BIOTECHNOLOGY APPLICABLE TO ANIMAL PRODUCTION AND HEALTH IN ASIA

Proceedings of the Regional Workshop on Biotechnology in Animal Production and Health in Asia Bangkok, Thailand, 17-21October1988

Kasetsart University Food and Agriculture Organization of the United Nations Bangkok, Thailand Ill

TABLE OF CONTENTS

I. INTRODUCTION 1 Il. RECOMMENDATIONS 3

A. General Recommendations 3

B. Biotechnology in Animal Production 5 1. Embryo transfer (ET) and manipulation 6 2. Open nucleus breeding systems (ONBS) 6 3. Animal feeding and ruminant nutrition 7 4. Production related hormones 7 5. Immunology ofreproduction 8 6. Genetic engineering applied to farm animals 9

C. Biotechnology in Animal Health 9 1. Diagnostic Methods 9 2. Vaccine Production 10

D. FAO/UNDP Regional Project 11

Ill. ANNEXES 13

ANNEX A List of participants 15

ANNEX B Programme 21

ANNEX C Teclmical Papers Presented 25

1. Opening Statement H.A. Jasiorowski 27

OVERALL REVIEW

2. Application of Recombinant DNA K.S. Guise 32 Techniques in Animal Improvement 3. Review of Development of Biotechnology T.K. Mukherjee 53 in Animal ProduGtion and Health in Asia

NUTRITION AND FEED RESOURCE DEVELOPMENT

4. Breeding and Biotechnology for R.B. Singh 69 Improvement of Livestock Feeds 5. Application of Biotechnology in I. F. Dalmacio 82 Improvement of Fibrous Crop Residues 6. Application of Biotechnology in Y.W. Ho et al . 93 Improvement of Fibrous Crop Residues: Anaerobic Rumen Fungi in the Utilisation of Fibrous Crop Residues as Feed Resource for Ruminant 7. Potential of Biotechnology in Animal C. Natarajan 97 Production and Health in India 8. Present Status of the Use of Genetically T.K. Mukherjee 101 Engineered Growth Hormones, and growth hormone gene in animals

BREEDING/GENETICS/REPRODUCTION

9. Genetic Improvement of Livestock J. Hodges 116 in Developing Countries Using Open Nucleus Breeding System 10. Potential of Multiple Ovulation and P.N. Bhat 125 Embryo Transfer Technologies in Developing Countries in Asia 11. Embryo Transfer Technology in Cattle: M. Anwar et al . 145 Experience in Pakistan 12. Embryo Transfer Technology in Cattle: S. Singhajan et al. 149 Experiences in Thailand 13. Embryo Transfer Technology in Pigs: P. Chantaraprateep 153 Experiences in Thailand 14. Superovulation and Embryo Transfer W. Sharifuddin et al . 158 in Goats: The Malaysian Experience

VACCINE PRODUCTION AND DISEASE DIAGNOSIS

15. Genetically Engineered Vaccines for K. Y amanouchi 163 Animal Disease - Present Status 16. Biotechnology in Animal Disease B.W~J. Mahy et al. 167 Diagnosis in Developing Countries: New Types of Diagnostic Methods, 17. DraftFAO/UNDPProject B.K. Soni 182

ANNEX D Country Reports Presented 193

1. China, People's Republic of Wang Ruixiang 195 2. India P.N. Bhat 197 3. Indonesia G. Gunawan 210 4. Korea, Republic of K. S. Im 218 5. Malaysia (presented by W. Sharifuddin) T.K. Mukherjee 221 6. Pakistan M. Anwar et al. 235 7. Thailand C. Satayapunt 237 1

A Regional Workshop on Biotechnology in Animal Production and Health in Asia was held from 17 to 21 October 1988 at the PAO Regional Office for Asia and the Pacific in Bangkok, Thailand. It was organized jointly by the F AO and the Kasetsart University, Thailand.

Mr. S.S. Puri, F AO Assistant Director General and Regional Representative for Asia and the Pacific, in his opening address, said that more research in this highly promising and relatively new field should be carried out by the developing nations of this Region. Since problems of developing countries are very different from those of developed nations, sharing of high technology is becoming progressively difficult. Biotechnology as applied to livestock development of this Region can only be undertaken in this Region. He cited the case of the water buffalo as being particularly pertinent. The ubiquitous water buffalo is widely used throughout the Region as a draught animal for farm labour and as a source of meat and milk.

It is, therefore, quite logical that scientists in the Region must take the lead in all aspects of biotechnology that relate to the required research improvement of livestock in the Region.

Dr. H.A. Jasiorowski, DirectoroftheFAO Animal Production and Health Division told the Workshop participants that the improvement of livestock nutrition was another area where much work needs to be done within the Region. Without better nutrition for its livestock, Dr. Jasiorowski said, the production of adequate supplies of high-quality protein for the Region's population, which makes up 56 percent of the entire human race will continue to lag behind.

He added that the advanced technology is available to do this work relatively quickly, at least in comparison to past decades, but scientists of the Region must work together in a network and take necessary leap forward and share the labour to reach an achievable target.

Dr. S utham Areekul, Rec tor of the Kase ts art University, while addressing the Workshop, outlined the advantages of biotechnological research in developing countries. He identified genetic engineering and embryo transfer as major areas of thrust for developing countries. Another area of the thrust he identified was animal vaccines and diagnostics. He said embryo transfer and associated technologies were key to buffalo development. He cautioned the Workshop that while genetic engineering looked exciting and promising from the point of view of benefits, it also could do a lot of damage to environment and therefore it was imperative that researchers kept this in mind. The safeguard which are neededin undertaking genetic engineering work with bacteria and viruses must be enforced. The experiments should be so designed that facilities where such experiments are being done should be safe enough that there is no release of genetically engineered organisms in the environment and no problem addeq to already difficult situation for the ecology of the Region. 2

He welcomed the delegates from the countries abroad and hoped that they would have a very enjoyable Workshop. He hoped they would come up with specific recommendations which could be taken up in the institutions of the Region for the benefit of the poorest of the poor. He ·declared the Workshop opened.

There were 19 invited experts from 10 countries. Their names and addresses, as well as those of the Organizing Committee, F AO Staff and Observers are given as Appendix 1 of this paper.

The primary purpose of the Workshop was to enable selected experts in biotechnology applied to animal production and health from 8 Asian countries to consult and discuss current issues and to formulate a workplan for a Regional Network linking national institutes in the participating countries which are involved in biotechnology research and development. Biotechnology applied to animal production and health includes the topics of Breeding and Genetics, Reproduction, Nutrition, Lactation, Growth, Disease Diagnosis and Vaccine Production.

Specifically, the Workshop :

a) Reviewed the national capabilities for biotechnology applied to animal production and health. b) Reviewed the foreseen areas of biotechnology which are having or are likely to have an impact on the future development of animal production and health in the developing countries of Asia. c) Identified the specific topics of animal production and health in the countries of Asia which merit biotechnology research and development in the immediate, medium-term and long-term. d) Identified the special strengths of individual institutes engaged in biotechnology research applied to animal production and health. e) Formulated recommendations for institutionalized linkages of research for the de­ velopment of biotechnology for improving animal production and health in the Region. f) Drew up a 3-year workplan for a network which incorporates Technical Cooperation between Developing Countries in the areas ofresearch.projects, training, communica­ tions, equipment and pilot projects for application in practice.

The Workshop discussed seventeen (17) technical papers on 4 major thrust areas of biotechnology :

(a) Genetic Engineering for vaccine and diagnostic agent production.

(b) Augmentation of feed resources through engineered micro-organism on different substrates.

(c) Augmentation offertility - genetic improvement oflivestock through Multiple Ovula­ tion Embryo Transfer (MOET).

(d) Genetic engineering for animal improvement and seven (7) country reports on status of biotechnology research in the Region. Recommendations were presented by individual experts, evaluating the need for further development of the biotechnology in livestock 3

production and health in Asian and Pacific Region. The Workshop adopted the following recommendations addressed to FAO Member Countries as well as to F AO : IL Recommendation A) General Recommendations

It is recommended ToFAO

1. That in order to create conditions for sustained growth of research, development and large scale application of biotechnologies in the Region; it is necessary to improve research cooperation, coordination, planning, identification of priorities, training and exchange of informa­ tion. The Workshop recommended that a F AO network of collaborating centres in animal production and health biotechnology should be set up;

2. That following institutes should form the F AO network of collaborating centres in animal production and health biotechnology :

India (i) Indian Veterinary Research Institute Izatnagar, UP 24 3122

(ii) National Dairy Research Institute Kamal, Haryana

Indonesia (i) Research Institute for Animal Production P.O. Box 123, Ciawi-Bogor

(ii) Research Institute for Veterinary Science JI. Cimanggu Bogor

Japan Laboratory Animal Research Centre Institute of Medical Science University of Tokyo 4-6-1 Shino-gane-dai Mirato-ku, Tokyo

Malaysia Faculty of Veterinary Medicine and Animal Science Universiti Pertanian Malaysia 43400 Serdang, Selangor

Pakistan Animal Sciences Institute Pakistan Agricultural Research Council Plot 20, G-5, Post Box 1031 Islamabad 4

Philippines National Institute of Biotechnology and Applied Microbiology University of Philippines at Los Banos Laguna 3720

Thailand Kasetsart University Bangkhen, Bangkok 10900

The People's Republic of China (i) Institute of Animal Science Chinese Academy of Agricultural Science Malianwa, Hai-Dian Beijing

(ii) Harbin Institute of Veterinary Science Chinese Academy of Agricultural Science Harbui

Republic of Korea (i) Department of Animal Science College of Agriculture Seoul National University Suwon 170

(ii) Veterinary Research Institute Office of Rural Development An yang

3. The network should be understood as an open one, and institutions from other countries of the Region can join it later.

4. That the purpose of the network is to facilitate the national governments in the Region to strengthen the cooperation, advise on objectives of regional significance to plan together for research and application and to advise on priorities for research activities, to coordinate training programmes, to exchange information and to coordinate other activities.

5. That FAO should initiate the cooperation immediately through the mechanism of contracts from its regular programme funds as seed money to allow the countries in the Region to start the network at the institutions identified by the Workshop and follow it up with assistance from Technical Cooperation Programme (TCP) and UNDP.

6. That F AO should positively respond to the requests of the member countries for short term assistance under the provisions of Technical Cooperation Programme (TCP) with the specific objective of bridging the gap until more extensive trust funds can be obtained.

7. That FAO should explore the possibilities for obtaining long term financial support for the network from UNDP or other donor agencies and bilateral sources. 5

8. That F AO should take the initiative in the organization of training courses especially in the subjects identified by the Workshop. The organization of such courses should be drawn primarily upon the existing skills and professional experience within the Region, supplemented by additional human resources from other regions wherever needed.

9. That F AO should continue producing livestock biotechnology bibliography.

10. That FAQ should organize regional meetings of scientists to exchange information on projects taken up under the network and to receive new information relevant to the network from invited scientists from outside the Region. The network will, itself through such meetings organized by F AO, take responsibility for the future development of programmes of research and application, especially when long term funding from trust sources is assured.

11. That FAQ should support national governments in their efforts to give their scientists more advanced training; and facilitate procurement of chemicals, reagents, antigen and antibodies etc.

12. That F AO should be willing to provide consul tan ts when appropriate to address specific issues needing expert advice.

13. That FAQ should publish the report and proceedings of this Workshop.

14. That FAQ should continue to maintain close contact with sister organizations in the UN system whose responsibility include the briefs on legal, patenting, environmental and human food hazards which may arise from the applications of biotechnology to animal production and health, with the aim of maintaining the highest levels of international cooperation in the use of biotechnology products for the benefits of all people and nations, while also respecting the legitimate rights of nationals to formulate their own standards.

To Member Countries' Governments

15. That Member Countries in the Region wishing to take part in the network should be willing to contribute national resources to the network in the form of physical facilities, staff and, where appropriate, funding them in the spirit of the Technical Cooperation among Developing Countries (TCDC) and according to the normal procedures in trust funds such as UNDP projects;

16. That Member Countries' Governments should take advantage of the expertise and services supplied by the UN Organization, UNIDO, in its brief on biotechnology, with special reference to the Newsletter "Biotechnology Monitor" which is published regularly by UNIDO and covers technical, legal, economic, social and organizational aspects of biotechnology at national and international levels. B) Biotechnology in Animal Production

It is recommended that the following topics should be given priority for immediate ac- tion: 6

- Embryo transfer and associated technologies. - Open nucleus breeding systems with MOET. -Improvement in utilization of agro-industrial by-products in animal feeding and ruminant nutrition through biotechnology. - Production related hormones. - Immunobiology of reproduction. - Genetic engineering applied to farm animals (RFLP, genome analysis).

It is recommended that in each topic there should be a stage of immediate application and the development of research applied to regional problems and training.

1. Embryo Transfer (ET} and Manipulation

a) Background: ET is seen as a necessary adjunct for the genetic improvement of livestock in developing countries. It will supplement existing breeding practices and also offers the possibility of applying new genetic improvement methods e.g. open nucleus breeding systems for the evaluation of superior females and the production of elite males. The technique can be used both for the improvement of indigenous breeds and also for cross breeding with exotic germplasm.

b) Immediate application : Adaption of ET including Cryopreservation and Embryo Splitting for use in designated nucleus herds and flocks.

c) Research priorities : a) adaption of ET to cattle, buffaloes, sheep, goats and pigs in nucleus breeding systems. The nucleus herds of buffaloes are to provide i) elite males. ii) In vitro techniques for oocyte maturation and fertilization. b) E.T.. in buffaloes : In the case of buffaloes, studies should be taken up on the normal hormonal profile of both riverine and swamp buffaloes under varying field conditions. The structure of buffalo ovary should be investigated in detail, in terms of primary and secondary oocytes in various breeds. It is necessary that new regime for superovulation be introduced utilizing either FSH/LH or PMSG. Efforts should be made to investigate in vitro fertilization and develop this technique as an alternative source of buffalo embryos.

d) Training : Training courses in ET and its application to breeding programmes are a priority to provide basic training to scientists already experienced in female reproduction and also to offer updated courses where new technologies can be introduced. An immediate need is for a training course to be held in the next year for 8 trainess for 3 weeks. Additional courses should be arranged when appropriate. The already existing training facilities in the Region should be fully utilised.

2. Open Nucleus Breeding Systems (ONBS}

a) Background : The background to animal improvement is currently the use of superior males whose genes are spread through the population either by AI or natural service. A basic need is recording system which will provide accurate data for sire evaluation. In this Region, it has proved difficult to implement this standard system oflivestock improvement, ET is a newly available tool for avoiding the need for extensive field recording. In the open nucleus herd system, superior females 7

are brought to a controlled environment in a centrally managed location and are used to produce many superior offsprings. Thus ET and ONBS are linked together for genetic improvement of livestock.

b) Immediate application: Cattle, buffalo, sheep, goat and pig elite herds to be established by the identification and acquisition of outstanding females. The development of appropriate recording and documentation systems in the nucleus herds and flocks. The introduction of ET techniques suited to the herd or flock. The acquisition of superior males and or semen.

c) Research priorities: To identify superior males and females.

d) Training: A training course to be organized for 8 participants (last for 2-3 weeks) and to equip the participants in the genetic programme and practical management of ONBS.

3. Animal Feeding and Ruminant Nutrition

a) Background : The Region has many forage resources agro-industrial, by-products (paddy, wheat straw and palm processed fibers) suitable for ruminant production which are poorly utilized at the present time. Opportunities are being developed in other parts of the world for biotechnology to contribute to break down of fibrous materials (lignin) and increase digestibility of these by-products. The microorganisms can be used prior to ingestion or in the rumen. It calls for a concerted effort to research the areas of rumen ecology, physiology and genetics of micro­ organisms. Research and training in these basic subjects is needed for the effective application of these new possibilities in the Region.

b) Immediate application: Improved utilization of agro-industrial by-products.

c) Research priorities : To identify the most important areas for application of these emerging techniques, besides bio-degredation of straws. Establish a type culture collection centre for such organisms.

d) Training: Courses at several levels are necessary in view of the current lack of specialist research units on this topic in the Region. These are:

- Shod terms courses organized within the Region with specialists brought in from other regions.

-Longer term, high level, graduate degree and post-doctoral courses in developed countries where the technology is advanced.

- Courses for biotechnology laboratory support staff.

4. Production Related Hormones

a) Background : Biotechnology offers increasing opportunity to manipulate the life processes of growth and lactation by the provision of naturally occurring hormones in larger doses. 8

These hormones may be produced endogenously through the production of transgenic animals or used externally through periodic dosing of the animal during the periods of its growth or lactation.

The Region has a very large meat/milk production population of livestock where this therapy could be useful in enhancing meat and milk. In buffaloes, an increase by 20-30% in milk yield has been reported. It may, however, be desirable to make necessary adjustment to management of these animals.

Since an immediate increase in milk production is desired, growth hormone level modification/manipulation in the following ways should be considered.

1. Exogenous growth hormone (GH) administration.

Options:

a) Purchase of GI-I production technology or GI-I itself from outside the Region b) Regional GI-I production c) Examination of timed release technologies to ease repeated injections of GI-I

2. Alternative exogenous methods of GI-I level manipulation.

a) Synthesis and administration of GI-I releasing factor (GHRF) b) Anti-somatostatin immunization c) Somatomedin manipulation (lGF-1)

3. Creation of transgenic animals with increased GI-I productiori.

b) Possible Application: Identification of other products for which legal patent may have expired and the ones that may be available at reasonable prices which could be produced in the Region and even exported.

c) Research priority : Cloning/synthesis/expression/utilization of somatotrophins or its variants as well as its analogues could be developed using RDNA technique for commercial products.

d) Training: A training course for specialists in molecular biology of growth hormone and its cloning and expression in a suitable vector needs to be undertaken. The training course will need inputs from outside the Region.

5. Immunobiology of Reproduction

a) Background: New techniques are being creatively researched at the pure research level which appear likely to have an impact upon reproductive performance in the future by the use of immune technology. Such techniques will make possible the manipulation of reproduction in 9

mammals using systems within the animal to control the quantitative and time sequence occurrence of reproduction.

b) Immediate application: Techniques being developed with small laboratory mammals to be applied to livestock.

c) Research priorities : To identify one most promising area for transfer from the laboratory to livestock.

d) Training : Specialist level short courses.

6. Genetic Engineering Applied to Farm Animals

a) Background : There are several techniques, including RFLP which offer new methods of genome analysis and mapping. These are suitable in the longer term for use with domestic animal species to supplement conventional breeding and selection methods for economically important traits.

b) Immediate application: Although the techniques do not appear to offer any immediate application in developing countries, the research is being actively pursued in association with mapping the mammalian genome. It is likely that as genome libraries are built-up, application will become available.

c) Research priorities : NONE

d) Training : Advanced short courses from trained animal breeders and geneticists in the new techniques.

C) Biotechnology in Animal Health 1. Diagnosis Method.

The prospects for the application of biotechnology for animal health fall into two main areas:

- Disease diagnosis - Production of safe, inexpensive and effective vaccine

It is recommended that these areas should be given priority in the network for immediate action.

Disease Diagnosis

a) Background : There are immediate prospects for improvement in accuracy and rapidity of assays by the introduction of ELISA tests utilizing monoclonal antibodies. Restriction endonuclear mapping, nucleic acid probes and sequence analysis should also be developed as quick 10 aids to diagnosis of diseases. It is, however, desirable to develop simple tests which are suitable for field use. For this purpose, interchange of specialized reagents such as monoclonal antibodies and DNA clones and probes will be required.

b) Research priorities : The development of simple diagnostic tests based on monoclonal antibodies or DNA probes for use under field conditions or in laboratories without specialized equipment.

c) Improved disease diagnosis will require training and transfer of the following technolo- gies:

a) Development of ELISA techniques b) Monoclonal antibody production c) Gene characterization and cloning d) Nucleic acid probe hybridization e) Nucleic acid sequence analysis f) Non Isotopic DNA detection

d) Training :

- Proposed training activities : A training course on Production and Characterization of Monoclonal Antibodies should be held in the Region.

- A training course genome analysis should be held in the Region.

2. Vaccine Production

a) Background

Within the fie1d of vaccines, there are new developments through the use of live recombinant vectors expressing genes for immunogenic proteins (Rinderpest, Blue tongue, FMD), synthetic peptides, anti-idiotypes, subunit immunogens. It will be necessary to improve biotech­ nologies for large scale cultivation of bacterial and eucaryotic cells expressing these antigens. The development of vaccines will need in the longer term, a better understanding of immune response system of domestic livestock.

b) Research priorities

Production and characterization of monoclonal antibodies against antigens of relevant pathogens for use in diagnosis and antigen purification.

Characterization, cloning and sequence analysis of genes from relevant pathogens.

Development of expression systems from viruses, bacteria parasites or eucaryotic cells for these gene clones. 11

c) Training

Improved vaccines will require training and transfer of the following technologies :

- large scale cultivation of bacterial and eucaryotic cells infermentors and on micro­ carners.

- molecular characterization and cloning of genes encoding protective immunogens.

- Application of expression systems for these genes. D. FAO/UNDP Regional Project

Draft FAO/UNDP Project entitled "Biotechnology Development Network for Animal Production and Health in Asia" :

Dr. B.K. Soni presented the draft Project to the Workshop, and explained its elements.

The Workshop identified various areas of strength in the institutions of the Region for the purpose of cooperation under this project to be supported by UNDP. These are listed below :

Malaysia

i) Embryo culture and transfer. ii) Manipulation and control of rumen fermentation.

Pakistan

i) Embryo culture and transfer.

Philippines

i) Cloning of specific genes for vaccine production. ii) in-vitro treatment of feeds by manipulated microorganisms.

China

i) Embryo culture and transfer. ii) Production of monoclonal antibodies. iii) Gene mapping and recombinant DNA techniques. iv) Cloning of specific genes for production related hormones.

India

i) Embryo culture and transfer. ii) Production of monoclonal antibodies. iii) Gene mapping and recombinant DNA techniques. 12

iv) Cloning of specific genes for vaccine production. v) Cloning of specific genes for production related hormones.

Indonesia

i) Manipulation and control of rumen fermentation. ii) in-vitro treatment of feeds by manii;)ulated microorganisms.

Korea

i) Gene mapping and recombinant DNA techniques. ii) Cloning of specific genes for production related hormones.

Thailand

i) Embryo culture and transfer. ii) Production of monoclonal antibodies. iii) In-vitro treatment of feeds by manipulated microorganisms. iv) Manipulating and control of rumen fermentation.

