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Assisted Reproduction In Endangered Species Rick Mauldin & Collette Thepenier BS640, Fall 2006. What is an Endangered Species?. Defined by the Convention on International Trade in Endangered Species (CITES)

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Assisted Reproduction

In

Endangered Species

Rick Mauldin & Collette Thepenier

BS640, Fall 2006


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What is an Endangered Species?

Defined by the Convention on International

Trade in Endangered Species (CITES)

Appendix I includes species threatened with extinction. Trade in specimens of these species is permitted only in exceptional circumstances.

Appendix II includes species not necessarily threatened with extinction, but in which trade must be controlled in order to avoid utilization incompatible with their survival.


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What is an Endangered Species?

“A species that is in danger of extinction and whose survival is unlikely if the causal factors continue; included are species whose numbers have been reduced to a critical level or whose habitats have been so drastically reduced that the species are deemed to be in danger of extinction.”

www.artistwd.com.


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Causes of Extinction

  • Habitat loss or degradation

    • agriculture

    • development

  • Overexploitation (hunting)

  • Competition/predation by invasive

  • species

  • Combinations of the above,

  • frequently with a triggering event,

  • i.e. natural disaster


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    World Conservation Union 2006 Red List

    Mammals 1,093

    Birds 1,206

    Reptiles 341

    Amphibians 1,811

    Fishes 1,173

    Total 5,624

    Invertebrates 2,101


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    What is Assisted Reproduction?

    In humans:

    “The use of medical techniques, such as drug therapy, artificial insemination, or in vitro fertilization, to enhance fertility.”

    Expanded to include:

    Any directed action taken by humans to enhance reproduction in animals.


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    Assisted Reproduction Techniques

    Divide the topic into two parts:

    1) Assisted reproduction with a technical

    component (mostly mammals)

    2) Assisted reproduction using various

    forms of population management

    The two are not mutually exclusive


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    Assisted Reproduction Techniques

    • Artificial Insemination

    • Embryo transfer

    • In vitro fertilization

    • Semen/embryo sexing

    • Gamete/embryo micromanipulation

    • Somatic cell nuclear transfer (SCNT)

    • Genome resource banking


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    Five Steps Necessary for Adoption of an

    AR Technique

    (from Swanson, 2006)

    • Technique development in a domestic

    • animal counterpart, if available

    • 2) Characterization of species-specific reproductive

    • biology in a targeted non-domestic animal

    • 3) Assessment of technique feasibility for producing

    • offspring

    • 4) Demonstration of adequate efficiency for applied usage

    • 5) Application of new tool for population management


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    ‘Charismatic Megafauna’

    Animals that have popular appeal and so can form the basis of conservation campaigns and fundraising drives.



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    Before we even begin:

    • For endangered species, lack of basic knowledge

      of reproductive cycles is a major impediment

    • How can information concerning estrous cycles/

      pregnancy be obtained when individuals are rare,

      restrictions exist on what may be done with

      captives?

    • Fecal/urinary assays for steroid/hormone metabolites

      to indicate endocrine status

      • gorillas (Czekal, 1997)

      • pandas (Kersey et al. 2003)

      • killer whales (Pukazhenthi and Wildt (2004)

      • Ultrasonography also useful in following ovarian

      • cycles


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    Artificial Insemination (AI)

    • Wild female trapped and fertilized with

    • sperm from zoo male, released, or vice

    • versa.

    • Major application is to avoid genetic

    • depression in fragmented populations

    • Assumes sperm can be collected


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    “First, we are working with species that are

    often dangerous, if not homicidal” (Wildt, 2006)


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    Artificial Insemination

    • Standard collection methods, i.e.

      • artificial vagina

      • vaginal condoms

      • electroejaculation (under anesthesia)

      • post-mortem epididymal

  • These can be difficult for:

    • non-domestic equids,

    • some great apes,

    • canids

    • marsupials (Pukazhenthi &Wildt, 2004)

  • Large interspecific differences in freshvs.

    frozen


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    Artificial Insemination

    • Problems:

    • Ovulation must be induced or synchronized by

      exogenous gonadotropins which frequently do

      not work

    • Gross female anatomical differences, i.e two

      uteri in marsupials, complex cervix in oryx,

      rhinoceros, others.

