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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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slide1

Assisted Reproduction

In

Endangered Species

Rick Mauldin & Collette Thepenier

BS640, Fall 2006

slide2

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.

slide3

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.

slide4

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
slide5

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

slide6

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.

slide7

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

slide8

Assisted Reproduction Techniques

  • Artificial Insemination
  • Embryo transfer
  • In vitro fertilization
  • Semen/embryo sexing
  • Gamete/embryo micromanipulation
  • Somatic cell nuclear transfer (SCNT)
  • Genome resource banking
slide9

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
slide10

‘Charismatic Megafauna’

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

slide13

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
slide14

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
slide15

“First, we are working with species that are

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

slide16

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

slide17

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)

slide18

Artificial Insemination Successes

  • Conceptions:
  • gerenuk
    • (Litocranius walleri)
    • pacific white-sided dolphin
    • (Lagenorhynchus obliquidens)
    • Bottle-nosed dolphin
    • (Tursiops truncatus)
slide19

Artificial Insemination Successes

Live births:

Persian leopard

(Panthera pardus saxicolor)

Spanish Ibex

(Capra pyrenaica hispanica)

Giant panda

(Ailuropoda melanoleuca)

Cheetah

(Acinonyx jubatus)

slide20

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

slide21

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

slide22

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.
slide23

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)
slide24

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)
slide26

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)

slide27

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
slide28

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
cryopreservation
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

genome resource banking
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
genome resource banking32
Types:

Semen Banks

Spermatogonia

Embryo or Oocyte Banks

Primordial Follicles

Fertilized Oocytes

Tissue Graft Banks

Ovarian Tissue

Testicular Tissue

Genome Resource Banking
pros cons
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

ex situ conservation
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
ex situ conservation37
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
ex situ conservation38
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 $
slide39
“[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

in situ conservation
In situ 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
In situ 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
in situ conservation42
In situ 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

zoo biologists vs wildlife managers
Zoo Biologists vs. Wildlife Managers

So who is right?

…they BOTH are

strategies
Strategies
  • Captive Breeding Programs
    • Small # of Founder Individuals
    • Entire Population
      • Black Footed Ferrets

Mustela nigripes

strategies cont d
Strategies(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

strategies cont d46
Strategies(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

strategies cont d47
Strategies(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

strategies cont d48
Strategies(cont’d)
  • Habitat Acquisition & Restoration
    • Rainforest
      • Blue Poison Arrow Frog
    • Wetlands
      • Bog Turtle

Dendrobates azureus

Clemmys muhlenbergii

strategies cont d49
Strategies(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

trage dies
Tragedies
  • 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

financial considerations
Financial Considerations
  • There is no such thing as a Million-Dollar Ocelot
  • But there is such a thing as a Million-Dollar Bull
take home message
Take Home Message:

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

thanks
THANKS!

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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