The Workshop congratulated the F AO for having prepared the strategies of the network to be implemented in three phases (i) contract assignments with seed money to begin the cooperation, (ii) TCP assistance and (iii) Funding from a donor agency (UNDP) for a Regional Project after the termination of the network which will be a permanent cooperative network under the overall coor­ dination of F AO. The Workshop recommends that FAQ look for donors so that second and the third phases which are critical to the task are undertaken in the time frame approved by this Workshop. Annexes

Annex-A

List of Participants

The People's Republic of China Dr. R. Dharsana Veterinary Research Officer Dr. Liu Young Hui Research Institute for Animal Production (BPT) Director P.O. Box 123, Ciawi-Bogor Chinese National Centre for Biotechnology Development The Republic of Korea 54 .Sanlihe Road, Beijing Prof. Kyung Soon Im Dr. Wang Ruixiang Professor and Head Head, Dept. of Animal Reproduction Dept. of Animal Science Institute of Animal Science Agriculture Faculty Chinese Academy of Agricultural Sciences National University Malianwa, Hai-Dian Suwon 170 Beijing Malaysia India Prof. T .K. Mukherjee Dr. P.N. Bhat Institute of Advance Studies Director University of Malaya Indian Veterinary Research Institute Kuala Lumpur 59100 Izatnagar, 243122 (UP) Dr. W. Sharifuddin Dr. C. Natarajan Lecturer, Veterinary Faculty Director Universiti Pertanian Malaysia Department of Biotechnology 43400 Serdang, Selangor Ministry of Science and Technology Block 2 (7-8th Floor), CGO Complex Dr. Ho Ying Wan LodhiRoad Department of Biology New Delhi-110003 Universiti Pertanian Malaysia 43400 Serdang, Selangor Indonesia Pakistan Dr. B. Gunawan Director Dr.M.Anwar Research Institute for Animal Production (BPT) Director (Animal Production) P.O. Box 123, Ciawi-Bogor Pakistan Agricultural Research Council Plot 20, G-5, Post Box 1031 Islamabad 16·

Dr. M. Ashfaque Japan Associate Professor and Head Dept. of Veterinary Microbiology Prof. Kazuya Y amanouchi Faculty of Veterinary Science Laboratory Animal Research Centre Agriculture University Institute of Medical Science Faisalabad University of Tokyo 4-6-1 Shiro-gane-dai Philippines Mirato-ku, Tokyo

Dr. Ida F. Dalmacio U.S.A. Associate Professor Institute of Biological Sciences Dr. Kevin S. Guise University of Philippines at Los Banos Department of Animal Science Laguna3720 and Institute of Human Genetics University of Minnesota Thailand 1988 Fitch Avenue St. Paul, Minnesota 55108 Dr. Chamnean Satayapunt Department of Animal Science FAO Faculty of Agriculture Kasetsart University Mr. S.S. Puri Bangkhen, Bangkok Assistant Director General Thailand 10900 and Regional Representative for Asia and the Pacific Dr. Vanda Sujarit F AO Regional Office Department of Anatomy Phra Atit Road Faculty of Veterinary Medicine Bangkok Kasetsart University Bangkhen, Bangkok 10900 Dr. H.A. Jasiorowski Director Dr. Peerasak Chantaraprateep Animal Production and Health Division Department of Obstetrics, Gynaecology FAQ, Rome 00100 and Reproduction Faculty of Veterinary Science Dr. John Hodges Chulalongkorn University Senior Officer Henri Dunant Road Animal Breeding and Genetic Resources Bangkok 10500 Animal Production and Health Division FAQ, Rome 00100 Dr. Samphun Singhajan Ratchaburi A.I. Station Dr. R.B. Singh Nang Po Village Regional Plant Production and Protection Offi­ Amphur Potharam cer Ratchaburi 70120 F AO Regional Office for Asia and the Pacific Phra Atit Road Bangkok 17

Dr. M. Sasaki Dr. Suvichai Rojanasthien Regional Animal Health Officer Department of Veterinary Obstetrics, Gynae­ FA0 Regional Office for Asia and the Pacific cology and Animal Reproduction Phra Atit Road Faculty of Veterinary Medicine Bangkok KasetsartUniversity

Mr. M. Uotila Dr. Prachak Poomvises Regional Dairy Development Officer Fac,ulty of Veterinary Science F AO Regional Office for Asia and the Pacific Chulalongkorn University Phra Atit Road Bangkok Dr. Chainarong Lohachit Department of Obstetrics Gynaecology Observers and Reproduction Faculty of Veterinary Science Chulalongkorn University Thailand Dr. A. Kunavongkrit Dr. Supat Attatham Faculty of Veterinary Science Plant Genetic Engineering Unit Chulalongkorn University National Center for Genetic Engineering and Biotechnology Dr. Prachin Virakul Kasetsart University (Kamphaeng Saen) Faculty of Veterinary Science Chulalongkorn University Dr. Tipwadee Attatham National Center for Genetic Engineering and Dr. Mongkol Techakumphu Biotechnology Faculty of Veterinary Science Kasetsart University Chulalongkorn University

Mr. Pokkwan Hutangura Dr. Ratana Chotesangasa National Center for Genetic Engineering and Department of Animal Science Biotechnology Faculty of Agriculture Kasetsart University Kasetsart University

Miss Chutima Monchawin Dr. Dumrong Leenanuruksa National Center for Genetic Engineering and Department of Animal Science Biotechnology Faculty of Agriculture Kasetsart University Kasetsart University

Miss Orady Tanpatta-anun Dr. Pornsri Chairatanayuth National Center for Genetic Engineering and Department of Animal Science Biotechnology Faculty of Agriculture Kasetsart University Kasetsart University

Dr. Samutra Sirivejpandu Dr. Voravit Siripolvat Department of Veterinary Obstetrics, Gynae­ Department of Animal Science cology and Animal Reproduction Faculty of Agriculture Faculty of Veterinary Medicine Kasetsart University Kasetsart University 18

Dr. Kanchana Markvichitr Mr. Sompom Duanyai Department of Animal Science Sisaket Agricultural College Faculty of Agriculture Kasetsart University Dr. Suneejit Kongthon Department of Livestock Development Dr. Bandhit Thanindratarn Department of Animal Science Dr. Wilai Linchongsubongkoch Faculty of Agriculture Foot and Mouth Disease Vaccine Production Kasetsart University Centre,DLD Pakchong, Nakom Ratchasima 30130 Mrs. Lavanaya Kraidej Department of Microbiology Dr. Prasit Tanomkun Faculty of Science Artificial Insemination Centre Kasetsart University Dr. Sala Kongsmak Dr. Chatt Chamchong Department of Livestock Development Department of Agricultural Economics Kasetsart University Dr. Prasert Songsasen Artificial Insemination Division Dr. Orapin Bhumiphamon Department of Livestock Development Department of Biological Technology Faculty of Agro-industry Dr. Parishat Sukhato Kasetsart University Artificial Insemination Division Department of Livestock Development Dr. Malinee Limpoka Department of Pharmacology Dr. Somthep Tumvasom Faculty of Veterinary Medicine Faculty of Agriculture Kasetsart University KasetsartUniversity

Dr. Yanyong Intraraksa Dr. Malee Suwana-Adth Department of Physiology Rector's Office Faculty of Veterinary Medicine Kasetsart University Kasetsart University Mrs. Renu Vejaratpimol Dr. Sakol Panyim Faculty of Science Department of Biochemistry Silpakorn University Faculty of Science Mahidol University Dr. Sirichai Sripongpun Department of Animal Sciences· Dr. KanokPavasuthipaisit Faculty of Natural Resource Faculty of Science Prince of Songkla University Mahidol University Dr. Tip-Aksorn Sinchaisri Dr. S ukumal Chongthammakon Depmtment of Pathology Department of Anatomy Faculty of Medicine Faculty of Science · Chiangmai University Mahidol University 19

Dr. Supote Methiyapun Organizing Committee National Animal Health and Production Institute DLD Dr. Sutharm Areekul Rector Dr. Yongyuth Yuthavong Kasetsart University Director National Center for Genetic Engineering Professor Charan Chantalakhana and Biotechnology Vice Rector Ministry of Science and Energy Kasetsart University Rama 6 Road, Bangkok 10400 Dr. Balbir K. Soni Dr. Visuth Sukpatarapirome Regional Animal Production and Health officer Adsgo Co., Ltd. FAO Regional Office for Asia and the Pacific P.O. Box 129 Omnuch, Bangkok 10250 Dr. Pibul Chai-anan Faculty of Veterinary Medicine Mr~ Udom Vangtal KasetsartUniversity Dairy Farm 257/1 Songpol Road Dr. Thira Sutabutra Ban-Pong, Ratchaburi Director Kasetsart University Research and Mr. Adul Vangtal Development Institute Dairy Farm Ban-Pong, Ratchaburi M.L. Uemsook Kitiyakara Kasetsart University

Dr. Somtawil Dhanasobhon Assistant Director Kasetsart University Research and Development Institute

Annex-B Programme

Monday, 17October1988

08:30 Registration

09:00-09:45 Inauguration

Introduction to Workshop by Dr. H.A. Jasiorowski, Director of F AO Animal Production and Health Division

Election of Chairman

Address by Mr. S.S. Puri F AO Assistant Director General and Regional Representative for Asia and the Pacific

Address by Dr. Sutharm Areekul, Rector of Kasetsart University Vote of Thanks

09:45-10:00 Tea

10:00-11:00 Review of Development of Biotechnology in Animal Production and Health in Asia

11:00-12:00 Application of Recombinant DNA Techniques in Animal Improvement

12:00-12:30 Potential of Multiple Ovulation and Embryo Transfer Technology in Developing Countries of Asia

14:00-15:00 Embryo Transfer Technology in Goats: Experiences in Malaysia

15:00-15:30 Embryo Transfer Technology in Cattle: Experiences in Pakistan

15:30-15:45 Tea

15:45-16:30 Embryo Transfer Technology in Cattle and Pigs: Experiences in Thailand 22 16:30-17:00 Discussion and Summary on Scientific Review Papers

Tuesday, 18October1988

08:30-09: 15 Present Status of the Use of Genetically Engineered Growth Hormones in Pigs

09: 15-10:00 Present Status of the Use of Genetically Engineered Growth Hormones in Chicken, Cattle and Buffaloes

10:00-10:30 Discussion and Summary on Scientific Review Papers

10:30-11:00 Tea

11:00-11:45 Country Reports The People's Republic of China India

11 :45-12:30 Country Reports Indonesia The Republic of Korea

14:00-15:00 Application of Biotechnology in Open Nucleus Breeding System in Developing Countries

15:00-15:15 Tea

15: 15-15:45 Application of Biotechnology in Improvement of Fibrous Crop Residues

15:45-16: 15 Biotechnology and Improvement of Livestock Feeds

16: 15-17:00 Discussion and Summary on Scientific Review Papers

Wednesday, 19October1988

08:30-09:30 Genetically Engineered Vaccines for Animal Diseases -Present Status

09:30-10:30 New Diagnostic Methods based on Monoclonal Antibodies for Animal Diseases

10:30-10:45 Tea

10:45-11:45 Biotechnology for Production of Vaccines for Specific Animal Diseases

11:45-12:30 Discussion and Summary on Scientific Review Papers

14:00-15: 15 Country Reports Malaysia Pakistan 23 15:15-15:30 Tea

15:30-17:00 Country Reports Philippines Thailand

Thursday, 20 October 1988

08:30-09:30 Introduction to draft F AO/UNDP Project entitled "Biotechnology Development Network for Animal Production and Health in Asia"

09:30-12:30 Formulation of 3-year work plan in order to strengthen reseach in specialized fields of Biotechnology

14:00-17:00 Participants will be requested to join any one of the three working groups, A, B,orC

A 1. Multiple Ovulation and Embryo Transfer Technology ii. Gene mapping and recombinant DNA techniques

B iii. Manipulation and control of rumen fermentation for improvement of fibrous crop residues iv. In-vitro treatrnent of feeds by manipulated microorganisms c v. Production of monoclonal antibodies vi. Cloning of specific genes for vaccine production vii. Cloning of specific genes for production-related hormones

Friday, 21October1988

08:30-12:30 Preparation of the report

Those who are not participating in the preparation will visit research. institutes working on Biotechnology in Thailand

15:00 Final approval of the report and recommendations

Annex C

Technical Papers Presented

by

H.A. Jasiorowski Director of Animal Production & Health Division, Agriculture Department FAO, Rome, Italy

The primary purpose of this Workshop, Region to assess on-going work and the scope organized by FAO jointly with the University of for introduction, application and further de­ Kasetsart, is to enable selected experts in bio­ velopment of methods of biotechnology in the technology applied to animal production and countries of the Region in collaboration with health from 8 Asian countries to consult and those local institutes and scientists involved. In discuss current issues and to formulate a workplan the Latin America and Ca.ribbean region, a for an F AO Regional Network linking national parallel consultancy was carried out with similar institutes in the participating countries which are terms of reference and scope. involved in biotechnology research and development. The FAO initiative on biotechnology in animal production and health was highlighted FAO policy is to be involved in bio­ through the publication in 1986 ofa special issue technology development especially where it helps on biotechnology of the World Animal Review developing countries to make use of new tech­ which outlined the key concepts of the new nologies and to develop research in this field. technologies.

The Animal Production and Health To assist livestock scientists in de­ Division at F AO Headquarters started a series of veloping countries, a quarterly bibliography on activities in biotechnology for livestock in 1984, animal biotechnology was started in 1987 as a in which it analysed the situation, needs and service to the institutes concerned since up-to­ conditions prevailing and then defined its stra­ date information is often lacking in these tegy in this field. An informal technical meeting countries. held in October 1985 established the broad guide­ lines and provided a framework for the formal As a result of these earlier activities, Expert Consultation on Biotechnology in Ani­ FAO is now ready to assist in establishing a mal Production and Health in Rome in October network of biotechnology centres of excellence 1986. Taking into account therather diverse con­ in the Asia the Pacific Region. The objectives ditions in various developing countries, it was will be to prepare and distribute essential bio­ decided first to concentrate biotechnology ac­ technology reagents, promote exchange of in­ tivities mainly in Latin America and the Carib­ formation and ideas, arrange regional training bean and in the Asia and the Pacific regions. programmes and avoid either duplication or neglect of essential areas of opportunity. A In 1987, an FAO consultant visited comparable network has already been estab­ selected institutes in the Asia and the Pacific lished by FAO in Latin America and the Carib- 28

bean. We are seeking regional UNDP support for Although several embryo sexing tech­ these networks. niques are being intensively researched, to-date none of them is cmTently suitable for field appli­ Let me now describe briefly what we cation. When a practical method becomes avail­ understand by biotechnology in livestock de­ able, it will certainly ensure a more widespread velopment and what we expect it to contribute to use for embryo transfer of value at the commer­ developing countries. cial level. Research continues on sexing of sper­ matozoa but it has so far been without success I will start with reproduction. despite many years of effort. If it is eventually successful, then, combined with ET, it will Embryo transfer: Embryo transferin cattle has contribute effectively to yet further progress. been in existence since the early 1970's, and its application for elite breeding programmes is Thus where a developing country which established. For example, 50 percent of the dairy has· mastered the techniques of obtaining em­ bulls in Canadian A.I. Centres result from em­ bryos, splitting may well be an optional extra. bryo transfer. Nevertheless, the use of the tech­ However, as the sexing of embryos is not avail­ nique in developed countries has not, so far, able, it is too premature to include the sexing of increased substantially the rate of genetic pro­ embryos in breeding plans, but there are no gress. It is predicted that the expected additional reasons why research on these subjects should genetic progress in countries with efficient dairy not be undertaken in developing countries as progeny testing programmes will only be around well. 10 percent. However, the theoretical models of Nicholas and Smith, 1983, which proposed that Maturation of oocytes and in vitro fertiliza­ multiple ovulation embryo transfer (MOET) tion: Major steps forward may also be obtained should be linked to a new selection programme through mastering the maturing and fertilizing based on a nucleus herd breeding system and the of mammalian ova in in vitro conditions. The evaluation of bulls using the productivity of their latest reports from Ireland suggest that this can siblings, show that new perspectives are pos­ be achieved. Professor Ian Gordon of University sible. F AO considers this new approach for College, Dublin reports the production of zy­ genetic selection to be a promising method for gotes using oocytes from the ovaries of cows the future of animal breeding in developing obtained from a slaughterhouse. The embryos countries. This is because the lack of proper obtained after in vitro maturation and fertiliza­ infrastructure has often made it impossible to tion have been transferred in recipient cows or introduce the mass field performance recording deep frozen. This in vitro method could be used necessary in selection schemes currently used in to provide a cheap supply of embryos for de­ developed countries. veloping countries.

Microsurgery and sexing embryos: The tech­ Cloning embryos : The cloning of embryos nique of embryo splitting (into two), which is could well bring about a revolution in animal re­ well established in developed countries, increases production. Whilst it is true that the first reports the 60 calves born from 100 transplanted em­ of embryo cloning in mice at the beginning of the bryos to lOOcalvesbornfrom 100 split embryos. 1980's (Karl Ilmensee and independently Peter The identical twins produced can usefully be Hope) were not confirmed in later experiments, used in research programmes where they also it is the latest work of Steen Willadsen which increase the volume and precision of data seems to offer the most promise in sheep and obtained. cattle embryo cloning. The technical details have not been published. However, from general 29

reports in the scientific press it appears he has capable of producing milk containing human cloned embryos through linking embryo cells albumin. This type of milk can have wide and oocytes with their nuclei removed. It is said application, inter alia, for aiding the recovery of that cattle embryos can be used for cloning to the human patients following serious operations. 32 blastomere stage. Thus, theoretically, one embryo could give 32 identical individuals. It The most important breakthrough for was reported in Science (29 January 1988) that genetic engineering with mammals in the near Willadsen was able to clone cells of previously future will be in the development of successful frozen embryos as well as obtain calves from the methods for a more precise transfer of DNA. So second generation of embryos (where the em­ far, in pigs, sheep and goats, numbers of em­ bryo used for cloning itself was obtained by bryos surviving gene injection procedures are cloning). These are fascinating achievements. below 10 percent, and numbers of transgenic Intensive research continues and there is no offspring compared to number of embryos trans­ reason why it should be confined only to a few ferred are less than 1 percent. highly developed countries. A second breakthrough would be to Gene transfer: This is the new and exciting achieve the transfer of whole gene sequences technique to produce transgenic animals which which are responsible for quantitative character­ may have great potential for improving animal istics such as milk yield. There is still a long way breeding. to go to achieve that aim. For example, in the human genome which contains 50,000 to 100, The first successful transfer of genes 000 genes, only 300 have been studied suffi­ between different animal species was the now ciently to allow them to be the subject of genetic famous transfer of a rat gene regulating growth engineering. Our know ledge of the animal genes hormone excretion to mouse embryos wich re­ is even much smaller. sulted in a giant mouse (1982, Dr. Richard Palmiter and Dr. Ralph Brinster). Since then Animal nutrition : Nutrition is the main limit­ work has progressed very much. From 1982 ing factor in animal production especially in de­ onwards, 500 various genes as well as promoters veloping countries and therefore much hope is have been used and tried in a number of experi­ attached to the application of biotechnology. On ments. In 1986, the USDA Research Station in one hand, this focuses on improving the value of Beltsville (USA) produced pigs integrating and the fodder provided and, on the other, increasing expressing the gene for the human growth thefeed utilization by the animal. Biotechnology hormone. However, nearly all the transgenic in this field has found new horizons. Some of the animals produced with growth hormone to date examples are as follows: have had serious abnormalities in function or morphology. As is known, cereals, chiefly barley and maize, play a major role in animal nutrition In 1987, Dr. John Clark (AFRC) in the (mainly monogastrics) especially in intensive UK obtained sheep with human genes corre­ production systems. In developed countries, more sponding to "Factor 9" (substance producing than 90 percent of grain produced is used for blood clotting). These sheep produce "Factor 9" animal feed. However, cereals are low in essen­ in their milk which is then extracted for treating tial amino acids, including lysine (as well as haemophiliacs. threonine and tryptophan). As a result, there have been commercial efforts in industrial syn­ Rover, a US pharmaceutical company, thesis of these amino acids, for supplementing is close to the production of transgenic cows cereal rations. Now, however, efforts are being 30

made to transfer genes responsible for cereal established. proteins rich in lysine. New cereal species with increased 1ysine levels are appearing, but there is Apart from in vitro diagnosis MABs still much to be mastered before these achieve­ have an even wider application in in vivo diagno­ ments can be used widely in practice. sis as well as in therapeutics (treatment). Through the linking of antibodies with specific therapeu­ Another example of new possibilities is tic substances (toxins), it will be possible to link the modification of the microbiological ecosys­ diagnosis with therapeutics. Studies in this di­ tem in the rumen of animals. From time imme­ rection are extremely active, especially in the morial, man has tried by various means to influ­ multinational pharmaceutical companies, due to ence the fermentation process in the rumen. The the huge market expected. results achieved are extremely modest in regard to the effort invested. There is currently hope Application of biotechnological meth­ that genetic engineering can be applied to rumen ods in vaccine production represents another microflora. It seems possible to construct rumen important field of new technology in animal bacteria with greater amylase and xy lase activity health. Genetically engineered vaccines prom­ to breakdown starch and hemicellulose. Regula­ ise a higher grade of innocuity, and efficiency as tion through genetic engineering, of such bacte­ well as a lower cost of production. Promising rial functions as breakdown and synthesis of results of veterinary research in the preparation protein of feed, increased hemicellulose break­ of subunit vaccines for veterinary use, vaccines down and above all the possibility of the break­ based on peptide chemistry and live attenuated down of plant lignin not so far done by any vaccines have been achieved.DNA recombinant animal, is very complex. The genetic engineer­ techniques, expressed antigens, synthetic pep­ ing of microflora opens up enormous possibili­ tides, monoclonal antibodies and some other ties for the regulation of fermentation processes biotechnology procedures and products are in the rumen. Nevertheless, we are far from opening up a new future for the animal health exploiting the know ledge we have. Little is known immunization programme. These developments so far of the complicated enzymatic relation­ are only beginning and their importance for ships in the rumen which are influenced by developing countries cannot be overestimated. hundreds ofbac terial species. Even less is known We have encouraging examples from this Re­ of the genetics of these bacteria. Work in this gion that this type of research can be carried on field is of extreme importance for developing in developing countries as well. countries. Ladies and Gentlemen, these are only Veterinary applications: Biotechnology has examples of what biotechnology is bringing to brought veterinary practice new diagnostic us. methods for disease, and new treatment as well as vaccines. However, the application ofnew meth­ ods and the use of new products based on bio­ The basis of the new diagnostic meth­ technology in developing countries will have to ods lies in the discovery of ways to produce take fully into account, possible environmental monoclonal antibodies (MABs) on a large-scale implications and may have to operate within the and relatively cheaply using tissue culture. What framework oflegal constraints including patents is important is that the tests are usually employed and copyrights. These last aspects may well outside the body (in vitro) using small samples prove to be restricting, since more and more of urine, milk or blood for the analysis. In the biotechnology research is concentrated in large USA, over 100 tests based on MABs have been private companies. 31

It is to be hoped that the results of our We are convinced that this consultation meeting will lead to recommendations for the will contribute to further development of bio­ future development of biotechnology in live­ technology in the Region enabling improvement stock production and health in the countries of of animal production and health -one of the basic the Region. We should suggest methods for preconditions for improved living standards in immediate application, identify biotechnology Asia and the Pacific Region. I wish to thank you research priorities and regional networks of for your attention and I wish you a successful collaborating centres as well as training needs. discussion and pleasant stay in Thailand. The meeting should also suggestFAO's involve­ ment in this development within the limits of our programme and budget. Application of Recombinant DNA Techniques in Animal Iinprovement

Kevin S. Guise Department of Animal Science and Institute of Human Genetics University of Minnesota St. Paul, Minnesota 55108 U.S.A.

Introduction aspect of molecular genetics that has excited much speculation is the ability to transfer genes between and within species--adding novel genes Recombinant DNA technology is just to a species or simply artificially duplicating entering its second decade of existance. The term already existing genes and thus increasing syn­ "genetic engineering" has begun to move from thesis of a specific gene product. While not the the realm of science fiction to that of science fact. first instance of gene transfer in animals, the The concept of improvement of economic traits work by Palmiter et al. (1982a) which resulted in of domestic animals through genetic engineer­ a mouse twice normal size certainly was the ing orrecombinantDNA technology has excited stimulus for a worldwide effort in gene transfer. research groups around the world. The potential While still largely focused on growth hormone, for dramatic enhancement of production charac­ this transfer effort is beginning to spread out with teristics not only exists bu tis currently starting to an eve to introduction of other genes. DNA be realized. Three areas of recombinant DNA marker.assisted selection and gene mapping are technology will be outlined here. This technol­ attendant but still largely unexplored method­ ogy is being utilized for (1) gene mapping and ologies in domestic animals. DNA-level marker identification and selection, (2) gene cloning, and (3) gene transfer. Here we will explore three aspects of molecular genetics--" genetic engineering" --and As will be detailed below, the major seek to define what the fan shaped future of these impact of molecular genetics has been two-fold methodologies hold for animal production. to date--gene cloning and gene transfer. Through the cloning of specific genes, it is now possible Gene Mapping and DNA Polym to raise in bacteria, much as antibiotics are pro­ duced from molds, kilogram quantities of spe­ 1norphisn1s cific gene products, such as the peptide hormones, of which growth hormone is one, which previ­ Recombinant DNA technology has ously required many years of effort per gram. allowed rapid improvement of eukaryotic gene Sufficient quantities of pure gene products were maps and the development of DNA-level mark­ now potentially available for definitive testing to ers for use in selective breeding, gene identifica­ determine their regulatory effects. The second tion, and gene cloning. The goal is the detection 33 oflinkage of DNA marker alleles with segregat­ isms. A single HVR probe such as that isolated ing quantitative trait alleles. The development of by Jeffreys et al. (1985) or a region in the single recombinant DNA techniques has provided stranded phage M13 (Vassart et al., 1987) will methods for resolving polymorphisms at the detect a large number of alleles. The autoradi­ DNA level, generating numerous markers well­ ograph of a HVR probed sample shows multiple distributed throughout the genome. bands which in essenceconstitutea "fingerprint" of the DNA for that organism. The pattern is of DNA level markers sufficient complexity, due to size differences of the DNA restriction endonuclease fragments ThreemajorformsofDNA-levelmark­ upon which the repetitive DNA resides, that it ers are currently in use--restriction fragment may be used in forensic studies to positively length polymorphisms, hypervariable region identify individuals. It has been used in paternity probes, and oligonucleotide probes. testing in man and sparrows (Burke and Bruford, 1987; Helminen etal., 1988), and is being devel­ Restriction fragment length polymor­ oped for use in farm animals. The potential of the phisms (RFLP) are so named because of their system resides in the ability to correlate HVR mode of detection. The allelic differences de­ band presence/absence with a desired trait and tected are single base changes in the recognition thus gain a means of manipulation of that trait. sequence of a restriction endonuclease. Since restriction endonucleases are quite specific in Oligonucleotide probes for the detec­ their cleavage site recognition, a single DNA tion of DNA-level polymorphisms is a relatively base difference within a restriction' site will to­ new technique.Synthetic DNA sequences of 15- tally destroy the recognition and cleavage. De­ 20 base pairs, called oligonucleotides, can be tection of such site absences is usually per­ created by chemical synthesis to any known or formed by agarose gel electrophoresis ofrestric­ desired sequence. Detection of DNA polymor­ tion endonuclease digested DNA, followed by phisms using oligonucleotide probes depends Southern blotting and autoradiographic detec­ upon the ability of the probe to hybridize to its tion of the fragments through use of a radiola­ genomic complement under different tempera­ belled cloned DNA probe. Loss of a restriction ture/salt conditions. Probes hybridize most site will generate a larger frag'ment due to the strongly to precisely complementary DNA se­ cleavage failure. Detected by autoradiography quences--any single base difference between as a mobility difference of the fragments during that complementary sequence and the probe electrophoresis, the RFLP can be subsequently lowers the melting temperature of the probe/ treated in linkage analysis as any other polymor­ sequence hybrid. Unlike RFLP or HVR, poly­ phism. One power of the technique must be morphisms of which are manifested as fragment remem bered--w hat the gel analysis detects is the length differences, oligonucleotide differences genotype since both alleles in a heterozygote are may be detected as differing binding efficiencies individually detectable. Because of the number on DNA dot blots. Dot blot techniques are far of individual probes necessary to saturate a less complexandmorecosteffective when com­ genome with markers, little concrete progress paring the large number of samples necessary for has been made in domestic animals, but the such marker/trait correlations (Beckman, 1988). potential value exists (Soller and Beckman, 1982; 1986). Gene mapping

