Embryo Transfer Newsletter

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Embryo Transfer Newsletter A Publication of the

International Embryo Transfer Society Volume 24, Number 2

June 2006

In this issue... From the President ........................1 2006 Board of Governors..............4 2006 Committee Chairs ................4 Feature Article...............................5 Classifieds ...................................11 HASAC .......................................13 33rd Annual Conference .............15 Call for Abstracts ......................17 Main Conference Program........18 PreConference I Program..........19 PreConference II Program ........20 Post Conference Program .........21 Manual Order Form ....................22

Embryo Transfer Newsletter Matthew B. Wheeler, Editor e-mail: [email protected] Vol. 24, No. 2

June 2006 ISSN 1083-4699

Published quarterly by the International Embryo Transfer Society 1111 North Dunlap Avenue Savoy, IL 61874 USA (217) 398-4697, FAX: (217) 398-4119 E-mail: [email protected] Web Site: http://www.iets.org Letters to the Editor are welcomed. Letters must be signed and limited to 300 words and are subject to editing for length, style, and accuracy. Please include name, address, telephone, FAX, and E-mail contact information.

From the President JUNE 2006 “Time flies just like an arrow”. I can not believe that 5 months have already passed since IETS 2006 in Orlando. After coming back from Orlando I have been working on the project proposals and reports for what I have done during this fiscal year (in Japan the fiscal year begins from the 1st April) using my computer while smoking and listening to the Old Hippy Type music of 60’s and 70’s. Now I find myself writing this letter from the President to inform all the members mainly about IETS 2007 in Kyoto. Carol Keefer, program chair and Fulvio Gandolfi, program Co-chair, and have been working very hard on shaping up the program of 2007 annual conference in Kyoto, Japan. They have developed a very interesting scientific program with the title “Embryo Quality and Fetal Development - early determinants of developmental success”. We are also working on some other ideas that would lure back some practitioners from the embryo transfer businesses, who have not been attending our IETS conferences lately. For the main meeting, Dr Osamu Dochi is preparing Practitioner’s Forum entitled “Use of the embryo transfer for the improvement of fertility in dairy cows”, and the practical use of embryo transfer as the improvement strategy for low fertility of dairy cows will be discussed here. Also the Local Organizing Committee has been working very hard on behalf of the IETS and trying to get sponsors and funding from companies and societies related to Embryo Transfer. So far Japanese Society of Embryo transfer obtained a big grant form the Japanese Government! Furthermore, we are going to have two pre- conference symposia and one post-conference symposium. As pre- conference symposia, Hiroshi Imai, Harpreet Kochhar and Michel Thibier will organize the symposium entitled “Assisted reproductive technologies and food safety in farm animals”, and Jeremy Thompson and Gabor Vajta will organize the other one entitled “Innovative techniques in human and animal embryology”. The former is very important to show the increasing importance of products, obtained from somatic cell cloned or transgenic animals, in the food chain, and the latter is an important subject not only for domestic animals but also for human reproductive biology. Although the two pre-symposia will be held in parallel, the organizers are arranging their subjects in such a way so as to avoid an overlap between the two. As both scientific programs are so attractive, I would like to attend both of them. I wish if I could have my clone to do so! As a post-conference symposium, the Local Organizing Embryo Transfer Newsletter 1

Committee will be organizing the symposium entitled “Quality Control of Embryos for Embryo Transfer and Related Advanced Technologies in Domestic Species (a tentative title)”. We will tell you more about it in the near future. While the 2007 annual conference is shaping up, we are already working on the 2008 meeting in Denver, USA. We have recently confirmed the designation of George Seidel Jr and Patrick Lonergan as Program Chair and Co-chair. As you know George is a sort of a father of IETS and Pat is a world renowned scientist and a member of the Board of Governors. I am pretty sure that they will develop a very interesting scientific program. In addition to the annual conference planning, there are the Board’s efforts to ensure a sound financial foundation and a wider network for the Society; there are two additional items of note that have occurred during the past several months and I have described them below. Addition of Electronic Media to Printed Copies of the Proceedings It has been estimated that producing the Proceedings for the Annual meeting on CD will save the society about $8-12,000/per year. The Board of Governors have decided that CDs (or other electronic media as technology evolves) will be mailed to those members that do not attend the annual meeting, however, they will have the option of purchasing a hard copy at a cost determined by the society. The attendees of the annual conference will be given the choice of a hard copy or CD if pre-registered; all on-site registrants will be provided CD; all registrants will have the option of purchasing a hard copy, on a first come first served basis. Hard copies at the venue will be limited. The rationale for providing non-attendees with electronic copies of the proceedings is that the production of the proceedings is a cost borne by the annual conference budget. Printing and mailing of hard copies to non-attendees increases the registration fees for conference delegates. Therefore, in order to keep registration fees for the annual conference at reasonable rates the Board made the decision to send electronic copies to non-attendees, which will save on production and mailing costs. Printed copies, as stated above, will be available for purchase by non-attendees. This was a fiscally prudent decision that retains flexibility for the members. It was unanimously accepted by the Board. Review Articles from Asia As I mentioned in March Newsletter, I think that the establishment of wider and stronger net work to gather persons studying or working on ET is essential for the future of IETS. Thus I asked some of my Asian friends to review about Animal Biotechnologies in their countries to find the common interests in the field of studies among all IETS members. In this issue you will find the review article by Dr Nguyen Xuan Bui from Vietnam, and I am planning to have review articles in September and December Issues by Rangsun Parunpai form Tahiland and Qing-Yuan Sun from China, respectively. Although we will have a rainy season, which lasts for one and half months, ahead in Japan, most of IETS members will have a joyful summer (or winter for those of you in the Southern Hemisphere) as usual. Please plan to attend the 2007 annual conference in Kyoto, and please don’t forget to get your abstracts submitted early. The deadline is August 1, 2006! Best regards, Takashi (TAKU) Nagai President, IETS

June 2006 2

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2006 IETS Board of Governors President Takashi (TAKU) Nagai, PhD National Institute of Livestock & Grassland Science Ibaraki, Japan Phone: 81-298-38-8750 E-mail: [email protected]

Governors (cont): Peter W. Farin, PhD, DVM North Carolina State University Raleigh, NC Phone: (919) 513-6469 E-mail: [email protected] Pat Lonergan, PhD University College Dublin Newcastle, County Dublin, Ireland Phone: 353-1-6012147 E-mail: [email protected]

Immediate Past President Matthew Wheeler, PhD University of Illinois Urbana, IL Phone: (217) 333-2239 E-mail: [email protected]

Ed L. Squires, PhD Colorado State University Fort Collins, CO Phone: (970) 491-8409 E-mail: [email protected]

Vice President Naida M. Loskutoff, PhD Center for Conservation & Research Henry Doorly Zoo Omaha, NE Phone: (402) 733-8401 E-mail: [email protected] Secretary-Treasurer Richard A. Fayrer-Hosken, PhD, DVM University of Georgia Athens, GA Phone: (706) 542-6326 E-mail: [email protected] Governors: David C. Faber, DVM Trans Ova Genetics Sioux Center, IA Phone: (712) 722-3586 E-mail: [email protected]

Janneke Van Wagtendonk, MSc Fonterra Co-operative Group Ltd Palmerston North, New Zealand Phone: 64 6 350 4656 E-mail: [email protected] Christine Wrenzycki, PhD Institute for Animal Breeding Mariensee, Neustadt Germany Phone: 49-5034-871196 E-mail: [email protected]

IETS 2006 Committee Chairs Awards Committee Gabriel Bo Inst. De Reproduccion Animal Cordoba (IRAC) Cordoba, ARGENTINA Companion animal, non-domestic and endangered species committee (CANDES) Naida Loskutoff Henry Doorly Zoo Omaha, NE, USA Data Retrieval Committee Michel Thibier Ministere De L’Agriculture De L’Alimentation De La Peche Et Des Affaires Rurales Paris, France Finance Committee Edward L. Squires Colorado State University Fort Collins, CO, USA

Heath and Safety Advisory Committee Michel Thibier Ministere De L’Agriculture De L’Alimentation De La Peche Et Des Affaires Rurales Paris, France Membership Committee Christine Wrenzycki Institute for Animal Breeding Mariensee, Neustadt Germany Nominations Committee Matthew B. Wheeler University of Illinois Urbana, IL, USA Publications Committee Matthew B. Wheeler University of Illinois Urbana, IL, USA

