Arat, S. 2011. In vitro Conservation and

In vitro Conservation and
Preliminary Molecular
Characterization of Some Turkish
Native Domestic Animal Genetic
Resources-I
(TURKHAYGEN-I)
Assoc.Prof.Sezen ARAT
RBI 8th Global Conference on
Conservation of Animal Genetic Resources
4-8 October 2011
Genetic resources
• 27000 animal and plant species irreversibly
disappear per year
• Several endemic animal species and breeds
have been totally lost or under danger in
Turkey
Mammalian
Avian
Reptiles
Amphibian
Fish
World
4200
9000
5115
3025
21000
Turkey
160
453
106
23
504
Under risk
64
253
43
11
58
Turkish Ministry of Environment and Forestry, 2004
Sheep genetic resources
• There has been a 47% decrease in sheep
population in Turkey in the last two decades and
this decline continues
• Under risk
– White Karaman, Red Karaman, Cine Caparı,
Daglic, Karakul, Awassi, Herik, Kivircik, Tuj,
Karayaka, Imbros, Hemsin
• Under Heavy risk
– Sakız, Norduz
• Extinct
– Halkali, Karakacan
Genetic resources
• The archaeological and genetic data indicates
that domestication centers for cattle, sheep,
goat, and probably swine are in the southwest
Asia including a part of Anatolia
– (Bruford et al, 2003)
• Anatolian native breeds, being the closest
relatives of the first domesticates, must be given
priority in conservation
– (Bruford et al, 2003)
• Molecular genetics studies have shown that the
European cattle, sheep and goat breeds have
originated and spread from Anatolia
– (Loftus et al., 1999; Troy et al., 2001, Bruford and Towsend,
2004, Lenstra et al., 2005).
Preservation of genetic resources
• Conservation of the biological richness and
diversity is one of the main concerns in most of
the countries’ agenda
• Conservation schemes in animals (Simianer
2005)
Modern Noah’s Ark
Importance of genetic resources
• Conservation of genetic resources has been
discussed on various platforms in Turkey and
was declared as a priority
– TUBITAK (Turkish Scientific and Technological
Research Council)'s Vision-2023 Science and
Technology Prevision Project
– Turkish Academy of Sciences (TUBA) Molecular
Life Sciences and Technology Foresight Project
(2003-2023)
– II. Agriculture Assembly (2004)
International agreements
• Turkey took part as contractor in the
international agreements on conservation of the
biodiversity and genetic resources
– UN Convention on Biological Diversity
• The proposed TURKHAYGEN-I project will
contribute to fulfill the commitments resulted
from these agreements
FEBRUARY 2005
MARCH 2007
TURKHAYGEN-I consortium
Sezen Arat
Haydar Bagis, Berrin Erdag, Digdem Aktoprakligil, Evren Koban, Arzu
Taş, Tolga Akkoç, Fatih Karakaya, Ozlem Celasin, Melis Denizci,
Ozgur Aslan, Aylin Özdemir, Koray B. Balcioglu, Umit Ozturk, Ali
Daskin, Necmettin Tekin, Mustafa Kaymaz, Ongun Uysal,
Ergun Akcay, Okan Ertugrul, Bilal Akyuz, Ozlem, Gucuyener,
Ercan Kurar, Zafer Bulut, Vahdettin Altunok,
Mehmet Nizamlioglu, Dursun Ali Dinc, Ahmet Semacan,
Kenan Coyan, Mehmet Guler, Mehmet Ataman,
Aydin Guzeloglu, Serhat Pabuccuoglu, Kemal Ak,
Sema Birler, Serhat Aklan, Alper Baran, Mithat Evecen,
Ozen Banu Ozdas, Umit Cirit, Suleyman Bacinoglu,
Ihsan Soysal, Muhittin Ozder, Emel Ozkan, Hakan Sagirkaya, Yavuz
Nak, Deniz Nak, Zekariya Nur, Ahmet Gumen, Burcu Ustunel,
Umut Tasdemir, Barbaros Tuncer, Huseyin Kinet,
Numan Akyol, Hamdi Kizil, Tahir Karasahin,
Muharrem Satilmis, Inci Togan, Havva Dinc,
Bekir Ankarali, A. Oya Akin, Mustafa Kucukkebapci,
Abdulkadir Ozluturk, Sinan Kopuzlu, Deniz Soysal,
Tamer Sezenler, Adnan Unalan, Ayse Isik, Melik Aytac
Ilkay Demirhan
TURKHAYGEN-I organization
Sezen Arat
TÜBİTAK-MAM
Project Coordinator
Board Members
Inci Togan
Executive Member
Middle East Technical
University
Okan Ertugrul
Executive Member
Ankara University
Serhat Pabuccuoglu
Executive Member
Istanbul University
Hakan Sagirkaya
Executive Member
Uludağ University
Umut Tasdemir
ExecutiveMember
Lalahan Livestock Central
Research Institute
Ercan Kurar
Selçuk University
Executive Member
• Owner Institution:
– Turkish Ministry of Agriculture and Rural Affairs
Organization
• Administrative Institution:
