BALANCING CONSERVATION OBJECTIVES AND METHODS FOR AnGR: THE EMERGING ROLE OF EX SITU IN VITRO CONSER

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BALANCING CONSERVATION OBJECTIVES AND METHODS FOR
AnGRTHE EMERGING ROLE OF EX SITU IN VITRO
CONSERVATION

• Sipke J. Hiemstra Henri Woelders
• Balice, 1 June 2007

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In this paper

• The need for AnGR conservation strategies
• Conservation objectives and approaches
• State of the art in cryopreservation and
  reproductive technologies
• Decision making for ex situ in vitro strategies
• European and global issues

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Global status and trends what is the problem ?

• Some examples
• FAO (2007) 36 of breeds in Europe and Caucasus
  are categorized at risk(but what does this
  mean exactly?)
• Poultry, pig (and cattle) breeding industry only
  a few global players left
• Within-breed diversity often limited, e.g.
  Holstein Friesian Effective population size lt
  100
• Agro-ecosystems and associated local breeds
  threatened
• Tisdell (2003) major socio-economic causes to
  loss of AnGR diversity
• Economic globalisation and extension of markets
  (breeding material)
• Decoupling of animal husbandry from surrounding
  natural environment
• Increasing major threats to AnGR (e.g. Gibson et
  al. 2006)

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What to conserve ?

• Breeds ?
• Allelic diversity ?
• Knowledge ?
• Ecosystems, rural areas or livelihoods ?

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In situ or ex situ how to conserve genetic
diversity

• In situ conservation
• Conservation by utilization and sustainable
  breeding
• Maintaining a breed in a dynamic state,
  characterized by slow and balanced adaptation to
  conditions and changing functions
• But threatened by genetic drift, high selection
  pressure, inbreeding or extinction risks
  (diseases/disasters)
• Ex situ conservation
• Live (in vivo) - limited scale- will not
  guarantee maintenance of present day diversity-
  often not a clear distinction with in situ
• Cryo storage (in vitro/gene banks)- instrumental
  for in situ efforts, supporting breeding today
  (increase Ne)- long term objectives for rare
  breeds ánd commercial breeds (insurance
  market/production system changes/ calamities)
• In situ and ex situ are complementary Risk
  management! Long term ánd short term
  objectives!

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Types of genetic resource material in gene bank?

• Spermatozoa
• Oocytes
• Embryos/embryonic cells
• Somatic cells
• Feasible?
• Practical?
• Economical?

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Cryobiologic principles

• Cryopreservation suspended animation in liquid
  nitrogen (-196 C)
• All chemical and physical processes arrested
• Only Cosmic background radiation is a possible
  source of DNA damage.
• This is estimated to become relevant only after
  several thousands of years (Mazur, 1985)
• Consequently Storage is safe for several
  thousands of years, possibly longer

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Semen cryopreservation

• Semen of most species can be frozen adequately
• Fair post-thaw viability can be expected for most
  species
• Dedicated freezing media and equipment available
• Optimal freezing media, cryoprotectant,
  protocols, widely varies between species (i.e.
  mammals and birds)
• Large differences between species in
  insemination techniques and pregnancy rates
  using fresh or frozen semen
• Fertility of frozen semen lower than that of
  fresh semen
• In cattle, pregnancy rates with frozen semen are
  as good as that of fresh semen, but specific
  problems in other species

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Example improving freezing boar semen

• optimising cooling rate
• minimising cryo-damage
• less boar variation with new method
• elevation of level of poorest males

Woelders et al., 2005
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Example progress in cock semen preservation

• State of the art often low post-thaw survival
  and fertilized eggs
• Recent research
• A large number of cryoprotective agents (CPAs)
  have been tested (glycerol, DMSO, EG, DMA)
• A number of media have been compared
• A new medium was composed that combines the
  advantages of different media
• The interaction between cooling rate and CPA
  concentration was studied
• Good results with high cooling rate and DMA

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Dev embr. Underdev embryos
Dev. embryos Underdeveloped embryos
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Semen cryopreservation (dis)advantages

• Advantages
• Existing infrastructure for semen collection
  and for insemination in a number of species
• Sperm survival after thawing is adequate for most
  species
• But freezability varies among species,
  breeds/lines and males
• Disadvantages
• Back-crossing is required, at least for 6
  generations
• High re-generation costs (higher than embryos)
• Mitochondrial genes are not conserved
• In some species or countries no infrastructure
  is available or collection may be a problem

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Cryopreservation of oocytes

• Considerable progress with oocyte
  cryopreservation in last 10 years
• Viable oocytes after freezing and thawing in
  great number of species
• But.. chance of getting healthy offspring from
  cryopreserved oocytes is still much lower
  compared to embryos
• In vitro production of embryos is possible for
  major livestock species, but efficiency varies
  between species(collection of oocytes from
  slaughtered animals or by the use of ovum
  pick-up)
• Freezing oocytes of avian and fish species is not
  successful (large size, high lipid content, polar
  organisation)

