equine embryo transfer- dr. vasant parmar

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.