulation on equine embryo recovery rate
Reference Day
6(%) 7(%) 8(%) 9(%)
Luliano et al., (1985) 21/32 (66) 68/90 (76) 50/61 (82) ----
Castleberry et al., (1980)
3/13 (23) 15/22 (68) 4/8 (50) ----
Squires et al., (1985) 86/137 (63) 73/96 (76) 218/293 (74) 43/53 (81)
Meira et al., (1993) 70/127 (55) 23/41 (56) ---- ----
Wade and Gallagher (1989)
---- 26/45 (58) 31/47 (66) ----
Bowen et al., (1985) 12/23 (52) ---- 25/31 (81) ----
Fleury and Alvarenga (1999)
---- 106/215 (49) 388/669 (58) 18/33 (55)
Total 192/332 (58) 311/509 (61) 716/1109 (65) 61/86 (71)
(Aguilar and Woods 1997)
No. of ovulation
50% Embryo Recovery
(Squires, 1995)
> 50% Embryo Recovery
(Squires, 1987)
Thoroughbreds
Draft Horses
Warmbloods
Age & Reproductive History of Donor
History of Infertility Lower Embryo Recovery
(Squires 1995)
(Dauglas, 1982) 34.3%
(Dauglas et al., 1985) 28%
(Woods et al., 1986) 19%
(Squres et al., 1992) 28%
(Vogelsang et al., 1995) 40%
Quality of semen
Type of Extender
Cooled Semen
Frozen Semen
(Francl et al., 1987)
(Amann and Pickett 1998)
Type of Semen Recovery Rate
Raw Semen 78%
Skim Milk Extended Semen 62%
EDTA Lactase Extended Semen
48%
(Squires et al., 1988)
Mare Category
Overall Fresh Semen
Chilled Semen
Frozen Semwen
All Mares
59.6% 87.6% 47.4% 46.6%
< 14 yrs 67% 98% 48% 51%
> 14 yrs 43% 47% 47% 32%
Effect of Semen and Age of mare
on Recovery rate
(Meadows et al., 1999)
Factors affecting Pregnancy Rate
Method of Transfer
Synchrony of Donor and Recipient
Quality of Embryo
Age of Donor
(Squires et al., 1999)
Method of Transfer
Synchrony of Donor and Recipient
Quality of Embryo
Age of Donor
(Squires et al., 1999)
Method of Transfer
Surgical Transfer Non-Surgical Transfer
Comparison of Pregnancy Rate
Surgical Non-Surgical
Imel, 1981 53.3% 26.6%
Iuliano et al., 1985 72% 45%
Mc Kinnon et al., 1998 66% 39%
Squires and Seidal 2003 74% 52%
(Castleberry et al., 1980; Squires et al., 1982)
Synchrony of Donor and Recipient
Most Critical Factor
Synchrony Transfers Pregnancy at
50 day (%)
-3 23 69.6
-2 51 78.4
-1 118 67.8
0 174 69.5
1 69 71.0
2 13 53.8
(Martin et al., 1998)
Quality of Embryo
Embryo Quality grade
Fresh Cooled Combined
1 78 77 77
2 60 66 63
3 46 60 57
4 33 40 38
(Squires et al., 1992)
Morphologically Abnormal Embryos
Low Pregnancy Rate (3-5 %) (Mc Kinnon, 1988)
Age of Donor
Older Donor Mare Young Donor Mare
Early Embryonic Death(Vogelsang 1989)
Mare Category
Overall Fresh Semen
Chilled Semen
All Mares 80% 87% 73%
< 14 yrs 80% 86% 75%
> 14 yrs 76% 100% 63%
(Meadows et al., 1999)
ConclusionConclusion
Selection of recipient and donor are important in Equine Selection of recipient and donor are important in Equine Embryo Transfer.Embryo Transfer.
eCG, GnRH and Porcine FSH are not effective for eCG, GnRH and Porcine FSH are not effective for superovulation in mare. superovulation in mare.
BID administration of equine FSH increase ovulation rate and BID administration of equine FSH increase ovulation rate and embryo recovery.embryo recovery.
The degree of synchrony between recipient and donor appear The degree of synchrony between recipient and donor appear to be non critical.to be non critical.
Surgical methods have resulted in improved rates of embryo Surgical methods have resulted in improved rates of embryo recovery and survival but the technique is complicated and recovery and survival but the technique is complicated and more traumatic for the recipient mare.more traumatic for the recipient mare.
Embryos of quality score up to 2 results in good pregnancy Embryos of quality score up to 2 results in good pregnancy rate.rate.