Hypervariable region probes (HVR) The DNA-level marker detected poly­ depend upon the presence of "minisatellite" morphisms outlined here may be correlated in repetitive DNA regions in most higher organ- linkage studies with themselves or with protein 34 polymorphisms or phenotypic traits. The accu­ would thus be but steps aways. mulation of such linkage. data will eventually saturate the gene map with markers. A well Gene Cloning saturated gene map will enable the rapid correla­ tion of any newly discovered trait with a marker One of the many reminders of realism useful for selection. Gene mapping has two should be injected early in any gene cloning or of complexity. Linkage analysis will de­ le~els gene transfer project. To mak.e the effort of gene rive the linear order and relative recombina­ transfer worthwhile, you must have at our cur­ tional distance between genes or probe sites. rent technological stage, an isolated single gene Several techniques exist which also enable the that is known to produce the desired effect in the assignment of such linkage groups to specific whole organism. Unfortunately, not all single, physical chromosomes. The most elegant gene simple genes are open to isolation under gene mapping technique utilizes a basic recombinant cloning conditions. There are a number of desir­ DNA technology--in situ hybridization (Harper able, economically valuable single genes that and Saunders, 1981). A radiolabelled (usually many desire to clone and thus be able to dissect tritiated) cloned gene or DNA probe is hybrid­ gene action and perhaps transfer it to new species ized to metaphase chromosome spreads. After or strains. Once such gene is the Booroola (F) removal of unbound probe through extensive gene seen in the Booroola Merino sheep, that washing of the spreads, photographic emulsion controls the number of eggs shed from the ovary is applied to the slides. During exposure in the at each estrous cycle (Piper et al., 1985). But dark, the radiolabelled and hybridized probe unfortunately for any cloning efforts, no other produces upon development, silver grains over information is available. No specific protein has its locus on the chromosome. Usually statistical been identified nor correlated with specific al­ correlation of relative grain frequency versus leles of the gene, nor has a hormonal increase specific chromosomal region as identified by been observed indicating that the F gene encodes karyotypic banding, is necessary to determine a peptide hormone. A foundation of basic bio­ map position of single copy genes. Only rarely is chemical and physiological research must be it possible to simply observe a cluster of grains laid before a gene can be cloned. This is a major over one chromosomal region on one metaphase constrain tin the area of animal research, because spread and thus directly map a gene. of the paucity of information obtained to date. Some conclusions can be drawn from hurrian or DNA Sequencing murine work, but frequently these similarities are stretched when pushed into other mammal­ The ultimate in gene mapping is the ian species, and usually fail when applied to generation of the complete DNA sequence of an avian systems. organism. This is being considered for man at this time, using newly developed automated There are now a number of gene clon­ dideoxy sequencing instrumentation (Hood et al ing methods being used, but they fall into two ., 1987). It will be well worth monitoring this groups based in part on one's knowledge of the process, since the same automated systems would gene to be cloned. The most certain method of also be capable of generating the complete gene cloning is to actually chemically synthesize genomic sequence for any domestic animal. The the gene. Automatic DNA synthesizers, "gene ability to search a computer data base containing machines", are currently capable oflinking up to a complete genomic sequence would enable the 100 DNA bases together in a specific, defined "walking" from a RFLP, HVR, or oligonucleo­ and requested order. Genes for small proteins tide probe site to the linked gene of economic (under 30 amino acid residues) can be made in interest. Cloning and manipulation of that gene one pass--larger genes can be processed in parts 35 and chemical! y linked together. Genes encoding purposes, but in reality several limitations are small proteins such as insulin and growth met in practical usage. While 15,000 bases (15 hormone releasing factor (GHRF) have been kilobases or 15kb) is adequate for most peptide synthesized in similar fashion. This mode of hormones, such as growth hormone, genes are gene cloning requires them ost know ledge of the being currently identified that extend over 1 structure of a gene. It requires a complete and million bases (1 megabase or 1 Mb) in length. precise amino acid sequence of the desired pro­ Some of these megabase genes will be prime tein, which can then bereverse translated into the candidates for genetic manipulation and/or gene DNA sequence through standard codon usage transfer. In addition, to achieve certain genetic tables. But few proteins have been subjected to improvement goals, it will be necessary to the exhaustive amino acid sequencing which manipulate and transfer several intact genes in enables this technique to be used. one single piece of construct DNA. Thus there is a need for vectors for cloning and manipulation Most gene cloning is by a more labori­ of very large segments of DNA. Two such sys­ ous set of methods that involve isolation of the tems currently exist--cosmids and yeast artificial desired gene from a pool of genes, and then chromosomes (y AC)--thatwill support genomic proving procedures to show that the isolate is library construction using pieces of DNA 35-45 indeed the desired gene. The starting material for kb in size (cosmids)or up to several hundred these isolation methods are usually "libraries" of kilobase pairs (YACs). The cosmid vector is a DNA molecules. These libraries occur in two modified lambda phage where all that remains of forms, genomic or cDNA, with the c designat­ the original phage DNA after library construc­ ing the derivation of the DNA from RNA via tion are two small DNA segments termed cos­ reverse transcriptase. site (Collins and Hohn, 1978). Cos-sites, so named because they are the cohesive ends of the Genomic libraries are prepared by cleav­ lambda phage molecule, are all that are neces­ ing isolated chromosomal DNA with specific sary under the proper conditions, to package, restriction endonucleases. These endonucleases maintain, and propagate the intervening foreign cleave the DNA precisely at their recognition DNA segment. Elimination of the normal phage sequences--usually a four or six base palindromic genes allows room for insertion of a 35-45 kb sequence. This precise cleavage produces short foreign DNA segment and still be under the pieces ofDNA that are then enzymatically linked maximum size limit for a segment of DNA to larger pieces of vector DNA that are capable packageable into a phage protein coat (head). ofreplication in biological systems. For genomic The necessary functions· of these deleted genes DNA, because of its relatively large size, strains are supplied in vitro through use of a phage oflambda phage are the usual choice for cloning packaging extract. The name "cosmid" is de­ and propagation vectors, since they can be propa­ rived from the use of the cos -sites in the library gated in E. coli and can tolerate large foreign construction, and the fact that the cosmid phage DNA inserts. There are now hundreds of lambda upon reinfection of E. coli is not viable as a phage-based sys terns for cloning pieces of DNA phage but instead lives as a plasmid within the of up to about 15 ,000 bases in size. These phage bacteria. Plasmids are autonomously replicating systems vary as to which phage genes are re­ minichromosomes found in large numbers in tained in addition to the foreign DNA, and what many microorganisms, and frequently carry drug novel genes are added. Drug resistance markers resistance genes. They are circular pieces of and genes to promote expression of the inserted DNA several kilo bases in length and are of major DNA are frequently included in phage con­ use in recombinant DNA technology for the structs. An insert size limit of 15,000 base pairs mainpulation of small (up to 10 kb) pieces of may seem sufficiently large for any cloning DNA. Their major use is in cDNA library con- 36 struction, discussed below. By controlling the original phage or plasmid (cosmid) concentration, a field of individual Currently the only feasible method of plaques or colonies is produced after incubation. manipulating segments of DNA several hundred These plaques or colonies can then be bound to kilo bases in length is the Y AC (Yeast Artifical nitrocellulase by imprint lifts. Each plaque or Chromosome; Burke et al., 1987). They are colony is derived from one phage or one plas­ precisely that, artificial chromosomes composed mid, each with one foreign DNA insert. Thus, of the structural DNA elements required for one phage plaque or plasmid colony contains chromosome replication, segregation, and sta­ thousands of copies of one and only one foreign bility in yeast, artificially attached to foreign DNA sequence. The problem now of course is to DNA segments. Thus a large piece of foreign determine which plaque or colony has the gene DNA is inserted into a reengineered yeast chro­ of interest. mosome that retains in essence only the replica­ tion origins, centromeric, and telomeric regions There are numerous techniques that of the normal chromosome. This Y AC construct can be used to solve this problem. Perhaps the can now be propagated in yeast as an addition to easiest and mostpowerfulis DNA hybridization. the normal yeast chromosome complement, and DNA probes are radio labelled and hybridized to will, if properly constructed, express as proteins the lifts. Under proper salt and temperature of the foreign genes it contains. Thus this offers conditions, the probe will hybridize only with its a mechanism of cloning DNA molecules ten complement--the gene of interest if the probe is times large than those clonable in a cosmid chosen or created properly. DNA probes may be system. oligodeoxynucleotides created synthetically to match the known protein sequence of the desired Frequently, it is not necessary or even gene, or they may be segments of cloned genes desirable to seek the genomic version of a gene. from other species, i.e., using a bovine GHDNA A cDNA copy of the gene may be quite adequate probe to isolate the human GH gene. and will probably be more easily isolated. cDNA libraries are derived from mRNA extracted from This brings up the problem of informa­ a tissue that expresses the gene of interest through tion again. To clone a gene, one needs one or the use of reverse transcriptase. Since the fre­ more of the following "handles" on the gene of quency of a gene in a cDNA library is directly interest: (a) Protein sequence--all or part of the proportional to the frequency of its mRNA in the gene. (b) A cloned gene from other species. (c) sampled tissue, usually a tissue that highly ex­ An antibody to the protein that is highly specific. presses the gene of interest is used as the mRNA (d) A tissue that highly expresses the gene and source. A tissue, such as the pituitary, which one that expresses it not at all. (e) A mutant cell highly expresses growth hormone (GH), usually line that requires the gene of interest for growth. has a markedly increased mRNA level for that protein. cDNAs, since they are generally smaller, The above DNA hybridization tech­ are usually ligated directly into various plasmids nique uses either of the pieces of information in that will transform and be propagated at a high (a) or (b). The radioactive probe binds selec­ level in E. coli. tively to the region of the nitrocellulose lift wherein lies the gene of interest. Reference back Cosmid, phage, and plasmid libraries to the original colony or plaque plate allows are searched in approximately the same manner. isolation of the specific plaque or colony con­ The library is plated by spreading a solution of taining that gene. Of course, detection is by phage on a plate of E. coli or a solution of autoradiography of the hybridized and washed plasmid-containing E. coli on a nutrient plate. lift, producing a dark spot in the region of inter- 37

est. The antibody procedure is similar, save that of the mRNA) prevents or hinders other modes the original libraries are creat,ed in phage or of library search. The method of search is rela­ plasmids that allow expression of the foreign tively simple, and introduces a technique that protein in E. coli. The bacterial colonies now will be discussed in more detail under the topic

have small amounts of the foreign gene -encoded of Gene Transfer. This technique, CaPO4 medi­ proteins, which can be detected on lifts with an ated gene transfer, is basically the treatment of 125 1-labelled antibody to that protein. plasmid or genomic DNA with CaP04 which precipitates around and with the DNA. When

We have already seen how utilization layered over a cell monolayer, this CaP04-DNA of a highly expressing tissue for generation of a complex is taken up by the monolayer cells, and cDNA library is advantageous. This can be car­ in a certain small percentage of the cells, the ried one step farther by doing comparative hy­ genes on the introduced foreign DNA are ex­ bridization with a library created from a tissue pressed. Some of the expressing cells will incor­ that does not express the gene of interest. There porate the gene or geries into its own chromoso­ are several methods used for comparison, some mal complement, a process which usually re­ relying on highlighting colonies that bind one quires a number of cell generations. Of course, if but not the other pool of mRNA made from the selective conditions are applied during or after two tissues, while others rely on a column sepa­ the gene transfer process, only cells that have ration of the portion of onemRNA-turned-cDNA taken up the gene that complements the defi­ pool that finds no or little counterpart in the non­ ciency will survive, given that the foreign gene expressing pool. The advantage of this set of survives transfer in an expressible condition. procedures is that it requires the least amount of Stable integration of the expressing gene pro­ information to clone a gene--simple identifica­ duces a phenotypically normal cell line that will tion of tissues that highly express or not express pass on its acquired trait. The foreign gene iso­ the gene is sufficient. The disadvantage is that it lated from the rest of the library, can now be re­ still requires more information to prove the iden­ isolated by a number of methods--the simplest tity of what has been isolated. Any gene showing being the construction of aDNAlibraryfrom the similar expression differentials will be selected transfected cell line using a DNA probe to a along with the desired gene. One of the major intentionally included flanking sequence (plas­ uses for this technique is the cloning of genes mid or other foreign gene) that was in each of the correlated with a change in physiological or original library plasmids. Indeed, often the origi­ developmental state. By comparative hybridiza­ nal plasmid into which the foreign gene was tion techniques, all genes that are turned on, for inserted is used as a probe, since the only seg­ instance, upon the induction oflactation could be ments of DNA in the transfected cell library isolated by comparison of pre-and post-induc­ which contain plasmid sequences also carry the tion samples of the same tissue. sought gene. The CaP04 transfection technique has been used most successfully to isolate genes Finally, there is the approach using a that cause transformation of cell lines--onco­ mutant cell line that can be complemented by a genes. Oncogenes were originally isolated from foreign gene from a DNA library, and thereby libraries prepared from tumor tissue, and confer enabled to survive under normal! y lethal or subop­ this unregulated growth on the recipient cell timal conditions. Severallow expression "house­ lines. Oncogenes have been shown to be modi­ keeping" enzymes have been isolated and cloned fied cellular genes--normal growth regulating in this manner. These are enzymes normally and mediating genes that through the process of present in every cell, but which may show major oncogenesis have been mutated and released differences in expression between tissues, whose from their normal controls. The cellular genes, normally low level of expression (less than 0.01 % cellular oncogenes, have been pursued as clon- 38 ing goals by many labs, and may prove important era of infatuation with complex molecular trans~ genes for consideration in schemes of genetic fer technologies. There are still many genes or engineering of gene transfer because of their traits for which classical selection and cross­ roles in basic growth regulation. breeding is the only feasible mode of manipula­ tion or transfer. Current molecular techniques Thus, we have outlined the basic gene are unable to efficiently improve many of the cloning technologies. Elementary information desirable economic quantitative traits--traits that on the protein product of the desired gene clon­ have been effectively improved by rnillenia of ing targets is a necessity, both to enable the initial selective breeding. The present inability of cloning, but also to prove the putative identity of molecular or recombinant DNA technology to the isolated cloned gene. Since many genes exist make a mojor impact on the improvement of in closely related gene families, verification of a quantitative traits can be attributed solely to the gene's putative identity is a must. Many of the lack of basic knowledge of the biochemical, above techniques will isolate genes that are physiological, and genetic basis of these traits. closely related to the desired gene as well as the Presently, only major genes for which cloning desired gene itself. In cases where there is any and isolation information is available, are good doubt, one or more independent lines of proof candidates for gene transfer. Successful gene must be used in addition to that used in the transfer depends upon not only the knowledge of original cloning. Thus, there are cases where the exact nature, identity, or sequence of the there is enough background information to clone candidate gene, but also what factors influence a gene--one "handle"on it--but no supportive its temporal, developmental, or special expres­ information to prove, in a non-circular manner, sion in an organism. Depending on the specific its identity. Since sometimes the ultimate proof gene, the reproductive physiology of the target of a gene's identity is its action in a whole organism, and the desired expression profile of organism--gene transfer and the analysis of the the gene, some transfer techniques will prove transgenic organism can provide the ultimate more effective than others. We will examine the confirmation of a gene's identity. major techniques individually, since each has strengths and weaknesses. Gene Tran sfer CaP04wMediated Transfer An early effort to change the animal genome with exogenous DNA was attempted by Calcium phosphate-mediated DNA Munro (1968) in chickens. Ovaries and testes of transfer or uptake was originally developed by white leghorns were injected with DNA isolated Graham and van der Eb (1973) for assaying from bantam fowl, which are colored and have adenoviral DNA, but was soon modified by other morphological differences. Some offspring Wigler et al., (1977) in order to insert a fragment · had patches of dark feathers and the bantam claw of DNA carrying the herpes simplex thymidine morphology, but because of the lack of molecu­ kinase(TK) gene into a TK- mammalian cell lar technology at the time, no evaluation of gene line. A large number of genes have been inserted transfer could be performed. Gene transfer within via this technique, which has, for instance, en­ the molecular era has involved five basic meth­ abled the cloning of the two dozen or more oncogenes known to date. odologies: CaPO 4-mediated, electroporation, lipofection, microinjection, andret:roviral inser­ tion. Somatic cell fusion, a powerful but non­ Transfection is accomplished by pi­ molecular technology, should also be consid­ petting a suspension of DNA, complexed with CaP0 into a fine particulate precipitate, on to a ered in any strategic plan. The original method of 4 gene transfer--sex--must be remembered in this monolayer of cells in a culture flask. Various 39

methods may be used to enhance the stability fer has been observed, there is still hope that it and uptake of the foreign DNA. Glycerol shock, may be possible to mimic cell culture conditions treatment with polyethylene glycol (PEG) and because of the large number of recipient eggs substitution of DEAE dextran (diethylaminoethyl available at one time. Experimentation of the

dextran) or polybrene for CaP04 are among the proper scale to substantiate this hope has yet to variations that can be used. Efficiency varies be performed. The other system involves macro­

with the method and the recipient cell line. injection of CaP04 DNA into the inner cell mass Under optimal conditions and using the best, ofnewly laid chicken eggs. No stable integration most receptive cell lines, transfection rates of 10 was observed in 3 ,000 recipient eggs (F. C. Leung, 2 3 - to 10- per cell can be obtained. This is based on BattelleLaboratories, personal communication), the integration and expression of the foreign although transient expression was observed. DNA, and should be compared to the usual Currently, due to the low efficiency, only fish 5 7 efficiency of 10- to 10- • Because of this low with their high fecundity are prospective candi­ level of transformation, selection systems are dates for this mode of gene transfer. The ability usually used to identify the successful recipients. to manipulate multiply, and transfect in cell These selection systems may be a gene that culture primordial germ cells is an area of very complements a genetic defect in the recipient active research. Combined with the develop­ cell line, i.e., TK or hypoxanthine phosphori­ ment of techniques allowing colonization of the bosyl transferase (HPRT), or may be a dominant gonads by these reengineered germ cells, or drug resistance gene such as neo that confers regeneration of a whole animal from such a resistance to the drug G-418. A nonselectable to ti potent cell would allow transfer to be accom­ gene is linked to the selectable gene and the two plished in cell culture under selection, and only co-transferred into the cell line. Selection is the successful transformants manipulated into a applied and produces a set of surviving cells that fully transgenic or gonadal chimera/transgenic express the selectable marker gene, and most animal. probably also the original gene of interest. Trans­

fection by CaP04-mediated transfer seems to Electroporation work best on monolayer cells, with rates for suspension cells being estimated in the order of Gene transfer by electroporation can be 5 7 10- to 10- • basically described as the formation of transient pores in the cell membrane by pulsing an electric

CaP04-mediated transfer, and similar current across the cell while the cell is immersed straight DNA treatment procedures are easy and in a DNA containing solution. The key to suc­ inexpensive to perform, require little specialized cess is the determination of the proper duration, equipment, and can be done without an infec­ voltage, and current of the pulse, which must tious agent. They are also of low efficiency, the produce the transient pores long enough for uptake of DNA and its expression is low, and DNA to enter, but not irrevocably damage the there is a tendency for concatemers of the foreign cell. Electroporation has seen maximum utility DNA to be inserted to head-to-tail orientation. in plant protoplasts, bacteria, and animal cell Thus, the low efficiency necessitates the use of a cultures (Jastreboff et al., 1987). It suffers from selectable marker gene and/or a large number of the same deficiencies as other mass transfer indtviduals if attempted in animals. Two sys­ techniques, such as the CaP04 method--low tems have been explored with undetermined efficiency, need for selection system, concatena­ success to date. A standard CaP04-mediated tion, etc. It is currently being explored for gene transfer has been attempted at the time of fertili­ transfer :n fish, and for rumen bacteria. As in the zation in salmonid eggs using the neo drug resis­ CaP<\ methods, major use in generation of tance gene. While no conclusively positive trans- mammalian transgenics will await development 40

of totipotent cell manipulation technology. In this manner, it may be possible to have some of the advantages of a transgenic animal such as Lipofection controlled long-term expression of a gene with­ out the regulatory and proprietary problems posed Lipofection is a gene transfer techno­ by a transgenic animal. logy based on the encapsulation of DNA in a phospholipid bilayer. The encapsulated DNA is Microinj ection then delivered by membrane fusion of the liposome with the cell membrane. Efficiencies Microinjection is the most straightfor­ do not exceed other mass transfer methods such ward of gene transfer techniques and has a usu­

as CaP04-mediated transfer (Fraley et al., 1981), ally high efficiency (in fish over 80% of the eggs and it suffers from the same limitations as injected show foreign DNA integration); how­

CaP04-mediated gene transfer or electropora­ ever, only one cell at a time can be injected. Since tion. It too is being explored for use in fish, but integration of foreign DNA is a random event, its immediate most intriguing use has been in the and since the site of integration plays a major role area of in vivo transfer and expression of genes in the level and specificity of gene expression, it in adult animals. is desirable when seeking the optimal transgenic animal to have a procedure capable of producing Liposomes encapsulating a recombi­ a large number of transgenic individuals from nant plasmid containing the rat preproinsulin I which to choose. Microinjection requires a heavy gene were injected intravenously into adult rats. time investment to meet this criterion. This gene was found to be transiently expressed in a physiologically active form in the liver and The first success in animals in microin­ spleen of the recipient animals, and led to a jection was by Gordon et al. (1980), where plas­ significant effect on the blood glucose levels of mid DNA containing the herpes simplex TK those animals (Nicolau et al., 1983). There are gene was injected into one of the pronuclei of a indications that by insertion of glycolopids into recently fertilized mouse egg. The ovum was the liposome bilayer, the DNA-carrying liposome then placed into the oviduct of a pseudopregnant may be targeted to a specific cell type such as the female, where it developed into a normal pup non-phagocytosing cells of the liver or spleen. with theplasmidDNAineverycell. The injected The glycolipid appears to be recognized by the DNA is transmitted to offspring in a normal lectins on the cell surface, which in turn leads to Mendelian manner, creating a line of transgenic an increased uptake of the encapsulated DNA mice carrying the exogenous DNA in every cell. (Nicolau et al., 1987). While the prime interest in this technique has been for gene therapy in The work of Palmiter et al. (1982a), man, it may also be of interest in farm animals. where a rat growth hormone gene linked to a It may be possible to develop a peptide hormone mouse metallothionein promoter was microin­ gene construct (such as growth hormone) encap­ jected into the male pronucleus of a newly ferti­ sulated in a glycolipid bilayer liposome. Upon lized mouse egg, excited the scientific commu­ intravenous injection the liposome, targeted for nity with mice twice normal size. Again these the liver, would transiently express the growth mice passed on the trait in a true Mendelian hormone gene in the liver. This may be an easier manner and have shown its stability through a mode of growth hormone eQ.hancement than number of generations. The mouse has proven to straig~t hormone injection since it would not be an excellent model system for gene transfer, require repetitive hormone injections or the al­ with its primary uses being the refinement of ternative slow-release systems. The target organ physical injection techniques and the testing of would act as the production and release system. plasmid constructs. As mentioned above, the 41

early Palmiter's work was with a rat GH gene copies being variable. Integration is at a single linked to a mouse metallothionein promoter. site, apparently at random location on the chro­ Such fusion genes are quite common--with the mosome, and when two genes are coinjected, promoter (the on/off switch) being isolated from they usually integrate at the same site. Brinster a different gene of the same or different species et al. (1985) have suggested that initial integra­ than the gene from which expression is desired. tion is by a single linear copy at a spontaneously The metallothionein gene, and its promoter has generated break in the chromosomal DNA. This been given much attention due to its inducibility is followed by recombination of the integrated by heavy metals (Zn, Cu) and other agents, and foreign DNA with molecules circularized after because in a normal mouse its primary expres­ injection to form the tandemly repeated arrays. sion site is the liver. In the transgenic mice produced, the GH was turned on by Zn in the The site of integration, which appears water supply, but was expressed at a low level to be random, influences gene expression. In two before Zn addition, and could not be shut off by transgenic mouse lines, microinjectedrabbit beta­ Zn withdrawal. This differs from the nonleaky globin was expressed in inappropriate tissues sheepmetallothionein promoter used by Ward et (Lacy et al., 1983). Also, not all animals that al. (1986) which shows no such low level ex­ have integrated an apparently functional gene pression of the fusion gene before addition of the will express it. Palmiter et al. (1983) report only inducing Zn, and appears to be turned off upon 70% of the transgenic mammals express the removal of Zn after induction. foreign gene. This has been found to be true in the transgenic fish, with about 50% of transgenic TheexperimentofPalmiteretal. (1982) goldfish and northern pike showing expression. has been repeated with human GH rather than rat Finally, major differences in the levels of expres­ GH with the same effect--large mice (Palmiter sion have been observed by all experimenters. et al., 1983). Within the last few years, a wide These data indicate that the site of integration-­ variety of genes have been transfen-ed into mice the surrounding chromosomal DNA--plays a (see Table I) and most express to some level in a major role in the expression of an inserted gene, fraction of the transgenic mice. There is consid­ including suppression of its expression and erable variation in the efficiency with which changing its specificity of expression. mice are born from transferredinjectedeggs,and in the percent of micewhichintegrate the foreign It is now possible, as shown repeatedly DNA. Ward et al. (1986)reportintegrationrates in mice, to transfer a wide variety of genes, and of from 0 to 18%. There are many unknowns in to specificaIIy express those foreign genes in the procedure. Why certain DNAs are lower in desired tissues with a high degree of reliability. integration rate by a factor of 10 is not apparent. Promoter/enhancer elements are being isolated Indeed the integration mechanism itself is largely that give tissue specific expression (e.g., beta­ a black box. Linear molecules have been re­ globin for erythroid cells, metallothionein for ported to integrate more efficiently than circular liver, elastase for pancreatic cells, etc. --see Table molecules, and Brinstcr et al. (1985) report that II), or are inducible under certain physiologic or linear DNA is readily circularized in the mouse environmental conditions (e.g., heat-shock, heavy pronucleus. Due to increased efficiency of incor­ metals, and glucocorticoids). In the future, de­ poration at certain stages in the cell cycle, it is velopmental stage specific controlling elements assumed that integration may be enhanced by the may come into usage allowing transferred genes normal DNA replication process. Here, as in the to be expressed only when optimally required in

