POTENTIAL IMPACT OF EMERGING TECHNOLOGIES ON AnGRCONSERVATION AND USE

Ann Van Soom & Katrien SmitsDepartment of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Belgium

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Questions to answer in this talk

1. Assisted reproductive technology (ART)?

2. Emerging technologies in the horse?

3. Impact on AnGR conservation and use?

4. Future perspectives and conclusions

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Assisted reproductive technology

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History of ART

1900 20001960 19901980

IVF

OPU

SCNT

1890

2 Angoras4 Belgian Hares

Heape: ET in rabbits

1950

Kanitsky : ET in pigsWillet : ET in cattle

Surgical

1970

MOET

Allen : ET in horses

Non-surgical

Mother of all ART : embryo transfer (ET)

In vivo produced embryos (fresh or frozen)In vitro produced embryos (fresh or frozen)Cloned embryos (fresh or frozen) produced after somatic cell nucleus transfer (SCNT)

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Embryo transfer in cattle

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• Transfer of an embryo from donor to recipient

1. Synchrony between donor-recipient

2. Superovulation

3. Insemination

4. Flushing of uterus to retrieve embryos

5. Transfer of embryos to recipient (65 % calf)

In vivo embryos

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1. Collection of oocytes (Living or dead donor)

2. In vitro maturation

3. In vitro fertilization

4. Culture for 7 days to produce in vitro embryos

5. Transfer of embryos to recipient (55 % calf)

In vitro embryos

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1. Collection of donor somatic cells (Living or dead donor)

2. Collection of recipient mature oocytes

3. Enucleation of recipient oocyte

4. Transfer of donor cell to recipient egg

5. Culture for 7 days to produce embryos

6. Transfer of embryos to recipient

Cloned embryos

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Emerging technologies in the horse?

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Difficulties associated with ART in horses

1. No assisted reproduction allowed in thoroughbreds

2. Difficult superovulation in mares

3. Difficult in vitro fertilization in horses

4. Cloning in horses?

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No assisted reproduction

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Eclipse : April 1, 1764 - February 26, 1789 George Stubbs

Superovulation and embryo transfer in horses?

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Superovulation and embryo transfer in horses?

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In vitro fertilization (IVF) in horses?Intracytoplasmic sperm injection (ICSI)

• Only two foals born after IVF with in vivo maturedoocytes (Palmer et al., 1991)

• First foal born after ICSI (Squires et al., 1996)

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Oocyte collection

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Technical aspects of ICSI

Immature oocyte

Mature oocyte after 24-28 h

ICSI

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Results in terms of embryo culture

70 % cleavage at day 38-10 % blastocyst rate at Day 9

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Birth of SMICSIon 27th of October 2009

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Cloning in horses?

• Prometea: the world’sfirst cloned horse (7th cloned species) was born in 2003 and has given birth to a foal in 2008.

22Galli et al., 2003

Skin biopsy from donor horse

Culture of somatic cells

Collection of horse oocytes

Transfer of donor nucleus to recipient oocyte : fusion and activation by electrical pulse

Enucleation of mature oocyte

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Culture of the embryo to the blastocyst stage

Intrauterine transfer to recipient marePregnancy is monitored by ultrasound

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ICSI horseSCNT horse

ICSI horseSCNT horse

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Viagen (USA)

Cryozootech (F)

Avantea (I)

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Texas A&M University

Colorado State University

Ghent University?

Goulburn Valley Equine Hospital (AU)

Impact on AnGR conservation and use

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What is needed for ICSI?

• One (im)mature oocyte, fresh or frozen, collected from

dead or living mare

• One sperm cell, fresh orfrozen (even frozen in -20°C) collected after ejaculation (living stallion), or from the epididymisor testis after dead or castrationof the stallion

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So in theory …

1. You can collect oocytes from a genetically valuable mare :– after her death and use them fresh or freeze them (freezing of

cortical strips)– after she became sub- or infertile (removal of one ovary,

adhesions of oviduct, ovulation problems, uterineproblems….)

– …..

2. You can use semen from a genetically valuable stallion– which has semen of very low quality– of which very few frozen semen straws are left (and refreeze

the sperm cells after use)– of which the testicles have been kept chilled or frozen– ….

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And in practice….