    • Actual site of semen deposition frequently not

      known, usually transabdominally by

      laparoscope (invasive surgery)


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    Artificial Insemination Successes

    • Conceptions:

    • gerenuk

      • (Litocranius walleri)

      • pacific white-sided dolphin

      • (Lagenorhynchus obliquidens)

      • Bottle-nosed dolphin

      • (Tursiops truncatus)


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    Artificial Insemination Successes

    Live births:

    Persian leopard

    (Panthera pardus saxicolor)

    Spanish Ibex

    (Capra pyrenaica hispanica)

    Giant panda

    (Ailuropoda melanoleuca)

    Cheetah

    (Acinonyx jubatus)


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    Embryo Transfer (ET)

    Most important aspect is to pave the way for

    interspecies transfer

    Gaur born to a holstein

    (Stover, et al. 1981)

    Indian desert cat to a

    domestic cat (Loskutoff, 2003)

    Urial to a domestic sheep

    (Ullah et al. 2006)

    Little is know about embryo development or

    feto-maternal recognition in most species


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    In vitro Fertilization

    Many attempts, few successes

    Requires oocyte collection, knowledge of ovarian

    cycles. Can use ovaries from dying animals.

    Pumas, tigers, cheetahs, Indian desert cats, gaur, elephants, gorillas, zebras, marmosets,

    minke whales, ocelots, springboks, many others

    Live births in only tigers, Indian desert cats, gorillas,

    and European mouflon


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    Gamete/Embryo Micromanipulation

    • Intra-cytoplasmic sperm injection (ICSI)

    • Sub-zonal insemination (SUZI)

      • sperm placed between zp and vitelline membrane,

        • can lead to polyspermy

    • In clouded leopards and cheetah, up to 70% of

    • sperm may be deformed or abnormal.

    • ICSI allows selection of healthy sperm, but has

    • not been applied to endangered species yet.


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    Semen Sexing

    • Great potential in single-sex dominated social

    • structures (O’Brien et al. 2002)

    • Excessive males can play havoc with small

    • population management

    • Used successfully in gorillas in conjunction

    • with IVF and ICSI (O’Brien et al. 2002)


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    Somatic Cell Nuclear Transfer (SCNT)

    • Has been attempted in:

    • giant panda (Chen, et al. 2002)

    • Argali sheep (White et al. 1999)

    • gaur (Lanza et al. 2000)

    • but these attempts did not result in viable offspring

    • Apparently healthy offspring resulted in trans-

    • species cloning in mouflon sheep (Loi, et al. 2001)

    • (mouflon granulosa nuclei to enucleated sheep oocyte)


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    Small Cat Species

    Survival Plan

    Black-footed cat

    Fishing cat

    (Prionailurus viverrinus)

    Arabian sand cat

    (Felis margarita)

    Pallas’ cat

    (Otocolobus manul)

    Brazilian ocelot

    (Leopardus pardalis mitis)

    (Felis nigripes)


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    ART Success Story

    The Wild European Mouflon sheep

    (Ovis musimon)

    • Oocytes collected, then matured and fertilized in vitro

    • After cryopreservation, 10 pairs of blastocysts were

    • transplanted into 10 recipient domestic ewes.

    • Results: 4 live offspring


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    The Story So Far……..

    • ART shows great promise for enhancing

    • reproduction in endangered species

    • To date, reality has not kept up with “hype”,

    • results are inconsistent, usually failure. These

    • techniques require much more R&D

    • Many species do not have the time to wait, and

    • ART is expensive and represents an investment

    • that can be used elsewhere

    • Disparity between promise and deliverable

    • results has caused conflict between ART

    • researchers and wildlife biologists


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    CryoPreservation

    • Inhibits all biological activity, esp. that which leads to cell death or deterioration

    • Use Vitrification Agent to prevent crystalline formation via the conversion of cellular material to an amorphous glass-like solid

    • CONS:

      • Species differences in cryosensitivity

        - Nieman and Rath, 2001

      • No standard Vitrification method

        - Andrabi & Maxwell, 2006


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    Genome Resource Banking

    • Goal: To create depositories of germplasm as an interface between ex situ and in situ conservation programs- Holt and Pickard, 1999

