Ampika Thongphakdee (DVM, PhD)

Wildlife Reproductive Innovation Center, Research Department Bureau of Conservation & Research, Zoological Park Organization, THAILAND

Crisis of wildlife loss

Causes of this extinction crisis

- loss & degradation of natural habitats

- climate change

- invasive species

- pollution

- unsustainable exploitation

- illegal wildlife trade

(IUCN Red List of Threatened Species)

Extinction rates 100 to 1,000 times

above their natural level!!

Maintaining genetic diversity of captive wildlife population of global importance

Strengthen research programs on reproductive science and establish genome resource bank

Conducting reintroduction programs for the ‘extinct-in-the-wild’ species

Fighting Extinction!

Eld’s deer Malayan tapirGoralEastern Sarus crane

5

Suitable Captive Management Facilitates Natural Breeding.

Understanding of Basic Reproductive Science is a Key!!

Sperm morphology and semen collection by electroejaculation in white rhinocerous

Reproductive hormone estimation by noninvasive technique from fecal and urine samples

CU-ZPO MOU since 2007

Fishing cat Asian golden cat Clouded leopard

Leopard cat Flat-headed cat Jungle cat

Investigation of Semen Characteristic in Asian Wild Felids

Credit picture # Nattachai Choipeng, Arnuparp Yhamdee, Piyapong Chinnadate

1) macrocephalic sperm, leopard cat

2) bent and coil tails, fishing cat

3) double head sperm, flat headed cat

0

10

20

30

40

50

60

70

Head

Tail

Percentage of abnormal sperm morphology (teratospermia)

1 2 3

Electroejaculation in clouded leopard

Sperm cryopreservation and in vitro fertilization

Sperm binding

Embryo development

Frozen sperm

Populationsurvey

Habitat, ecology

Health surveillance

Genetic preservation

Captive breeding

Fishing cat research project: Intensively managed population (IMP) model

Fishing cat (Prionailurus viverinus)

• Medium sized wild cat

• Wetland habitats

• Endangered

- wetland destruction and degradation

Camera trap and carnivore case trap

Population survey

• about 28 fishing cats in 30 Km2 of investigated area

• Male: 10-13 Kg., 7-10 Km2 of habitat size

• Female: 7-9.5 Kg., 4-7 Km2 of habitat size, 2-3 cubs per litter

• Main feed from 119 feces samples

**Some of fishing cats were killed by local people because fishing cats hunt chickens and ducks in the community.

Health surveillance and hematological value of fishing cats

White blood cell (WBC) 14587.5 ± 5829.3 x 109/L

Red blood cell (RBC) 6.7 ± 1.0 x 106/µL

Hematocrit (Hct) 14.1 ± 2.8 g/dL

Platelet 162.0 ± 66.5 x103/µL

Albumin (Alb) 12.7 ± 28.2 mg%

Alkaline phosphatase (ALP) 26 ± 23.2 IU/L

Alanine aminotransferase (ALT) 96.2 ± 62 IU/L

Aspartate aminotranferase (AST) 87.1 ± 17.8 IU/L

Blood urea nitrogen (BUN) 33.1 ±12.7 mg%

Cholesterol 126.8 ± 49.2 mg%

Creatinine 14.5 ± 40.9 mg%

Creatinine kinase 311.4 ± 203.3 IU/L

Total billirubin 1.45 ± 0.9 mg%

Total protein 8.35 ±1.5 mg%

Disease detection!!

%Toxoplasmosis

Semen characteristic of fishing cat

parameters value

semen volume 209± 138.6 µL

pH 8.2± 0.6

motility 76.7± 17.6 %

sperm concentration 128.4 ± 182.8 x 106

spermatozoa/mL

normal sperm head 89.5± 5.0%

normal sperm tail 51.8± 19.8 %

• 2/5 males had good semen quality and the samples was cryopreserved.

• AI captive females with wild males has been conducting.

• Banked semen and ART can play important roles on genetic exchange.

Monitoring radio signal from fishing cat

Genome Resource Bank

Oocyte (egg)

cell

sperm

embryo

Insurance against losses of genetic diversity or entire species

- disaster; tsunami, flooding, earthquake

- infectious disease outbreaks

Reduce risk from transportation

(Howard and Wildt, 2009)

Biological material could be recovered from free-living animals, stored & used without removing more individuals from native habitats.

Cryopreservation= important tool for establishment of GRB, as it maintains

the genetic material viability for an indefinite time.

Liquid nitrogen -196ᵒ C

ZPO Genome Resource Bank

32 wildlife species

- Asian elephant- Malayan tapir- guar, serow, goral - deer & wild cat sp.

5,810 samples- sperm: 5,113 straws- somatic cells: 668 tubes- embryos: 29 straws- ovarian tissues

Semen characteristics of Eastern sarus crane is influenced by seasonality.Cold storage of Eastern sarus crane semen & the routine production of chicks at Nakhonratchasima Zoo by AI.

Eastern sarus crane (Grus antigone sharpii)

• Extinct-in-the-wild species.

• 8-year reintroduction program (2008-2016)

Tharasanit et al., Reprod Domest Anim (ICAR, 2012)

• Artificial insemination (AI)

• In vitro fertilization (IVF)

• Nuclear transfer (NT/cloning)

• Embryo transfer (ET)

ARTs: methods used to achieve pregnancy by artificial or partially artificial means

ARTs help to achieve…

74% of adult male panda fail to mate naturally!! (poor mating interest)

What would we do if natural mating failed repeatedly?