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Feature Article Current Status and Trends of Animal Reproductive Biotechnology in Vietnam Bui Xuan Nguyen Vietnamese Academy of Science and Technology, Hanoi, Vietnam Abstract The reproductive biotechnologies that have been developed in Vietnam include technical activities from artificial insemination (AI) to embryo production through in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). Although the investment for research in this field is still modest, there is a great potential to develop such reproductive technologies in Vietnam. The country is characterized by the riches in native animal biodiversity, the large need of rapid passage from the traditional animal breeding to more efficient modern breeding, and the application of new biomedical activity to meet the actual rapid economic and demographic development. In this paper, the research that has been done in the domain of animal biotechnology in Vietnam is reviewed. Artificial Insemination and Embryo Transfer Artificial insemination (AI) was introduced in Vietnam during 1960’s. A large network of AI with central stations for semen production from selected males and the peripheral stations with local inseminators working at villages was set up. The practice of AI was so rapidly developed in pigs that the pig population was estimated more than 22 million and AI produced most of them with fresh semen. While AI was less used for cattle; around 20 % of a population, approximately 4 million, and very few buffalo; only several hundred of a population of more than 4 million head. The first successful embryo transfer (ET) was reported in rabbits (1), cattle (17) and goats (44). These studies were carried out to develop ET methods, which were adapted to animals under tropical and non-standardized breeding conditions in Vietnam. Interesting results were obtained with buffalo showing that the ovaries of swamp buffalo have a poor number of antral follicles, only 20% of those obtained from cow’s ovaries (11). Moreover, when applied to buffalo, the ovarian response to the gonadotropin treatment generally used for superovulatory induction in cattle was found to be not effective. Mean numbers of corporus luteum (CL) and non-ovulated follicles in these cases were only 0.66 and 0.0, respectively, for the anoestrus buffaloes and 2.3 and 1.0, respectively, for the cyclic buffaloes. However, the optimal method of superovulatory induction with estradiol and hCG supplementation was developed and resulted in the increasing level of ovarian stimulation up to that normally obtained in cattle; mean ovarian response of 8.7 CL per donor, embryo recovery rates of 46% by non-surgical collection and of more than 90% by postmortem flushing, 70% of recovered embryos with good quality (9, 10, 13, 23). The application of gonadotropin-releasing hormone (GnRH) and Pregnancy-associated glycoproteins (PAGs) in early pregnancy diagnosis to improve results of estrus synchronization and pregnancy in buffalo is being carried out in cooperation with University Parma – Italy and University Liege-Belgium (20, 51). The most representative cattle breeds in Vietnam are local Yellow; crossbreed Yellow-Zebu, Holstein-Zebu-Yellow cross (Ha-an) and Holstein. Thanks to there improving body weigh (more 300 kg for selected animals) and big population (more than one million), crossbreed yellow zebu can be served as recipients for embryos from dairy Holstein or Ha–an breeds. Estrus synchronization was investigated with the different combination of PGF2a, pregnant mare gonadotropin (PMSG), estrogen, SynchroMate B, human chorionic gonadotropin (hCG) and gonadotropin-releasing hormone (GnRH) in different areas and seasons. When treated with equine chorionic (eCG) and estradiol-17b, a high rate of more than 90% of cows with ovulation and estrus synchronized at the same day was obtained (15, 20). Non-surgical transfer of good quality embryos into recipients treated with this method for estrous synchronization gave pregnancy rates of 65-70% within 3 months after ET (17, 48). The possibility and the method to set up a mini- Open Nucleus Breeding Systems with Multiple Ovulation Embryo transfer (ONBS-MOET) using modern technique of embryo production by IVF, sexing, freezing to accelerate genetic progress in cattle - breeding programs was reported (5, 6, 24, 44). Superovulation in local goat and pig populations were induced using the combination of eCG plus hCG and PGF2a. The treatment of goats with 2000 I.U. eCG and 1000 I.U. hCG resulted in mean ovarian response with 15 CL/donor. Recovery rate by surgical method was more than 80%. Furthermore, more than 80% of recovered embryos developed to Embryo Transfer Newsletter 5

hatched blastocysts after culture in vitro in medium B2 plus 10% SVF at 39oC and under gas phase of 90%N2, 5%CO2, 5%O2. Surgical transfer of fresh and frozen embryos was applied in local goats and the production of pure Sannen kids by this technique was established (44). Cryopreservation and Cryobanking Long-term gamete and embryo preservation based on the associated treatments of partial dehydration and freezing has been developed (2, 3, 22). It was reported that dehydration of most of cellular water is the way by which the lower organisms can survive hyper- or hypothermia for unlimited period. Research was undertaken to apply this principle to a long-term preservation of mammalian gametes and embryos. Results of investigations in mice, rabbits, cattle and sheep showed that mammalian oocytes and embryos at different stages of development have ability to tolerate the loss of most of their cellular water for a short duration (10-30 minutes) when they were exposed to 0.5-1.0M sucrose. The increasing of the tolerant duration up to 24 hours can be achieved if dehydration was carried out at low temperature or in the solution consisted in sucrose and gelatin 10%. These embryos can be used for storage in liquid nitrogen after further treatment with appropriate permeable cryoprotectants. Observation of embryos after thawing did not show any interruption in their ultrastructural integrity and cytoskeletal organization (4, 8). The development of original principle of embryo preservation by rapid and ultrarapid freezing after partial dehydration at room temperature was performed successfully with rabbit embryos; more than 92% of frozen-thawed embryos developed normally in vitro, and the rate of live young obtained after transfer to recipients was similar with control unfrozen embryos (3). Based on results of these investigations, methods were adapted for rapid freezing of embryos of different species including cattle, buffalo, sheep, goat, and mouse. Recently, when bovine cloned embryos were vitrified, more than 90% of survival rate after thawing was reported (32). Successful freezing was obtained also in case of semen preservation in pig, goat, cattle, human (14, 28) and wild minipig (unpublished data, 2005). In Vitro Maturation and Fertilization of Oocytes In vitro production of embryos has been studied in cattle and buffalo since 1994 with the technical assistance from BOMED-USA. The first calves from IVF embryos and from IVF-sexing embryos were born in 2002. For the research, the main oocyte resource was collected from yellow and yellow-Zebu cattle’s ovaries at a local slaughterhouse. Ovaries from these breeds are characterized by small size and poor number of follicles with diameter of more than 2 mm. The method of in vitro maturation suitable to these oocytes was established based on the experienced methods of in vitro maturation without or with different hormonal combination. Cross breed embryos Zebu-Yellow or Holstein-yellow were produced using frozen semen; the rate of maturation, fertilization and development to the morula-blastocyst stage were more than 80%, 59% and 30%, respectively (12, 41, 42, 52). For application, an approach for production of tropical diary calves by ET using local Laisind (Bos indicus) recipients and intercontinental fresh IVF embryos shipped from USA was developed. Embryos were produced at BOMED laboratory using the oocytes from Holstein cows and semen from Gir bull. The further culture, biopsy by micromanipulation, sexing by PCR and embryo transfer were followed for embryos at the morula or early blastocyst stage at day 6-7 of IVF. More than 99% of manipulated embryos were developed in vitro to normal blastocysts and 100% of PCR with clear indication of sex was obtained (25-27). The average calving rate of more than 50% was obtained for transfer of fresh IVF-sexing embryo into local Zebu- yellow recipients (46, 48, 54). The production of IVM-IVF embryo was obtained for swamp buffalo. It is interest to notice that the rate of in vitro maturation for swamp buffalo was lower of 30% when the maturation was carried out in the medium currently used for cattle. The addition of estradiol to IVM medium significantly increased the rate of maturation to more 60% and the number of morula-blastocyst in consequence (12, 19, 21, 36, 59). Somatic Cell Nuclear Transfer The study of somatic cell nuclear transfer has been carried out at Vietnamese Academy of Science and Technology (Laboratory of Biology of Reproduction and Development) since 1998 with the cooperation from French INRA and Kinki University, Japan. This study is now applied to other species and oriented for the major direction such as conservation of biodiversity, improvement of animal production and for bio-medical application. During last ten years, the last New World large mammal endangered species such as Kouprey (Bos sauveli, 1937), Saola (Pseudoryx nghetinhensis, 1993), and giant muntjac (Megamuntiacus vuquangensis, 1994) were discovered in Vietnam. Regarding these newest and greatest challenges, cell samples were collected from two new discovered species: Pseudoryx nghetinhensis and Megamuntiacus vuquangensis; also a mini cryobanking of somatic cell for