CaP04-mediated gene transfer, injected DNA development. usually integrates as a tandem, head-to-tail array of multiple copies, with the exact number of One aspect of the promoter/enhancer 42

element research has allowed the term "Molecu­ cies. It is anticipated that in cattle, higher GH lar Farming" to be coined. Molecular farming levels will increase milk production, and in pigs, uses specific promoters to express a foreign gene as expected based on GH injection studies, there in a harvestable context such as milk, serum, or is repartioning from fat to protein, producing a egg white. Under this scheme, the gene for a leaner hog. In transgenic pigs, while increased complex biomolecule of pharmaceutical or in­ GH has produced a leaner pig with better feed dustrial interest is combined with a specific conversion, it also apparently causes arthritis, promoter, such as human clotting Factor IX with anestrous and early mortality. It must yet be the beta-lac to globulin promoter. In a transgenic determined whether finer control of GH expres­ animal with this construct, Factor IX would be sion will alleviate these negative aspects (Pursel expresse~ and secreted into the milk, and hence et al., 1987). could be isolated from the milk. Based on the current high costs and attendant health risks of As stated before, microinjection is a isolation of such biopharmaceuticals from human highly efficient means of transfer, albeit a labo­ donors, a cow producing less than 1 % of its milk rious one. It is more suited to animals with low

protein in the form of one of these biophar­ fecundity than is CaP04-mediated transfer, but maceuticals could generate revenues in excess the fertilized eggs must be manipulable and of $100,000 U.S. per day. Of course this is culturable in vitro for a period of time--tech­ predicated on the cmrnnt prices for such com­ nolgies that need improvement in some farm pounds, and prices would fall given a larger species. supply, but it can be easily demonstrated that despite the large expenses in generating such Retroviral transfection animals, the project is financially feasible and scientifically sound. Transgenic mice and sheep Retroviruses are RNA viruses with an have already been produced that express sheep RNA-protein core and glycoprotein envelope, beta-lactoglobulin (mice) and human Factor IX which upon entry into a cell, produce a DNA (sheep) in their milk (Simons et al., 1987; Si­ copy through reverse transcription of its RNA mons et al., 1988). The expression levels are template. This double stranded DNA copy can currently low, but there is no reason that further integrate into the host genome as a single copy manipulation of the control sequences should (the provirus) at a randomly chosen site. During not solve that problem. the proviral stage, the virus is treated by the cell as it would any other gene-- it is replicated as the More general gene transfer in non­ cell divides and can express its contained foreign murine species--fish, cattle, pigs, sheep, and genes. It is possible through recombinant DNA rabbits has been proceeding in the past few technology to re-engineer a retrovirus, deleting years. Integration frequencies range from nearly genes responsible for the production of infective 100% (fish) to 15-25% (mice) to 2-5% (sheep) particles, replacing them with a foreign gene that with rabbits and pigs in the 8-15 % range (Kraeus­ is desirable for transfer. Through use of a "helper" slich, 1986). This work has been performed with virus, defective viral particles are raised which various metallothionein-GH constructs, and are then used to infect the recipient cell line or higher circulating levels of GH have been ob­ animal. The recombinant virus integrates and served. Only fish (carp and a related species, the expresses its incorporated foreign genes. loach) (Z.Y.Zhu, Institute of Hydrobiology, People's Republic of China, personal communi­ The advantages of such a system are cation) showed growth enhancement, suggest­ obvious. Up to 100% of the cells can be infected ing that responses observed in mice cannot be and can express the viral and foreign genes. automatrcally applied to other mammalian spe- Since masses of cells can be infected at once with 43

high efficiency, it is possible to transfect at later leukosis virus (ALV) to produce transgenic chick­ stages of embryonic development, or indeed, in ens that carry a defective AL V provirus insert. theory, adult animals. Integration while at nm­ These transgenic chickens express a subgroup A dom, is generally a single precise copy, thus envelope glycoprotein and are found to be thus eliminating the head-to-tail repeat structures seen very resistant to subgroup A leukosis-sarcoma in other transfer systems. So far, the infection virus infection. This transfer andconstitutiveex­ and harboring of the retro viral provirus does not pression of a viral envelope protein gene in a seem to harm cells. The main disadvantages are transgenic animal can create an artificial domi­ the availability of a well-characterizedretroviral nant gene forresistance to a specific virus. While vector for the chosen species (e.g., none are this probably will only be successful where there available for fish), and the integration of the are virus specific receptors that can be blocked provirus at a different site in each separately by the envelope protein, this opens the door for infected cell. The former argues for more re­ the production of animal strains inherently resis­ search into the gene structure of animal viruses, tant to viruses of major economic impact. so that they can be modified to delete infective Retroviral transfer developments are capacities and incorporate foreign genes. The being pressed at a rapid rate by biomedical latter is based on the observation that as many as research for use in human gene therapy. This 20% of the random integration events in gene mass of research must be constantly monitored transfer may cause amutagenic event by integra­ for use in farm species. tion in a vital past of the genome. While most are masked due to integration on only one chro­ Gene transfer~general considerations matid, some will integrate opposite an existing recessive mutation. This is less of a potential Each of the gene transfer systems out­ problem in microinjection, since integration is lined here have specific advantages or disadvan­

possible while there are few cells in the organ­ , tages. Mass transfer techniques (CaP04 electro­ ism--a poor integration site generally causes poration, and lipofection) are oflow efficiency, embryo death. In a retroviral transfection at a but can transfer large genes. Microinjection is later stage of development, the potential is much laborious but highly effective, and can be per­ greater to have an organism that is viable but formed in most non-avian species. Retroviral chimeric for the mutation creating possible subop­ transfer potentially works at any stage of devel­ timal performance. So far, the retro viral proce­ opment but requires specialized species specific dure has been used successfully to transfect bone viral vectors. Additionally, retro viral transfer marrow cells which were reimplanted into the cannot transfer very large DNA segments due to adult mouse. Expression of the transferred genes packaging constraints of the viral head, there was detected in the chimeric mouse. exists a potential of viral recombination creating an infectious recombinant virus from a defective Retroviral transfer is of greatest inter­ construct, and the integration of viral sequences est in avian systems where other forms of gene during transfer of the target gene may be undesir­ transfer are extremely difficult due to the physi­ able because of governmental regulations. It ology of the avian reproductive system. So far, may be necessary for easy approval by govern­ successful retroviral transfer in poultry has pro­ ment regulatory bodies of a transgenic animal duced chickens with increased GH levels but no for human consumption, that the animal only phenotypic changes. Of more importance for the contain extra copies of genes or sequences al economic improvement of farm animals in gen­ ready existing in that animal. That is, the optimal eral and poultry specifically, is the work of animal may be, for instance, a transgenic carp Salter et al. (1987) and Salter and Crittenden that has an extra carp growth hormone gene with (1988). They have used a recombinant avian a carp promoter sequence and no other foreign 44

' DNA--plasmid, viral,orotherspecies~Currently the cloning of which is a goal for many labs. microinjection is the key technique to insert such constructs and only those desired sequences. Low-expression enzymes

Which Genes to Pursue? The housekeeping or low expression enzymes form a class of genes that may prove The next logical question, given the quite desirable to manipulate. These are basic ability to clone and transfer a gene, is what gene? enzymes necessary for food utilization, protein There are a number of classes of genes that synthesis, etc. Increased levels of a rate limiting present potential--immediate or promised--as enzyme would serve to enhance the efficiency of objects of cloning and transfer. its whole system. This has been shown in trout where one strain of rainbow trout expresses Hormones and growth factors phosphoglucomutase in the liver to a high level compared with the normal state. This strain shows enhanced growth compared to the nonex­ Peptide hormones and growth factors pressing strain, a condition thought to arise in are numerous, and have been discussed in part more efficient yolk utilization in the fry (Allen­ above (GH and oncogenes). GH levels in dorf 1983). Certainly there are other rate transgenic mice have also been increased not by et al., limiting steps that could be enhanced if identi­ transfer of GH but of growth hormone releasing fied. factor (GHRF) (Hammer et al., 1985). Growth hormoneandGHRFhavestimulatedinterestnot only from those wishing an absolute size in­ Disease resistance crease in animals, but also those interested in nutrient partitioning and in increasing milk pro­ Immunoglobulingenes have been trans­ duction substantially. Skeletal growth is also ferred into mice and the potential exists for the affected by insulin-like growth factor I. Prolactin indentification of a functional immunoglobulin is clearly :;i. key to milk induction and affects gene from a resistant species and the transfer of osmo-regulation and smoltification in fish. Thus, this gene back into the host species. Identifica­ these two hormones are obvious candidates. tion of viral envel()pe genes for transfer must be Reproduction is under complex hormonal con­ of major interest after the results of AL V in trol, and can be enhanced by treatment with chickens (Salter et al., 1987; Salter and Critten­ specific hormones. One of the cautions that must den, 1988). be remembered is that increasing hormonal lev­ els via gene transfer may have adverse effects on Structural or specialized proteins other systems e.g., transgenic mice with in­ creased GH have reduced fertility and transgenic This category includes milk proteins, pigs are less than totally healthy. Many more wool proteins, etc. Alteration of the genes re­ systems than reproduction are under hormonal sponsible for such processes may affect produc­ control, but the paucity of know ledge as to the tion rate or quality--wool length might be in­ control systems prevents any modification pro­ creased, etc. More immediately, as in the low posals at this moment. expression enzyme system, identification and enhancement of rate limiting steps could signifi­ Increasing hormone levels via gene can t1 y increase growth rate of, for instance, wool. transfer is a sledge hammer approach to regula­ We have already discussed the potential of tion. Much finer control might be had by control molecular farming, of for instance, turning the of numbers and location of hormone receptors, dairy cow into a biochemical factory. 45 Novel genes ductiofl. An anti-sense mRNA to beta-galactosi­ dase in E. coli blocked 99% of the normal beta­ With the ability to cross species barri­ galactosidase production (Pestka et al., 1984). ers, it is possible to introduce new capabilities Plasmids expressing an antisehse gene to protein into farm animals. An example is a current effort 4.1 have been injected into Xenopus eggs, .and by Ward in Australia to introduce into sheep a extinguished endogenous protein 4.1 transcrip­ pathway to synthesize cysteine from serine. Since tion (Giebelhaus et al., 1988). Such antisense m it is known that the circulating blood level of RNA genes should be transmitted in a standard cysteine is a limiting factor in wool production, Mendelian fashion like any other transferred an effort is being made to transfer the cysteine gene. In addition to providing a mechanism to synthesis pathway from E. coli to sheep, with a block functional genes, this approach would target of expression in the rumen epithelium. allow the dissection of complex gene systems by While no serious proposal has been selective blocking of one component at a time. made to transfer whole metabolic, developmen­ tal, and physiological systems--such as to pro­ Summary duce feathers on cows--such a complex system transfer, while impossible now, may not be out Above we have explored molecular of the realm of possibility in the future. biology and its potential for use in animal agri­ culture. This is by necessity an overview. Only One novel gene glass that has great the major techniques were presented and an promise is that of the anti-sense gene. It is attempt was made to limit the presentation to possible to create byrecombinantDNAmethod­ proven methodologies. It is easy to move from ologies, an "inverse" copy of an unwanted gene genetic engineering current to genetic engineer­ that will block the expression of that gene. For ing future, and from science to science fiction. example, if prolac tin were identified as the cause One can reasonably argue that almost anything is of broodiness in turkeys, an antiprolactin gene technologically possible given resources and could be created and transferred into turkeys. time--this is certainly true in biology, especially This antigene would produce an anti-sense molecular biology. Need a purple cow, a wooly mRNA for prolactin which is exactly comple­ hog or a sheep that lays eggs? These are not just mentary to the normal prolactin mRNA. The around the C()rner but if desired strongly, they anti-sense mRNA and normal mRNA, being may someday exist. They will not exist, nor will complementary, will form a double stranded most of the much more practical goals, without anti-sense RNA/mRNA complex, which cannot a vast increase in basic know ledge of the biol­ be translated by ribosomes. The net effect is a ogy, physiology and genetics of the very animals marked decreased in, in this case, prolactin pro- we have had under domestication for millenia.

Table I. Genes transferred to micea.

Transgene Species References a-Actin/globin Rat/human Shani (1986) aa (I) collagen/CAT Mouse/bacterial Khillan et al. (1986) aA-crystallin/CAT Mouse/bacterial Overbech et al. (1985) a-1 antitrypsin Human Kelsey et al. (1987) a-1-acid glycoprotein Human Dente et al. (1988) atrial natriuretic factor/large T Mouse/SV40 Field (1988) 46

Table I. Genes transferred to micea. (cont'd)

Trans gene Species References BKV early region BKV Small et al. (1986b) C-myc Mouse Schoenenberger et al. (1988) C-reactive protein Human Ciliberto et al. (1987) ~-casein Rat Lee et al. (1988)

E a MHC class II Mouse Pinkert et al. (1985), Yamarnura et at. (1985), Le Meur et al. (1985) Elastase I Rat Swift et al. (1984) Elastase I/growth hormone Rat/human Ornitz et al. (1985a,b) Elastase I/SV40 early region Rat/SV40 Ornitz et al. (1985b) Factor IX Human Choo et al. (1987) a-Fetoprotein Mouse Krumlauf et al. (1985a) ~-Glob in Rabbit T. Wagner et al. (1981), Lacy et al. ~-Globin Human E. Wagner et al. (1981), Stewartetal. (1982), Towneset al. (1985), Costantini et al. (1986) o-~-Globin Mouse/human Humphries et al. (1985) Gonadotropin-releasing hormone Mouse Mason et al. (1986) Granulocyte-macrophage colony Mouse Lang et al. (1987) stimulating factor Growth hormone Human Hammer et al. (1984), Wagner et al. (1985) rans gene Species Reference Growth hormone Rat Hammer et al. (1984) Growth hormone/diptheria toxin Rat/diptheria Behringer et al. (1988) HB V surface antigen HBV Chisari et al. (1985), Babinet et al. (1985) HIVLTR/CAT HIV/bacterial Khillan et al. (1988) Immunoglobulin k Mouse Storb et al. (1984, 1986) Immunoglobulin µ Mouse Weaver et al. (1985, 1986), Storb et al. (1986) , Immunoglobulin µ Mouse Rusconi and Kohler (1985), Storb et al. (1986) Immunoglobulin µ Human Yamarnura et al. (1986) Insulin Human Burki and Ullrich (1982), Bucchini et al. (1986) Insulin/MHC class I Rat/mouse Allison et al. (1988) Insulin/SV40 early region Rat/SV40 Hanahan (1985) Interleukin 2 Human Nishi et al. (1988) JCV early region JCV Small et al. (1986a,b) ~-lactoglobulin Sheep Simons et al. (1987) Luciferase Firefly Di Lella et al. (1988) MHC class I Pig Frels et al. (1985) MHC class i Mouse Bieberich et al. (1986) 47 Table I. Genes transferred to micea. (cont'd)

Trans gene Species References Middle T oncogene Polyomo virus Williams et al. (1988) MMTV L 1R/thymidine kinase MMTV/HSV Ross and Solter (1985) MT-1/calcitonin Mouse/rat Crenshaw et al. (1987) I MT-I/growth hormone Mouse/rat Palmiter et al. (1982a) MT-I/growth hormone Mouse/human Palmiter et al. (1983) MT-I/growth hormone Mouse/bovine Hammer et.al. (1985) MT-I/growth hormone releasing factor Mouse/human Hammer et al. (1985) MT-I/growth hormone/SY40 early region Mouse/human/SY40 Messing et al. (1985),Palmiter et al. (1985b) MT-I/HPRT Mouse/human Stout (1985) MT-I/RB V surface antigen Mouse/HBV Chisari et al. (1985) MT-I/low density lipoprotein Mouse/human Hofmann et al. (1988) MT-I/ornithine transcarbamylase Mouse/human Kelley et al. (1988) MT-I/placental lactogen Mouse/human Palmiter (1985a) MT-1/somatostatin Mouse/rat Low et al. (1985) MT-I/thymidine kinase Mouse/RSV Palmiter et al. (1982b) MT-1/transthyretin Mouse/human Sasaki et al. (1986) MT-IINgrowth hormone Human/human Hammer (1985) MMTV/c-myc MMTV/mouse Stewart et al. (1984) Myelin basic protein Mouse Readhead et al. (1987) Myosin light chain Rat Shani (1985) Neurofilament Human Julein et al. (1987) Pancreatic amyl..~se Mouse Osb(}\n et al. (1987) Phenylethanolamine-N-methy ltransferase Human Baetge et al. (1988) Prealbumin (mutant) Human Shimada et al. (1987) Protamine-2/c-myc Mouse/mouse Stewart et al. (1988) Protamine-2/SV40 early region Mouse/SV40 Stewart et al. (1988) Renin-2 Mouse Tronik et al. (1987) RSVL1R/CAT RSV/bacterial Overbeek et al. (1986) SV40 early region SV40 Brinsteretal. (1984),Palmiter et al. (1985b), Small et al. (1985) SV40 large T antigen SV40 Palmiter et al. (1985b) SV40/ormthine transcarbamylase SV40/rat Cavard et al. (1988) Super oxide dismutase Human Epstein et al. (1987) T-cell receptor ~ Mouse Uematsu et al. (1988) Thymidine kinase HSY T. Wagner et al. (1981) Transferrin Chicken McKnight et al. (1983) Vasopressin/large T Cow/SV40 Rindi et al. (1988)

8Abbreviation: BKV, BK virus; CAT, chloramphenicol acetyltransferase; HBV, hepatitis B virus; HPRT, hypoxanthine phosphoribosyltransferase; HSV,herpes simplex vius; JCV, JC virus; L TR, long terminal repeat; MHC, major histocompatibility complex; MMTV, mouse mammary tumor virus; MT, metallothionein; RSV, Rous sarcoma virus; SV40, Simian virus 40. 48

Table Il. Tissue-specific gene expression.

Gene Cell Type Specificity References

Rabbit B-globin Erythroiq Wagner, T et al. (1981) Human B-globin Erythrbid Townes et al. (1985) Mouse/human B-globin Erythroid Chada et al. (1985) Rat elastase l Pancreatic Swift et al. (1984) Rat myosin light chain Skeletal muscle Shani (1985) Mouse alpha-fetoprotein Yolk sac and liver Krumlauf et al. (1985b) Mouse k light chain (lg) B cells Storb et al. (1984) Mouse µ heavy chain (lg) Band Tcells Grosschedl et al. (1984) Insulin/T-antigen Pancreatic cells Hanahan (1985) Human a-1-acid glycoprotein Liver, serum Dente et al. (1988) Human C-reactive protein Liver Ciliberto et al. (1988) Sheep B-Iactoglobulin Mammary Simons et al. (1987)

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T.K. Mukherjee Institute for Advanced Studies University of Malaya Kuala Lumpur Malaysia

Summary to perform functions more efficiently that is to produce more or better products or to do so more Biotechnological developments affect­ efficiently. Prospective application of genetic ing livestock improvement during the last few engineering/recombinant DNA technology for years in North America and Europe have been livestock development and forage and vaccine marked by biological developments that are production has been discussed in many confer­ absolutely amazing. Against this background, ences. In general there is tremendous optimism development of this new technology in Asia concerning its future impact in animal produc­ (excluding Japan) is remarkably slow although tion and health. sporadic attempts have been made by individual scientists to investigate certain aspects of em­ In addition to these, new developmept bryo transfer, molecular biology, feed biotech­ of reproductive technologies forms the basis of nology and production of immunogens. applied biotechnology in livestock in North America and Europe, which feature high per­ The present paper discusses briefly formance livestock with an advanced level of some of the above aspects and presents current existing livestock technologies (A.I., high preci­ status of biotechnological developments in 8 sion in quality control of feed, advanced veteri­ countries of Asia. A detailed report on this sub­ nary diagnostic and control methods and devel­ ject has been presented to F AO on whose request oped allied industries with a high technical pre­ this review was undertaken in October- N ovem­ cision). ber, 1987. Although much of the application of Introduction biotechnology has focussed on the needs of the developed world, the most urgent needs are To a biologist, or more specifically for clearly in the developing world where the oppor­ this paper, to an animal scientist or a veterinar­ tunities and possibilities for the development of ian, biotechnology refers to use of microbial, biotechnology are numerous. Therefore, it was animal or plant cells to synthesize, break-down timely for FAQ to conduct a survey on the or transform materials into a new product. One present status of biotechnology in selected coun­ of the most important areas of biotechnology is tries of Asia (South Korea, Philippines, China, genetic engineering which is actually a combi­ Indonesia, Malaysia, Thailand, India and Paki­ nation of some modern techniques that alter cells stan). This paper is actually an abbreviated form 54 of the report submitted to F AO on the present progress using recombinant DNA technology. status of biotechnology and future plans of these Scientists at the bacteriology division have pre­ countries, based on visits to 39 institutes in 7 sented DNA profile of enterotoxigenic E. coli countries. A short report on China's develop­ through agarose gel electrophoresis, and subse­ ment of biotechnology in animal production and quently plasmid DNA profile of transconjugants health is also presented in the appendix 1. The K 88 ab and K 88 ac, which when digested with areas covered in this review include: Hind III were found to be different. Plasmid DNA profile of E.coli (K99 and F41) and the i) r-DNA technology for genetic im­ transformant (K 99) were also found to be provement and genetic resistance to diseases in different. This institute is presently engaged in animals. the gene cloning techniques using E. coli as vector. However during the visit scientists ii) Embryo transfer technology and engaged in the study (Drs. Kim and Yun) were allied techniques. not available to discuss about the gene constructs and cloning methodologies. iii) Biotechnology for enhanced feed production. India:

iv) Production of monoclonal antibod­ Major work has been undertaken by ies and vaccines. Indian Veterinary Research Institute (IVRI), Izatnagar and Bangalore campus and National r~DNA Technology Institute oflmmunology (NII), New Delhi, in the following areas: RecombinantDNAtechnologyresearch in Asia is mainly confined to basic research in (a) Molecular characterization of pox which molecular biologists are engaged in re­ virus especially caprine virus (both virulent and search involving prokaryotes. This involves non virulent types) is in progress using gene discovery of suitable vectors (plasmids and sequencing techniques. Identification of pro­ nonplasmids) for propagation of genes and moter region in the genome is being made in expression of these genes in selected hosts. Some collaboration with Virus Research Institute, biological laboratories are also engaged in DNA Pirbright, U.K. (P. Bhat - Personal communi­ fingerprinting or restriction fragment length cation). polymorphism work. Most of these biological laboratories are adequately equipped to under­ (b) Studies on molecular cloning in take major research and train scientists from clostridial species for vaccine production have agricultural disciplines. been initiated. Early work involved DNA isola­ Only a few animal science/veterinary tion of nonpathogenic clostridial species ( C. per­ laboratories were found to be engaged in mo­ fringenes, type C and type D) its characterization lecular biology research. There are very few and isolation of plasmid DNA from host vector published reports on the work done, hence most (Bhat et al., 1986). of the report here is based on personal communi­ cation or annual reports of the institutes visited. Another group of workers has been engaged in the characterization of gene sequences Korea: in Clostridium welchii type D and location of gene blocks responsible for production of "afla At the Veterinary Research Institute, toxin" (Srivastava, Singh and Ashok Kumar - Anyang, vaccine development programme is in Annual Report ofIVRI, 1986). 55

( c) Molecular characterization ofFMD fingerprinting andrecombinantDNA techniques virus type 0, A and Asia I has been attempted. for xylanase and other enzymes are available at (Suryanarayana et al., 1986). Complimentary UPM One of the researchers at UPM's biotech­ DNAs for viral RNA type A and 0 were pre­ nology group, Dr. Ubdullah Sipat has cloned pared. A c-DNA probe- has been successfully xylanase gene for Bacteroides succinogens, used for the identification of virus specific using E.coli HB 101 as the host, while he was sequences (Rao et al., 1986). working at University of Guelph, Canada.

(d) Cloned-DNA for the major antigen iv) Mitochondrial and nuclear RFLP of FMD disease has been shown to be expressed work as markers for the identification of diffe­ in E. coli (Surayanarayana et al., 1986). The rent breeds of buffaloes (at UPM) and chickens c-DNA has been cloned in the Barn Hl site and (at UM). Cloning of pituitary growth hormone at the Pst 1 site of expression vector, PUR 222. gene using retroviruses as a vector has been planned. ( e) A partial library of Y chromosome derived DNA sequence of bovine odgin in E. v)AstudyhasbeencompletedatUMto coli has been constructed at NII (Khandekar et relate the transformable antibiotic resistance traits al., 1986). inE. coli strains of bovine, avian porcine sources from slaughter houses with the frequency of R (f) Characterization of extra chromoso­ plasmids, which are extrachromosomal genetic mal plasmids responsible for desired characters markers, and are suspected to be responsible for of starter culture (Batish-personal communica­ drug resistance (Koh et al., 1988). tion) and identification of plasmids for lactose utilization in strains of streptococci (Dutta and vi) Integrated, well equipped laboratoa Sinha - NDRI Annual Report, 1986). ries had been set up forr-DNA technology work for research at UPM and UM. UPM's B.Sc Malaysia: (Biotechnology) students and UM's, M.Sc (Biotechnology) students will use their respec­ Genetic engineering research for ani­ tive laboratories for their practical work as well. mal production and health has been undertaken by Veterinary Faculty of University Pertanian Philippines: Malaysia (UPM) and two Departments of Uni­ versity of Malay (UM)-Department of Genetics Basic work on sevel'al aspects of bio­ and Cellular Biology and Institute for Advanced technology is being conducted at the National Studies. Institute of Applied Microbiology and Biotech­ Research in progress includes: nology, Los Banos, Philippines. The Institute uses protoplast fusion and recombinant DNA i) Molecular characterization of New technology in the genetic improvement of anti­ Castle Disease (NCD) virus at UPM biotic producing microbial strains. Research on local production of tylosin and antibiotics for ii) Collection of baseline data on diver­ animal feed is also in progress. sity of rumen bacteria and fungi has been com­ pleted (UPM mainly; bacterial work at UM also). Thailand, Pakistan and Indonesia: At UPM, studies on genomic and plasmid DNA in selected microbes and fungi have begun. The Veterinary Faculty of Kasetsart University, Thailand, Central Research Institute iii) Facilities for gene mapping and for Animal Sciences, Bogor, Indonesia, and 56

