Artificial Insemination in Endangered Species

Artificial Insemination in
Endangered Species –
Challenges and Successes
Budhan Pukazhenthi
Center 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
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
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
Reptile, 7,500
13%
Mammal,
5,500
10%
48%
18%
Bird, 10,200
11%
Amphibian,
5,921
Fish, 27,000
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)
197
58 (29.4)
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)
1,852
259 (13.9)
76
69 (90.8)
12,118
1,196 (9.9)
Cryobiology
Journal of Mammology
Theriogenology
Zoo Biology
Total
90% of publications are on common, already-studied species
Wildt 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 breeding
Behavioral incompatibility
Poor reproductive performance
Anatomical defects
• Maintain genetic diversity in small populations
Low number of founders
Limited gene pool in population
• Movement of gametes for conservation strategies
Species Survival Plans (SSPs)
Genome Resource Banks (GRBs)
Information Critical for Developing
Artificial Insemination
•
•
•
•
•
Male
Seasonality
Age
Semen collection
Gene diversity and
ejaculate quality
Cryopreservation
•
•
•
•
•
•
•
•
Female
Seasonality
Age
Ovulation mechanism
Control of ovarian
function
Time of insemination
Site of insemination
Sperm concentration
Impact of stress and/or
anesthesia
• Seasonality – Pallas cat (distinct
seasonality), cheetah (seasonality
not evident)
• Age – onset of puberty; impact on
sperm DNA integrity
• Semen collection –
electroejaculation; rectal massage
(rarely artificial vagina)
• Low gene diversity – poor semen
quality
• Cryopreservation – low post-thaw
sperm survival
Androgens (ng
(ng/g)
/g)
Male Reproduction
1800
Pallas cat
1200
600
0
Apr
Jun
Aug
Oct
Dec
Feb
Apr
Major Challenges Working with
Endangered Species Sperm
High incidence of
abnormal sperm
Diversity in sperm morphology
Equidae
Avian
Bovidae
Felidae
Poor sperm cryosurvival
Motility
Clouded leopard
Black footed ferret
Elephant
~30%
~30%
~50%
Acrosome
Integrity
~20%
~30%
~60%
Cheetah
600
600
Bred
400
Birth 400
200
200
0
-20
0
0
20
40
60
Days
80 100
120
Progestogens (mg/g feces)
• Non-invasive
hormone monitoring
(feces, urine, saliva,
hair)
• Ultrasonography
• Laparoscopy
Estradiol (ng/g feces)
Tools for Understanding Female
Reproductive Biology
Female Reproduction – Control of
Ovarian Function
•
•
•
•
•
Felids (eCG-hCG; eCG-GnRH; pFSH-pLH)
Canids (GnRH agonist)
Bovids (CIDR + PGF2α; PGF2α at 12 d interval)
Cervids (CIDR for 12-14 d +/- PMSG)
Equids (Altrenogest + GnRH)
120
180
100
160
140
80
120
100
60
80
40
60
40
20
20
0
Spontaneous
0
1 11 21 31
41 of
51 sample
61 71 81collection
91 101111121131141151161171
Day
200
120
180
100
160
140
80
120
Induced
100
60
80
40
60
40
20
20
0
Day of sample collection
0
Progesterone metabolites
(mg/g feces)
200
Progesterone metabolites
(µg/g feces)
Ovulation Mechanisms
Sensitivity of Felids to Exogenous
Gonadotropins
Leopard cat
Domestic cat
Ocelot
Clouded leopard
Snow leopard
Puma
Cheetah
Tiger
Weight
(kg)
3
4
9
15
30
35
35
250
eCG
(iu)
100
100
500
75
600
200
200
1,000
hCG
(iu)
75
75
225
50
300
100
100
750
Diversity in Optimal Time of
Insemination
After hCG
• Leopard cat (36-38 h)
• Ocelot (39-43 h)
• Cheetah (44-48 h)
Scimitar horned oryx
(Morrow et al., 2000)
Follicle Diameter +/- GnRH
• White rhinoceros (>26 mm)
• Indian rhinoceros (>130 mm)
After 2nd PGF2α
White rhinoceros
(Hermes et al., 2009)
• Mhorr gazelle (48-60 h)
• Scimitar-horned oryx (56-72 h)
• Gerenuk (66-96 h)
• Blackbuck (72-96 h)
anLH
ovLH
30
20
0.4
10
0.2
0.0
0
0
20 40 60 80 100 120 140
Days
200
350
300
160
250
120
200
80
150
100
40
0
-40 -20
50
0
0
20
40
60
80 100 120 140
Day from Estrogen Peak
Urinary Progestins (ng/mg Cr)
Urinary Estrogens (ng/mg Cr)
40
1.0
0.8
0.6
LH (ng
(ng/ml)
/ml)
Progestagens (ng/ml)
ng/ml)
Hormone Profile as Cue for
Insemination Timing
Integration of Hormone Monitoring/
Ultrasonography and AI
•
•
•
•
•
Elephant
Marine mammals
Rhinoceros
Equids
Wolves
10000
AI #1 6:00 p.m.
AI #2 3:30 p.m.
9000
200
8000
Ovulation
5000
100
4000
P4 ng/mg Cr
6000
3000
2000
1000
0
Date of Urine Collection
Estrogen Conjugates (E1C)
Day 81
9/
2/
06
0
8/
23
/0
6
8/
24
/0
6
8/
25
/0
6
8/
26
/0
6
8/
27
/0
6
8/
28
/0
6
8/
29
/0
6
8/
30
/0
6
8/
31
/0
6
9/
1/
06
E1C ng/mg Cr
7000
Progesterone (P4)
(Stoops et al., 2007)
Insemination Techniques
Urogenital Sinus Insemination
Endoscopic Cervical/Intrauterine
Insemination
(Allen et al., 2008)
(Hildebrandt et al., 1998)
Laparoscopic Intrauterine Insemination
(Howard et al., 1992)
Intramagnal-Intraovarian Pocket Insemination
(Blanco et al., 2009)
Other ‘Consistent’ Species
Ocelot
Cheetah
(Howard et al., 1997)
(Swanson et al., 2009)
Giant panda
(Howard et al., 2009)
Other ‘Consistent’ Species
Eld’s deer
Scimitar-horned oryx
(Monfort et al., 1993)
(Morrow et al., 2000)
Other ‘Consistent’ Species
Koala
Elephant
25 calves
produced
by AI
(Allen et al., 2008)
White rhinoceros
(Saragusty et al., 2009)
(Hermes et al., 2009)
Repeated AI
successes
Killer whale
Bottlenose dolphin
Pacific white-sided
dolphin
Robeck, O’Brien, Steinman
Busch Entertainment Corporation's SeaWorld and Busch Gardens
Repeated AI
successes
Killer whale
Bottlenose dolphin
Pacific white-sided
dolphin
Calves from
sex-sorted
sperm
Robeck, O’Brien, Steinman
Busch Entertainment Corporation's SeaWorld and Busch Gardens
AI Successes in Marine
Mammals
Species
Live Offspring Produced
Killer Whale
(Orcinus orca)
Bottlenose Dolphin
(Tursiops truncatus)
4
11*
Pacific White-Sided Dolphin
(Lagenorhynchus obliquidens)
5
Beluga Whale
(Delphinapterus leucas)
3
* Six calves with sex sorted sperm
Robeck, 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
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
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)
• Terri Roth, William Swanson, Monica Stoops
(Cincinnati Zoo & Botanical Garden)
• Juan Blanco (Centro de Estudios de Rapaces Ibericas,
Spain)