– TUBITAK Marmara Research Center GEBI
• Partner Institutions:
–
–
–
–
–
–
–
–
–
–
–
Adnan Menderes University
Ankara University
Ataturk University
Istanbul University
Lalahan Livestock Central Research Institute
Middle East Technical University
Mustafa Kemal University
Namık Kemal University
Ondokuz Mayıs University
Selçuk University
Uludağ University
Objectives
• Main objective is ex-situ in vitro preservation of
endemic domestic animal species
• Establishment of cryobanks
– Preservation of animal genetic resources from 1500
animals,
– DNA, somatic cell, embryo and semen samples
• Genetic characterization of endemic domestic
animal species,
• Utilization of gained knowledge in registration
studies,
• Expand national researcher capacity in animal
genetics and biotechnology areas
• Combination and dissemination of knowledge
Work packages
• Collection of animal material
• Development of new technologies in preserving
live material
• Conservation of the genetic resources
• Genetic characterization of the species
• Distribution of the information and
standardization
Work packages
WP-I Collection of animal material
• Data of the flock are systematically recorded to
standard files
• Digital fotographs are taken
• Location data will also be recorded by using
GPS devices
Work packages
WP-II Technological development
• In order to develop and improve the
technologies of live material preservation
techniques includes several sub work packages
–
–
–
–
–
Tissue and cell cryopreservation
Utilization alternative cell types
Alternative reproduction techniques,
Embryo and gamete cryopreservation
NT technologies
Work packages
WP-III Preservation of Genetic Resources
• Preservation of samples of 1500 animals
according to methods defined by international
sources and organizations
–
–
–
–
Genomic DNA
Somatic cell
Embryo
Sperma
• Duplicate samples are kept in two gene banks
constructed in LLCRI and GEBI
Work packages
WP-IV Characterization of species
• After comprehensive sample collection, all
animals are characterized at the molecular level
• Genetic diversity and phylogenetic analyses are
accomplished using molecular markers
– 20 Microsatellites
– mtDNA
Work packages
WP-V Distribution and standardization of data
• Distribution of data by constructing a database and
web page
http://www.turkhaygen.gov.tr
• Collaboration with national and international
organizations
• Arrangement of seminars, congresses and media
declarations to represent the project
• Standardization
–
–
–
–
DNA Genotyping
Domestic standardizasyon panel
ISAG comparison tests
Cryopreservation protocols
Educational activities
• Training and workshops
– Sample collection techniques
– Cell culture techniques
– Cryopreservation (embryo and sperm)
– Embryo applications
– Embryo transfer
– Molecular biology techniques
– Bioinformatics
– Breed identification and genetic diversity
– Current statistical applications on genetic
diversity data
Animal Material
• Ovis aries (sheep)
– White Karaman
– Cine Çaparı
– Daglic
– Hemsin
– Herik
– Awassi
– Imros
– Karakul
– Karayaka
– Kivircik
– Red Karaman
– Norduz
– Sakız
Anatolian sheep
White karaman
Red karaman
Kıvırcık
Karagül
İvesi
Dağlıç
Animal Material
• Capra hircus (goat) • Bos taurus (cattle)
– Angora goat
– Honamli goat
– Native Hair goat
– Kilis goat
– Norduz goat
– South Anatolian Red
– Anatolian Black
– Anatolian Grey
– East Anatolian Red
– Zavot
– Native Southern
Yellow Cattle
Anatolian goats
Hair
goat
Angora goat
Honamlı
Norduz
Anatolian cattle
Anatolian Grey
South Anatolian Red
Anatolian Black
Anatolian Buffalo
Animal Material
• Equus caballus (horse)
– Canik
– Hinis horse
– Malakan horse
– Cukurova horse
– Ayvacık Pony
• Bos bulalis (water buffalo)
– Anatolian water buffalo
Anatolian Horses
Malakan
Ayvacık Pony
Hınıs
Canik
TURKHAYGEN-I Details
Start :
March 15, 2007
Duration:
4.5 years
Budget:
~4.800.000 EURO
Funding Agency: TUBITAK
TURKHAGEN-I current status
Specie
Breed
DNA
Somatic
Embryo
Sperma
13
13
13
13
13
5
5
5
5
5
6
6
6
3
6
5
5
5
NA
NA
1
-
-
NA
NA
CRYOBANK
There are two
banks containing
same
infrastructure.