    • GRB can be tool for managing the exchange of genetic diversity among endangered species by facilitating the creation of a global gene pool- Hanks, 2001

    • One of the most effective ways to conserve genetic resources is by establishing cryocollections and cryobanks of endangered and industrially important species- Katov, 2006



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    Types:

    Semen Banks

    Spermatogonia

    Embryo or Oocyte Banks

    Primordial Follicles

    Fertilized Oocytes

    Tissue Graft Banks

    Ovarian Tissue

    Testicular Tissue

    Genome Resource Banking


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    Pros & Cons

    • Semen Banks

      • (+): Heterozygosity; DEC in captive ♂ and in mvmt of animals

      • (-): Low efficiency in sperm production – Paris, et al., 2004

    • Embryo or Oocyte Banks

      • (+): Storage of full or half complement of DNA to preserve heterozygosity

      • (-): Cryobiology of Embryo & Oocyte viability relatively unknown – Andrabi & Maxwell, 2006

    • Tissue Graft Banks

      • (+): Age, Cycle doesn’t matter; Can collect from carcasses of unknown etiology; Future with SCNT – Cleary et al., 2006; Snow et al., 2001

      • (-): More research needed to identify sources of suitable cells

        – Andrabi & Maxwell, 2006




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    Ex situ Conservation

    Entrance to the Bronx Zoo in New York

    • “Off-site” Conservation

    • Protection via the removal of a species from unsafe or threatened habitat and placing the entire population (or some portion of) under human care


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    Ex situ Conservation

    Methods:

    • Zoos & Breeding Centers

      • Maintain whole, protected specimens for breeding and reintroduction into the wild when necessary and possible

      • Education of Public

    • “Frozen Zoo”

      • Gene Banks, consisting of cryogenic facilities used to store living sperm, eggs, or embryos


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    Ex situ Conservation

    Cons:

    • Helpful, but insufficient to save species from extinction

      • Cannot recreate natural habitat or the adaptation/evolutionary selection that would occur therein

    • Assisted Reproductive Technologies are very $ COSTLY $


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    “[T]he long-term survival of most endangered species depends not only on our ability to prevent further losses but also on our ability to increase their populations by restoring degraded habitats, often on private lands.”

    - Wilcove & Lee, 2004


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    In situ depends not only on our ability to prevent further losses but also on our ability to increase their populations by restoring degraded habitats, often on private lands.” Conservation

    • “On-site” Conservation

    • Protection of a species in it’s natural habitat either by protecting or cleaning up the habitat itself, or by defending the species from predators


    In situ conservation41 l.jpg
    In situ depends not only on our ability to prevent further losses but also on our ability to increase their populations by restoring degraded habitats, often on private lands.” Conservation

    Benefit:

    • Allow species population to recover in its natural environment, in which adaptations have evolved naturally

      Essentially Involves Habitat Protection from:

    • Human Intrusion & Destruction


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    In situ depends not only on our ability to prevent further losses but also on our ability to increase their populations by restoring degraded habitats, often on private lands.” Conservation

    Methods:

    • Endangered Species Act

      • Prevent Gov’t Agencies or Private Individuals from inflicting further harm on a species once it has been declared Endangered

      • BUT it does NOT require private citizens to undo past deeds for the sake of recovery – so…

    • Incentive-based Programs

      • Safe Harbor (>>> success)

      • Environmental Defense’s Landowner Conservation Assistance Program (>>> success)

      • Conservation Banking (> success)

        - Wilcove & Lee, 2004


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    Zoo Biologists vs. Wildlife Managers depends not only on our ability to prevent further losses but also on our ability to increase their populations by restoring degraded habitats, often on private lands.”

    So who is right?

    …they BOTH are


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    Strategies depends not only on our ability to prevent further losses but also on our ability to increase their populations by restoring degraded habitats, often on private lands.”

