Assisted Reproduction In Endangered Species Rick Mauldin & Collette Thepenier BS640, Fall 2006

1. Assisted Reproduction In Endangered Species Rick Mauldin & Collette Thepenier BS640, Fall 2006

2. 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.

3. 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.

4. 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

5. 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

6. 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.

7. 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

8. Assisted Reproduction Techniques • Artificial Insemination • Embryo transfer • In vitro fertilization • Semen/embryo sexing • Gamete/embryo micromanipulation • Somatic cell nuclear transfer (SCNT) • Genome resource banking

9. 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

10. ‘Charismatic Megafauna’ Animals that have popular appeal and so can form the basis of conservation campaigns and fundraising drives.

12. World Wildlife Fund Logo

13. 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

14. 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

15. “First, we are working with species that are often dangerous, if not homicidal” (Wildt, 2006)

16. 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

17. 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)

18. Artificial Insemination Successes • Conceptions: • gerenuk • (Litocranius walleri) • pacific white-sided dolphin • (Lagenorhynchus obliquidens) • Bottle-nosed dolphin • (Tursiops truncatus)

19. Artificial Insemination Successes Live births: Persian leopard (Panthera pardus saxicolor) Spanish Ibex (Capra pyrenaica hispanica) Giant panda (Ailuropoda melanoleuca) Cheetah (Acinonyx jubatus)

20. 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

21. 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

22. 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.

23. 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)

24. 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)

26. 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)

27. 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

28. 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

29. 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

30. 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

31. - Holt and Pickard, 1999

32. Types: Semen Banks Spermatogonia Embryo or Oocyte Banks Primordial Follicles Fertilized Oocytes Tissue Graft Banks Ovarian Tissue Testicular Tissue Genome Resource Banking

33. 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

34. - Holt and Pickard, 1999

35. Assisted ≠ Artificial

36. 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

37. 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

38. 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 $

39. “[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

40. 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

41. 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

42. 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

43. Zoo Biologists vs. Wildlife Managers So who is right? …they BOTH are

44. Strategies • Captive Breeding Programs • Small # of Founder Individuals • Entire Population • Black Footed Ferrets Mustela nigripes

45. 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

46. 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

47. 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

48. Strategies(cont’d) • Habitat Acquisition & Restoration • Rainforest • Blue Poison Arrow Frog • Wetlands • Bog Turtle Dendrobates azureus Clemmys muhlenbergii

49. 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

50. 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