Department of Animal Sciences, Agriculture From the survey of 7 Asian countries, University at Faisalabad have plans to start work South Korea and India have been found to have on identification of genetic markers for produc­ undertaken large scale integrated plans for genetic tion and disease traits in livestock. improvement. Research effort in different insti­ tutions of both these countries are centrally Embryo Transfer (E.T.) Technology cooperated through the efforts of Livestock and Allied Techniques Experiment Station, Suweon, Korea and Department of Biotechnology, Ministry of Science and Technology in New Delhi, respec­ Embryo transfer technology seemed to tively. Several aspects of embryo transfer re­ be the most investigated area in the countries search in these two countries are undertaken by visited. Therefore a greater part of my review IVRI, NDRI and NII in India, and Faculties of submitted to FAO deals with embryo transfer Agriculture and Veterinary of South Korean research and planned programme for genetic National University at Suweon and Livestock improvement through embryo transfer. For the ExperimentalStation,Suweon.Embryotransfer present paper, a brief outline of embryo transfer research in swamp buffalo, cattle and goats at work is presented. UPM, Malaysia, and in Nili-Ravi buffaloes and cattle at National Agriculture Research Council Basic steps of embryo transfer inclue: (NARC), Islamabad, Pakistan is in progress. Successful transfer in goats and cattle have been i) donor selection, made in UPM and NARC respectively, and ii) superovulation, research on superovulation and embryo recov­ iii) mating ery techniques have been strengthened. iv) collection of ova, v) examination and evaluation of ova In Thailand, both Kasetsart University and embryos and and Chulalongkorn University have long term vi) transfer and freezing of embryos. programmes for embryo transfer in cattle and Special processing techniques e.g. sexing, split­ swamp buffaloes. Embryo transfer in buffaloes ting and cloning are normally undertaken when were not found to be successful yet due to poor the work on the above basic steps of embryo recovery of embryos. Similar experience has transfer are completed. been recorded at NARC, Islamabad, NDRI, Kamal and UPM, Malaysia. Prasit (1987) sug­ In Asia, commercial embryo transfer is gested E.T. to be acceptable, more research very low. As evidenced from Table 2 (lritani, concerning recovery of embryo in proper deve­ 1984), number of embryo transfer is only 1.4 lopmental stages, and a greater understanding of percent of the transferin U.S., a great majority of swamp buffaloes are necessary. Sharifuddin and which was done in Japan (Table 1). In Philip­ Jainudeen (1984) found neither the uterus nor pines,privateE.T.companiesareadvocatingthe the uterine horn could be exteriorized for non propagation of these new technology since early surgical collection ofbuffaloes. This view is also 1980's. Amongst them are ANSA farms, Mag­ supported by Madan at NDRI, Karnal. Therefore nolia Dairy Farm (MDF) of San Miguel copora­ further detail studies on the anatomy and tion and Biogenetics (Phil), commonly known as reproductive tract in relation to sperm and embryo Philgene. Similar private efforts have been made transport in buffaloes are in progress at all the in Indonesia and Thailand. However initial above institutions. success, especially in Philippines, has not been vigorously pursued in later years. 57 Basic research on various aspects of Biotechnology for Production of Feed, embryo transfer in laboratory animals (in~vitro Feed Ingredients and Biogas fertilization, freezing, cryopreservation .. .,etc.) have been attempted earlier by Dr. Im and his as­ Almost all the countries visited are sociates at Faculty of Agriculture, National Uni­ involved in some kind of work on biodegrada­ versity of Seoul, and by Drs. Bhattacharya and tion of organic wastes for production of animal Dr. Agrawal atl.V.R.I., Izatnagar. Some of their feed or biogas. Majority of these studies can not experimental procedures are now extended to be categorically called as biotechnological work small and large ruminants. since these involve known and old methods of degrading lignocellulosic materials for improve­ Malaysian Agricultural Research and ment of nutritive value of feed. Therefore only Development Institute (MARDI) has drawn up those projects/laboratory researches, which have large scale plans of embryo transfer with the either microbiological treatment for bi odegrada­ objectives of improving milk yield in Sahiwal­ tion or deal with huge machineries/fermenters/ Friesian herd and improving beef production in power plants are recorded here: Kedah Kelantan (KK) or KK crossbreds. Similar plans for improvement of cattle have been made Philippines: by Central Animal Production Research Insti­ tute, Bogor, Indonesia. 1. National Biotechnology Institute, Los Banos:

Some of the salient features of embryo a) Production of tylosin and feed anti- transfer found by most of the institutions visited biotics are: b) Low cost biogas systems using crop residues i) Surgical transfer yielded better re­ c) Production of genetically improved sults than that of nonsurgical transfer. lignolytic microorganisms. d) Enzyme engineering techniques for ii) More thf].n half the embryos recov­ synthesis of ligninases. ered are transferable. Pregnancy with rriorula e) A low technology tumbler process stage embryos is better than early blastocyst for producing microbial proteins using selected stage. strains of fungi and yeast. This can be used as a feed ingredient in poultry and pigs. iii) There is no significant difference between frozen and fresh embryos in terms of 2. Maya Farms (near Manila): pregnancy. South Korea's found no significant difference in pregnancy when transfer with one a) Production of biogas from farm embryo was compared with 2 embryos at Live­ wastes - this farm produces 94 tonnes of manure stockExperiment Sta ti on, Suweon, Korea (Table and produces 7400 cubic meters of biogas that 3). generate 7,400 kw -hours a day if all are used to run electric generators. This farm is considered iv) Small ruminants' embryo transfer to be most efficient in biogas production in Asia. seem to be more successful compared to large ruminants. Malaysia:

v) No pregnancy is yet recorded in 1. Universiti Pertanian Malaysia: buffalo. a) Utilization of agro-industrial wastes 58 as animal feed using biotechnological method- ogy, New Delhi). ologies (Jalaludin, 1986). 2. NDRI Kamal: b) Role of rumen in the digestion of fibers in ruminants (Ho et al., 1987) with a view a) Enrichment of wheat straw using to develop in-vitro system of digestion of fi­ fungal (coprinus) and urea treatment (Dr. B.N. brous materials. Gupta).

2. Dunlop (Malaysia) Sdn. bhd. Pakistan:

a) Fermentation of POME using fil­ 1. NARC, Islamabad amentous fungi and subsequent use of dehy­ drated fermented slurry (45.6 percent crude a) Fungal treatment of agricultural protein) as an ingredient of poultry/pig feed. wastes (cereal straws and sugar cane bagasse) This industrial process was developed by Dun­ (Dr. Amanat Ali). lop (Malaysia) but subsequently stopped for not being cost effective. 2. Department of Animal Nutrition, University of Faisalabad: 3. University of Malay. a) Mycelial biomass protein produc­ a) Characterization of rumen bacteria tion from rice husk, rice polish and rice straw with a view to genetic manipulation of bacteria using Trichoderma harzanum (Abu Saeed in future for effective digestion of fibre mate­ Hashmi, 1987). rials. Indonesia: b) Production of protein biomass utilizing palm oil mill effluent as substrate for 1. Central Research Institute for Animal Sci­ thermophilic andmesophilic fungi (Kuthubuteen, ence, Bogar. 1988). a) Protein enrichment of cassava tuber Thailand: using yeast inoculum (Candida ingenuosa and Canandida utalis) (Dr. Jinadasa Darma) 1. Kasetsart University, Bangkok: b) Protein enrichment ofrice straw and a) Enhancement oflignocellulosic bio­ wood chips using Pleurotus sp. (Dr. Jinadasa conversion through coupling actions of com­ Darma) mercial micro-organism in mixed culture fer­ mentation (Dr. Pomsri). 2. National Centre for Research in Biotechnol­ ogy: India: a) Protein enrichment ofrice straw using 1. Indian Institute of Technology, New Delhi: Coprinus sp. (Dr. Triadi Basuki). Korea: a) Biochemical engineering research for development of single cell proteins from There are some Universities/Institutions waste biomass (Drs. S .K. Mukhopadhaya and engaged in work on single cell protein produc­ Dr. T.K. Ghosh at Indian Institute of Technol- tion, according to Director of Livestock Re- 59 search Station, Suweon. Due to lack of time, techniques are quite common in most of the visits were not possible to these institutions. laboratories visited.

Production of Monoclonal Antibodies Work on production of monoclonal and Vaccines antibodies had been quite intensive in South Korea. Hybridoma techniques are also being Most of the Asian laboratories dealing used in some laboratories oflndia, Malaysia and with the diagnosis of viral and bacterial diseases, Philippines. Similar work has also been planned and production of vaccines have improved their in Indonesia, Thailand and Pakistan. Table 3 techniques during the past few years. Use of shows a list of diseases for the diagnosis of which immunofluorescence microscope, radioimmu­ either Elisa or monoclonal antibody is used in noassay, enzyme linked immunosorbent (Elisa) different countries.

Table 3 Elisa and monoclonal antibody (MCB) tests available for diagnosis of diseases in Asian Countries

Country Test/genetically Viral or bacterial engineered Vaccine (GEV) diseases

S. Korea M.C.A. (Monoclonal a) Infectious Bovine antibody Rhinotracheitis vims (IBRV) b) Bovine Rotavirus (BR) c) Transmissible Gastro­ enteritis Virus (TGEV) d) Japanese Encephalitis Virus (JEV) e) Porcine Parvovirus (PPV) f) Enterotoxigenic E. Coli (ETEC) g) Pseudorabies Virus (PRV) h) Newcastle Disease Virus (NCDV) i) Herpes Virus of Turkeys (HVT)

Elisa BR, IBRV (Cattle) IB, NCD, HVT, ILT, IBD, (Chickens) Rabies (Dogs). (Chickens) cholera, Pseudorabies, Japanese encephalitis, Porcine parvovirus (Pigs) 60

GEV (genetically Piglet Diarrhoea engineered vaccine) (in progress)

Thailand Elisa Swine fever (Work in progress) at National Inst. of Animal health and production, Bangkok

HVT,ILT MCA pig pneumonia (Work in progress at the same institute)

MCA Assay of progesterone (Work in progress at Chulalongkorn U.)

Philippines MCA&ELISA Pasture/la haemolytica (Work in progress at Inst. Biotech, Los Banos)

Elisa Newcastle Disease Virus (Work in progress at Bureau of Animal Industries, Manila)

Indonesia Elisa IB, NCD (Chickens), Brucella, Anthrax, Babesiasis (Cattle) Anaplasmosis (Cattle &Buffalo)

Malaysia Pelleted field vaccine (UPM - testing in different countries- in progress) UPM IB, NC (Chickens) HS, Brucella (Cattle) Detection of progesterone level under field condi- tions in bovines.

MCA NCD (Chickens) (Work in progress) 61

India Elisa Sheep pox, Buffalo pox, (IVRI) FMD, NCD, Marek's disease

MCA Horseraddish peroxidase (IVRI) enzyme

MCA FMD (Type 0, A, Asia 1) (Work in progress at IVRI)

Elisa Leptospirosis (dogs) (NII) Detection of pregnancy

M.C.A Rabies (NII - Work in progress

Pakistan Elisa Pasturella multocida (NARC, Islamabad)

Elisa Pasturella multocida (Univ. Agriculture, Faisalabad - work in progress)

MCA Pasturella multocida (NARS, Islamabad Work in progress)

General Discussion in this paper, embryo transfer seemed to be the most actively planned area in all the countries. Most of the scientists in Animal Pro­ Large scale Government funds are available in duction and Health research at the institutions India and Pakistan for the development of E.T. visited felt the need for a vigorous approach to South Korea's efforts were very practical which undertalce biotechnology based research because included a cooperative approach of Govern­ they felt keeping up with the modern techniques ment, Research Institutions, Universities and is vital for development. However there are Veterinary clinics. Initial private initiatives to some scientists who argue for the nescessity of develop E.T. had been successful but not sus­ consolidating the present work for animal tained. More recently respective Governments impro vement through conventional means are showing greater interest in E.T. Malaysia's before undertaking very highly sophisticated programme is still the resear~h level. techniques. Genetic engineering research in rela­ Of the areas of biotechnology discussed tion to domestic animals is just starting to de- 62 velop in South Korea, India and malaysia where ested in the applications of Biotechnology to substantial commitment has been made by the animal production and health. R & D funds of Government. There are capable scientists in all 7 countries visited who could The government has created a new undertake molecular biology work but because National Centre for Biotechnology and De­ of lack of funds, such work has not be pursued velopment within the State Science and Tech­ yet by many institutions visited. nology Commission. This Centre is responsible for overall development of Biotechnology in all It is strongly believed in Asian coun­ areas. The address of the Centre is: 54 San-Ii-he tries that biotechnology can utilize raw materials Road, Beijing. from various wastes to convert it to renewable resources. Microbial digestion of such raw Discussions with the Directorate of the materials using fungi and bacteria had been the Centre indicated that there is special interest in basis of research in many of the processes dis­ the following topics. covered as viable commercial processes will take time. Characterization of rumen microbes a) Training of scientific staff and also in relation to its digesting properties of various of bench technicians. fibrous material in the feed will continue to be an b) Upgrading of scientific equipment important research in the animal biotechnology. and installation of new equipment. c) Cooperative projects to identify re­ The discovery of monoclonal antibo­ search and development areas. dies and Elisa techniques for diagnosis of dif­ d) Linkages of research to practical ferent diseases in South Korea will lead to many applications. industrial activities. Similar plans and continu­ e) Training of administrators in Bio­ ous vigorous researchis necessary in other coun­ technology uses and needs. tries to improve their own potentials for diagnos­ The Centre also indicate a strong inter­ tic facilities, and perhaps later, vaccine produc­ est in taking part in the proposed FAO/UNDP tion against specific antigens. Regional Biotechnology Network for applica­ tion to animal production and health. In general, one could conclude by say­ ing that the interest for development of research During the time available for the visit, potential for various aspects of biotechnology it was not possible to obtain details of the specific exist in all the countries but due to lack of funds interests of each research institute and univer­ and facilities, concrete cooperative efforts have sity. However, the Director of the Centre kindly not been made. In light of these facts, some re­ provided the following list of organizations at commendations have been made to FAO. which Biotechnology for animal production and health is already in progress. Appendix 1

Report on People's Republic of China (Prepared by Dr. John Hodges, FAO, Rome)

The development of Biotechnology in the People's Republic of China is being given high priority. In the areas of animal production and health, the Ministry of Agriculture, Animal Husbandry and Forestry is particularly inter- 63

Beijing Institute of Animal Science, Harbin Inst. of Vet. Sci, China Academy of Agriculture Science China Academy of Agricultural Science.

Lanzhou Institute of Veterinary Institute of Genetics, Science, China Academy of Agriculture Academia Sinica Science

Institute of Development Biology Institute of Zoology, Academia Sinica Academia Sinica

Beijing Agricultural University North-West China Agricultural Univ.

North-East China College of Agronomy Inner-Mongol College of Agricultural and Animal Science.

Jiangsu College of Agronomy Heilongjiang Institute of Animal Science

Shanghai Dairy Company No. 1. Xingjiang Academia of Animal Science.

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Jo, C. H., C. K. Cheong and W. S. Hwang. Kean, C.H., S. H. An, Y. H. Kim, J.B. Lee, B. (1987). Studies on the effects of ethylene glycol H. Kim and Y. S. Lee. (1986). Production and on the survival of frozed-thawed mouse em­ characterization of monoclonal antibody against bryos. Korean J. Vet. Res. 27(2): 331 - 334. Japanese encephalitis virus. Seminar Prod. & utilization MAB (RDA & FFTC/ASPAC, Se­ Jun, M. H. (1986). Production of monoclonal oul, Korea). pp. 8 - 1 to 8 - 19. antibodies against infectious bovine rhinotra­ cheitis virus. Seminar Prod. MAB (RDA &FFTC Latif, A. Ibrahim. (1986). Vaccination of free (ASPAC) pp 44 - 1to4 - 11. range chickens against New Castle Disease. 35th. Proc West Poultry Diseases. Peutro Vallartta, Khandekar, P., andR. Chaudhry. (1987). DNA Mexico. pp 85 - 86. 66

Lee, J. H., H. Y. Park and M. H. Cho. (1986). Res. Inst. Izatnagar, U.P. p. 73. Interactions between recipients and embryos affecting pregnancy rates in bovine embryo trans­ Rao,K. V.,andO.Ramakrishna. (1987). Oestro­ fer. Korean J. Embryo Transfer. 1(1): 83 - 89. gen and progesterone changes in superovulated prepuberal buffalo calves. Workshop Embryo Luckins, A. G. (1983). Development of sero­ Biotech.Nationalinst.,Immunology,NewDelhi. logical assays for studies of trypanosomiasis of p 12. livestock in Indonesia. Baktiwan Project. Fac­ ulty of Veterinary Science, Institut Pertanian Renard, J. P. and C. Babinet. (1987). Genetic Bogor, Indonesia. pp. 68. engineering in farm animals: The lessons from the genetic mouse model. Theriogenology27(1): Manan, A. (1987). Anaerobic cellulose diges­ 1811 - 200. tion of palm oil mill effluents by rumen micror­ ganisms. M.Sc thesis, University of Malaya, Rohani A. K., 0. Olhman and A. A. a. Saiful Kuala Lumpur, Malaysia. pp. 214. (1988). Degradation of cellulosic agriculture wastes by Aspergillus terreus SUK - 1. Proc. Martin, 0. C., A. V. Nocom, E. C. Sanchez and 11th. Ann. Conf. MSAP. pp 84 - 90. Juan M. Malabanan. (1985). Bovine embryo transfer at ANSA farms - A case report. Philip­ Sharifuddin, W., and M. R. Jainudeen. (1984). pine J. Vet & Anim. Sci. 11: 324. Superovulation and non-surgical collection of ova in the water buffalo (Buba/us bubalis) Proc. Mukherjee, T. K., and S. Bhubanendran. (1986). 10th Int. Cong. Anim. Reprod. & A.I., Univ. Application of biotechnology for improvement Illionis, Urbana - Champaign, pp. 240 - 242. oflivestockproduction and health in Asia (Paper presented at F AO Expert consulation on Bio­ Shaiful, A. A. A and T. K. Mukherjee. (1987). technology for Livestock Production and Health, Microbial growth, protein and energy contents Rome, 6 - 10 October, 1986). pp. 25. of stored goat feed pellets. Biological Wastes.

Razak N., Yusof Samah, Mohamad, N. Embi, Singh, B., A. K. Sinha and B. K. Singh. (1987). 0. Omar and G. Ismail. (1987). Pili of Pseudo­ Superovulatory response in circumpuberal and monas pseudomalleic and its haem agglutinating adult Black Bengal goats. Proc. Workshop and adherence properties: Proc 10th Malaysian Embryo Biotech., National Inst. Immunology, Microb Conf. (Kuala Lumpur, Malaysia) pp 10. New Delhi, p. 8.

Padolina, W. G. (1987). Development and utili­ Supar. (1986) .. Penggunaan metode enzyme­ zation of biotechnology in Agriculture. (Paper linked immunosorbent assay (ELISA) untuk presented during the seminar on quality through deteksi antigen Pili K 99 dan K 88 padaEscher­ appropriate technology sponsored by the Philip­ ichia coli dari anak babi diare. Penyakit Hewan pine Productivity Movement through?CARRD, 18(32) : 159 - 168. PCIERD and NEC; held on 1st. October, 1987 at Bicutan, Taguig, Metro Manila). Suryanaryana, V.V. S., B. U. Rao and J. D. Padayatty. (1986). Expression in E coli of the Rao, B.U., V. V. S. Suryanaryana, C. Butchaiah cloned cDNA for the major antigen of fool and and N. Banumathi ( 1986). Research on moleclar mouth disease virus Asia 1 63/72. J. Genet. 655 characterization of foot and mouth disease virus (1 & 2) : 19 - 30. and biotechnological aspects of immunogens and diagnostics. Annual Repeort Indian Vet. Wagner, T. E., P. C. Hoppe, J. D. Jollick, D.R. 67

School, R. L. Hodinka and J. B. Gault (1981) Wani, G. H., B. A. Buchoo and N. A. Sudan. Microinjection of rabbit B-globin gene into (1987). Superovulation in sheep. Proc. Work­ zygotes and its subsequent expression in adult shop Embryo Biotech, National Inst. Immunol­ mice and their offspring. Proc. Natl. Acad. Sci. ogy, New Delhi. p. 6. U.S.A. 78: 6376 - 6380. Yang, B. S., K. S., Im and Y. B. Lee. (1985) Wagner, T. E. (1985). The role of gene transfer Studies on the solubility of zona Pellucida and in animal agriculture and biotechnology. Can. J. developmental potency of isolated blastomere in Anim. Sci. 65 : 539 - 552. mice. Korean J. Anim. Sci. 2700: 706 - 710.

Ward, K. A., I. R. Franklin, J. D. Murray, C. D. Yeong, S. W. (1981). Biological utilization of Nancarrow, K. A. Raphael, N. W. Rigby, C.R. palm oil byproducts by chickens. Ph. D thesis, Bryne,B. W. WilsonandC.L. Hunt. (1986). The University of Malaya, Kuala Lumpur, Malaysia. direct gene transfer of DNA by microinjection. pp 168. Proc. 3rd. World Cong. Genetics applied to livestock production, Lincoln, Nebraska. Vol 12. pp 6 - 21. 68

Table 1 : Summary of survey of bovine and caprine embryo transfer (238 resposes - Iritani, 1984)

Country/ No. of embryos % frozen % collected % transferred region transferred of farm nonsurgically

Asia/Africa+ 1438 10 95 21 Australia 5399 6 73 87 N. Zealand Europe 6293 24 63 67 Canada 5262 1 76 52 U.S 121855 18 57 80

+Mainly from Japan but a few were transferred in Korea (200 +),India (70 +),Philippines (60 + ), Indonesia (30 +) , Thailand, Malaysia and Pakistan (20 + each).

Table 2: Pregnancy of embryo transfer by different methods at the Livestock Experiment Station, Suweon, Korea

Method of Embryo No. Recipient No. Pregnancy Transfer (%)

1 embryo Fresh 8 4 (50.0) Frozen 73 27 (40.0)

AI+ 1 embryo Fresh 12 8 (66.7) Frozen-thawed 10 5 (50.0)

2 embryos Fresh 5 2 (40.0)

Total 108 46 (42.6)

Table 3 : Pregnancy of embryo transfer by different methods at the Livestock Experiment Station, Suweon, Korea

Method of Embryo No. No. No. Transfer Recipient Pregnancy Abortion

1 embryo Fresh 8 4 (50.0) Frozcd-thawed 73 27 (40.0) Subtotal 81 31 (38.2)

AI+ 1 embryo Fresh 12 8 (66.7) Frozen-thawed 10 5 (50.0) 2 twins Subtotal 22 13(59.1)

2 embryos Fresh 5 2 (40.0)

Total 108 46 (42.6) reeding and Biotechnology for Improvement of Livestock Feeds

R.B. Singh Regional Plant Production and Protection Officer FAQ Regional Office Bangkok

I. Introduction discusseed at this Workshop, the animal scien­ tists in the Region are moving ahead with latest techniques for improved production oflivestock, Gaps in Livestock and Feed Production but such research and development efforts are lacking for improvement of livestock feed. In 1. The livestock products index in the order not only to make up the wide gap in produc­ world as a whole increased by 17% between tion and availability but also to meet the future 1980 and 1987. During the same period in the projected growth (about 4% per annum) oflive­ Asia-PaGific Region, the index rose by 47%. stock production in the Region, all-out efforts Yet contribution of animal production to the must be made to intensify improvement and total agricultural production in most of the de­ production of livestock feeds. veloping countries is only about 10%, and the productivity, especially of ruminants, contin­ Scope of the Paper ued to be rather low. The Asia-Pacific Region, which accounted for abour 56% of the world's 3. The present paper aims to analyse the population, accounted only for about 25% and following aspects: (i) the type of feedstuffs (ii) 17 % of the world's total meat and milk produc­ the status of breeding improved varieties of for­ tions, respectively. The per caput meat produc­ age and pasture species through conventional tion in the developing countries of the Region methods of genetic improvement, (iii) current was only about 12 kg/year against 51 kg/year in status of use of biotechnology in improvement of the developed countries. Likewise, per caput livestock feeds, primarily coarse grains, grain milk production in the developing countries was legumes, tropical forage grasses and legumes, as only 26 kg/year against 109 k$f'year in case of well as cassava, and (iv) future strategy of using the developed countries. conventional and biotechnological methods for 2. One of the main reasons for poor improved production and utilization of animal performance of the animal subsector in the de­ feeds. veloping countries of the Region has been in­ adequate supply of nutritive feed. Tropical for­ 4. It may be clarified that this paper ages, particularly grasses, have lower quality concentrates primarily on genetical restructuring and consequently lower intake and digestibility of feed crop resources. There are other enormous resulting in low yields of meat and milk. On the possibilities of improving the feed quantity and other hand, as evident from the topics being quality through biotechnological methods used 70

in processing and utilization of by-products 8. In view of the above, what the devel­ including straws and stovers, and wastes, pro­ oping countries of the Region require the most is duction of single cell-proteins, harnessing pho­ intensive pasture/fodder production on small tosynthetic bacteria - as a feed supplement, patches of land with and between other crops. developing feed from algae, particuhjtly Spirul­ Consequently, the cut and carry type of fodder ina, and processing copra meal and palm oil by­ production is most common and is the main products for producing feeds. These hold great source of green fodder which provides balanced promise, but are out of the scope of this paper. nutrition, including vitamins, substitutes the costly concentrates and improves the palatabil­ II. The Feedstuffs ity and intake of roughages.

5. Pastures and grasslands, communal 9. Production of cut and carry type grazing lands, wayside grazing, crop by-prod­ fodder must be considered in relation with the ucts and residues, fodder crops, non-conven­ production of food and other agricultural crops. tional by-products ofagro-industrial processing, Given the limited land resources of majority of and concentrate feeds comprise various feed­ the farming households in the Region and the stuff resources. About 940 million ha, more than ever-increasing pressure of increased and sus­ double of the total arable land in the Region are tained food and commercial crop productions, classified as permanent pastures. Bulk of it falls fodder and feed grain crop production must fit in Australia, China and Mongolia. Except in into cropping patterns without disturbing the certain pockets in a few countries, most of the production of main food and industrial crops. pasture is probably unmanaged pasture land and This will call for choice of forage species and is available for grazing only for 4 to 5 months in development of varieties which may fit into a year. multiple and inter-cropping patterns.