Photographs from CSUhttp://www.news.colostate.edu/Release/4174 29

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20 oocytes collected 14 mature oocytes ICSI with sperm of cremello Quarter

Horse stallion

Tuesday fatallyinjured after

tornado in 2008

8 embryos cleaved

4 blastocysts transferredto 4 recipients

Buckskin and cremello foal born at CSU in 2009

Rescue of a valuable mare

How feasible is this?

• Hinrichs 2010: “In the past 4 years, we have produced 21 embryos from the ovaries of 16 client mares postmortem, for 13 pregnancies. The ovaries should be recovered from the mare as soon as possible post mortem, and held at room temperature during shipment to the laboratory. The ability to achieve embryos or viable pregnancies with the recovered oocytesdecreases with time after about 6 h post mortem (Ribeiro et al. 2008).”

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Rescue of a subfertile stallion

A. Post-thaw motility > 30 % and > 40 % pregnancy /cycle

B. Post-thaw motility > 30 % and no pregnancies

C. Post-thaw motility 10-30 % and < 20 % pregnancy/cycle

D. Post-thaw motility < 10 % and no pregnancies

32Lazzari et al., 2002

ICSI with semen of stallions of different fertilities

Stallion N of oocytes injected % cleaved % morulae/blastocysts per cleaved*

A 117 75a 48a

B 78 63a 43a

C 81 79a 46a

D 46 9b 0b

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Lazzari et al., 2002

* : In vivo culture of cleaved embryos from D2-D7 in sheep oviduct

Rescue of a dead or castrated stallion

• The two caudal epididymides of a normal, sexually rested, adult stallion should contain 54x109 sperm or approximately 61% of the sperm in the excurrent duct system

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Rescue of a valuable stallion

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54x109 sperm

Sperm harvested from stallion tissuesafter storage for 24 hours at 5°C remain viable.The tail and ductus deferens are then isolated.

Bruemmer, 2006

Rescue of a valuable stallion

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Retrograde flushing15-20 billion sperm cells

Float-up method4-5 billion sperm cells

cauda

ductus

Bruemmer, 2006

Pregnancies and live foals of frozen epididymal semen after AI and ART (low dose, ICSI)

What is needed for cloning?

1. No viable gametes of the chosen individual need to be collected !

2. The collection and banking of somatic cells is less invasive and easier to implement

3. Somatic cell nuclear transfer allows preservation and screening of the entire genome from an individual

37Mastromonaco and King 2006

So in theory…

You can preserve the genetics from individuals:

1. who have died prematurely or unexpectedly;

2. in their prime reproductive years, experiencing reproductive dysfunction;

3. in their post-reproductive years who have failed or lacked the opportunity to breed;

4. who have been castrated.

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And in practice…

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Commercialization of semen/offspringfrom clones of dead/castrated top horses

• Pieraz (world champion endurance- gelded)

• Chellano Z (Jumping champion died at age11)

• Gem twist (jumping champion gelded)

• Grand Dame (Jumping champion)

• Poetin (dressage world champion )

• And many more….

41Source : Cryozootech website

Cryozootech offers somatic cellfreezing and gene banking for

• Rare or endangeredequids such as Baudet du Poitou, Poitevin...

• Geldings which are top performers

• Old and subfertilestallions

• Exceptional mares witha low number of offspring

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Influencing factors : life span of celltype and chromosomal constitution

43Mastromonaco and King 2006

Influencing factors : fusion method

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Zona-free method is about 3.2 times more effective than the zona-enclosed method in the case of cumulus-derived NT-embryos Zona –free method is 2.3 times more effective in the case of fibroblast-derived NT-embryos.

Lagutina et al., 2005

Pregnancies and foals after SCNT

Donor cell Horse No of embryos

No ofrecipients

Pregnant at 3 months (%)

Offspring(%)

Cum 22h IVM * 3 3 0 0

Fetal fibroblasts C 33 18 1 (6) 0

Adult fibroblasts A 4 2 0 0

Adult fibroblasts B 71 23 3 (13) 2 (9)

Adult fibroblasts C 26 12 1 (8) 0

45Lagutina et al., 2005B=Clone of Pieraz for Cryozootech

Future perspectives and conclusions

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How can ART in the future be appliedfor AnGR?

