Equine Embryo Transfer,By:Dr.Dhiren Bhoi

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Equine Embryo Transfer Presented by DR. DHIREN B. BHOI M. V. Sc. (Gynaecology and Obstatrics) Anand Veterinary College, Anand, Gujarat E-Mail: drdhirenvet@gmail.com

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Introduction

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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)

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1st Successful Embryo Transfer was done in Rabbit by Walter Heape in 1890. Surgical Embryo Transfer by Allen and Rowson in 1972. Equine Non-Surgical Embryo Recovery by Oguri and Tsutsumi in 1972. Successful Non-Surgical Embryo Transfer by Oguri and Tsutsumi in 1974.

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In India 1st Embryo Transferred Foal By Lt Gen S Pattabhiraman ARMY PRIDE (GAURAV) February 10, 2006 at Equine Breeding Stud, Babugarh

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Applications To Obtain foals from Performer older mares. To provide a Genetically promising foals. To increase the No. of foals/mare/lifetime To obtain foals from physically disable mares Use as a Research tool To allow Freezing, Cloning, Embryo Sexing etc To obtain the foals during non-breeding season (Squires and Seidel, 1995: Aguilar and Woods, 1997)

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Selection of Donor

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Selection of Donor Genetic superiority Ability to produce large number of usable embryos Size and tone of Uterus and Cervix Normal fertility and genitalia Regular estrus cycles (Squires et al., 1999)

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Selection of Recipient

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Selection of Recipient Body weight 400 to 550kg. Age 3 to 10 yrs. Normal Estrus cycle. Free of Ovarian and Uterine abnormalities. (Squires et al., 1999)

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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

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Comparison of Cycling and Ovariectomised mares As Recipient (Rocha filho et al., 2004)

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Estrus Synchronization

Estrus Synchronization : 

Estrus Synchronization The degree of synchrony between recipient and donor appears to be not critical Synchrony between ovulation in recipient and donor mares is +1 to -3 days. (Allen et al.,1976; Imel et al.1981; Douglas, 1982; McKinnon and 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: (Yardaydin et al., 1993 ;Allen, 2001)

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(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 )

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Superovulation

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Equine chorionic gonadotropin GnRH Porcine FSH Immunization against inhibin () (Allen 1982 ) (Squires et al. 1989) (McCue et al., 1992) (Irvine 1981; Squires et al. 2003)

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ECG Superovulation in ruminant Even in large doses, less effective (Allen,1982) GnRH Multiple follicle in seasonally anoestrus mare (Johnson, 1987) Ineffective to cyclic mare

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Porcine FSH High levels of porcine FSH twice daily 1.2 – 1.7 ovulations (Squires et al., 2003)

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Inhibin Immunization against inhibin Suppression of FSH (Ginther et al., 2001) Inhibin α subunit doubling of ovulation rate (McCue et al., 1992)

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On day 0 and day 35 Ovulations 1.86 Then goes to 2.29 (Mc Kinnon et al., 1992)

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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)

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D-0 D-5 D-7 hCG (3000IU) Ov D-0 D-7 Ovulation Start EPE treatment 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

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No of treatment days – 6.6 days

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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)

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(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

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Factors affecting on Superovulation Day of initial treatment Size of largest follicle at initial treatment Frequency of injection Hormone used

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Follicle population Before appearance of a dominant follicle Day of initial treatment Treatment Day 15-19 19-23 Ov. 2.9 1.3 (Woods and Ginther, 1983) Day 5 12 Ov. 2.9 1.1 (Dippert et al., 1992)

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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)

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Frequency of injection Twice daily Once daily EPE (Douglas et al., 1974) (lapin and Ginther 1977; Douglas, 1979) Compare once with twice daily inj. of EPE (Alvarenga et al., 2001)

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(Scoggin et al., 2002) SID- Once a day BID – Twice a day Mare treated with EPE Once or Twice daily

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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)

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Embryo collection

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Embryo collection Equine embryo 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 collection day : 

Size of embryo on different collection day (Fleury and Alvarenga, 1999)