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nuclear transfer was set up for other different domestic species (cattle, buffalo, goat, pig) and wild species (bear, gaur, monkey, minipig) (18, 33). A model of interspecies nuclear transfer using bovine enucleated oocytes as universal host ooplasts for reprogramming of somatic cell nuclear was studied for somatic cell from buffalo (30). Actually, the cloned embryos were obtained in cattle, buffalo, monkey, mouse by homo-species nuclear transfer (29, 37, 58) and in Saola, Gaur, and bear by interspecies nuclear transfer (39, 40, 45, 60). Saola Saola (Pseudoryx nghetinhensis) is a new mammalian species discovered in Vietnam (Dung et al. 1993). Cell samples were collected from two females and one male young animal, which were captured by local hunters. Molecular phylogeny was carried out on Saola and suggested the placement of this species within Bovinae (49,55,56). The cloned Saola embryos were obtained by interspecies nuclear transfer using bovine enucleated oocytes as host ooplasts (39, 40) and Saola somatic cells which were collected in different status; muscle tissue was collected 20 h after the animal died and skin explants were collected from a live young female. Karyotypes examined at different passage provided the following distribution of chromosome number: n = 51 (3%); n = 50 (78%); n = 49 (10%); n = 48 (5%); n = 46-47 (3.5%). This result showed that reconstructed embryos from Saola cells (SLNT) can develop normally into the blastocyst stage with a similar rate as compared to homo-species bovine reconstructed embryos using cultured skin fibroblast cells; 75% of fused, 79% of divided, and 37% of reconstructed embryos developed to blastocyst stage. The average cell numbers observed for inner cell mass (ICM) and trophoblast were 36 and 52, respectively (39, 61). The study regarding transcriptional activity of cloned Saola and cloned bovine embryos by monitoring the incorporation of BrUTP into membrane-permeabilized embryos was undertaken in cooperation with Institute INRA, French. The results show that somatic nuclear transfer can be used to routinely generate cloned Saola embryos whose early global transcriptional activities were observed as that seen in cloned bovine embryo (39). Recently, in order to assess the somatic nuclear reprogramming in cloned embryos with regard to a normal reprogramming of the IVF embryos, a subtracted cDNA library relevant to the onset of genome activation in IVF bovine embryos was firstly established using SSH (Suppression Subtractive Hybridization) technique. This dedicated library was then subjected to differential screening with different samples corresponding to different types of bovine embryos obtained after somatic nuclear transfer using distinct fibroblast cell lines (unpublished data). In order to identify genes that are differentially regulated between two biological situations (homo or hetero species NT embryos), further different screening of this library will be realized with Saola embryo obtained by nuclear transfer of Saola fibroblast cell into bovine cytoplasm (50, 62). In other hand, the assessment of the applicability of bovine microsatellite markers on Saola (Pseudoryx nghetinhensis) was also carried out. A total of 127 microsatellite markers were tested on a male and a young female Saola. An efficient amplification was observed for 123 markers (96.8%), 73 markers (59.3%) were polymorphic. Four loci (BM2304, BMS1928, BMS779 and ILSTS006) on cattle chromosomes 1, 4, 7 and 8 respectively, failed to amplify in Saola. The results show that most of the bovine microsatellite markers are applicable in Saola and therefore they can be used to study the phylogenetic relationships and the genetic diversity of the Saola population (56). Minipig Vietnam has a splendid resource of porcine breeds with non-PERV pure miniature breeds such as Ban, Meo and Co Soc breeds. Some of these breeds have adult body weigh of 5 kg and body long of 60 cm at 2 years old. SCNT has been applied to this breed as a tool for its conservation to keep biodiversity as well as to multiple the mini-pigs as models for the research and application in human xeno-transplantation. This work has developed recently with the cooperation with research team of Dr. Takashi Nagai of National Agriculture and Bio-oriented Research Organization, Tsukuba and Dr. Teruhiko Wakayama from Riken Center. Somatic cells obtained from culture of minipig skin sample were transferred into enucleated oocytes prepared from domestic pig (Landrace) and fusion was done by direct current (DC) pulses of 30v for 25 µs in Zimmerman medium with 0.3 M Mannitol. More than 72% of reconstructed oocytes were fused, 81.2% cleaved to 4-8-cell stage, 50% developed to the morula stage and 12.5% developed to the blastocysts stage. Karyotyping showed a normal distribution of chromosome number (2n = 40) for cell population after passage and for reconstructed embryos (60). Monkey The nuclear transfer has been done in non-human primate (Macaca fascicularis and Macaca mulata) using somatic cells developed from skin explants obtained from adult monkey and more than 2,200 oocytes collected from FSH treated animals. The current nuclear transfer method with the modification to avoid direct effect of UV on the oocyte was applied.

Embryo Transfer Newsletter 7

It has been shown that monkey oocyte is more sensitive to the treatment of fluorescence. The time of exposure to ultraviolet light was restricted to less than 10 sec. The cloning method without the direct treatment of oocyte with Hoechst 33342 and fluorescence was used to improve the developmental potential of reconstructed embryos. Maternal chromosome removal was confirmed in this case by epifluorescence imaging of removed cytoplasm. When the conventional cloning was applied, the rate of successful enucleation confirmed was 70% to 80% and rate of fused oocytes was varied from 55% to 80%. Out of the fused oocytes, more than 65% cleaved and developed to 4-8-cell stage at day 2-3 after nuclear transfer. Good quality embryos were transferred to 7 recipients and 3 pregnancies was confirmed by ultrasonography at 2 and 3 months after embryo transfer (37, 38). The study of effect of combination of lower dose of gonadotropin treatment (7-8 days instead 10 days as full dose schedule) and long in vitro maturation up to 48-72 h is recently caring out (43, 57).

Summary The results presented in this paper indicate numerous aspects of the current state of embryo technologies in Vietnam. Current and future work is continuing along similar lines of investigation. References 1.

Nguyen B.X., Uoc N.T., Ty L.V., Binh D.V. (1980). First result of rabbit production by embryo transfer. Bulletin of Biology, National Scientific Institute of Vietnam, 203-213.

2.

Nguyen B.X., Heyman Y., Renard J.P. (1984). Direct freezing of cattle embryos after partial dehydration at room temperature. Theriogenology. 22,4,389-399

3.

Renard J. P., Nguyen B.X., Garnier V (1984). Two–step freezing of two-cell rabbit embryos after partial dehydration at room temperature. J. Reprod. Fert. 71, 573-580.

4.

Nguyen B.X. (1989). Effect of partial dehydration on the viability of mammalian embryos: study at room temperature and at low temperature. Thèse de Doctorat d’Etat, Paris, Université Pierre et Marie Curie. 166p.

5.

Nguyen B.X., Ty L.V., Uoc N.T. (1990). Model of ONBS-MOET for buffalo genetic improvement in Vietnam. Proceeding of the FAO Workshop on Open Nucleus Breeding Systems, Bulgaria, Nov 18-23, p 283-286.

6.

Nguyen B.X., Ty L.V., Uoc N.T. (1990). Model of ONBS-MOET for buffalo genetic improvement in Vietnam. Proceeding of the FAO Workshop on Open Nucleus Breeding Systems, Bulgaria, Nov 18-23, p 283-286.

7.

Nguyen B.X., Ty L.V., Uoc N.T. (1990). Water Buffalo in Vietnam 2 Buffalo breeding in Vietnam. Proceeding of the FAO Workshop on Open Nucleus Breeding Systems, Bulgaria, Nov 18-23, p 210-216.

8.

Gerard E., Nguyen B.X., Renard J.P. (1991). Rheological changes in mammalian egg cytoskeleton in response to cold adaptation. American Institute of Physics. Conference Proceeding: The living cell in four dimensions. G. Paillotin (Ed), New York, 226, 37-54.

9.