1. Frozen oocyte banking ?

2. Interspecies cloning?

3. Resurrection of extinct species/breeds?

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Frozen oocyte banking? YES but…

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Table 1. Developmental potential of somatic cell nuclear transfer embryos derived from fresh and cryopreserved oocytesa, bValues within the same columns with different superscripts differ significantly (P < 0.05)

Chang et al., 2008

Interspecies cloning? YES but…

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Domestic catAs recipient oocytefor donor wild cats

Sand cat17 % blastocysts

1 % offspring

African wild cat27 % blastocysts1 % offspring

Black-footed cat3 % blastocysts

No offspring

Rusty spotted cat 26 % blastocysts

No offspringGomez et al., 2009

Resurrection of extinct species and breeds?

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Cloning with frozen (no cryoprotectant!) mouse cells that were non viable.

Li and Mombaerts, 2008 : mouse cells at –80°C for up to 342 daysWakayama et al., 2008 : complete mouse body in -20°C for 16 years!

Resurrection of extinct species and breeds?

51Li and Mombaerts, 2008

Serial nuclear transfer after generation of embryonic stem cells from the donor cells!Jeopardizes ease of application in species other than mice, but may be circumvented byinterspecies nuclear transfer

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Resurrection of extinct species? YES but….

Woolly mammoth Pyrenean ibex

Acknowledgments

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•Colleagues of the department of Reproduction , Obstetrics and Herd Health•Research Fund of Ghent University, grant no. 01D04806•Slaughterhouse Rekkem•Equine ET centres : Keros (B), Hof Ter Leeuwe(B), Animal Embryo Centre (NL)

References• Allen WR (2005) The development and application of the modern reproductive

technologies to horse breeding. Reprod Dom Anim 40, 310-329

• Bruemmer, JE. (2006) Collection and Freezing of Epididymal Stallion Sperm. Vet Clin Equine22 (2006) 677–682

• Chang, C., Sung, L., Amano, T., Tian, X.C., Yang, X., and Nagy, Z.P. (2008). Nuclear transfer and oocyte cryopreservation. Reprod. Fertil. Dev. 21, 37–44.

• Choi et al (2006) Equine blastocyst development after intracytoplasmic injection of spermsubjected to two freeze-thaw cycles. Theriogenology 65:808-19

• Galli et al (2003) Pregnancy: a cloned horse born to its dam twin. Nature. 2003 Aug7;424(6949):635.

• Galli et al. (2007) Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer. Anim Reprod Sci 98; 39-55

• Gómez et al. (2009) Cloning endangered felids using heterospecific donor oocytes and interspecies embryo transfer. Reprod Fertil Dev. 2009;21(1):76-82.

• Lagutina et al. (2005) Somatic cell nuclear transfer in horses: effect of oocyte morphology, embryo reconstruction method and donor cell type. Reproduction 130 559-567

• Lazzari et al. (2002) Equine embryos at the compacted morula and blastocyst stage can be obtained by intracytoplasmic sperm injection (ICSI) of in vitro matured oocytes with frozen-thawed spermatozoa from semen of different fertilities. Theriogenology 58, 709-712. 54

References

• Li J, Mombaerts P. (2008) Nuclear –transfer mediated rescue of the nuclear genome of nonviable mouse cells frozen without cryoprotectant. Biol Reprod 79(4):588-93

• Mastromonaco A &King WA (2007) Cloning in companion animal, non-domestic and endangered species: can the technology become a practical reality? Reprod Fertil Dev 19, 748–761

• Palmer et al., (1991) In vitro fertilization in the horse. A retrospective study. Journal of Reproduction and Fertility Supplement 44, 375-384.

• Piña-Aguilar RE et al., (2009)Revival of extinct species using nuclear transfer: hope for the mammoth, true for the Pyrenean ibex, but is it time for "conservation cloning"?Cloning Stem Cells. 2009 Sep;11(3):341-6

• Ribeiro BI et al. (2008). Transport of equine ovaries for assisted reproduction. Anim Reprod Sci108:171-179.

• Squires et al 1996. A pregnancy after intracytoplasmic sperm injection into equine oocytes matured in vitro. Theriogenology 45, 306.

• Stroud B (2009) IETS foundation Bovine in vivo ova tutorial. www.iets.org

• Smits et al., (2010) Birth of the first ICSI foal in the Benelux. Vlaams Diergeneeskundig Tijdschrift 79, 134-138

• Vanderwall D (2000) Current Equine Embryo Transfer Techniques- IVIS

• Wakayama et al., 2008. Production of healthy cloned mice from bodies frozen at -20° C for 16 years. Proc Natl Acad Sci U S A.105(45):17318-22.

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