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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

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Procedure Most common method Non-surgical Transvaginal Embryo recovery Extended two way Foley catheter (French size 30 or 33) (Imel et al., 1981) (Kuzan and Seidel, 1986)

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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

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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

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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,

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75 micron filter positioned over a collecting cylinder Maintain atleast 20ml of fluid Recover majority (93 – 98%) of fluid Inject PGF2α : 10 mg I/M. (Iuliano, 1983)

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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

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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)

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Embryo Transfer

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Two methods Surgical Transfer Non-Surgical Transfer

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Surgical Embryo Transfer Via a Ventral midline laparotomy performed under general anesthesia. (Allen, 1982) Via a Flank laparotomy carried out under local infiltration anesthesia. (Squires et al., 1985)

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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.

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Advantages Invasive Impracticality Increased costs Disadvantages High pregnancy rates.(75-90%) (Allen, 2003) McKinnon et al., 1988)

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Non Surgical Embryo Transfer

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Preferable to surgical for its 1. Speed, 2. Simplicity, 3. Economy, 4. lack of post transfer complications

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Transcervical Transfer (Lascombres and Pashen, 2000) C Standard Artificial insemination Pipette B Disposable plastic “ Insemination gun “ A Reusable stainless steel “Insemination gun “

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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). Procedure

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Wilsher and Allen’s method Polansky’s duck billed vaginoscope Modified Vulsellum forceps Embryo loaded disposable transfer pipette

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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)

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Microsurgical transfer

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Endoscopy 60 cm non-flexible, Long optical section of Endoscope and a needle Flank of standing mare Abdominal cavity Uterine lumen (Muller, 2001)

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Ultrasound guided transfer Transvaginally Transducer-needle guide introduce by puncturing vaginal Wall Abdominal cavity. Lumen of Uterus (Muller, 2001)

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Comparison of Microsurgical, Surgical and Non surgical method of Embryo Transfer (Muller, 2001)

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Factors affecting embryo recovery Day of recovery No. of ovulation Age of the donor mare Quality of semen (Squires et al., 1995) (Squires et al., 1987) (Vogelsang et al., 1989) (Francl et al., 1987)

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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)

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(Fleury and Alvarenga 1999) Comparison of Recovery rates

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Effect of day post-ovulation on equine embryo recovery rate (Aguilar and Woods 1997)

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No. of ovulation Single Ovulation Double Ovulation 50% Embryo Recovery (Squires 1995) > 50% Embryo Recovery (Squires 1987) Thoroughbreds Draft Horses Warmbloods

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Age & Reproductive History of Donor History of Infertility Lower Embryo Recovery (Squires 1995)

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Quality of semen Type of Extender Cooled Semen Frozen Semen (Francl et al., 1987) (Amann and Pickett 1998) (Squires et al., 1988)

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Effect of Semen and Age of mare on Recovery rate

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Factors affecting Pregnancy Rate Method of Transfer Synchrony of Donor and Recipient Quality of Embryo Age of Donor (Squires et al., 1999)

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Method of Transfer Surgical Transfer > Non-Surgical Transfer Comparison of Pregnancy Rate

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Synchrony of Donor and Recipient Most Critical Factor (Martin et al., 1998)

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Quality of Embryo (Squires et al., 1992) Morphologically Abnormal Embryos Low Pregnancy Rate (3-5 %) (Mc Kinnon 1988)

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Age of Donor Older Donor Mare Young Donor Mare > Early Embryonic Death (Vogelsang 1989)

Conclusion : 

Conclusion selection of recipient and donor are important in Equine Embryo Transfer. eCG, GnRH and Porcine FSH are not effective for superovulation in mare. BID administration of equine FSH increase ovulation rate and embryo recovery. The degree of synchrony between recipient and donor appear to be non critical Surgical methods have resulted in improved rates of embryo recovery and survival but the technique is complicated and more traumatic for the recipient mare. Embryos of quality score up to 2 results in good pregnancy rate.

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THANK YOU