•-80 oC
•-150 oC
• -196 oC
SOME SCIENTIFIC ACTIVITIES AND OUTCOME OF THE PROJECT
•14 practical courses
•29 presentations in several international congresses
•32 presentations in several national congresses
•6 master thesis
•4 PhD thesis
•5 paper in the several international journals
•4 papers in the several national journals
CONSERVATION OF ENDANGERED SPECIES
•After all succesful cloning reports on several species,
somatic cell nuclear transfer has been suggested as a
potentially integral part of conservation programs for
endangered mammalians.
•Therefore instead of cryobanks involved conservation
programs contaning only gametes and embryos previously,
now it has been suggested to contain other biological sources
such as somatic cells (FAO, 2007).
•For this reason, the Turkish Ministry of Agriculture and
Rural Affairs has supported the national TURKHAYGEN-1
project (http://www.turkhaygen.gov.tr) to establish cryobank
containing different type of somatic cells beside germ cells
and embryos from native Anatolian livestock breeds.
Hungarian
Grey
Maremmana
Turkish Grey
same podolic
group of cattle
similar external
conformation
recently
underwent a
similar
demographic
reduction
CELL BANKING
CELL BANKING (www.turkhaygen.gov.tr)
Somatic cells of several individuals from
each 29 breeds of livestock containing
Anatolian Grey Cattle were stored in liquid
nitrogen.
MAIN MATERIALS FOR NT
•OOCYTE (MII)
•DONOR CELL AT G1-G0
- naturally (cumulus cells)
- serum starvation
- contact inhibition (confluency)
- cell cycle inhibitor (roscovitine)
www.turkhaygen.gov.tr
ANIMALS USED IN THE STUDY
DONOR ANIMALS : ANATOLIAN (TURKISH) GREY
CATTLE (1 male, 1 female)
CYTOPLASM SOURCES : HOLSTEIN
RECIPIENT MOTHERS : HOLSTEIN and SWISS BROWN
Oocyte aspiration
PREPARATION OF
RECIPIENT CYTOPLASM
Oocyte
aspiration
MI
oocyte
16-18 hr culture
MII
oocyte
Oocyte
enucleation
DONOR CELL TYPES
Various cell types, such as embryonics cells, fibroblasts,
mammary gland cells, cumulus cells, oviductal cells,
leukocytes, granulosa cells, germ cells, muscle cells and liver
cells have been used as donors for production of cloned
animals so far.
We used three different cell types for nuclear transfer in the
present study.
•Granulosa
•Fibroblast from ear
•Cartilage from ear (first time)
Electro-Fusion
-
+
TRANSPORTATION OF EMBRYOS
Portable incubator
Clone embryos
in the straws
Embryo Transfer
Embryo Development
7 days
Uterin Maturasiyon
Synchronized
ostrus
Ovulation
Transfer to recipient
CALVING
The calves were
delivered by
cesarean surgery on
day 274-285
gestation.
Recipient mothers of
one male cloned and
twin female clones
were Swiss Brown.
Other two recipient
mothers of two female
clones were Holstein
FIRST CLONE CALF “EFE” (STRONG MAN)
MALE CLONE and ITS DNA SOURCE
4 years old bull
20 days old male clone
7.5 months old male clone
Clone female calves and
their DNA source
5 years old cattle
KIRAZ
ECE
ECEM
TWINS
NİLÜFER
Male clone 12 months and females 7-8 months old
ECE and ECEM
EFE
NILUFER and
KIRAZ
Conclusion
Somatic cells cryopreserved in cryobank can be used for
nuclear transfer and result in the production of viable
cloned calves.
Endangered domestic native cow breeds can be cloned by
using oocytes and recipient mothers from dairy or meat
cow breeds which are genetically different than donor
animal.
Further information
Please visit TURKHAYGEN-I web pages
http://www.turkhaygen.gov.tr
Sezen ARAT D.V.M,PhD (project coordinator)
Turkish Scientific and Technological Research Council
Genetic Engineering and Biotechnology Institure
Kocaeli / TURKEY
[email protected]
[email protected]
www.turkhaygen.gov.tr
THANKS for YOUR PATIENCES
“Clone EFE”