Nguyen B.X., Ty L.V., Uoc N.T., Long D.D. (1991). Buffalo breeding in Vietnam: status of research and development activities in reproductive biotechnology. Proceeding of the FAO Workshop on Open Nucleus Breeding Systems, Bulgaria, Varna, May 13-18, 39-43

10. Uoc N.T., Ty L.V., Long D.D., Becker J-F. Nguyen B.X., Chupin D., Renard .J.P. (1992). Effect of estradiol supplementation on superovulation in swamp buffalo. Theriogenology. 38, 471-478. 11. Ty L.V., Nguyen B.X., Son H.N., Driancourt M.A. (1993) Superovulation and ovarian follicular population of infant buffaloes and calves. J. of Animal Sci. 102, 23-28. 12. Nguyen B.X., Ty L.V., Duc N.H., Uoc N.T. (1994). Preliminary results of in vitro maturation and fertilization in native yellow cattle and in Swamp buffalo. Bulletin of Institute of Biotechnology, 166-167. 13. Uoc N.T., Ty L.V., Duc N.H., Long. D.D., Nguyen B.X. (1994). Research on cattle embryo transfer under tropical conditions of Vietnam: 1. results of embryo production by superovulation. Bulletin of Institute of Biotechnology, 160-165. 14. Duc N.H, Uoc N.T, Ty L.V, Nguyen B.X. (1995). Preliminary results of freezing of human sperm and its activation for IVF. Bulletin of institute of Biotechnology, 195-204. 15. Uoc N.T., Ty L.V., Duc N.H., Long. D.D., Nguyen B.X. (1995). Effect of supplementation in estradiol and PMSG on estrus response in native yellow and Laisind cattle to estrus synchronization by prostaglandin. Bulletin of institute of Biotechnology, 179-185. 16. Nguyen B.X., Duc N.H., Ty L.V., Long D.D., Uoc N.T. (1996) Cattle embryo transfer under tropical conditions of Vietnam. Proceedings of 3rd Asia-Pacific Congress on Biotechnology, Huahin, Thailand, 22-30 17. Nguyen B.X., Duc N.H., Ty L.V., Long D.D., Uoc N.T. (1996). Embryo technology in Vietnam: Contemporary results obtained in a National project. Proceedings of 2nd Animal Biotech. Symposium, Nanjing, China. 21-28 18. Nguyen B.X., Duc N.H., Ty L.V., Long D.D., Uoc N.T. (1996). Preliminary study of preservation of buffalo genetic biodiversity by embryo technology. 1996. Proceedings of 2nd ABA Congress, Manila, Philippines , 215-220 19. Uoc N.T., Duc N.H., Nguyen B.X., Ty L.V. (1996) Nuclear maturation of swamp buffalo’s oocytes cultured in TCM-199 supplemented with FSH and Estadiol-17b. Bulletin of Institute of Biotechnology, 274-278 20. Uoc N.T. (1996). Control of estrus and superovulation for embryo transfer under tropical breeding conditions of Vietnam. Ph.D. thesis, Hanoi, 1996.

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21. Nguyen B.X., Duc N.H., Ty L.V., Long D.D., Uoc N.T. (1997). Quantitative and qualities of aspects of oocyte collection for in vitro maturation and fertilization: a comparative study in swamp buffalo and in native yellow cattle. Bulletin of Biology, 517-525 22. Nguyen B. X. (1997). Long-term conservation of gametes and embryos using the associated treatment of dehydration and freezing. Proceeding of Best Capsule 2001 Conference, Japan, Nov.1997. H 4-11. 23. Uoc N.T., Duong D.L., Ty L.V., Chupin D., Renard J.P., Nguyen B.X. (1997). Effect of supplementation of estradiol-17b to supeovulatory regime on ovarian response and embryo quality in cycling swamp buffalo (Bubalus bubalis). J. of Animal. Science. 47, 181-187. 24. Nguyen B.X., Ty L.V., Duc N.H., Uoc. N.T. (1998). Potential of genetic diversity and embryo biotechnology for improvement of meat-milk production under tropical conditions. Proceedings of the 4th Asia-pacific Conference on Agricultural Biotechnology, Darwin, Australia, p 292-294. 25. Bui L. C., Duc N.H., Ty L.V., Uoc. N.T., Nguyen B.X. (1999). Production of bovine embryos with selected sex using PCR. communication, 3rd National Congress on Biotechnology, Hanoi, Dec,1999, 1085-1090 26. Bui L.C., Uoc N.T., Ty L.V., Duc N.H., Luong L.D., Nguyen B.X. (1999). Sexing of bovine embryos: effects of PCR regime and sample collection. J. of Genetics and Applications, 4, 5-10 27. Uoc N.T., Ty L.V., Duc N.H., Hai N.V., Nguyen B.X. (1999). Sex determination of preimplatation bovine embryo by DNA amplification. Bulletin of Biology, 21(1): 34-38. 28. Xiem N.T., Lan L.P., Duc N.H., Uoc. N.T., Nguyen B.X. (1999), The first results of application of capacitation and freezing in treatment of human infertility. Bulletin de Medicine, 9,1999. 29. Nguyen B.X., Chi B.L., Duc N.H., Ty L.V., Uoc. N.T. (2000). Cloning of Ha-an bovine embryos by nuclear transfer with adult somatic cells. J. Biology, 1, 10-15. 30. Nguyen B.X., Chi B.L., Duc N.H., Ty L.V., Uoc. N.T. (2000). Comparison between the efficiencies of using bovine and buffalo oocytes as host ooplasts for embryo production by adult cell nuclear transfer. Theriogenology, 53 (1), 235. 31. Nguyen B.X. (2000). Micromanipulation and its application to animal research. Sixth advanced course on micomanipulation. Soon Chye Ng (Ed.), Singapore, 18:25. 32. Nguyen B.X., Sotomaru Y., Tani T., Kato Y., Tsunoda Y. (2000). Efficient cryopreservation of bovine blastocysts derived from nuclear transfer with somatic cells using partial dehydration and vitrification. Theriogenology, 53:1439-1448 33. Nguyen B.X., Chi B.L., Duc N.H., Ty L.V., Keo N., Khanh N., Nhon N., Son H.N., Uoc N.T. (2000). Application of cryobanking and cloning technology to conservation of animal biodiversity in Vietnam. Proceedings of Conference on Biodiversity, Hanoi, 8-2000:10-14. 34. Hassanin A., Seveau A., Thomas H., Bocherens H., Billiou D., Nguyen B.X. (2001). Evidence from DNA that the mysterious ‘linh duong’ (Pseudonovibos spiralis) is not a new bovid. C R Acad Sci III. 2001 Jan. 324(1): 71-80. 35. Nguyen B.X. (2001). Current status of Somatic Nuclear transplantation and animal cloning. Seven advanced course on micomanipulation. Soon Chye Ng (Ed), Singapore, 30:36. 36. Ty L.V., Chi B.L., Duc N.H., Son H.N., Uoc N.T., Nguyen B.X. (2001). The effect of supplementation with Estradiol-17b on the in vitro maturation of swamp buffalo oocytes. Theriogenology, 55:406 37. Nguyen B.X., Tan E.K.J., Paolo M., Uoc N.T., Liow S.L., Hebert S., Oh S.H., Ng S.C. (2002). Development of embryos cloned from fibroblasts in Long-tailed Macaque (Macaca fascicularis) Theriogenology, 57:438 38. Nguyen B.X. (2002). The new approaches in monkey reproduction. In, Joel Fagot (Eds). Primatology. Volume 5, 109-138. ADRSC, Marseille. 39. Bui L.C., Vignon X., Campion E., Laloy E., Lavengne Y., Ty L.V., Nguyen B.X., Renard J.P. (2002). Use of interspecific nuclear transfer to study the early embryonic development and nuclear activities of the endangered species Pseudoryx nghetinhensis (Saola). Theriogenology, 57:427 40. Uoc N.T., Chi B.L., Duc N.H., Ty L.V., Keo N., Tuoc D., Thanh N.T., Hanh N.V., Laloy E., Renard J.P., Nguyen B.X. (2002). Effect of tissue sampling conditions on the in vitro multiplication and reprogramming potential of somatic cells obtained from different specimens of the Saola (Speudoryx Nghetinhensis) species. Theriogenology, 57:437 41. Duc N.H. , Uoc N.T., Ty L.V., Hanh N.V., Thanh N.T., Bui L.C., Anh N.T., Huu Q.X. and Nguyen B.X. (2003). Potential for in vitro production of embryo from follicular oocyte of yellow and redsindhi-yellow cattle. Theriogenology. 59 (1) , 442 42. Duc N.H., Uoc N.T., Ty L.V., Huu Q.X., Thanh N.T., Bui L.C., Hanh N.V., Anh N.T., Linh N.V., Nguyen B.X. (2003). Results on the in-vitro embryo production and embryo transfer in Laisind cattle. Proceedings of Vietnam National Conference of Biotechnology, 699-702. 43. Hanh N.V., Uoc N.T., Ty L.V., Duc N.H., Thanh N.T., Bui L.C., Anh N.T., Huu Q.X. and Nguyen B.X. (2003). Oocyte production in non-human primate: effect of the combination of prior in vitro hormonal treatment and in vitro culture on the maturational competence. Theriogenology. 59 (1) , 488 44. Xuan N.B., Ty L.V., Duc N.H., Bui L.C., Thanh N.T., Linh N.V., Hanh N.V., Huu Q.X. Son H.N., Uoc N.T. (2003). Advanced results in Embryo - Cell Biotechnology and their perspectives of agriculture-bio-medical applications. Proceedings of Vietnam National Conference of Biotechnology, 712-716. 45. Ty L.V., Hanh N.V., Uoc N.T., Duc N.H., Thanh N.T., Bui L.C., Huu Q.X. and Nguyen B.X. (2003). Preliminary results of cell cryobanking and embryo production of black bear (Ursus thibetanus) by interspecies somatic cell nuclear transfer. Theriogenology. 59 (1), 290 46. Uoc N.T., Ty L.V., Duc N.H., Bui L.C., Thanh N.T., Linh N.V., Hanh N.V., Huu Q.X. Anh N.T., Son H.N., Long D.D., Nguyen B.X. (2003). Production of dairy tropical calves by transfer of IVF and sexed embryos. Proceedings of Vietnam National Conference of Biotechnology, 717-719. 47. Linh N.V., Ty L.V., Uoc N.T., Duc N.H., Hanh N.V., Huu Q.X., Nguyen B.X., (2003). Using immuno-surgery to isolate Inner Cell Mass (ICM) for Embryonic Stem cell culture. Proceedings of Vietnam National Conference of Biotechnology, 695-698.

