Artificial Insemination in Endangered Species –

Challenges and Successes

Budhan PukazhenthiCenter for Species Survival

Smithsonian National Zoological Park

Objectives of this Lecture

• Importance of wildlife research• Role of artificial insemination in management of

endangered species• Tools for understanding male and female • Tools for understanding male and female

reproductive biology• Semen collection and cryopreservation• Artificial insemination techniques• AI successes in endangered species• Remaining challenges• Conclusions

Wildlife Research Challenges

• Lack of knowledge

• Small, geographically dispersed collections

• Animals - timid to • Animals - timid to dangerous

• Requires genetic management to retain viability

Usually 90% gene diversity for the next 100 years

Numbers of Described Mammals, Reptiles, Fishes, Amphibians and Birds

Mammal, 5,500

Reptile, 7,500

13%

10%

Bird, 10,200

Amphibian, 5,921

Fish, 27,00048%

10%

18%

11%

Amazing Diversity in Reproduction

< 1% of annual lifespan reproducing

Sperm storage (mo) No males required

Reproductive Diversity Among Species

Species Seasonality Distinguishing features

Domestic dog None • Spontaneous ovulation

Bush dog None • Male reduces inter-estrous interval

Maned Wolf Strict(Oct – Feb)

• Ovulation only in presence of a male

Gray wolf Strict(Jan – Apr)

• Behavioral dominance regulates reproductive success (alpha female)

African wild dog Strict(Apr – Jul)

• Reproduction in alpha female, behavioral and estrogen suppression of subordinates• Litter size up to 20 pups

Island Fox Strict(Jan – Mar)

• Ovulation only in presence of a male

Number of Reproduction Publications for Wildlife Species, Jan 1999 - Sep 2009

Journals Total reproductive science manuscripts

Wildlife -related (%)

Animal Reproduction Sciences 1,434 125 (8.7)

Biology of Reproduction 3,991 286 (7.2)

Cryobiology 197 58 (29.4)

Journal of Mammology 36 32 (88.9)Journal of Mammology 36 32 (88.9)

Molecular Reproduction and Development 1,598 53 (3.3)

Reproduction 1,604 149 (9.3)

Reproduction of Domestic Animals 680 31 (4.5)

Reproduction Fertility and Development 650 134 (20.6)

Theriogenology 1,852 259 (13.9)

Zoo Biology 76 69 (90.8)

Total 12,118 1,196 (9.9)

90% of publications are on common, already-studied speciesWildt et al., 2009

Taking this Basic Information to Affect Species Management

• Consistency

• Contributing to maintaining gene diversity while moving the ‘population needle’

Role of Artificial Insemination in Managing Wildlife Species

• Management tool for enhancing conservation breedingBehavioral incompatibilityPoor reproductive performanceAnatomical defects

• Maintain genetic diversity in small populationsLow number of foundersLimited gene pool in population

• Movement of gametes for conservation strategiesSpecies Survival Plans (SSPs)Genome Resource Banks (GRBs)

Information Critical for Developing Artificial Insemination

Male• Seasonality• Age• Semen collection

Female • Seasonality• Age• Ovulation mechanism

• Gene diversity and ejaculate quality

• Cryopreservation

• Control of ovarian function

• Time of insemination• Site of insemination• Sperm concentration• Impact of stress and/or

anesthesia

Male Reproduction• Seasonality – Pallas cat (distinct

seasonality), cheetah (seasonality not evident)

• Age – onset of puberty; impact on sperm DNA integrity

• Semen collection –

0000

600600600600

1200120012001200

1800180018001800 Pallas catPallas catPallas catPallas cat

And

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AprApr Jun Aug Oct Dec Feb AprJun Aug Oct Dec Feb AprAprApr Jun Aug Oct Dec Feb AprJun Aug Oct Dec Feb Apr

• Semen collection –electroejaculation; rectal massage (rarely artificial vagina)

• Low gene diversity – poor semen quality

• Cryopreservation – low post-thaw sperm survival

Major Challenges Working with Endangered Species Sperm

Diversity in sperm morphology High incidence of abnormal sperm

Equidae

Poor sperm cryosurvival

Clouded leopard ~30% ~20%Black footed ferret ~30% ~30%Elephant ~50% ~60%

AcrosomeMotility Integrity

Avian Bovidae Felidae

Tools for Understanding Female Reproductive Biology

• Non-invasive hormone monitoring (feces, urine, saliva, hair)

200

400

600

200

400

600

Bred

Birth

Est

radi

ol(n

g/g

fece

s)

Progestogens

(mg/g feces)

Cheetah

hair)• Ultrasonography• Laparoscopy

-20 0 20 40 60 80 100 1200 0

(mg/g feces)

Days

Female Reproduction – Control of Ovarian Function

• Felids (eCG-hCG; eCG-GnRH; pFSH-pLH)

• Canids (GnRH agonist)

• Bovids (CIDR + PGF2α; PGF2α at 12 d interval)• Bovids (CIDR + PGF2α; PGF2α at 12 d interval)

• Cervids (CIDR for 12-14 d +/- PMSG)

• Equids (Altrenogest + GnRH)

Ovulation Mechanisms

40

60

80

100

120

140

160

180

200

20

40

60

80

100

120

Pro

gest

eron

e m

etab

olite

s (µ

g/g

fece

s)

Spontaneous

0

20

40

1 11 21 31 41 51 61 71 81 91 1011111211311411511611710

20

Day of sample collection

Pro

gest

eron

e m

etab

olite

s

0

20

40

60

80

100

120

140

160

180

200

0

20

40

60

80

100

120

Day of sample collection

Pro

gest

eron

e m

etab

olite

s (m

g/g

fece

s)