6. As regards crop by-products, resi­ 10. Another agro-ecosystem which dues and stovers, such as straws ofrice, wheat, offers unique opportunity for promotion of fod­ barley, maize, sugarcane, etc. comprise a major der crops in the Region is in association with part of the feed of large ruminants in most of the plantation crops. The plantation crops, mainly developing countries. However, availability of coconut, rubber and oil palm, occupy about 50 these resources is dependent on extent of pro­ million ha in the Asia-Pacific Region. The land duction of these crops for grains and other pri­ under grown-up plantation trees is usually un­ mary products. Some of these crops, especially used or underutilized. Generally, such lands are coarse grains and legumes are major feed re­ infested with native grasses and weeds. Experi­ sources and are deliberately improved and pro­ ences in Southeast Asian and Pacific Island duced for feed purposes. countries have shown that lands under plantation crops could be sown to promising grass and 7. In Asia-Pacific Region, unlike other legume pasture species and can carry 5 to 7 regions, in most of the developing countries, beasts per ha against one on unmanaged lands. more than half of the holdings are below one ha. Crop-livestock integration was promising not And more than 50% of the ruminants are raised only from the point of view of additional income by smallholders. Bulk of the animals are raised to the farmer but also improved soil fertjlity and under mixed farming - crop plus livestock pro­ weed management of the plantations. For differ­ duction. Therefore, pasture-based livestock ent plantation crops and agro-ecological settings production, as common in Australia, New Zeal­ suitable species and varieties shouldbe selected and and U.S.A., is rather a rare or limited phe­ to ensure synergistic inter-action between the nomenon in most of the developing countries. pasture and plantation crops. \ 71 11. Ley farming is another agro-eco­ legume pastures in the tropics could be as suc­ system which has a transitional role to play cessful as the temperate legume pastures and particularly in the interior lands which have forages. recently been brought under plough and are devoid of intensive inputs. Mountain slopes and III. Breedings for Genetic remotely connected areas may also benefit from ley farming. But, this system, although impor­ Improvement of Feeds - tant under certain situations, would be of mar­ ConventionalApporach ginal significance in the overall fodder produc­ tion systems of the Region. 15. The improvements relate to in­ creased and sustained yield, wide adaptability, 12. Savannas, vast marshy and coastal and improved quality in terms of palatability, lands, hill slopes, highly acidic or alkaline soils, nutritional elements, and digestibility. To bring scrub lands, vast arid and semi-arid non-irri­ about the desired genetic improvements, ade­ gated tracts and other marginal lands, where quate variability and effective method (s) of production of food grains is uneconomical, ·screening, selecting and multiplying the desired comprise habitats where grasses and legumes variables are necessary. have proven to be efficient colonizers and many of them are actually and potentially valuable 16. In terms of genetic constitution and fodder sources. Development of forage varieties population structure, fodder species fall under suited to the marginal settings will go a long way two categories. The first category comprises in not only supporting animal production under types which have been developed through breed­ stress conditions but also in improving the de­ ing programmes as in case of cut and carry forage graded lands. crops like maize, sorghum, millet, napier grass, alfalfa, clover, cowpea, and even Leucaena 13. Grasses are the predominant vege­ hybrids. In addition to improved cultivars, tative plants of most sown pastures and native inbreds,hybrids, and synthetics of these crops, and naturalized grazing lands. Legumes usually their land races and wild relatives continue to do not contribute significantly to the herbage in constitute important genepool reservoirs for most grazing lands. Cultivated types, however, further improvement. Breeding procedures such provide a tremendous potential in improving as introduction, pedigree, back cross, mass se­ forage quality, increasing animal forage intake, lection, single seed descent, modified bulk, and maintaining a high level of digestibility, extend­ recurrent selection methods used for handling ing the grazing period, and additing nitrogen to populations derived from intra-specific and in­ the soil-plant-animal system. ter-specific crosses have generally been used for these crops and new cul ti vars released regularly. 14. Fewer legume species are culti­ vated than grass species, even though the legume 17. High heterosis for both forage and family is larger. Significance of legumes in grain yields in Fl hybrids of maize, millet and pastures, both as forage species and their benefi­ sorghum are well-known and should continue to cial effect on the growth of associated grasses, be exploited through choice of good combiners. was high-lighted with the high productivity of Furthermore, there are wide differences in ra­ pastures in the temperate countries. However, it tooning abilities of sorghum hybrids, synthetics received little attention in the tropical countries and standard varieties which should be exploited. until some 25 years ago when the Australian New accessions should be screened as potential forage improvement project in the tropical con­ parents for Fl production. For instance, in pearl ditions of Queensland demonstrated that the millet, Burton and Davies (1984) used mixed 72 pollen from the accession plants in the quaran­ is lightly grazed during this period and then tine planting and placed them on the cytoplasmic utilized as Foggage (standing hay) in the dry male sterile heads of Tift 23DA to produce Fl season. This one legume aided considerably in hybrid seed. These hybrids were tested against the transformation of natural grazing lands, where the standard hybrid Gahi 3 in replicated yield one animal roamed over 15 to 20 ha of poor trails. Those accessions giving the highest yield­ quality native grass to pastures that now support ing hybrids were considered the best sources of one animal unit on slightly more than one ha. genes for yield and were used in several breeding Townsville stylo seeds are produced commer­ schemes. cially and several new cultivars have been se­ lected among variants occurring in the diverse 18. The second category of species are population (Hutton, 1970). generally pasture and range land species which comprise wild, weedy and semi-wild forms. In 20. Ecotypic selections and mass selec­ recent years, some of these have become domes­ tions in introduced populations have been quite ticated, usually in the countries other than their successful. Burt,Reidand Williams (1976), based own, as for example Mediterranean, African and on a study of an extensive collection of Stylosan­ South American pasture grasses and legumes in thes spp. showed that there was a close relation­ Australia. The superior eco-types were given ship between groupings formed purely on mor­ specific cultivar names and released for general phological-agronomic classifications, and the production. For example, Greenleaf (Desmodi um climates of the countries of origin. Such studies intortum ) is a mixture of three introductions were helpful in identifying the most promising from El-Salvador and Guatemala and Silverleaf sites for locating introductions, but also for indi­ (D. uncinatum) represents a single introduction cating the climate, hence the region for obtaining from Brazil (Hutton, 1970). These two cultivars promising introductions. have greatly contributed not only to Australian livestock industry, but their seeds were shipped 21. From the above it is clear that the to almost all parts of tropics, including the coun­ pasture and rangeland species are mostly straight tries from which the introductions came and ecotypic selections from wild and weedy forms where common genotypes of both cultivars still and little genetic upgradation of these species exist in the native vegetation. has taken place even through conventional breed­ 19. Several cultivated species came to ing programmes let alone biotechnological tools. the attention of cattlemen or pasture agronomists Therefore, there is tremendous scope for genetic as propitious introductions. Under favourable improvement of these feed resources by collect­ conditions, seeds scattered and became an im­ ing, evaluating and selecting from the natural portant component of the natural vegetation. populations and further using them in breeding Such was the case of Townsville sty lo in north­ programmes. The accessions collected from eastern Queensland, Australia (Humphreys, diverse agro-ecological and climatological con­ 1967). This legume was found to be widely ditions should be critically characterized, docu­ spread around Townsville in the 1920's where it mented and conserved for future use. was relished by cattle. It is thought that seeds arrived by ships from Brazil in the early 20th 22. Following predominant forage leg­ century, and the environment was suitable for umes have South and Southeast Asia as their natural selection and development of an ecotypic primary or secondary centres of diversity: population. Townsville sty lo is an annual which produces 2 to 3 tons per ha of dry herbage as well as a heavy seed crop during the rainy season in northeast Queensland and Northern Territory. It 73

Primary Centre of Diversity Secondary Centre of Diversity

Alysicarpus regosus Calo po gonium mucunoides 11 vagina/is·. Canavalia ensiformis Desmodium biarticulatum Centrosema plumieri 11 heterocarpon 11 pubescens 11 heterophyllum Clitoria ternatea 11 ovalifolium Desmodium canum Macrotyloma uniflorum II intortum Mucuna pruriens 11 scorpiarus Pueraria phaseoloides 11 triflorum (in PNG) 11 thunber giana 11 uncinatum Teramnus labialis Glycine wightii Vigna hosei Lablab purpureus 11 radiata Lablab purpureus II unguiculata Stylosanthes guianensis 11 humilis

23. Of the above listed species, the under marginal lands and stress conditions and selected species of only four genera, namely, mixed, relay and companion craping; the condi­ Stylosanthes, Centrosema, Desmodium and tions under which these have evolved. There­ Pueraria, predominate the pasture and forage fore, for judicious exploitation of the vast mar­ cultivation. Since all these species are self-polli- ginal cropping conditions, collectionoflegumes natedandtheselectedgenotypesarespecifically from such habitats is important for identifying fitfordefinedagro-ecologicalhabitats, themani- and utilizing the desirable genes. Further, sev­ fold wildforms are beingselectively eliminated. eral legume forms co-inhabit the rich tropical Therefore,collectionandconservationofnative rain-forests and put up lexurious growth even and wild populations of these four genera de- under shades. Sampling of such populations serve top priority. would yield valuable genotypes to be grown 24. Information on population archi- under the sizeable plantation and orchard acre­ tecture, breeding system, ecotypic adaptation age in the Region. The associated variation in strategies, responsiveness to Rhizobium strains, Rhizobium should also be conserved and evalu­ ability to utilize limiting macro- and micro- ated. elements, seed dormancy, seed storage ability, 26. In this Region, Australia has been a reaction to pests and diseases, soil pH, domesti- pioneer in collecting, conserving, evaluating, cability, freedom from toxic substances,photop- and utilizing pasture species. Through its inter­ eriodic responses, tolerance to drought or ill- national programmes the country has been as­ drained conditions, competitive aoility and per- sisting several countries in the Region in pasture sistence, etc., is extremely valuable for efficient research and. management, including genetic sampling of the natural populations as well as for resource activities. Several of the developing utilization of the collected germplasm. Such , countries have also lately been giving some information are generally missing in case of \ attention to this activity. Through collection pasture and forage legumes. ·expeditions carried out since 1979, Thailand, Malaysia, Indonesia, Papua New Guinea, and 25. In the years to come, legumes as China, netted 1500 samples of several forage forage crops are likely to gain greater favour legumes (Table 1; Schultze-Kraft, 1986). The 74

collection missions indicated that (i) only a small forms are eroding fast due to extension of arable fraction of the natural variability has been col- cropping. lected in the form of germplasm, and (ii) the

Table 1: Collection of Native Forage Legume Germplasm in Southeast Asia

YEAR COUNTRY PRINCIPAL GENERA COLLECTED NO.OF SAMPLES

1979 THAILAND DESMODIUM 77

1982 THAILAND, MALAYSIA DESMODIUM,DENDROLOBIUM, 362 PHYLLODIUM, PUERRARIA TADEHAGE

1983 INDONESIA DESMODIU, URARIA 56 ALYS/CARPUS

PAPUA NEW GUINEA DESMODIUM, CODARIOCALYX, 138 FLEMING/A, PUERARIA, PYCNOSPORA, URARIA

1984 CHINA: HAINAN ISLAND DESMODIUM, PHYILODIUM, 92 TADEHAGI, PUERARIA, ALYS/CARPUS

THAILAND DESMODIUM,DENDROLOBIUM 154 PHYLLODIUM, TADEHAGI, PUERARIA

INDONESIA DESMODIUM,AESCHYNOME, 28 ALYS/CARPUS

INDONESIA DESMODIUM, CALOPOGONIUM, 494 CENTROSEMA,PSEUDARTHRIA, PUERARIA, URARIA

27. The primary objectives of the above­ such as Pueraria phaseoloides, mentioned collections were two fold (Schultze­ Desmodium ovalifolium, D. Kraft, 1986): he terophyllum and D. heterocar­ pon whose genetic bases have been i) To increase the variability in the extremely narrow, the expectation well-known 'traditional' species, being to find genotypes with which 75

any major constraints in the spe­ fully. Some of the promising but non-traditional cies could be overcome; species are: Arachis pintoi, Centrosema brasili­ anum, C. macrocurpum, Zornia glabra, Z. Lati­ ii) To make available for evalu­ folia, S. capitata, S. guianensis var. pauciflora ation purposes a broad range of and S. macrocephala. These should also be new species, which may be introduced and evaluated systematically. agronomically as yet unknown or only little known, the expectation 30. As regards grasses,Jmperata cylin­ being to identify new species with dric a is the dominant native grass species, a forage potential. Such species although another 10 or 12 grass species such as are: Desmodium strigillosum, D. Arundanaria spp., Dactyloctenium aegypti - styracifolium, D. gangeticum, D. cumDigitaria adscendens, Eragrostis viscosa, sequax,D. velutinum, Codarioca­ Callipedium parviflorum, Chrysopogon acicu­ lyx gyroides,Dendrolobium spp., latus, Themeda triandra, T. anstralis,Axonopus Dicerma biarticulatum, hegnera compressus, I schaemum muticum, !. timorense, obcordata, Phyllodium spp., Ottochloa nodosa, Paspolum conjugatum, Tadehagi spp., Uraria spp.,Dun­ Arundinella setosa, Dicanthium caricosum, baria spp., Flemigia spp. These Pennisetum polystachyon, Cenchrus ciliaris, species should be evaluated under and Pennisetum pedicellatum occurred widely diverse conditions to ensure their in several countries of the Region. But, variable fuller exploitation. forms/ecotypes of these and other species are poorly represented in the national collections. 28. Among the potential species, Adequate collection of germplasm should re­ Desmodium strigillosum and D. styracifolium ceive highest priority through carefully planned are resistant to little leaf mycoplasm and root­ expeditions within and outside the countries. knot nematode (CIAT, 1984). Besides, these Jack R. Harlan (1981), a pioneer in genetic species are tolerant to low-fertility and drought resources and breeding of grasses, underlined conditions. Further, D. sequax, because of its that several plant breeding programmes have vigor and lack of disease problems, holds good failed due to inadequate collections. promise, in the tropics, andD. velutinum shows good growth on acid and infertile soils. 31. While there are serious gaps in 29. Tropical America is generally re­ collections and there is an urgent need of rescu­ garded as the major centre of diversity of tropi­ ing the genetic resources which are eroding fast cal legumes and most of the cultivated species under population, socio-economic and environ­ areoftropicalAmericanorigin(Williams, 1983). mental pressures, the accessions which are al­ Although Africa has provided three commercial ready being maintained/conserved in different forage legumes, namely, Lablab purpureus, genebanks and repositories should be evaluated, Lotononis bainesii, andNeonototia wightii, the documented, and information as well as selected New World tropics continue to be considered a materials shared among interested countries and particularly promising cource of legume institutions. Guidelines for this work already germplasm. Much of the recent plant explora­ exist (Sing and Williams, 1984; IBPGR, 1981) tion activities have concentrated on tropical and should be widely followed for harmoniza­ America. Species like Calo po gonium mucunoi­ tion of collecting and sharing the information. des, Centrosema pubescens,Macroptilium atro­ purpureum, Stylosanthes guianensis, S. hamata, 32. No doubt, the evaluation patterns S. humilis, and S. scabra are traditional species will be determined on the basis of the intended of known potential and should be exploited use of the species. However, while evaluating 76 forage species, attention should particularly be come this difficulty, it would be desirable to paid to persistency, herbage yield, herbage transfer an apomictic gene into pearl millet quality and nutritive value, legume/grass com­ from one of its allied species either through patibility, propagation and establishment abil­ conventional breeding or biotechnological ity, seed production ability adaptation to stress methods. conditions and resistance to pests atid diseases and other stress conditions such aS problem 34. Like in forage legumes, in forage soils, drought and frost. In legumes, particu­ grasses also there is great scope for selecting larly, shortened internodes, increased nodal promising cultivars from germ plasm collections/ rooting, reduced alkaloid contents etc., are introductions. To cite an example, thePanicum important characteristics. maximum collections of ORS TOM, numbering 400 accessions, were sent to Brazil and per - 33. In grasses, where apom1x1s is formance of 156 accessions and means for the common, mode of reproduction should be best 25 accessions based on evaluations over checked carefully and the accessions should be two years are given in Table 2 (Savidan et al., grouped into following three categories: (i) 1985). obligate or nearly obligate apomicts, (ii) facul­ tative apomicts, and (iii) obligate sexuals. This 35. It may be seen from the table that grouping is extremely useful for further there is immense variation in the accessions breeding programmes and for production of Fl introduced and new varieties superior to the best seeds of selected hybrids. Some of the cultivated locally available variety could be selected from grass species such as pearl millet (Pennisetum the introductions. Besides, the variability could typhoides ) are obligately sexual and their Fl be channelized into further breeding program­ seed is to be produced and distributed for every mes. fresh crop, which is a costly operation. To over-

Table 2: Variability for components of forage quality and leaf dry matter production in 156 accessions of Panicum maximum, compared to performance of the cultivar "Coloniao"

Agronomic Range of Meanof25 Coloniao Component Variation

% leaf/DM 25.4 - 84.3 79.1 62.0 % CP/leafDM 11.5 - 17.7 15.9 16.1 %IVDMD 52.2 - 69.2 64.7 66.5 Leaf DM (t/ha/yr) 2.4 - 37.8 28.0 14.3

IV. Biotechnology for Improve .. ideas and techniques, popularly referred to as biotechnology, for improving the production, ment of Animal Feeds productivity, quality, and utilization of agricul­ tural and other biological products. The term 36. Front-line researches in the bio­ "biotechnology" has been used in widely logical sciences have been generating novel different contexts, from the production of bi- 77

ogas to the in vitro synthesis of food and other niqueformicro-propagation. However, the tech­ bio-products. In the present paper, only those nique is useful in case of cassava for initial rapid biotechnological techniques which have actu­ multiplication of selected clones for distribution ally or have the potential to increase the effi­ and organizing initial yield evaluation trials but ciency of conservation, exchange, identifica­ not for commercial production as the crop can tion, selection and cross-transfer of desirable easily be propagated through stem cuttings. genes for improvement of livestock feedstuffs have been considered. In Vitro Culture for Germpiasm Exchange 37. Before the use of the individual biotechnological techniques.in improvement of 40. Micropropagation is already prov­ feeds is described, the writer would like to ing as a valuable tool for quarantine purposes emphasize that he views the biotechnological while exchanging germplasm and transferring techniques as an aid to conventional breeding planting materials. In vitro cultured materials techniques to help in attaining certain desired from healthy explants are the most accepted outputs more efficiently and quickly than other­ materials from the point of view of exclusion of wise. Table 3 summarises the possible contribu­ new pests and diseases and this approach is tions from biotechnology to practical plant breed­ already being extensively used in case of cas­ ing (Cohen et al., 1988). Described below are sava and other root and tuber crops. Planting the techniques which are already being applied, materials prepared through micropropagation or have potential for application for feed im­ of Brachiaria germplasm collected in Africa by provenment: CIAT could be distributed to several institutions In Vitro Clonal Propagation in Latin America, free of diseases, while Pani­ cum maximum collections of ORSTOM intro­ 38. This technique is used for the rapid duced in Brazil in usual manner were infested true-to-type multiplication of selected genotypes. with nematodes and fungal - diseases (Savidan, Successful somatic cell cultures and efficient 1988) which is objectional from the quarantine regeneration methods for several forage grasses point of view. and coarse grain cereals have been established (Vasil, 1985, 1987). Similar successes have 41. Often, through in vitro culture, it is also been attained in several of the forage possible to free a diseased material of the patho­ legumes as well as root and tuber crops. But gen, even viruses. Suitable techniques, such as majority of the grasses produce plenty of seeds ELISA test, have been developed for pathogen and at the same time could be propagated vege­ indexing and should be used widley. FAO RAP A, tatively (rhizomes, runners, etc.). Furthermore, through an expert consultation, developed guide­ a large number of the forage grasses produce lines for the use of tissue culture for quarantine apomictic seed hence can be clonally multiplied purposes in exchange of germ plasm and transfer through seed - the most efficient method of of planting materials in 1987 and commended its clonal propagation. Therefore, there is little use widely. advantage of in vitro clonal propagation of forage grasses for the purpose of rapid multipli­ In Vitro Conservation cation. 42. Clonally propagated species and 39. As regards forage legumes, gener­ those producing recalcitrant seeds are best con­ ally these are self-pollinated and set abundant served in in vitro. This technique is being rou­ seed, hence can benefit little from in vitro tech- tinely practised in case of cassava. However, 78

somaclonal variation could cause problem. stabilizing unique gerrnplasm in homozygous Therefore, further research is needed to ensure form as the case may be in polyploid forage genetic stability of cultures. grasses. Through haploidy and haploidization selection efficiency can be increased. Fmther, Somadonal Variation haploid vegetative tissues can be used to prepare protoplasts from species to be hybridized by 43. Ordinarily, plants regenerated from somatic cell fusion to produce diploid hybrids. cells derived from a single plant are supposed to be identical. But, sometimes considerable vari­ Embryo Rescue ations are found for a range of characteristics. This variation, called as somaclonal variation 46. In distant crosses, sometimes seeds can be and has been used by plant breeders in fail to form due to embryo abortion. Before the selecting new varieties, such as in sugarcane. abortion sets in, embryo can be isolated and Among forage species, Davies et al. (1986) re­ cultured on suitable media to the stage of viable ported somaclonal variations for vigour, growth seedling. Phillips et al (1982) used this tech­ habit, leaf width and leaf chlorophyll concentra­ nique for production of a perennial interspecific tion in Paspalum dilatatum. Somaclonal vari­ hybrid between Trifolium pratense and T. ations have also been reported in maize, barley, sarosiense. This technique is rather relatively and alfalfa. The variations concern amino-acid simple but highly effective and should be used over-production, resistance to herbicides and wherever necessary. diseases, and tolerance to heat and problem soils. Somatic Hybridization

44. Somaclonal variation, like muta­ 4 7. Fusion of two distinct somatic cells tion, is random and nonpredictable. This ap­ and regeneration of plants from the somatic proach may be useful in those cases where there hybrid cell offers unique opportunities for dis­ is dearth of variability in the naturally occurring tant hybridization and circumventing normal forms. Considering that most of the forage spe­ reproductive barriers, thus facilitating transfer cies have not undergone intensive breeding and of genes from remotely related or even unrelated selection, and are generally still near-wild or plant species. Following the initiative of Cock­ weedy types, there exists impressive natural ing (1960),Nitsch and Ohyamaforthefirsttime variability which is yet to be collected, char­ regenerated normal plants from tobacco proto­ acterised and utilized. Therefore, there is gener­ plasts in 1971. Since then, plant regeneration ally no justification in resorting to somaclonal from protoplasts and isolation of somaclonal variation while not even a fraction of the impres­ variants has been reported in several crops, more sive naturally occurring variability has been frequently in solanaceous species. As regards exploited. feed crops, regeneration from protoplast cul­ tures oflegumes is rather difficult, yet successes Use of Haploids have been reported for Medicago satava, Trifo­ lium repens, Trigonellafoenum-graecum, and 45. Anther culture and other techniques Crotalaria juncea, and Vigna aconitifolia (in have been used to produce haploids in several Krishnamurty et al., 1984). crops, including, maize, rye, barley and fescue. Kasha and Rein bergs ( 1980) used this method to 48. Parasexual plants have been lim­ develop a new barley variety only within five ited primarily to those families which are more years. Haploids can be used for production of amenable to protoplast culture. The production homozygous inbred lines for Fl hybrids and for of true somatic hybrids between Medicago sa- 79 tiva and M. falcata (in Pelletier and Chupeau, for barley, maize, rye, oats and other cereals. 1984) are encouraging and can provide new Similarly, based on single seed test (without dimensions for improvement of the cultivated destroying the seed which could be used for species. Protoplastfusions already demonstrated growing the next generation) protein content in case of barley, maize, and several legume and amino acid profile of the desired material species, with standardization of techniques for could be known and the information may be plant regeneration, would be extremely helpful used for rejecting or retaining the material for in limited gene transfer forresistance to diseases further progeny testing. and pests, tolerance to problem soils, drought, and frost, and also for improvement of protein 51. Monitoring the presence of genes, content, amino-acid profile and other nutritional either directly or indirectly, is being further factors both in grain and vegetative parts. developed using arange of new diagnostic tech­ niques, such as probes for specific nucleic acids 49. Rapeseed and mustard cake is an and their sequences, monoclonal antibodies, and important feed in the Indian subcontinent and in labelling. In practical plant breeding, however, other regions. Normal rape protoplasts were the application of these highly valuable tech­ fused with rape protoplasts having a cytoplasm niques is still oflimited use. However, as know l­ from radish. The regenerated plant combined edge accumulates, the use of these techniques favourable attributes of normal chlorophyll for identification of gene products and genes synthesis, normal nectaries and cytoplasmic male themselves will greatly speed up the breeding sterility and could be utilized for heterosis breed­ process, particularI y for disease resistance as the ing ofrapeseeds (in,Rajnchapel-Messai, 1983). expression of one gene may mask the expression Further, through three-way somatic fusion of other genes. involving Brassica campestries (wild turnip), a normal male sterile rape line with resistance to Selection at Cell Level herbicide atrazine was produced. With suitable modifications, these rape hybrids could be intro­ 52. For those species which can be duced in sub-tropical and even warmer regions regenerated through in vitro culture, selection which will produce not only protein rich cake techniques can be applied at cell level in the but also edible oil which is in short supply in culture, in the same way as done for bacteria, and several countries of the Region. Furthermore, the scale of screening can be enhanced rape variaties with low content of sulfur com­ enormously. In maize, herbicide resistant lines pounds (glycosinolates) have been developed by exposing cells to sub-lethal doses of herbi­ and are suitable for feeding even young animals, cide in the culture media were selected (Shaner and because of the good palatability of rape­ et al, 1985). Applying toxins produced by fungi cake they are preferred by milk-cows. and bacteria, cell lines resistant to the toxins of HelminthosporiummaydisraceT(Southemcorn Screening for Desirable Traits leaf blight) were isolated which developed into resistant maize plants. Similar approaches are 50. Screening of segregating popula­ being used widely in barley, sugarcane, oats, tions, which is a common practice in breeding sorghum and pearl millet and hold great prom­ programme, can be made more efficient through ise. techniques applied at early stages or even to the seed. Biochemical tests for height gene can 53. The technique is helpful also in select dwarf and tall types at early seedling selecting lines with higher contents of specific stages (Gale and Gregory, 1977; sharma and amino-acids, particularly the essential amino­ Singh, 1984) and the technique can be applied acids. Using this approach, maize lines with 80

33% to 59% increased threonine content and it was possible to increase amounts of free 1ysine barley lines with 10% to 20% higher lysine and and threonine (Bright et al 1984) or by isolation tryptophane contents have been isolated. and modification of the genes for aspartate ki­ nase, other enzymes, or both. Genetic Engineering 57. Cassava is a major feed crop ex­ 54. Raw materials of plant breeding ported from this Region to EEC countries and is can be manipulated through genetic transforma­ a major potential feed crop of this Region. Its tion as single genes can be identified, isolated, feed value can immensely be improved if its cloned, and in some instances, inserted into protein content could be increased. Recently, other organisms and made to express them­ work on potato has led to development of potato selves. Important physiological traits such as lines with as high as 4% protein and the line is photo-synthesis, photorespiration, biological named as Protato. Such a possibility exists in nitrogen fixation, and protein synthesis and cassava also. Further, work in Indonesia (Na­ storage, could be altered to maximise economic tional Centre for Biotechnology Research, yield and the efficiency of resource utilization. Bogor) showed that cassava protein content A plant can be engineered to produce its own could be increased five-fold within 48 hours by insecticide, such as transfer to solanaceous plants growing Rhizopus mould on ground cassava the gene for the insect toxins produced by tuber in a medium containing 2.03 gm of urea Bacillus thuringiensis (Vaeck et al., 1987.) per 100 gm of ground cassava tuber.