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48. Nguyen B.X., Uoc N.T., Ty L.V., Monson R.L., Leibfried-Rutledge M.L., Duc N.H., L.C. Bui L.C., Huu Q.X., Hanh N.V., Thanh N.T., Linh N.V., and Rutledge J.J. 2004 Production of tropical diary calves by embryo transfer using local Laisind (Bos indicus) recipients and intercontinental fresh IVF shipped embryos. Reprod. Fert. & Development, 16 (1,2), 249-250. 49. Ahrens E., Graphodatskaya D., Nguyen B.X., Stranzinger G.. Cytogenetic comparison of Saola (Pseudoryx nghetinhensis) and cattle (Bos taurus) using G- and Q-banding and FISH. Cytogenet Genome Res. 2005; 111(2): 147-51. 50. Bui L.C., Leandri R., Renard J.P. and Duranthon V. (2005) SSH adequacy to preimplantation mammalian development: Scarce specific transcripts cloning despite irregular normalisation. BMC Genomics 6, 155-177. 51. De Rensis F., Ronci G., Guarneri P., Nguyen B.X., Presicce G.A., Huszenicza G., Scaramuzzi R.J. (2005). Conception rate after fixed time insemination following ovsynch protocol with and without progesterone supplementation in cyclic and non-cyclic Mediterranean Italian buffaloes (Bubalus bubalis). Theriogenology 63(7): 1824-31. 52. Duc N.H. (2005). In vitro maturation and fertilization in local yellow cattle and yellow –zebu cattle; Ph.D. Thesis, Hanoi, 2005. 53. Nguyen B.X. (2005). Somatic cell cloning in monkeys. Proceeding of the 2nd Asian Reprod Biotech. Conference, Bangkok, Nov 2005, 77-83. 54. Nguyen B.X. (2005). Report of project of production and transfer of IVF-sexing embryos for dairy cattle breeding. Hanoi, VAST, 180p. 55. Thanh N.T., Nguyen B.X. and Stranzinger G. (2005) Characterization of G - banded chromosomes of a female Saola (Pseudoryx nghetinhensis, 2n = 50) and X chromosome identification by means of fluorescent in situ hybridization (FISH). Cytogenet Genome Res. 109(4): 502-6. 56. Thanh N.T, Ménétrey F., Genini S., Nguyen V.L., Vögeli P., Nguyen B.X. and Stranzinger G. (2005) Application of bovine microsatellite markers on Saola (Pseudoryx nghetinhensis). J Anim Breed Genet. 122(3): 195-8. 57. Uoc N. T., Bavister B.D., Hanh N.V., Bui L.C., Thanh N.T., Duc N.H., Huu Q.X., Linh N.V., Nguyen B.X. (2005) Somatic cell nuclear transfer in non-human primate: the possibility of using oocytes matured in vitro for up to 3 days as host ooplasts. Reprod. Fert. Development, 17:177-178. 58. Duc N.H., Brochard V., Jouneau A., Renard J.P., Nguyen B.X. (2005). Development of cloned mice embryos using microinjection (Piezoelectric) of different passage of embryonic stem cells. 2nd Asian Reprod. Biotech Conference, Thailand Bangkok, Nov 2005, p35. 59. Linh N.V., Uoc N.T., Huu Q.X., Hanh N.V., Duc N.H., Thanh N.T., Chi B.L., Tich N.K, Long D.D., de Rennis and Nguyen B.X. (2005). Effect of 17b-Estradiol supplementation on the in vitro maturation and embryo production in swamp buffalo. 2nd Asian Reprod Biotech Conference, Thailand Bangkok, Nov 2005, p167. 60. Nguyen B.X., Uoc N.T., Nagai T., Linh N.V., Huu Q.X., Hanh N.V., Thanh N.T., Duc N.H., Bui L.C., (2006). Production of Ban minipig embryo by somatic cell nuclear transfer. Reprod. Fert. Development 18:1, 147. 61. Nguyen B. X., Uoc N.T., Duc N.H., Bui L.C., Thanh N.T., Linh N.V., Hanh N. V., Huu X. (2005). Somatic cloning in Vietnam: state and perspectives. Proceedings National Conference on Basic Biotechnology, Hanoi, 269-274. 62. Bui L.C., Faure C., Henaut A., Vignon X., Lebourhis D., Renard J.P., and Duranthon V. Genome reprogramming after nuclear transfer included both correct and excursive gene activation and repression (in preparation).

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Classifieds Upcoming Events-Continuing Education Opportunities

Upcoming Events-Continuing Education Opportunities

TRIENNIAL REPRODUCTION SYMPOSIUM July 9, 2006

7TH INTERNATIONAL RUMINANT REPRODUCTION SYMPOSIUM

The Triennial Reproduction Symposium will be held in Minneapolis on Sunday, July 9 from 8:00 am to 5:30 pm, immediately before the ADSA/ASAS annual meeting. Topics include: 1.

2. 3.

4.

Minisymposium I. Follicle and oocyte – 3 speakers on The dominant follicle, Cytoplasmic maturation, and Meiotic maturation of oocytes. (M. Lucy, University of Missouri; A. Watson, University, Western Ontario; F. Richard, Laval University) 2006 Casida Award for Graduate Education. To Be Announced. The USDA-NRI grants program in Reproduction – Panel discussion on the balance between translational and basic research funding. (M. Mirando, USDA; J. Reeves, Washington State University; W. Thatcher, University of Florida) Techniques sessions on: RNAi (R. Anthony, Colorado State University); Microarrays (G. Smith, Michigan State University); Statistical power calculations (R. Lenth, University of Iowa) and Analysis of binomial and categorical data (R. Quaas, Cornell University). 5. Minisymposium II. Reproductive immunology – 3 speakers on Interface with the endocrine system, The enigma of the fetal allograft, and Sperm and seminal plasma and the female immune system.(P. Hansen, University of Florida; C. Davies, Washington State University; S. Robertson, University of Adelaide)

Registration is $100; $30 for students. Details are at http://adsa.asas.org/meetings/2006/

It is with great pleasure that the Organising Committee for the 7th International Ruminant Reproduction Symposium invites the submission of abstracts for poster presentation in this scientific event to be held between 13-17th August 2006 in Wellington, New Zealand. The Ruminant Reproduction Symposium is the most prestigious international reproduction conference for ruminants with a major emphasis on sheep and cattle, but also includes keynote presentations on other species. It has been held approximately every 4 years in various countries around the world including Leura, Australia (1980), Ithaca, USA (1986), Nice, France (1990), Townsville, Australia (1994), Colorado Springs, USA (1998), Crieff, Scotland (2002) and now, New Zealand. Twenty seven international speakers have been invited to attend the Ruminant Reproduction Symposium. They will highlight current knowledge and state-of-the-art reproductive biology and technology applicable to both developed and developing nations, in a range of ruminant species. Please visit the website to view the proposed scientific programme (www.ruminantsymposium2006.co.nz). This stimulating meeting will be complimented by the wonderful opportunities for both outdoor activities and cultural experiences that the vibrant capital of New Zealand offers. Symposium Registration: Earlybird registration will be available from February 2006 and will close 30th April 2006. Otherwise, normal registration is available right up to the time of the Symposium.