    • Captive Breeding Programs

      • Small # of Founder Individuals

      • Entire Population

        • Black Footed Ferrets

    Mustela nigripes


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    Strategies depends not only on our ability to prevent further losses but also on our ability to increase their populations by restoring degraded habitats, often on private lands.”(cont’d)

    Lynx canadensis

    • Reintroduction

      • Translocation of Wild Members

        • Canadian Lynx

          • Continuous release of wild caught members into wild to compensate for high initial mortality

      • Captive Bred

        • Whooping Crane

          • Show the birds their migration path

    Grus americana


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    Strategies depends not only on our ability to prevent further losses but also on our ability to increase their populations by restoring degraded habitats, often on private lands.”(cont’d)

    • Reduction in Disease/Pollution

      • California Winter Run Chinook Salmon

        • Clean up heavy metal leaching into water

      • Peregrine Falcon

        • Ban DDT pesticide

    • Predator Removal

      • California Least Tern

        • Common Crow

    Oncorhynchus tshawytscha

    Sterna antillarum browni

    Falco peregrinus


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    Strategies depends not only on our ability to prevent further losses but also on our ability to increase their populations by restoring degraded habitats, often on private lands.”(cont’d)

    Enhydra lutris

    • Laws Against Poaching & Illegal Collecting

      • Southern Sea Otter

      • Galapagos Tortoise

    • Development Legislation & Restrictions

      • Grizzly Bears

        • Banning development in areas that would fragment habitat and thus increase human interaction

    Geochelone nigra

    Ursus arctos


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    Strategies depends not only on our ability to prevent further losses but also on our ability to increase their populations by restoring degraded habitats, often on private lands.”(cont’d)

    • Habitat Acquisition & Restoration

      • Rainforest

        • Blue Poison Arrow Frog

      • Wetlands

        • Bog Turtle

    Dendrobates azureus

    Clemmys muhlenbergii


    Strategies cont d49 l.jpg
    Strategies depends not only on our ability to prevent further losses but also on our ability to increase their populations by restoring degraded habitats, often on private lands.”(cont’d)

    • Reducing Inbreeding

      • Florida Panther

        • Introduce Texas Mountain Lion to wild population

    • Public Cooperation

      • Karner Blue Butterfly

        • Don’t mow lawns later in summer to avoid interference with butterfly eggs

    • Ranching Program

      • American Alligator

        • Eggs are purchased from landowners

    Puma concolor coryi

    Alligator mississippiensis


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    Trage depends not only on our ability to prevent further losses but also on our ability to increase their populations by restoring degraded habitats, often on private lands.”dies

    • Unavoidable…

      • Accidents/Disaster

        • Puerto Rican Parrot

          • 1989 Hurricane Hugo

        • Leatherback Sea Turtle

          • 2004 Tsunami

      • War

        • Mountain Gorillas

          • Civil War in Rwanda

    Amazona vittata

    Dermochelys coriacea

    Gorilla berengei berengei


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    - Holt and Pickard, 1999 depends not only on our ability to prevent further losses but also on our ability to increase their populations by restoring degraded habitats, often on private lands.”


    Financial considerations l.jpg
    Financial Considerations depends not only on our ability to prevent further losses but also on our ability to increase their populations by restoring degraded habitats, often on private lands.”

    • There is no such thing as a Million-Dollar Ocelot

    • But there is such a thing as a Million-Dollar Bull


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    - Holt and Pickard, 1999 depends not only on our ability to prevent further losses but also on our ability to increase their populations by restoring degraded habitats, often on private lands.”


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    Take Home Message: depends not only on our ability to prevent further losses but also on our ability to increase their populations by restoring degraded habitats, often on private lands.”

    Synergistic Approach!

    • Use both ART and ARM:

      • Neither are sufficient by themselves (at present)

    • ART has potential for success in the FUTURE

    • ARM can keep problems from getting worse TODAY

      ART + ARM=Success


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    Questions? depends not only on our ability to prevent further losses but also on our ability to increase their populations by restoring degraded habitats, often on private lands.”


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    THANKS! depends not only on our ability to prevent further losses but also on our ability to increase their populations by restoring degraded habitats, often on private lands.”

    If you would like copies of the slides

    or a list of citations, please email:

    Collette Thepenier

    [email protected]

    Rick Mauldin

    [email protected]


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