Induced

Sensitivity of Felids to Exogenous Gonadotropins

Weight eCG hCG(kg) (iu) (iu)

Leopard cat 3 100 75Domestic cat 4 100 75Ocelot 9 500 225Clouded leopard 15 75 50Snow leopard 30 600 300Puma 35 200 100Cheetah 35 200 100Tiger 250 1,000 750

Diversity in Optimal Time of Insemination

• Leopard cat (36-38 h)• Ocelot (39-43 h)• Cheetah (44-48 h)

• White rhinoceros (>26 mm)• Indian rhinoceros (>130 mm)

After hCG Follicle Diameter +/- GnRH

• Cheetah (44-48 h)

• Mhorr gazelle (48-60 h)• Scimitar-horned oryx (56-72 h)• Gerenuk (66-96 h)• Blackbuck (72-96 h)

After 2nd PGF2α White rhinoceros(Hermes et al., 2009)

Scimitar horned oryx(Morrow et al., 2000)

Hormone Profile as Cue for Insemination Timing

0.2

0.4

0.6

0.8

1.0

Pro

gest

agen

sP

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stag

ens

((ngng

/ml)

/ml)

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10

20

30

40

LH (

LH (ngng/m

l)/m

l)

anLHanLH ovLHovLH

Urin

ary

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ns(n

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(ng/

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Urinary P

rogestins(ng/m

g Cr)

Urinary P

rogestins(ng/m

g Cr)

-40-40 -20-20 00 2020 4040 6060 8080 100100 120120 14014000

4040

8080

120120

160160

200200

00

5050

100100

150150

200200

250250

300300

350350

Day from Estrogen PeakDay from Estrogen Peak

0 20 40 60 80 100 120 1400.0P

roge

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Pro

gest

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DaysDays

0

Integration of Hormone Monitoring/ Ultrasonography and AI

• Elephant• Marine mammals• Rhinoceros• Equids• Wolves• Wolves

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

8/23/

068/2

4/06

8/25/

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6/06

8/27/

068/2

8/06

8/29/

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8/31/

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/06

9/2/0

6

Date of Urine Collection

E1C

ng

/mg

Cr

0

100

200P

4 n

g/m

g C

r

Estrogen Conjugates (E1C) Progesterone (P4)

AI #1 6:00 p.m.AI #2 3:30 p.m.

Ovulation

Day 81

(Stoops et al., 2007)

Insemination Techniques

(Allen et al., 2008)

Urogenital Sinus Insemination Endoscopic Cervical/Intrauterine Insemination

(Allen et al., 2008)(Hildebrandt et al., 1998)

(Blanco et al., 2009)

Laparoscopic Intrauterine Insemination Intramagnal-Intraovarian Pocket Insemination

(Howard et al., 1992)

Other ‘Consistent’ Species

Cheetah Ocelot

(Swanson et al., 2009)(Howard et al., 1997)

Giant panda

(Howard et al., 2009)

Other ‘Consistent’ Species

Scimitar-horned oryx

Eld’s deer

(Monfort et al., 1993)

(Morrow et al., 2000)

Other ‘Consistent’ SpeciesKoala

(Allen et al., 2008)

Elephant

25 calvesproducedby AI

(Hermes et al., 2009)

(Allen et al., 2008)

White rhinoceros

(Saragusty et al., 2009)

by AI

Repeated AI successes

Killer whaleBottlenose dolphin

Pacific white-sided dolphin

Robeck, O’Brien, SteinmanBusch Entertainment Corporation's SeaWorld and Busch Gardens

Repeated AI successes

Killer whaleBottlenose dolphin

Robeck, O’Brien, SteinmanBusch Entertainment Corporation's SeaWorld and Busch Gardens

Pacific white-sided dolphin

Calves from sex-sorted sperm

AI Successes in Marine Mammals

Species

Killer Whale(Orcinus orca)

Bottlenose Dolphin

Live Offspring Produced

4

11*Bottlenose Dolphin (Tursiops truncatus)

Pacific White-Sided Dolphin(Lagenorhynchus obliquidens)

Beluga Whale(Delphinapterus leucas)

11*

5

3

* Six calves with sex sorted spermRobeck, O’Brien, Steinman

Busch Entertainment Corporation's SeaWorld and Busch Gardens

AI Success in Birds

Consistently Benefiting from Reproductive Science

Whooping crane (G. Gee, G. Archibald)

White-naped crane(W. Lynch)

Artificial Insemination in Birds(Species Managed with Artificial Insemination)

• Houbara bustard• Whooping crane• Hispaniola parrot• Philippine eagle• Philippine eagle• Spanish imperial eagle• Bonelli’s eagle• Peregrine falcon• Kiwi (free-ranging)

Challenges/Opportunities

• Low numbers of animals• Optimize methods to control ovarian function• Improve sperm collection and cryopreservation • Improve sperm collection and cryopreservation

technologies• Develop novel tools for insemination• Optimize conditions for maintaining pregnancy• Pregnancy diagnosis

Acknowledgments

• David Wildt, Nucharin Songsasen, Janine Brown, JoGayle Howard (National Zoo)

• Justine O’Brien, Todd Robeck (Busch Entertainment Corporation's SeaWorld and Busch Gardens)Corporation's SeaWorld and Busch Gardens)

• Terri Roth, William Swanson, Monica Stoops (Cincinnati Zoo & Botanical Garden)

• Juan Blanco (Centro de Estudios de Rapaces Ibericas, Spain)