55. As mentioned earlier, coarse grains V. Future Prospects are major animal feed. Protein and amino-acid content of these feed resources determine their 58. To meet the ever-increasing de­ nutritional value. Storageproterins (prolamines) mand oflivestockproducts, production and utili­ in these grains are usually low in essential zation of feeds must be intensified. The develop­ amino-acid content. For instance barley is defi­ ing countries in the Region have so far been cient in lysine, threonine and histidine as feed mainly concerned, and understandably so, on for pigs (Fuller et al 1979). For maize, limiting increasing food production. In the process, the amino-acids are lysine and tryptophane (Bres­ improvement of feed resources got neglected. sani, 1975). Lately, after having attained considerable suc­ cess in food crop production, the countries are 56. In maize, two important genes, Op- paying little more attention than in the past to 2 and Floury, were identified and used in con­ improved production of feeds in order to ventional breeding programmes to develop ly­ strengthen their livestock production. sine and typtophanerichmaize varieties without sacrificing the storage protein content. In barley 59. In most of the developing coun­ also several protein mutants were isolated but tries, even the basic elements of improved pro­ were not as favourable as Op-2 and Floury of duction of feeds are, yet not in place. There is: maize. But work of Showry and Kreis (1987) has lack of improved varieties specifically bred for shown that barley seed quality can be improved feed purposes, paucity of seed of recommended by genetic engineering. While maintaining a varieties at the right time and in the desired high level of storage protein synthesis in grains, quantity, and poor use of rhizobial inoculants balanced decrease of prolamine and increase of and other mangement practices. Redressed of lysine-rich salt soluble proteins could be at­ these shortcomings is fundamental to the im­ tained, provided the latter did not affect the grain provement of livestock feed. development. Further, through genie mutations 81

60. As regards genetic improvement of advisable to identify priority feed crop species, livestock feed, the conventional and biotechnol­ two to three grasses and lwo to three legumes ogical approaches should be seen together in a and may be cassava to be improved through balanced manner. Most of the grass and legume biotechnological techniques. Based on national forage and pasture species have not been sub­ strengths, centres of excellence for biotechnol­ jected to intensive breeding. The cultivars are ogical work on specific feed crops/commodities still pretty close to their wild progenitors and could be assigned to specific centres/countries. weedy relatives. The vast naturally occurring Based on their capabilities and interests, other variability is largely untapped. Systemtic utili­ countries may participate in activities of one or zation of the variability is bound to give reward­ the other or all the centres. ing results. Therefore, the countries should first pay attention to collecting, conserving, evaluat­ 63. As regards coarse grains, work on ing and utilizing indigenous and exotic grain protein content and amino-acid profile is germ plasm to enable them to establish appropri­ already engaging attention of the North. In the ate breeding programmes. Development and South also a few laboratories are working on large-scale adoption of location-specific man­ these aspects. Greater interaction among these agement practices, including varieties, should groups should be strengthened to share the tech­ be emphasized. nology and to avoid reinventing the wheel. Such laboratories in the Region should be encouraged 61. As regards biotechnology for im­ to intensify their biotechnological work related provement oflivestock feed, no doubt, the tech­ to increased accumulation of good quality pro­ nique is proving effective in several cases and tein in the sinks of coarse grains, other grasses, holds great promise as an aid to the conventional and legumes. breeding approaches. The use and degree of adoption of biotechnological techniques would 64. Of the several aspects of biotech­ vary from crop to crop and country to country. nology, such as in vitro culture for micropropa­ Depending on circumstances, even simple tricks gation, germplasm conservation, exchange, at times could be highly topical and useful. The somatic hybridization, selection at cellular level, introduction to Africa by ILCA of the bacteria gene and gene product monitoring, and genetic responsible for degrading mimosine (a toxin engineering have topical uses for solving spe­ found in leucaena)is an example of how the use cific problems of specific crops. Each of these of bacteria has made it possible to make 'leu­ must be assessed in terms of activities to be caena an excellent fodder crop (Buttle, 1986). undertaken under the regional network arrange­ All countries, especially the developing ones, ment. Each aspect is important in its own right. need not establish a full-fledged national pro­ But, all the aspects need not be pursued at this gramme on genetic engineering of forage, pas­ stage by everyone, otherwise the very spirit of ture and feed crops. Given the limited research cooperation is marred and there is a tendency of and development resources, most of the coun­ undertaking avoidable duplicative work. tries may like to concentrate on conventional breeding, agronomy and extension activities, 65. With the interface of plants and but keep abreast of the development in the field animals, each with its own conventional and of biotechnology for feeds and take up to a few new-technology scientists, It may be empha­ selected aspects ofbiotechnology based on their sized that, in case of feeds for Iivestock, applied felt needs. and fundamental research should not be done entirely in separate compartments. In establish­ 62. Given the diversity of feed crops, ing the network, such compartmentalization for a networking regional programme, it may be should be avoided. Ida F. Dalmacio University of the Philippines at Los Banos College, Laguna, Philippines

Introduction considered. However, pretreatment of rice straw with urea as a source of ammonia seems to be of practical application in small farms in Asia Fibrous crop residues are agricultural (Doyle, 1988). by-products which have low digestibility and low nitrogen content; hence, have poor feeding Physico-chemical treatments have been value. The low nutritional quality can be attri­ proven to increase digestibility better than physi­ buted to the presence of insoluble substances in cal or chemical treatments alone (Table 2). the cell wall which prevents the access of micro­ Apparently, the physical treatment increases the organisms and enzymes to the cell components surface area (Jalaludin, 1988). Attempts to increase the of the plant material which comes in contact with the chemical thereby enhancing its feeding value of such residues have been effectiveness. made(Figure 1) and are well documented. The methods include physical, chemical and biologi­ cal treatments or their combinations. Biological and BiotechnologyaBased Treatment Processes Biological and Chemical Methods of Composting and ensiling are com­ Treating Fibrous Crop Residues mon practices which allow development of or­ ganisms in the residues in order to improve their Of the physical treatments available, quality. However, composting has been cited to chopping and soaking are the simplest and would unlikely improve the feeding value ofrice straw seem to have the greatest potential for large scale (Doyle, 1983) and ensiling has likewise been use in developing countries, although more severe reported to unlikely improve the in vivo dry physical treatments can also result to higher in­ matter digestibility of straw (Vanselow, 1983). creases in in vitro digestibility -of fibrous mate­ rials (Table 1) (Doyle, 1988). In 1979, Burrows et al., reported the Chemicals which solubilize the indi­ increase in the digestibility of barley straw from gestible cell wall constituents particularly so­ 45%-about 55% after growth of Coprinus cin­ dium hydroxide, calcium hydroxide and ammo­ ereus for 10 days. A similar experiment on the nium hydroxide/anhydrous ammonia have been continuous recycling of rice straw in mushroom used for chemical treatments. Although effec­ cultivation for animal feed has been tried by tive in improving the digestibility of straw, the Quimio (1988). Results of her experiment cost and potential hazard to the user as well as to showed that Volvariella growth for 4 weeks the environment are factors which have to be caused a reduction in the crude fiber ofrice straw 83

Figure 1. Treatments available for improving the nutritive value of crop residues.

I CROP RESIDUES I

I PRETREATMENT I

I I I PHYSICAL CHEMICAL PHYSICO-CHEMICAL BIOLOGICAL

Soaking Sodium Hydroxide Particle Size/Chemicals Addition of Enzymes

Chopping Calcium Hydroxide Na OH/Pelleting White Rot Fungi

Grinding Potassium Hydroxide Urea/Pelleting Mushrooms

Pelleting Ammonium Hydroxide Lime/Pelleting

Boiling Anhydrous Ammonia Chemicals/Steaming

Steam Urea/Ammonia Na OH/Temperature Under Pressure Sodium Carbonate

Gamma- Sodium Chlorite Irradiation Chlorine Gas

Sulphur Dioxide

I RUMINANTS I

HUMAN I From: Ibrahim, 1982 84

Table 1. Effect pf physical treatments on potential (in vitro ) organic matter digestibility of fibrous residues.

Treatment Substrate Digestibility Reference Untreated Treated

Ball milling Aspen 11 55 Moore et al. (1972)

Gamma irradiation Barley straw 38 61 Ibrahim & Pearce (1980b)

Steam under pressure Barley straw 38 59 Ibrahim & (170 °C for 1 hour) Pearce (1982)

Grinding Barley straw 40 53 Coombe et al. (1979b)

From Doyle (1988).

Table 2. In vitro and in vivo digestibility and organic matter intake (OMI) and digestible organic matter intake (DOMI) of barley straw by steers.

Treatment In vitro In vivo DMD DMD OMI DOMI (%) (%) (kg/day) (kg/day)

Chopped straw 43 55 3.49 2.00

Water soaked chopped straw 38 56 2.72 1.60

Ground pelleted straw 53 47 6.01 2.99

NaOH treated straw 77 66 4.49 3.01

Ground, NaOH treated

pelleted straw 66 59 6.23 3.74

From Coombe et al. (1979b) 85

from 25.9%-20.22% (Table 3). Further, she substrate, and the development of hypercellu­ reported that the lignin remaining after Volvari­ lase-producing organisms through mutation e lla growth can be degraded by growing another (Joglekar and Karan th, 1984 ). Pro top last fusion mushroom, Pleurotus sajor-caju (Table 4). of cellulase-producing penicillia is also cur­ Although simultaneous application of cellulase rently being worked on in this regard (Santos, and ligninase have been suggested as a better 1988). approach (Kim, 1981), the use of mushrooms seems to be the most economical among the Some agro-industrial fibrous residues methods used to improve feeding value or sat­ in Asian countries and their composition are isfy the nutritional needs of ruminants (Quimio, listed in Table 6. Although copra meal, the 1988). ground residue obtained after the extraction of part of the oil from the dried meat of the coconut Biotechnology, with respect to the (Seiden, 1957), is not included in the list, this improvement of fibrous crop residues for use in commodity has lately been given attention by animal feeds, would denote the use of micro­ biotechnologists in the Philippines. organisms or parts of cells such as enzymes to produce commercial quantities of useful sub­ Copra meal is by far the most abundant stances (Schneiderman, 1987). Compared to the feed stuff ingredient that the Philippines can physical and chemical methods, the use of offer. However, only a small fraction of the microorganisms is a relatively new approach. locally produced copra meal is used for live­ With the use of microorganisms, specific iso­ stock and poultry feeds. A deterrent to the exten­ lates have to be identified for particular sub­ sive use of copra meal as feedstuff is its low strates and optimum conditions for the growth digestibility due to its fiber content. Past efforts and enzyme production of the organism have to have not been very successful because of the be established in order to achieve the desired common belief that copra meal was mainly cel­ result. Likewise, improvement of selected or­ lulose. However, with the elucidation of the ganisms through muta.tion, hybridization, proto­ chemical composition of copra meal that the plast fusion or recombinant DNA technology carbohydrate consists mainly of mannan (Balas­ have to be done in order to further increase the ubramanian, 1976), isolation of mannanase­ degradative capability of selected organisms. producing organisms was intensified. The ap­ proach then taken at the National Institutes of A number of microbial genera are Biotechnology and Applied Microbiology capable of utilizing cellulose, hemicellulose (BIOTECH) atthe University of the Philippines and lignin (Table 5) which are abundant in plant at Los Banos (UPLB) was to hydrolyze the by-products. Enzymatic hydrolysis of cellulose mannan by using mannanase from Streptomyces compares favorably to acid and alkali hydrolysis spp. The theoretical effects are three-fold: (1) because enzymes are recoverable, specific, low marked reduction in fiber content increasing the in energy requirements and nonpolluting (Mac­ digestibility of copra meal; (2) increased energy ris, 1984). However, the economy of the cellu­ value by making simpler sugar molecules more lose bioconversion process remains unattractive available to the animal and (3) improved nutri­ due to the high cost of enzyme production and tional quality by making proteins that are bound the low yield of cellulases. Researches have, by the polysaccharide molecules also more therefore, been conducted in an effort to develop available (Teves, 1987). an efficient process of enzymatic hydrolysis of cellulose. Two approaches have been followed: After 24 hours of mannanase treatment, the variation of fermenta.tion engineering para­ a significant decrease was observed in the crude meters such as pH, temperature and nature of fiber (12.68%-6.32%) of the meal. Significant 86

Table 3. Proximate analysis of rice straw composts after Volvariella growth.

%Crude % Crude %Ash Protein Fiber

Crude rice straw (control) 5.92 25.97 23.83

Rice straw after 2 weeks 6.55 27.32 23.94

after 4 weeks 6.59 20.22 30.18

Rice straw/banana leaves

after 2 weeks 6.44 26.82 17.51

after 4 weeks 6.26 24.62 22.27

From Quimio (1988).

Table 4~ Lignin, protein and crude fiber contents (%) of different rice straw composts.

Lignin Protein Crude Fiber

Crude rice straw 9.57 5.56 26.82

Volvariella-spent compost 13.77 6.27 24.62 (30 days after)

Volvariella-Pleurotus - 8.31 6.39 19.42 spent compost (another 2 months after)

Volvariella-P leurotus - 7.71 6.92 15.00 Pleurotus -spent compost (total of 5 months)

From Quimio (1988). 87 Table 5. Some microbial genera capable of utilizing cellulose, hemicellulose and lignin (from Alexander, 1977).

Cellulose Hemicellulose Lignin Degraders Degraders Degraders

Fungi Fungi Fungi

Alternaria Alternaria Collybia Aspergillus Aspergillus Fornes Chaetorniurn Chaetorniurn Pleurotus Coprinus Fusariurn Polyporus Fornes Glornerella Poria Fusariurn · Penicilliurn Trarnetes Myrotheciurn Trichoderrna Penicilliurn Polyporus Bacteria Rhizoctonia Rhizopus Bacillus Trarnetes Cytophaga Trichoderrna Erwinia Trichotheciurn P seudornonas Verticilliurn Streptornyces Zygorhynchus

Bacteria

Bacillus Cellulornonas Clostridiurn Corynebacteriurn Cytophaga Polyangiurn P seudornonas Sporocytophaga Vibrio M icrornonospora Nocardia Streptornyces Streptosporangiurn 88

Table 6. Some agro-industrial fibrous residues in Asian countries

Dry Crude Crude Feedstuff matter protein fiber Ash (%) (%) (%) (%)

Sugarcane Bagasse (Saccharum officinarum) 92.7 2.5 39.7 4.5 Bamboo (Bambusa spp.), green var. 42.0 7.6 12.2 5.2 yellow var. 37.9 6.7 11.7 4.0 Banana stem/trunk (Musa sapientum) 5.3 0.2 1.5 1.1 Cassava leaves (Manihot esculenta ) 100.0 23.2 21.9 7.8 Corn cobs (Zea mays ) 92.2 3.0 34.6 2.4 Corn husks 88.4 7.1 27.1 1.7 Corn silage, well-eared 35.0 2.8 8.4 4.0 Corn stover 89.0 4.5 32.0 7.1 Corn stover silage 28.4 1.9 8.2 3.6 Cottonseed hulls (Gossypium hirsutum) 100.0 4.4 38.0 2.8 Groundnut hulls (Arachis hypogea) 100.0 6.2 45.7 11.1 Groundnut straw 100.0 8.1 40.6 8.7 Rice hulls (Oryza sativa ), huller mill 90.4 2.5 38.0 19.3 Rice hulls, 'cono' mill 90.1 2.5 39.4 29.2 Rice straw 92.6 3.7 31.4 21.2 Rice straw silage 31.6 1.9 9.5 3.6 Sincamas forage (Pachyrrhizus erosus) 89.2 15.5 27.0 7.8 Sorghum stover (Sorghum vulgare) 90.8 8.3 33.3 7.3 Sugarcane tops silage 24.8 1.7 7.7 2.2 Sweet potato leaf-vine silage 27.8 2.8 4.4 . 7.3 Tamarind leaf meal (T amarindus indica ) 90.6 13.3 22.0 6.9 Tapilan hay (P haseolus calcaratus ) 84.5 13.5 31.1 9.8

Source: Gerpacio & Castillo 1979; Devendra 1977; Ranjhan 1980; Yeh 1963.

improvements were noted in the apparent di­ Conclusion gestibility of dry matter, crude protein, crude fat, crude fiber and NFE of diets containing man­ Biotechnology in the developing coun­ nanase-treated meal compared to those with tries is still in its infancy; hence, isolation, screen­ untreated meal. The metabolizable energy of ing, identification and improvement of enzyme­ diets with treated meal also increased signifi­ producing microorganisms as well as establish­ cantly compared to diets with untreated copra ment of conditions for maximum enzyme pro­ meal (Tables 7, 8 and 9). Moreover, weight gain duction in parti-cular substrates are currently and efficiency of feed utilization of the broilers being worked on intensively. Some of the re­ were significantly improved when fed diets searches have passed laboratory scale and the containing mannanase-treated meal than when techniques are now being tried o.n a bigger scale. fed diets with untreated meal (Figure 2). · Based on limited research results, biotechno- 89

Table 7. Proximate analyses of untreated and mannanase-treated copra meal

Copra meal % Ash Crude Crude Crude NFE protein fat fiber

Untreated 6.46• 19.44• 7.68• 12.68• 53.74•

Mannanase-treated 12.00b 24.36b 10.72b 6.32b 46.60b

Values with different superscripts within columns are significantly different at 1 % level of probability.

From Teves et al. (1988).

Table 8. Coefficients of apparent digestibility of dry matter and organic matter of diets with increasing levels of untreated and mannanase-treated copra meal.

EXPERIMENTAL DIET DRY CRUDE CRUDE CRUDE NFE MATTER PROTEIN FAT FIBIER

Tl 1 (5% UCM)* 67.94 45.16 81.31 25.04 84.77 T12 (10% UCM) 66.45 49.94 86.54 25.62 80.80 T13 (15% UCM) 70.53 53.94 81.96 25.88 83.31 Average 68.3P 49.68b 83.27b 25.5lb 82.96b T21 (5% MTCM)** 81.75 64.53 91.46 49.69 94.12 T22 (10% MTCM) 80.50 59.70 88.48 49.29 92.78 T23 (15% MTCM) 80.68 59.24 94.47 86.65 92.67 Average 80.98• 61.16• 91.47• 61.88• 93.19•

Averages with different superscripts within columns are signficantly different at 5% level of probability. *UCM = untreated copra meal **MTCM = mannanase-treated copra meal

From Teves et al. (1988) 90

Table 9. Apparent metabolizable energy (AME) of experimental diets (Kcal/Kg).

EXPERIMENTAL DIET AME

Tll (5% UCM)* 2562 T12 (10% UCM) 2593 T13 (15% UCM) 2632 Average 2595.67b T21 (5% MTCM)** 3086 T22 (10% MTCM) 3067 T23 (15% MTCM) 3081 Average 3078.ooa

Averages with different superscripts within column are significantly different at 5 % level probability.

*UCM = untreated copra meal **MTCM = mannanase-treated copra meal

From Teves et al. (1988).

Figure 2. Bar graph of body weight (g) of broilers fed different types of experimental diets.

800

700

,..-.. 600 ~..... "Sb. 500

~ 400 .s 300 0 p:) 200

100

0

0 2 3 Feeding Time (Weeks)

~ . t>J Tll Tl2 T13 ~ T21 &J T22 mT23· (5% UCM) * (10% UCM)' (15% UCM) (5% MTCM)** ( 10% MTCM) (15% MTCM),

*UCM =untreated copra meal **MTCM = mannanase-treated copra meal 91 logy seems to be promising as a means of im­ ADAB. James Cook Univ., Australia. proving fibrous crop residues; however, a lot of DOYLE, P.T. 1983. Digestion of treated crop effort still has to be exerted and a sizable amount residues and the need for nutrient additions in of money still has to be invested before the balances rations using such residues. In : The technology can be suited to farm conditions. Utilization of Fibrous Agricultural Residues. G.R. Pearce, ed. Australian Government Print­ The use of enzymes for large-scale ing Services. treatment of crop residues still is uneconomical at this stage. However, this should not be a DOYLE, P.T. 1988. Options for the treatment reason for us to abandon this idea. While we are of fibrous roughages in developing countries. A trying to find means of making the technique review. Proc. 2nd Annual Meeting of Austra­ more cost-effective, let us try other ways of lian-Asian Fibrous Agricultural Residues Re­ optimizing the utilization of our fibrous crop search Network. Serdang, Malaysia, 3-7 May residues, e.g., development of "inoculants" for 1988. particular substrates. Hopefully, if the microor­ ganisms can thrive well in the residues, they will GERPACIO, A.L. andL.S. CASTILLO. 1979. be able to degrade a significant amount of the Nutrient composition of some Philippine feed­ insoluble carbohydrates. This method will cir­ stuffs. Tech. Bull. 21, 117 pp. 4th rev. cumvent the problems attendant to enzyme ex­ traction and purification and might be a more IBRAHIM,M.N.M.andG.R.PEARCE.1980b. practical alternative to the treatment of fibrous Effects of gamma irradiation on the composition agricultural by-products. and in vitro digestibility of crop by-products. · Agric. Wastes. 2: 2543-259. References IBRAHIM,M.N .M. 1982. The effects of boiling ALEXANDER, M. 1977. Soil Microbiology. and of steaming under pressure on the chemical New York: John Wiley and Sons. 467 p. composition and in vitro digestibility of crop by-products. Agric. Wastes. 4: 443-452. BALASUBRAMANIAN,K.1976.Polysaccha­ rides of the kernel of maturing and matured IBRAHIM, M.N.M. 1983. Physical, chemical, coconuts. J. Food Sci. 41: 1370-1373. physico-chemical and biological treatments of crop residues - an overview. In : The Utilization BURROWS, I., K.J. SEAL and H.0.W. EG­ of Fibrous Agricultural Residues. G.R. Pearce, GINS. 1979. The biodegradation of barley straw ed. Australian Govenment Printing Services. by Coprinus cinereus for the production' of ruminant feed. In: Straw Decay and its Effects JALALUDIN, S. 1988. Treatment of fibrous on Disposal and Utilization. E. Grassland (ed.). residues under farm conditions in Asia. Proc. 2 John Wiley and Sons. N.Y. nd Annual Meeting of Australian-Asian Fibrous Agricultural Residues Research Network. Ser­ COOMBE, J.B., D.A. DINIUS and W.E. dang, Malaysia, 3-7 May 1988. WHEELER. 1979b. Effect of alkali treatment on intake and digestion of barley straw by beef JOGLEKAR, A.V. andN.G. KARANTH.1984. steers. J. Anim. Sci. 49: 169-176. Studies on cellulase production by a mutant­ Penicillium funiculosum UV - 49. Biotech. DEVENDRA, C. 1977. Ruminant nutrition and Bioeng.26: 1079-1084. productivity in the ASEAN region. Seminar on Animal Health and Nutrition in the Tropics. KIM, C.S. 1981. Processing of lignocellulosic 92 materials for animal consumption. Advances in SEIDEN,R.1957. TheHandbookofFeedstuffs. Food Producing Systems for Arid and Semi­ New York: Springer Publ. Co., Inc. 59 p. Arid Lands. Acad. Press Inc. TEVES, F.G. 1987. The Nutritive Value of MACRIS, B.J. 1984. Enhanced cellulase and copra meal treated with bacterial mannanase in B- glucosidase production by a mutant of Alter­ broiler diets. M.S. Thesis. Univ. of the Phils. naria alternata. Biotech. Bioeng. 26: 194-196. 93p.

MOORE, E.W. M.J. EFFLAND and M.A. TEVES,F.G., A.F. ZAMORA, M.R. CALA­ MILLETI.1972. Hydrolysis of wood and cellu­ PARDOandE.S.LUIS.1988.Nutritional value lose with cellulolytic enzymes. J. Agric. Food of copra meal treated with bacterial mannanase Chem. 20: 1173-75. in broiler diets. In : Recent Advances in Bio­ technology and Applied Biology. The Chinese QUIMIO, T.H. 1988. Countinuous recycling of University Press. rice straw in murshroom cultivation for animal feed. In : Recent Advances in Biotechnology VANSELOW, D.G. 1983. Microbial treatment and Applied Biology. The Chinese University of rice straw for improved ruminant nutrition - Press. preliminary results. In : The Utilization of Fi­ brous Agricultural Residues. G.R. Pearce, ed. RANJHAN, S.K. 1980. Animal Nutrition in Australian Government Pringting Services. Tropics and Subtropics. New Delhi: Vikas Publ. House Pty. Ltd. XVI- 446 pp. YEH, J.P.1963. Nutrient composition tables of the feeding stuffs in Taiwan. J. Chinese Agr. SANTOS, M.E. 1988. Personal communica­ Chem. Soc. 1: 56-69. tion.

SCHNEIDERMAN,H.1987. What biotechno­ logy has instruct for us. Ohio J. Sci. 87(5): 182- 185. Application of Biotechnology in provement of Fibrous Crop Residues

1 2 3 Y.W. Ho , N. Abdullah and S. Jalaludin 1. Department of Biology 2. Department of Biochemistry and Microbiology, 3. Department of animal Science, Universiti Pertanian Malaysia, 43400 Serdang, Selangor, Malaysia.