Concentration of urine estrogen and progesterone excretion in female panda evaluated by competitive enzyme linked immuno sorbent assay (ELISA).

Milky ejaculated semen in a warm water-jacketed glass cup (A), and sperm morphology ofpanda (B) William’s staining, x 1000

Transcervical Artificial Insemination

6 days old

Lin Bing was born on 27th May 2009

Siriaroonrat et al., 2009. Thai J Vet Med 39: 199-204.

Lin Bing gave birth on 27th July 2015

AI cubs born on 10 June 2015

Challenges in clouded leopard breeding

• most difficult felid to breed• largest canines for size• size difference male to female• severe male aggression• fatal attacks• high cub mortality

AI cubs, 2015

(Tipkantha et al., 2015)

LH 1

LH 2

8-11 weeks

6 weeks

18-20 days

Time to AI

Ovulation detection and semen collection for AI in Asian elephants

Morphology of normal sperm, Diff-Quik®stainingSemen collection

AI using endoscopy

Endangered Eld’s deer (Brow-antlered deer; Rucervus eldii)

Subspecies Wild Captive

A) Sangai <200 200

B) Thamin 2,500 2,000

C) Siamensis <200 <100

Thailand: Extinct in the wildCaptivity: Thamin 1,000/ Siamensis 80

Due to fragmented distribution and small population, the specie are therefore at risk from inbreeding and loss of genetic variation.

Thongtip et al., 2010

AI Eld’s deer reintroduction at Salakpra Wildlife Sanctuary 2012

frozen straws to the forest…

Thailand: Extinct in the wild

Frozen semen

Intrauterine AI in Thamin Eld’s Deer

In vitro fertilization (IVF)

Benefits:• multiple embryos and offspring

(if natural breeding, only 1 fawn/dam/year)

• selected genetic representation

= fertilization of sperm and egg occur outside female body

follicles

Ovarian response after hormonal stimulation

Embryos were produced in VitroSafe system.

Laparoscopic oocyte aspiration

Eld’s deer oocytes (n=81) develop to 2-cell (A), stained embryos at 2-cell (B), 4-cell (C) 16-cell (D) morula (E) blastocyst stages (E) and stained blastocyst (F). Blastocyst cell number 86.5 ± 28.2 (n=4)

D E F

A B C

Thongphakdee et al., manuscript in preparation

Test-tubed fawn born on 17th October 2011

IVF Eld’s deer gave birth 22nd October 2014

Somatic Cell Nuclear Transfer (Cloning)

Marbled cat and flat-headed cat cloned embryos

(Thongphakdee et al., 2006, 2009, 2010)

Donor cells= technique in which the nucleus of a somatic cell(donor cell) is transferred to the cytoplasm ofanenucleated oocyte ( nucleus removed).

• Medical therapies for degenerativediseases i.e. diabetes

• Produce eggs & sperm which couldbe used in captive breeding program

Drill primate (San Diego Zoo)

The rhino stem cells were shown to be capable of forming neurons (red) Snow leopard

(Verma et al., 2014)

Could it save endangered species?

Induced Pluripotent Stem Cells; iPS

Future challenge….

The quality of fresh and chilled ejaculates diluted in three extenders in wild boars (Sus scrofa)

Kakanang Buranaamnuay, Supaluk Kaitsomboon, Saifon Yaphila, Nudthakamol Kajornklin and Ampika Thongphakdee

Objective: To examine the quality of fresh and chilled

ejaculates diluted in three commercial extenders

D0

D1 D2 D3 D4 D5

I) Beltsville

Thawing Solution (BTS)

II) Androstar® Plus (Andro)

III) ModenaTM

(Modena)

15°C

Parameter Mean SD

Motility (%)

• Total 89.7 8.7

• Progressive 43.3 12.1

Viability (%) 85.7 9.3

Normal

morphology (%)

• Acrosome 88.7 3.2

• Head 91.3 2.5

• Tail 37.7 27.3

Table 1 Characteristics of fresh spermatozoa obtained from 3 wild boars

Superovulatory response of ovarian follicles using a split-single intramuscularly of

follicle-stimulating hormone in rusa deer

Vibuntita Chankitisakul, Ampika Thongphakdee, Chavin Chaisongkram, Kanda Ponsrila, Anuchisa Phojun, Nudthakamol Kajornklin,

Yosvaris Semaming, Wuttigrai Boonkum, and Mongkol Techakumphu

• Objective: To evaluate the follicular stimulation after treatment by the novel

program includes Presynch-Ovsynch and split-single doses of FSH injection protocols

that allowed for estrus synchronization and superstimulation.

TreatmentsOvulation

(No. of CLs)

Unruptured

follicle

Total

follicular

stimulation

Group 1 4.3 4.0 8.3

Group 2 3.0 4.3 7.3

Table 1 The ovarian follicular response of rusa deer

Presynch-Ovsynch and split-single doses of FSH injection protocols

could be used effectively for superovulation in rusa deer.

Presynch-Ovsynch with slow released FSH stimulation would be

more beneficial in terms of reduce animal stress and human

handling, which is feasible to be applied in other endangered

Cervidae.

Integration-free derivation of induced pluripotent stem cells from

endangered wild animals

Woranop Sukparangsi, Santhita Karoon, Ruttachuk Rungsiwiwut, Ampika Thongphakdee

• Recent reproductive science and progress of ARTs show possible promisingapplication in wildlife conservation.

Conclusion

• Harmonization of novel science and technologies combined with intensivepopulation management would bring “hope” for protecting threatened species.

Research collaboration and fundingKey of the success….