Position Announcements VETERINARIAN Frontier Genetics International is seeking a veterinarian with experience in beef cattle medicine and a passion for beef cattle and equine reproduction. FGI is a full service AETA certified and USDA approved, embryo transfer company with a 100+ donor capacity facility, embryo laboratory, indoor working facility including double equine stocks and phantom mare, Aloka 500, HeatWatch system, portable breeding chute and an experienced, competent staff. Benefits package includes competitive salary, vacation, retirement, dues, licenses, generous CE and insurance stipend. Associate will be involved in all aspects of our in clinic and on the farm ET programs. We are willing to train the right person. Send resume and letter to Lee Jones, DVM, Frontier Genetics International, RR 1 Box 66, Curtis, NE, 69025; 308-367-5690.

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AETA and CETA/ACTE Invites You! Joint Annual Scientific Convention The Westin Ottawa Hotel, Ottawa, Ontario, Canada October 5-7, 2006 Presentations will include: x Sexed semen technology x Field superovulation trial results x Ultrasound prediction of superovulation responses x Nutritional topics with the latest on chelated minerals and fatty acids and their effects on reproduction and fertility. x Trans Ova presentation on the applications of new technologies in embryo transfer practice The following additional topics/events will also be included in the program: x Labs by Drs. John Hasler and Reuben Mapletoft x Wet Labs: ultrasound, semen evaluation, and vitrification of embryos x Practitioner’s Forum x Certification update x Banquet x Exhibits x Golf tournament & Companion Tours x Breakout sessions Visit: http://www.aeta.org/06mtg.asp for more information!

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Health And Safety Advisory Committee (HASAC) THE IETS HEALTH AND SAFETY ADVISORY COMMITTEE (HASAC): What you want to know about the main conclusions of its meetings held in Orlando, Florida, January 2006 M Thibier – Chairman The IETS Health and Safety Advisory Committee and its subcommittees only met once this year, as in the previous year but in contrast to previous custom (i.e. a mid year meeting plus one at the IETS Conference). This reduces the cost of such meetings but of course slightly slows down the speed at which the various files are being handled and led to term. However quite a number of significant moves have been achieved this year at the Orlando meeting and the main points reported to the Board of Governors of IETS are herein summarized. One constant point being reiterated at each meeting of HASAC is that the members of IETS have the right to know exactly the huge amount of work that is being done by the HASAC members. It should it be reiterated here that all members are involved without any support but their own. This is true for all IETS committees and the BOG. Communication to the members of the work done by HASAC is thus a first priority of HASAC and the present report as well as those regularly edited in the IETS Newsletter is consistent with that goal. 1. New developments about the animal health issues. As a follow up to last year problem dealing with exports of bovine embryos to the European Union, and thanks to the reactivity of IETS HASAC, it has been possible to obtain from the Brussels authorities the correction needed in order to permit embryos from third-world countries (the ten third-world countries permitted to export embryos to the EU) to enter the European Union. This was approved by the EU Commission in the fall and has now been published in the “Official Journal of the European Union” under Decision of the Commission of 4 January 2006 – 2006/168/CE (OJEU 1 28.2.2006 – L 57/19). It was in connection with this problem raised in late 2004 that HASAC had decided to write and publish a paper on “Evaluation of risks of viral transmission to recipients of bovine embryos arising from fertilization with virus infected semen”. This has been achieved thanks to AE Wrathall, HA Simmons and A Van Soom ( Theriogenology, 2006, 65, (2) 247-274). As every year, the liaison officer of IETS to OIE (HASAC chair) has reported, at that meeting, the work that had been done and passed on to the OIE. It is of great interest to all IETS members to note that the OIE (World Animal Health Organization) Terrestrial Animal Health Code now includes not only the 4 Appendices dealing with embryos but also one additional Appendix: Appendix 3.3.5. This appendix reports the work of HASAC and now includes the criteria selected to categorize the disease risks associated with trade of in vivo produced embryos in addition to the lists of the diseases in the 4 categories. Several disease chapters of this Code are being revisited by HASAC for the articles of those chapters relevant to embryo transfer. Those on the three following diseases, as proposed by HASAC in early 2005, are now incorporated in the OIE Code: Blue tongue, Tuberculosis and Rift Valley Fever. Some new chapters are being tackled in the current year: Caprine contagious Pleuro Pneumonia, (CCPP), Lumpy Skin Disease and Scrapie. In parallel HASAC, under the leadership of George Perry (Australia), is working diligently on a risk assessment for abattoir derived IVF bovine embryos. Attempts to evaluate the risk of abattoir-derived IVF embryos do create unique challenges. Such an analysis is needed in particular in some regions where IVF produced embryos are being transferred in large numbers. HASAC encouraged George to have the risk assessment completed and asked anyone willing to help to get in contact with George. The disease model he works on is that of BVD and some precise figures of prevalence in some areas around the world for example are requested. Regarding the research update that is being reviewed by HASAC members every year, the number of scientific reports is now close to 400 and the review papers over 125. This update is now being posted on the IETS web site and can be consulted at any time. From the new publications under review (n = 22), it appeared that Bovine Viral Diarrhea disease continues to be a “hot” topic of interest in embryo production. A discussion at large on the categorization to which paratuberculosis could enter into took place and the Research subcommittee will prepare a position paper for next year taking into account the recently published papers. The Forms and Certification Subcommittee also achieved quite a number of points. A more detailed report will be given in a subsequent issue of this IETS Newsletter by the subcommittee chairperson 2. In the mean time, the main conclusions are being posted on the IETS website from the HASAC report. Let us just mention as an example that after considerable discussion, but in the final analysis, the subcommittee agreed that another color straw should be used for vitrified embryos that are either frozen for direct transfer or for non-direct transfer. Light blue has so been selected 3. Last but not least, it was decided and endorsed by the IETS Board of Governors that a 4th edition of the IETS Manual should be undertaken. The two co-editors in charge of that important task, are Professor D Stringfellow and Dr D Givens. They are currently preparing their strategy and timetable with the target of end 2007 to have the Manual ready. The principles on which the previous

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editions were done will remain identical for this new edition: i.e. “A procedural guide and general information for the use of embryo transfer technology emphasizing sanitary procedures.”

2. What about the food safety issue, could clones or clones offspring enter the food chain one day? The answer could be “yes, it is coming”. Indeed this subcommittee had one full day meeting with a number of remarkable presentations from USDA, Health Canada, OIE, Japan, from Food Standard Australia-New Zealand (FSANZ), University of Guelph, Monash and from one member of the Pew Initiative on Ag Biotechnology. The latest and most recent points referring to clones and transgenic animals were reviewed. Regarding some of the considerations, two resolutions were passed and later approved by the Board of Governors which are stated as such: Statement on the food testing of meat or milk from clones and their progeny: Traditional toxicological testing consisting of very high doses administered repeatedly is not suitable method for assessing the safety of any whole food, including that from clones and their progeny for several reasons. Chief among these is accepted observation that it is not possible to sufficiently exaggerate the dose of test material without so perturbing the physiology of test animals as to render the result difficult to interpret. This conclusion has been reached by several national and international expert bodies. IETS agrees with these conclusions. Approaches to evaluating data for assessing the food safety of clones: The most efficient and effective approach to evaluating the safety of food from animal clones is to take a weight of evidence, common sense approach. Given that the nature of the technology does not allow for the production of large numbers of genetically identical clones at any one time, and that each individual clone arises from an independent event, it is not possible to generate large populations of sufficient statistical power from which to draw conclusions. Rather, looking at smaller populations across similar life stages for common outcomes, and evaluating the weight of these data will yield the most meaningful results. The weight of evidence presented from several international sources thus for indicates that food safety risk is minimal. There is still no formal regulation as such that covers clones or/and clones offspring to eventually enter the food chain although the US FDA are in their final stage of a position document on “Food consumption risks associated with animal clones”. It was stated, in January, that such a document should be released in the coming months. From the IETS HASAC standpoint, it was decided to constitute a data base on transgenesis in addition to that of clones to be built up by this HASAC Food Safety subcommittee under the leadership of its chairperson Dr H Kochhar. In addition and after a proposal from the Board of Governors, an ad hoc group was set up from this Food Safety Subcommittee, dealing with the animal health care of clones. A first meeting took place during this conference and this group will prepare a document during the course of the present year. This is quite an important point to be of notice in particular in the concept of animal welfare and acceptance by the public of the animal reproductive biotechnology. Finally the proposal by the 2007 Local Organization Committee of the IETS Annual Conference of Kyoto, to organize a pre-conference symposium on Food safety issues was received with enthusiasm by the HASAC members and later accepted by the Board of Governors. This is one more reason for all IETS members to attend this next and fantastic IETS venue. This pre-conference symposium in Kyoto previous to the main Annual Conference itself will be one full day session and will be a unique opportunity to know where the world stands in those areas of cloning and transgenesis, particular in terms of risk assessment and regulations. Again the HASAC members have done an excellent job and they should all be commended .It was however pointed out that some regions of the world are not represented so much as such in those subcommittees particularly from South America, some countries in Asia, and Oceania. All volunteers to help in continuing this challenging work for the benefits of all IETS members will be more than welcome in the future meetings starting with that in Kyoto in January 2007. (Footnotes) 1 Official Journal of the European Union 2 Thanks to Irma Robertson, a full set of forms of recommended embryo record systems is now available, including in vivo collected embryos, in vitro produced embryos, clones and transgenics. 3 Since then there has been a concern as such light blue straws tended, at least for some groups, to rupture during vitrification. It is then recommended to use a blue plug inserted into the straw of identification of a vitrified embryo.

