potential impact of emerging technologies on angr...
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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
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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
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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
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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
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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
C
Viagen (USA)
Cryozootech (F)
Avantea (I)
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Texas A&M University
Colorado State University
Ghent University?
Goulburn Valley Equine Hospital (AU)
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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
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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
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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
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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
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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)
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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
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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.
38Provided that sufficient numbers of viable somatic cells have been frozen!
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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
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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
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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
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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
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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
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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
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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!
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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
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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)
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