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”