OPS vitrified horse COC Tharasanit et al.
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Oocytes ánd semen (dis)advantages

• Full complement of chromosomal and mitochondrial
  genes
• Flexibility in combining oocytes and semen
• Overall costs of ovum pick-up and IVF probably
  higher than using embryos
• Efficiency and reliability of using oocytes for
  generating embryos and young is still much lower
  compared to cryopreserved embryos

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Cryopreservation of embryos/embryonic cells

• Cryopreservation of embryos is adequate for
  mammalian livestock species (highly successful in
  cattle, recent good results in pigs)
• Success dependent on stage of the embryo (good
  results with blastocysts)
• Not successful in birds and fish (large size,
  high lipid content, polar organisation of early
  embryo)
• Surgical ET possible in all mammalian livestock
  species
• Non-surgical ET possible in cattle, horses (and
  pig)

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Embryo cryopreservation (dis)advantages

• Full complement of chromosomal and mitochondrial
  genes
• In some species infrastructure for ET is
  available (cattle), developing (pig) and is
  possible in other species
• Costs of embryo collection and freezing is in
  general much higher than semen cryopreservation
• No backcrossing needed low re-generation costs
• Especially interesting for species with long
  generation intervals and low reproductive rates

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Cryopreservation of somatic cells

• Collection of suitable somatic cells is possible,
  easy and cheap !
• Different cell types, e.g. skin fibroblasts, can
  be readily cryopreserved
• Using relatively simple cryopreservation
  techniques
• Use of somatic cells to generate offspring
  requires reproductive cloning

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Somatic cells an alternative for semen/embryos?

• In mammals, live offspring reported in sheep,
  cattle, mice, pigs, goats, horses, rabbits and
  cats
• No successful cloning reported in poultry
• Full complement of chromosomal, but no
  mitochondrial genes, after transfer of nuclei
• Low up-front costs of freezing somatic cells
• Reproductive cloning not efficient and not safe
  (yet)(lt4 transferred embryos develop into
  viable offspring)(abortions and malformed young)
• Ethical issues (but also clear differences in
  societal discussion between e.g. EU and USA)
• Future developments on longer term are likely

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Emerging reproductive technologies

• Transplantation of ovarian tissue and germ cells
• Successful germ cell transplantation in mammals,
  poultry and fish
• These emerging technologies may enable production
  of gametes or offspring of rare or extinct breeds
  by abundantly available individuals of related
  common breeds

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Costs of cryopreservation

• Limited published information on costs of AnGR
  conservation
• Investments today (collection, freezing) versus
  future regeneration/use costs
• Long term storage costs relatively cheap
  (compared to in situ)
• Technical developments further cost reduction
  expected
• Less collections/ejaculates needed
• Less doses needed for re-generation
• i.e. extraction of semen from the epididymus
  (after slaughter/castration)
• Boetcher/Gandini combination of embryos and
  semen is cost-effective

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In summary
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Choosing the technique (GandiniOldenbroek, 2007)

• Steps
• Which conservation objectives apply ?
• Rank techniques for their efficacy to reach the
  objectives
• Rank techniques for risk of failure
• Rank techniques for costs
• Choose the technique

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Choosing the donors (sampling)

• Decision-making at two levels
• Breeds to be included (prioritization of breeds)
• Sampling of individuals within selected breeds
•  

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Prioritising breeds from a genetic point of view
Total genetic diversity of a group of breeds
Contribution of a single breed to the total
diversity of the group of breeds

• contribution to the between-breed diversity
• contribution to the within-breed diversity

Centre for Genetic Resources, The Netherlands
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Poultry

• Estimation of Marker
• Estimated Kinships

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Sampling, not only technical/genetic
considerations..

• Interest of stakeholders
• Funding limitations
• Research costs lt-gt sampling-inefficiency costs?
• Cultural, historical reasons
• Legal issues/ownership
• Infrastructure and existing capacity
• Sanitary/veterinary aspects
• Quality standards

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Emerging role for ex situ (long term short
term) ?

• Long term in situ is often more expensive than ex
  situ in vitro
• In situ is vulnerable to serious threats
• Diseases and disasters
• Genetic risks (drift, strong selection, etc)
• Age of current farmers
• Etc.
• We can not control decisions of farmers,
  breeders and other stakeholders (unpredictable
  future)
• Fast technological (omics) developments will
  trigger/change conservation and future use
  (exploitation)
• Developments/genetic erosion go very fast better
  be quick, especially for species with short
  generation interval

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International issues

• Local versus regional transboundary versus
  international transboundary ? breed specific
  (cross-border) strategies needed
• Detailed assessment of current status and roles
  of ex situ conservation by country/species/breeds
• National sovereignty but need for regional (and
  global) collaboration and arrangements/support
• Mechanism to be developed to involve wider group
  of stakeholders (financially, national and
  international)
• Technical/research cooperation
• Regional (or global) gene bank?
• Rights and ownership?
• Rationalization/optimization of gene bank
  collections !

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Thank you for your attention