June 2006 14

33rd Annual Conference 2007 Annual Conference of the IETS January 7-9, 2007 Kyoto, Japan Letter of Invitation from the LOC To: IETS Members From: Akira Iritani and Hiroshi Imai, LOC member IETS 2007

On behalf of the Local Organizing Committee, we are pleased to formally invite you to the 33rd Annual Conference of the International Embryo Transfer Society at Kyoto, Japan, in the year 2007. Kyoto city was the formal capital of Japan for more than 1200 years and is gifted with abundant cultural and historical treasures. The city has a very convenient subway system so that it gives a ready access to the meeting venue, downtown, hotels and the major sightseeing locations. We are looking forward to our meeting at the scientific program and the special program in the New Year 2007. Please check sometime at the IETS web site at www.iets.org/2007 for updates. Sincerely yours, IETS 2007 Local Organizing Committee

Additional Program and Meeting information will be posted on the website (www.iets.org/2007) as it becomes available.

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Letter of Invitation from the Program Co-Chairs To: From: Subject:

IETS Members Carol Keefer, IETS 2007 Program Co-Chair Fulvio Gandolfi, IETS 2007 Program Co-Chair Call for Abstracts

On behalf of the IETS and the Chairman of the Local Organizing Committee, Dr. Akira Iritani, we would like to extend a warm invitation to all IETS members to join us at the next annual conference of the IETS, scheduled for 7-9 January 2007 in Kyoto, Japan. The theme of the IETS 2007 program is: “Embryo Quality and Fetal Development – Early Determinants of Developmental Success”. The program will highlight the latest scientific findings on the cellular and molecular mechanisms determining oocyte and sperm quality, and on its role in fertilization and early development. Presentation topics will include the long term consequences of in vitro culture and of nutrition on embryo development as well as the process of pregnancy recognition and an analysis of the genes involved in its maintenance to term. The program will include a practitioner’s forum and short communications showcasing the latest research results from leading laboratories around the world. The program will conclude with a keynote lecture by Dr. Janet Rossant on Cellular and Molecular Mechanism of Early Differentiation. The LOC is planning some exciting social events and will provide opportunities to explore Japanese culture. Details on the program, including the invited speakers and titles of their presentations, are currently available on the IETS website at:

http://www.iets.org/2007 In addition to the main program, there are plans to hold two Pre-Conference Symposia on Saturday “Assisted Reproductive Technologies And Food Safety In Farm Animals” and “Innovative Approaches in Embryology” (6 January 2007) and a Post-Conference Satellite Symposium on Wednesday “Quality Control of Embryos for Embryo Transfer and Related Advanced Technologies in Domestic Species “ (10 January 2007) organized by the Local Organizing. All of these events will also be held at the Kyoto International Conference Hall, Kyoto, Japan. Registration information will be posted on the website as soon as it is available. We are once again urging all members to utilize the electronic submission format in order to help keep costs to a minimum. We sincerely hope that you will all be able to join us in Kyoto to participate in IETS 2007!

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Call for Abstracts for Poster Presentations 2007 Annual Conference of the International Embryo Transfer Society January 7-9, 2007 Kyoto, Japan General Information: Free communication will be presented as posters, abstracts of which will be published in the January 2007 issue of Reproduction, Fertility and Development. The first author or a representative is required to present an expanded report of the data in poster format at the Annual Conference. Students may enter their abstract in a competition sponsored by the IETS Foundation and are also eligible to compete for travel awards. Details on eligibility, judging procedures, and prizes are available on the IETS website. Abstract Requirements: Abstracts must be in English and prepared strictly according to the instructions for submission. The language should be concise and avoid jargon. Abbreviations must be defined and should be used sparingly. Abstracts should consist of unpublished, original data that contains objectives, experimental methods (including statistical methods), results, and conclusions. The Program Chairman and two other referees will subject each abstract to rigorous scientific review. Rejection by the Program Chairman is final. If withdrawal of an abstract becomes necessary, notify the Program Chairman by September 15, 2006. Submission Restrictions: Due to space restrictions, submissions are limited. An individual may not be first author of more than one abstract. Space limitations allow a maximum of 3250 keystrokes (including characters, spaces, and punctuation). Begin count at the title and end count with the last word. Abstracts that are too long will be rejected automatically by the system. Coding for special characters is not included in the total keystroke count. If you must use boldface, italic, or sub-/superscript type anywhere in the abstract, consult the Text Attributes Coding information below. This special coding is not counted in the 3250 allowed keystrokes. Special characters such as Greek letters and math symbols are available on the electronic submission form itself. If your abstract includes a table the keystroke limit will be 2750 for the abstract and 500 for the table. The electronic submission form does allow submission of tables. The printed abstract width allows for 70 keystrokes per line, including spaces; keep this in mind when formatting the table. Tables that are too wide are not printable. You must also include coding for text attributes in the table. This includes the italicization, superscripting, and subscripting of numbers and letters in the table and the footnotes. Submission Deadlines: Abstracts must be submitted via the web site only. No fax or email copies will be accepted. The firm deadline for receipt of the abstract via the web site is August 1, 2006 by Midnight CST. Abstracts received after this date will be rejected. Author Agreements: By submitting an abstract, the author concedes (1) that the information in the submitted manuscript has never been published and is the work of the named authors, who all agree to be listed as co-authors, and (2) that the first author or a representative will present an expanded report of the data in poster form at the annual conference and that the work reported has not been published elsewhere. Failure to present a poster after your abstract has been published in the conference proceedings can result in rejection of abstracts submitted by your laboratory in future years. Authors need to be aware of patent considerations and copyright considerations before submitting an abstract for publication. All questions and correspondence should be directed to: IETS Headquarters 1111 North Dunlap Avenue Savoy, IL 61874 USA Phone: (217) 698-4697, FAX: (217) 398-4119 E-mail: [email protected] Embryo Transfer Newsletter 17

Main Conference Program January 7-9, 2007 Embryo Quality and Fetal Development – Early Determinants of Developmental Success Session 1* - OOCYTE QUALITY ™ Estimation of oocyte quality: Qing-Yuan Sun (China), ™ Oocyte quality and cryopreservation: Sergio Ledda (Italy) Session 2 - EARLY EMBRYO DEVELOPMENT ™ Maternal gene expression in oocytes and cleaved embryos: Nam-Hyung Kim (Korea) ™ Pattern of early gene expression: genome activation and cell differentiation: Tiziana A.L. Brevini (Italy) Session 3 - LONG TERM CONSEQUENCES ™ Long-term effect of embryo culture conditions: Jeremy Thompson (Australia) ™ Long term effects of nutritional programming of the embryo and fetus: Mechanisms and critical windows: Michael Symonds (UK) Session 4 - Implantation/Gestation ™ Regulation of pregnancy recognition and conceptus implantation by progesterone, interferons and endogenous retroviruses: Thomas E. Spencer (USA) ™ Gene expression and maintenance of pregnancy in bovine: roles of trophoblastic binucleate cells-specific molecules: Kazuyoshi Hashizume (Japan) Session 5 - SPERMATOZOA ™ State of the art in farm animal sperm evaluation: Heriberto Rodriguez-Martinez (Sweden), ™ Interactions of sperm with the female reproductive tract: “In vivo” is not “in vitro”: Susan Suarez (USA) Session 6 - KEYNOTE ADDRESS ™ Cellular and molecular mechanisms of early differentiation: Janet Rossant (Canada) *Sessions 1 through 5 will also feature 1 to 3 short presentations selected from submitted abstracts.

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Assisted reproductive technologies and food safety in farm animals Preliminary Program for the Pre-conference Satellite Symposium Kyoto, Japan Saturday, January 6, 2007 Organizers: Dr. H. Kochhar, Dr. M. Thibier, & Dr. H. Imai Features of program Background: Genetically modified livestock in agriculture are already an experimental reality and can rapidly become a commercial reality. The challenge is to see whether the existing science based safety-assessment model will work and whether the current marketing practices will be up to this challenge. The acid test of industry practices will be their capacity to build consumer confidence. Scientists, on the other hand, engaged in development of transgenic livestock intended to supply food must recognize that regulators and society consider the transgenic technology as a considerable shift from the traditional animal breeding practices. Livestock breeding is and will continue to be a balancing act of multiple trait selection, and it is naïve to believe that transgenes will become so important as to monopolize the selection process. Food safety and regulatory requirements for transgenic livestock are not yet definitive, but clearly have the potential to affect important areas such as trade certifications, animal identification, product identity and traceability. Hence this symposium will be an effort to flush out the issues pertaining to the science, food safety data from experiments in animal cloning and transgenesis, regulatory initiatives in this direction and the public perception of this technology. Hence we will have three components to the program: 1. Newer Assisted Reproductive Technologies – Cloning, Transgenesis etc.: The presenters are going to provide an overview of the technologies and how we are at a stage where there is a potential to market the food and products derived from these animals. 2. Food Safety components: The presenters in this sub-theme will provide valuable data in terms of safety of food derived from animal clones or transgenics as well as the parameters and strategies employed to generate the data. 3. Regulatory considerations: A tough decision to approve or not to approve the food based on the scientific information guides the regulators (which at present time are unclear for the biotechnology-derived animals). The presenters will look at the specific approach for assessments by different countries and the public perception to the food derived from such animals. 8.15 A.M.Opening address – Dr. Michel Thibier Section 1: Newer Assisted Reproductive Technologies 8.30 A.M. Animal Clones and methodology of cloning : Dr. I. Wilmut 9.00 A.M.Transgenic animal production and animal biotechnology – Dr. Jim Robl Section 2: Newer Assisted Reproductive Technologies and impacts on food safety 9.30 A. M. Comparison of clone and non-cloned cattle and their product evaluation over a 3 year period – Dr. Y Heyman 10.00 A.M.Health of animal clones and their progeny – present data – Dr. Ray Page 10.30 A.M. - Health Break 11.00 A. M. - Physiological character and health of clone cows. – Dr. Miharu Yonai 11.30 A.M. - Clones and progeny of Clones – safety and nutritional data – Dr. David Wells 12.00 A.M. - Food safety of products derived from cattle clones – Dr. Seiya Takahashi 12.30 A.M. - Lunch 2.00 P.M.Food safety of products derived from pig clones – Dr. J-H. Kim Section 3: Regulatory considerations in Animal Biotechnology 2.30 P.M. Regulatory considerations in transgenic livestocks from the aspect of Cartagena Protocol in Japan – Dr. K Yamanouchi 3.00 P.M. – Regulatory initiatives for biotechnology- derived animals in Canada – Dr. H.P.S. Kochhar and Dr. B. Evans 3.30 P.M. Health Break 3.45 P.M. Regulation of animal clones in the United States – Dr. L. Rudenko or Dr. J. Matheson 4.15 P.M. Food Safety Australia New Zealand (FSANZ) and regulation of animal clones: Dr. P Brent 4.45 P.M. Concluding remarks 5.00 P.M. Adjournment. Embryo Transfer Newsletter 19

Innovative techniques in human and animal embryology Preliminary Program for the Pre-conference Satellite Symposium for IETS, Kyoto, 6th January 2007 Organizers: Jeremy Thompson (Adelaide, Australia) Gábor Vajta (Tjele, Denmark) Timetable 08.00 – 08.05 Introduction 08.05 – 08.30 Keynote lecture 08.30 – 10.10 Section I 10.10 – 10.30 Coffee break 10.30 – 12.10 Section II 12.10 – 13.00 Lunch 13.00 – 14.40. Section III 14.40 – 15.00 Coffee break 15.00 – 16.40 Section IV 16.40 – 17.10 General discussion and Conclusion Speakers and Topics Keynote lecture Stanley Leibo Maturation and culture (4 lectures) Robert Gilchrist – Oocyte maturation: emerging concepts and technologies to improve developmental potential Jeremy Thompson – Alternatives to culture in the petri-dish Henrik Callesen, Ana Lopes – Quantification of embryo quality by respirometry Daniel Brison – Metabolic profiling of human embryos in culture; improving selection for transfer Stem cells (4 lectures) Teija Peura – Derivation of human embryonic stem cell lines Maisam Mitalipova – Maintaining epigenetic and genetis intergrity of human ES cells Kevin Sinclair – Embryonic stem cells and animal models of early human development and adult disease Fulvio Gandolfi – Isolation and characterization of pluripotent cell lines from pig embryos of different origins. Cryopreservation (4 lectures) David Gardner – Analysis of oocyte physiology to improve cryopreservation procedures Vladimir Isachenko – Aseptic technology of the small amount vitrification of human and bovine oocytes Masashige Kuwayama – Highly efficient vitrification for cryopreservation of human oocytes and embryos :The Cryotop method Amir Arav – Toward verification of vitrification parameters- what is really important for oocyte cryopreservation Embryo manipulation (4 lectures) Eric Overstrom – Oocyte induced enucleation reveals spindle-associated regulators of cytoplast developmental competence Cesare Galli – Comparative approach to nuclear transfer with the zona free method in cattle, horse, pig and sheep. Zsolt Peter Nagy – Artificial gametes: when and how? Jan Motlik – The porcine epidermal stem cells as a biomedical model for wound healing and normal/malignant epithelial cell propagation

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IETS Post-Conference Symposium January 10, 2007 “Quality Control of Embryos for Embryo Transfer and Related Advanced Technologies in Cattle“ Program: 08:00 Opening address：FLocal Organizing Committee Chair, Dr. A. Iritani 08:15 Introduction：FDr. Y. Izaike Session I: Oocyte collection following superstimulation and ovum pick-up (OPU) Chair: Dr. G. Seidel and Dr. H. Kanagawa 08:30 Superovulation and the control of the follicular wave: Dr. R.J. Mapletoft (Canada) 09:00 The Efficiency of Embryo Production by OPU: Dr. K. Imai (Japan) 09:30 Application of ultrasound guided follicular aspiration (OPU) in prepubertal and adult cattle.: Dr. H. Niemann (Germany) 10:00 Coffee Break Session II: Quality control of oocytes and embryos for in vitro production systems Chair: Dr. B. Bavister and Dr. Miyake 10: 30 The role of growth factor signaling on oocyte quality and maturation: Dr. K. P. McNatty (New Zealand) 11:00 Embryo quality in bovine embryos: influence of oocyte origin and culture environment on gene expression and developmental competence of IVF embryos: Dr. P. Lonergan (Ireland) 11: 30 Non-invasive quality assessment of IVP embryos: Dr. H. Abe (Japan) Lunch Break Session III: Embryo cryopreservation and commercial application of frozen embryos Chair: Dr. T. Kojima and Dr. G. Vajta 13:30 Cryopreservation of manipulated embryos: Dr. S. P. Leibo (USA) 14:00 Essential methods of freezing embryos for application in animal reproduction management: Dr. O. Dochi (Japan) 14:30 Vitrification and direct transfer of bovine embryos: Dr. G. Seidel (USA) 15:00 Mass production of cattle from IVM, IVF and cryopresevation of in vitro-produced embryos in Japan: Dr. Hamano (Japan) 15:30 Coffee Break Session IV: Early embryonic-loss and maintenance of early pregnancy with manipulated embryos Chair: Dr. K. Imakawa and Dr. G. Bo 16:00 What drives the formation of trophectoderm during early embryonic development? Dr. R. M. Roberts (USA) 16:30 Interaction between fetal and maternal environments during early pregnancy in domestic species: Dr. T. Ezashi (Japan) 17:00 Failure of uterine-conceptus interactions in cattle: Dr. T. R. Hansen (USA) 17:30 Improving pregnancy maintenance in dairy cows: Dr. W. W. Thatcher (USA) 17:30 Adjournment

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The 3rd Edition of the IETS Manual is available in French, Spanish, and English. The IETS publishes a manual with guidelines for general procedures for bovine embryo transfer, minimum standards for hygienic handling of embryos, an updated summary of results on embryo-pathogen interactions, and recommended standardization methods of labeling of containers of frozen embryos. To order your copy, please send this completed form with payment to: IETS Headquarters 1111 North Dunlap Avenue Savoy, IL 61874 USA Phone: (217) 356-3182, Fax: (217) 398-4119 E-mail: [email protected], Web site: http://www.iets.org Printed Book: (Please circle language desired) French Spanish (Printed English version of 3rd Edition no longer available)

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