Summary holdings from crops to pasture and thus tropical livestock farming will remain underdeveloped A major constraint limiting the growth unless mor~ profitable animal production sys­ of the Iivestock industry in Malaysia is the shor­ tems can be evolved (Jalaludin, 1986). tage of suitable feedstaff. The utilization of fi­ brous crop residues offers the best opportunity The production of fibrous crop residues for improving livestock production. At present, in most tropical countries is enormous and the the potential of these residues is not being used most effective way of exploiting these agricul­ to support animal production because of low tural wastes would be to use them as animal digestibility and poor nutritive value of the feeds, particularly for ruminants. At the mo­ materials. In order to find out whether these ment, the potential of these fibrous crop residues problems can be overcome by the manipulation is not used to best effect to support ruminant of microorganisms, a multidisciplinary research production because: (i) the intrinsic nutritive project for the manipulation of rumen microbes value of the materials is low and it is necessary with the aim of improving the utilization of to develop methods for physical, chemical or agricultural by-products has been initiated at biological treatments to improve their digestibil­ Universiti Pertanian Malaysia. This paper re­ ity and nutritional value to ruminants, and (ii) ports the studies on one of the rumen micro­ knowledge is lacking about how such feed mate­ organisms - the anaerobic rumen fungi and their rials can be digested optimally by the rumen role and potential in the utilization of fibrous micro-organisms (Jalaludin, 1986). Fibrous crop crop residues. residues cannot be successfully incorporated into any feeding system for ruminant production unless solutions for these problems are found. It Introduction is in this context that biotechnology can be applied to improve the utilization of fibrous crop A major constraint to the growth of the residues for ruminant production. livestock industry in many developing countries in the tropics is the shortage of easily-available Thedevelopmentofmethodsforphysi­ and suitable feeding materials as most of the cal, chemical or biologkal treatments of the animals are raised by small holders or landless fibrous crop residues to improve their nutritive farmers. Pasture production is low because ani­ value and digestibility for ruminants has been mal feed competes economically with other cash well documented and reviewed and will not be crops. Land owners are reluctant to convert their discussed here. In contrast, information on how 94

feed matedals are digested by rumen micro­ role in fibre digestion. organisms is lacking. At present, feed strategies are planned without much knowledge on the A study of the anaerobic fungi on three ecology, biology and performance of rumen types of fibrous feed materials (rice straws, palm micro-organisms, their precise role in the degra­ press fibres and guinea grass) in the rumen of dation of fibrous feed matedals and the interac­ cattle and swamp buffalo at ,UPM showed that tion between the vadous rumen micro-organ­ fungal. attachment on the plant fragments by isms. means of zoospores occurred very quickly - about 15-20 min after incubation in the rumen. The rumen is a complex ecosystem in The common sites for attachment on guinea which the supply of nutrients to the animal is grass and rice straws were stomata, cut-ends of determined by dynamic interactions between fragments and damaged surfaces. Similar sites feedstuff, micro-organisms and the animal. The for zoospore attachment have been observed by anaerobic fermentation of plant fibre by micro­ other workers (Bauchop, 1979a, 1981; Orpin, organisms in the rumen involves bacteria, proto­ 1977a&b, 1981). Attachment on the palm press zoa and fungi. The cellulolytic bacteria have fibres was mainly on cavities left by dislodged long been known to play an important role in the silica bodies, cut-ends and cracks. The attached degradation of feed materials in the rumen. zoospores shed their flagella very quickly, en­ However, in the last decade or so, anaerobic cysted and germinated with a single germ tube rumen fungi have been recognized to play a which very quickly branched to form a network significant role in the digestion of fibres in the of rhizoids. rumen. By 1h after rumen incubation, the fun­ Universiti Pertanian Malaysia (UPM) gal rhizoids were observed in the soft, thin­ has recently initiated a multidisciplinary project walled mesophy 11 and parenchyma cells of rice for the manipulation of rumen micro-organisms straws and guinea grass. Many of the rhizoids on with the aim of improving the utilization of contact with undamaged cell walls developed agricultural by-products. This paper highlights special appressorrium-like structures for pene­ the studies on one of therumen micro-organisms tration. A description of the "appressoria" and - the anaerobic rumen fungi and their role and method of penetration of the cell walls by the potential in the utilization of fibrous crop resi­ "appressoria" have been reported by Ho et al. dues. (1988a). Development of "appressoria" by the fungi for penetration of cell walls probably ac­ The anaerobic rumen fungi and their celerates the spread of rhizoid from cell to cell role in fibre digestion enabling maximum colonization of plant tissues within a short time. The anaerobic rumen fungi was first discovered by Orpin (1975) in the rumen of As more rhizoids colonized and pene­ sheep. Since then, they have been isolated from trated the plant cells, disruption and break-down a number of herbivorous animals consuming a of the cells occurred. After 6h of rumen incuba­ high fibre diet (Orpin, 1981; Bauchop, 1979 tion, the sclerench yma and vascular tissues in the a&b). The population density of the anaerobic rice straw and guinea grass were extensively rumen fungi is high when the host animal is fed colonized and areas of degradation occurred in with fibrous diets but they are absent or occur in the mesophyll, ground parenchyma and scler­ low numbers when the animal is fed with soft, enchyma (Ho et al., 1988b). Colonization of the leafy diets (Bauchop, 1979a). The heavy coloni­ sclerenchyma tissues in the palm press fibres zation of the fibrous plant materials by these also occurred. fungi in the rumen dig es ta indicates a significant 95 After 24h of incubation in the rumen, phologically and enzymatically to contribute to most of the thin-walled tissues of rice straws and fibre digestion and rumen fermentation. The guinea grass were degraded leaving mostly the development of methods to increase the popula­ vascular cylinders intact. Sections of these frag­ tion density of the anaerobic fungi; to select for ments showed lumina of cells filled with rhi­ superior fibre-degrading strains; and to manipu­ zoids and "appressoria" penetrating through the · late the superior strain of these fungi in therumen cell walls. Breakdown and disruption of cell are some possible means whereby the fungi walls were apparent in areas with profuse colo­ could be used to improve the utilization of fi­ nization of fungal rhizoids. Sclerenchyma tis­ brous crop residues as feed resource. for rumi­ sues of the palm press fibres were also heavily nants. colonized and in many cases, disruption and separation of the tissues occuned. References

The rumen fungi were also found to be Bauchop, T. (1979 a): The rumen anaerobic cellulolytic. In vitro culture of fungal isolates on fungi: colonizers of plant fibre. Ann. Rech. Vet. filter paper showed degradation of the filter 10: 246-248. paper in areas with fungal colonization. The fungi could also degrade cellulose in agar roll Bauchop, T. (1979 b ): Rumen anaerobic fungi of tubes, forming clearing zones around the colo­ cattle and sheep. Appl. Environ. Microbiol. 38: nies (Ho, unpublished data). 148-158.

Conclusion Bauchop, T. (1981): The anaerobic fungi in rumen fibre digestion. Agric. Environ. 6: 339- By nature of their growth, the fungi are 348. able to penetrate deeply into the plant tissues which are usually inaccessible to the rumen Bauchop, T. andMountfort,D.O. (1981): Cellu­ bacteria. The disruption of the plant tissues re­ lose fermentation by a rumen anaerobic fungus sults in a decrease of particle size which could in both the absence and presence of rumen enhance colonization of the particles by bacteria, methanogens. Appl. Environ. Microbiol. 42: thus accelerating fibre digestion in the rumen. 1103-1110. Besides being able to digest cellulose, the rumen fungi have also been reported to digest other Ho, Y.W., Abdullah, N. and Jalaludin, S. (1988 plant structural components such as pectin, a): Penetrating structures of anaerobic rumen hemicellulose and xylan (Orpin, 1981; Orpin & fungi in cattle and swamp buffalo. J. Gen. Micro­ Letcher, 1979; Bauchop & Mountfort, 1981) and bial. 134: 177-181. coum digest up to 45% dry weight of plant tissues (Orpin, 1981). Although the fungi cannot Ho, Y.W., Abdullah, N. and Jalaludin, S. (1988 degrade lignin, they appeared to be able to break b): Colonization of guinea grass by anaerobic the lignin-hemicellulose complexes and solubi­ rumen fungi in swamp buffalo and cattle. Anim. lise lignin (Orpin, 1981; Preston &Leng, 1987). Feed Sci. Technol. In Press. The removal of lignin from fibres which are physically protected by lignin enables them to be Jalaludin, S. (1986). Strategies for increased fermented by rumen bacteria (Preston & Leng, ruminant production in Malaysia. Keynote ad­ 1987). dress, Seminar/Workshop on Rumen Microbiol­ ogy, 22-24 April, 1986, Dept. of Animal Sci­ All these properties indicate that the ence, Universiti Pertanian Malaysia. anaerobic rumen fungi are well-equipped mor- 96

Orpin, C.G. (1975): Studies on the rumen Orpin, C.G. and Letcher, A.J. (1979): Utilization flagellateN eocallimastixfrontalis. J. Gen. Micro­ of cellulose, starch, xylan and other he- micellu­ biol. 91: 249-262. lose for growth by the rumen phycomycete Neocallimastixfrontalis. Cur,,Microbiol. 3: 121- Orpin, C.G. (1977 a): Invasion of plant tissue in 124. the rumen by the flagellate Neocallimastix frontalis. J. Gen. Microbiol. 98: 423-430. Preston, T.R. and Leng, R.A. (1987): Matching ruminant production systems with available Orpin, C.G. (1977 b): The rumen flagellate resources in the tropics and sub-tropics. Penam­ Piromonas communis: Its life-history and inva­ bul Books, Armidale, Australia. sion of plant material in the rumen. J. Gen. Microbiol. 99: 107-117.

Orpin, C.G. (1981): Fungi in ruminant degrada­ tion. In Agricultural Science Seminar: Degrada­ tion of plant cell-wall material, Agricultural Research Council, London, pp 37-46. Potential of Biotechnology in Animal Production and Health in India

C. Natarajan Director Animal/Aquaculture Biotechnology Division Department of Biotechnology Government of India New Delhi, India

Biotechnology, in the modern sense of research, and developmental programme have the term, describes the science of application to been initiated. The country is now poised to take discoveries in molecular biology for develop­ up demonstration of application of some of these ment in the broad sectors of veterinary and technologies into product plans for industrial medical care, agriculture, energy, environment, ventures wil!h emphasis on vertical integration of and industry. In respect to animal biotechno­ production, processing, marketing and econo­ logy, numerous specifically identifi~d areas of mics. Adequate attention commensurate with priority have emerged, such as, animal disease the available national resources has also been diagnostics and vaccines, antibiotics, hormones, paid for developing infra-structural facilities in­ immunotoxins,gene therapy, fermentation, bio­ cluding well equipped modern laboratories, tech­ degradation of agriculture and animal farm wastes nical expertise and information resource. and residues, single cell protein livestock feed supplements, and embryo transfer. This list is, It is clear that a vast potential exists in however, increasing day by day while new dis­ India for intensifying and expanding animal coveries are being made in protein, enzyme production and health programmes for (i) con­ and microbial engineering, and computer tech­ servation and multiplication of high producing nology. genetically defined breeds and strains of live­ stock for meat, milk, and eggs besides draught; The application of biotechnology, as a (ii) understanding the physiological and nutri­ total package of practices for enhancing animal tional aspects for optimal production and repro­ production through provision of adequate nutri­ duction; (iii) applying cheaper and more effec­ tion, health care, and management with sound tive animal disease diagnostic and prophylactic breeding programmes, has already found an measures; (iv) improving consumer safety in important place in India. There are several areas, respect of animal products and (v) developing such as Multiple Ovulation and Embryo Trans­ systems approach with a strong bio-informatics, fer (MOET), Immunodiagnostics, Anti-fertility computer and economics base. vaccines, Enzymes Hormones and fine chemical preparations, utilisation of non-conventional feed Some of the areas of potential biotech­ resources, DNA recombinant animal disease nology application in the Indian context have immuno-prophylactics, laboratory animal pro­ been discussed below. duction, cell-culture and bioinformatics on which 98

1. Multiple Ovulation and Embryo biological iQllplications". There are 7 centres Transfer (MOET): under this project including IVRI, NDRI, CIRB, HAU, APAU, GBPUAT and CIRG. The sub­ With the major objective of creating a project has proposed to cover studies on super­ 'nucleus' herd of highly productive dams and ovulation synchronisation, embryo collection, progeny tested sires to be used in national artifi­ preservation, genetic engineering and micro cial insemination programmes; in 1986-87 the manipulation, embryo environmental interac­ Department of Biotechnology has initiated a tion and their field application. There is a major national project entitled, 'Cattle herd Improve­ emphasis on developing the technology through ment for increased Productivity using Embryo adaptive research suitable to different geocli­ Transfer Technology' with a multi-agency net­ matic regions of the country. The ICAR also work of centres. The project also aims at stan­ supports a few other selected centres located in dardization and utilization of this technique in State Agricultural Universities other than those buffaloes. The aspects of intensive research and mentioned above for carrying out this activity. development under this project include superovu­ lation, oestrus synchronization, flushing and Concurrent with the MOET studies embryo recovery, in vitro oocyte maturation, emphasis is also being given to understand re­ embryo micromanipulation, cloning, splitting producti".e endocrinology, including immuno and sexing of embryos, purification and produc­ reproduction, and development of simple me­ tion of selected hormones involved in cattle and thods for oestrus and pregnancy detection espe­ buffalo reproduction, embryo cryopreservation cially in buffaloes. and embryo transfer in recipient foster cows owned by farmers in rural areas. The project has 2. Immunodiagnostics: made a significant progress and established necessary infra-structural facilities. A 700 ani­ Much headway has already been made mal donor/recipient herd has already been estab­ in India in the application of modern immuno­ lished. As many as 35 cow and 3 buffalo calves diagnostic methods, such as Radio/Enzyme have been born and success achieved in inducing immunoassay, immunofluorescence, and sev­ pregnancy in cows using cryo-preserved and eral modifications including adaptation of mi­ split embryos. Efforts are being made to over­ cro-methods of serodiagnosis. This has resulted come the poor superovulatory response in buffa­ in the detection of sub-clinical or latent infec­ loes and the heavy dependence on imported tions besides early diagnosis of clinical cases of hormones through intensive research. chronic and insidious nature. Competence has also been built up for developing diagnostic kits A network of infra-structural facilities which are simple, quick, accurate, and cheap; including the establishment of a main laboratory and therefore of field applicability. The tech­ at Bidaj; three R & D laboratories at NDRI niques of hybridoma and development of mono­ (Kamal), IVRI (Izatnagar) and NII (New Delhi); clonals have been successfully adopted for this four Regional Centres and 25 State Centres are purpose. As a result of these developments and being set up under this project. The ET project by the application of electron microscopy, im­ activities will have strong linkages with the proved histo-chemical/histo-enzymological national artificial insemination network. methods, fluorescent antibody technique, radio and enzyme immunoassays, ultra and differen­ The ICAR has also established sim ulta­ tial centrifugal analyses, and iso-electric fo­ neously an US AID assisted sub-project entitled, cussing several new causal agents, such as, those "Embryo Transfer Technology and Bio-engi­ ofVisna-Maedi of the slow virus infections, as­ neering in livestock species and their Patho- sociation of fungal toxins with 'Degnala' disease 99 in cattle and buffaloes and adrenal cortical hy­ tory models and in field trials. Successful at­ poplasia with horn cancer and possible etiology tempts have also been made to develop cDNAs of ethmoidal carcinomas in bovines have been for FMDV types, A, A22, and Asia-1 for the identified. Monoclonal antibodies have been production of genetically engineered vaccine. raised for specific diagnosis of Theilaria annu­ DNA plasmids have been isolated from Clos­ lata infection characterisation of foot and Mouth tridium welchii type D and experiments con­ disease virus types, Rinderpest, Rotavirus and ducted for their role in eliciting toxin production. Enterotoxigenic Escherichia coli infections to Among the priorities for development geneti­ mention a few. With the availability of equip­ cally engineered vaccines, FMD, RP, Rabies, ments for HPLC, Cytoflurometers and cell sor­ Sheep Pox, Enterovirus infections, Blue tongue, ters; and because of the training acquired in the and IBR deserve attention. field of immunology, efforts are underway to study the basic mechanisms, in disease pro­ 4. Infra~Structural Facilities: cesses including the complex antigen-antibody reactions. DNA based diagnostic probes as being In order to give an impetus to the esta­ used in the diagnosis of Mycobacterium tuber­ blishment and growth of infra-structural facili­ culosis infection in man are finding their use in ties in biotechnology, the government of India animal disease diagnosis as well. set up a National Biotechnology Board in 1982 and the Department of Biotechnology in the There is a considerable scope of apply­ Ministry of Science & Technology in 1986. This ing this knowledge for improving diagnosis of Department has mandate of planning, promot­ subclinical or latent infections, thus providing ing, and coordinating Biotechnology program­ tools for the study of molecular epidemiology. mes in plant, animal, and human medical sci­ ences. The major programmes which have been 3. Vaccines taken up so far by the Department include inte­ gratedman-power development,creation of infra­ On the vaccine front, outstanding re­ structural support to R & D programmes, post search accomplishments have been made in India graduate and post doctoral national, and over­ particularly in the development and use of con­ seas training programme of short and long term, ventional animal egg or cell culture based vac­ Visiting Scientists from Abroad Programmes, cines against several animal diseases including and introduction of Biotechnology teaching rinderpest, foot and mouth disease, rabies, sheep programmes even at undergraduate level. Sev­ pox, lung-worm disease, black disease, Marek's eral national facilities, such as, animal house disease, gumboro disease and theilariosis. In facility to maintain and supply disease free, recent years, efforts have been made to apply genetically and microbiologically defined labo­ modern innovations of nuclear techniques, ratory animals, centralised facility for produc­ molecular studies and genetic engineering me­ tion, import, and distribution of fine chemicals thods in animal disease vaccine development. and enzymes, and bioinformatics network link­ Using the technnique of growing BHK 21 cells ing specialised national centres, bilateral re­ in Micro-carriers (Cytodex-1) concentrated anti­ search and training programmes have already gen of FMDV in pigs has been obtained. In been initiated. Besides supporting a number of studies on non-specific immune response suc­ programmes at National Institutes like Indian cess has been achieved by demonstrating satis­ Institute of Science, Indian Institutes of Tech­ factory host specific immune response against nology, Centre for Cellular and Molecular Biol­ infections of varied nature, such as, Marek"s ogy, to name a few, the DBT has established its disease, FMD and rabies with an enzyme treated own research institutes viz. the NII and the preparation of Mycobacterium phlei in lobora- International Centre for Genetic Engineering 100

(ICGEB). 5.3 Dairy Technology:

Biotechnology research capabilities are Introduction and standardization of beingprobidedin theICARsystem at the UNDP energy saving methods in dairy technology, such Centres on Biotechnology in NDRI & IVRI and as, membrane processing for concentration of several State Agricultural Universities. milk in making 'Kho a' cheese, whey, butter milk, paneer, 'channa' casein and fermented milk pro­ 5. Others ducts, ultra high concentration of raw milk, me­ thods of packaging and transporting milk and 5.1 Genetics of Disease Resistance: milk products from the point of view of con­ sumer safety, enhancement of bio-protective properties of infant foods using certain bacteria In comparison to the knowledge avail­ and microbial rennet for cheese making. able in laboratory animal and man, the major histo compatibility complex in livestock has not 5.4 Livestock Products Technology: been well understood. The identification ofgenes involved in disease resistance and use of this A num her of biotechnological methods knowledge in the creation of specific diseaes are involved in the utilization of slaughter house resistant breeds of livestock needs attention. wastes into useful products of sports good, chitin 5.2 Poultry Production: oil (Neats foot oil), and glandular extracts for making hormones and enzymes which need to be Biotechnology has major application studied for economic value. in modern poultry production. Substantial prog­ ress has been achieved in India in raising inbred 5.5 Bio-conversion of Agricultural hybrid lines of poultry for layers and broilers on and Animal farm Residues and Wastes: scientific levels of flock management. It is time to go for intensive large scale production pro­ Microbial treatment ofligno-cellulosic grammes involving properly identified parent low quality roughages and crop residues, ma­ breeder stock, automatic hatchery operations, nipulation of rumen environment, development feed technology and post harvest processing, of single cell proteins and identification and use and packaging. There are scopes for using dwarf of growth promoters are some of the areas which varieties, applying molecular biology for identi­ require to be studied. Production ofbiogas from fying and converting non-conventional sources animal and other organic wastes utilizing of feed, and using hormones for hastening the methonogenic bacterial fermentation as well as growth rate. Developing disease resistant breeds production of alcohols from cellulose by using a and specific pathogen free flocks with better combination of microbial processes are to be feed formulation hold promise in increasing taken up for fuel land energy recycling. poultry production. Present Status of the Use of Genetically Engineered Growth Hormones and Growth Hormone Gene in Animals

T.K. Mukherjee Institute for Advanced Studies University of Malaya Kuala Lumpur Malaysia

), Summary somatomedins (IGF1 and IGF2 which in turn stimulates the growth of mesodermal tissues This paperreviews the present status of such as muscle, cartilage and bone. A schematic growth hormone gene (GHG) constructs, some diagram of the hormone control of growth per­ cloning procedures of the gene and transfer of formance is given inFig. 1(Su,1988).Injections genes in various domestic animals using GHG of of GH also increases milk production in dairy different origin. While possible application of cattle and yield a leaner carcass in pigs. GHG for enhancement of growth and other traits (e.g lactation in dairy cows, leanness of meat in Human growth hormone (HGH) is a pigs) has been advocated, yet there are still many single polypeptide chain that has 191 amino problems to be solved before its routine applica­ acids with 26 amino acids at the N-terminal as tion is recommended. signal peptide which is processed during secre­ tion. It is produced in the human pituitary gland, and like insulin, it is not glycosylated (Paladini Introduction eta!., 1986). TheDNAfragmentwererepurified and linked together to yield the complete DNA One of the most complicated but eco­ sequence for human growth hormone, beginning nomically important traits in animals is growth, with a methionine initiator codon, followed by which is actually dependent on its genotype; the sequence for the 191 amino acids in the environment (both macro and micro) in which mature protein and ending with signal to stop the genotype is distributed; diet; and the interac­ protein synthesis. The gene was then inserted in tion of genotype x environment and nutrient. an expression vector (PBR 322) and introduced Hormonal influence (microenvironmental) on into E. coli , where it directed the synthesis of growth is also considerable (Spencer, 1985). human growth hormone (Goedde! et al., 1979). Somatotropic hormones, insulin, sex steroids, thyroid hormones, and glucocorticoids are among Recent interest on growth hormones in those hormones which influence growth. Of domestic animals lies in the construction of these somatotropic hormone (somatotropin or growth hormone gene and the possible use of GH) is normally synthesized in the pituitary directinjections of growth hormones or microin­ gland and travels to the liver, where it promotes jection of growth hormone genes in nuclear the synthesis of a second set of hormones, the DNA of recipients, and the expression of the 102

genes. This briefreview is concerned with some 322. They contain a portion of the gene (amp') of the relevant work done in this regard in coding for the enzyme B-lactamase which is domestic animals. secreted into the periplasmic space and is re­ sponsible for bacterial resistance to the antibi­ Growth Hormone Gene Constructs otic ampicillin. The mammalian sequences were provided by cloned complimentary DNA con­ 1. A Simple Model trolling the entire coding region for the precursor form of rat pituitary GH. A simple model for the making of human growth hormone gene is given in Fig. 2 0/1[ atson Functional fusion of the two donor et al., 1983). A protein was produced through the DNA's was facilitated by knowledge of their combination of chemical synthesis of DNA primary structures (Seeburg et al., 1978). Both having first 24 amino acids and enzymatic syn­ DNA's possess a unique cleavage site for the thesis of c-DNA, a sequence that codes for this restriction endonuclease Pstl at which they can protein has been produced. A triplet of bases be joined to maintain the correct reading frame (ATG) specifying amino acid methionine was (Fig. 4 ). In the amp'gene the Pst 1 site is situated added immediately in front of the first codon. approximately two-thirds towards the end. In Once the beginning of the gene had been chemi­ RGH cDNA the corresponding site occurs a few cally synthesized to ensure a correct start to the base pairs distal to the initiator codone ofprepep­ protein, a DNA sequence coding for the remain­ tide. The resulting new gene codes for a chimaric der of the polypeptide chain (amino acid resi­ protein of 395 amino acids (Mol wt. 44,000) dues 25 through 191) was obtained by making containing the N-terminal 181 amino acids of c-DNA copies of m-RNA preparations from the B-lactamase. Sequencing of amino acids of human pituitary cells. These two DNA frag­ human growth hormone (h GH) showed that it ments were then separately cloned, precursor shares 95%, 91%and65% homology with bo­ covalently linked to 214 residues of rat pre GH. vine GH (b GH), ovine GH (o GH) and equine GH (e GH) respectively. Similarly Insulin like To enable selection of plasmids carry­ growth promoting factor (IGFl and IGF2) are ing the correct hybrid gene, the cloned GH c­ found to be homologous to that of proinsulin DNA, originally isolated tn plasmid (PBR 322), (Blundell et al., 1980). was first transferred from the Hind III sites of PBR 322 into the homologous site of PMB 9, a 3. Gene construct with mouse methal­ closely related plasmid which lacks the amp' lothionein gen I (MT) as promoter gene. In the resultant combination plasmid (PMB 9 - RGH) the gene for tetracycline resistance is Hammer et al., (1985) and Brem et al., expressed at reduced levels (Rodriguez et al., (1985) have followed Palmiter et al., (1982) in 1976). making gene constructs consisting of the mouse methallothionein gene I (MT) promoter/regula­ 2. Construc.tion of a hybrid gene torregion fused to either the rat or human growth hormone. For gene transfer in pigs, a 4.0 Kb The construction of a hybrid gene be­ linear Eco RI/Eco RI DNA fragment was used tween bacterial and mammalian DNA sequences, consisting· of the structural gene of the human capable of directing synthesis of rat growth growth hormone (h GH) and the promoter or hormone (RGH) sequences in bacteria has been regulatory region of the mouse methallothionein made by Seeburg et al., 1978 (Fig. 3). In this gene 1 (MT) fused in a B g 1 II site. This fragment construction the facterial sequences for this gene has been inserted into a 4.31 Kb hybrid vector were excised from the multicopy plasmid PBR containing a 2.35 Kb Eco RI/Barn H 1 fragment 103

of BPV and a 296 Kb fragment of PBR 327 For screening the DNA library, a probe to give the 9.31 Kb plasmid P x GH-1. This representing part of the DNA sequence of the plasmid was used to transform the E. coli strain hormone gene is required. Synthetic oligonu­ HB-101. cleotides are often used as probes. DNa-DNA hybridization (Sou them blotting) is usually used 4. Gene construct with sheep growth hormone for probing. and Methallothionien promoter Gene Expression Ward et al., (1986) used two different DNA fusion genes as constructs forinjections in Su (1988) has reviewed strategies for sheep. In the first case, they utilised a sheep efficient and controlled expression of growth growth hormone c-DNA linked to the regulatory hormone genes in both eukaryotic orprokaryotic sequence of a sheep methallothionein - I gene cells. A list of somatotropic gene cloning and (fig. 5). In the second case DNA used consisted expression has been tabulated by him (Table 1). of sheep growth hormone genomic sequence with the 5 - regulatory sequence replaced by the Efficient expression in eukaryotic cells sheep methallothionein - 1 gene regulatory se­ needs: quence (Fig. 6), which was purified on Elutip - d- columns. Many other gene constructs (Su et i) Promoter replacement : Promoters al., 1988)usinghumanorbovineorovine growth which have directed high level expres hormone gene have been made by: sion of hormone genes are: Simon virus 40 (SV 40) late region promoter, Min i) Introduction of a new translation start ute virus of mice (MVM) and Rous signal A TG for methionine next to the sarcoma retroviral long terminal re desired open reading frame peat.

ii) Creation of new restriction sites by ii] A 5 - noncoding AT - rich sequence substituting bases in order to facilitate optimizing the sequence and distance further manipulations between ribosome binding site · and initiation codon for enhancing transla iii] Nucleotide substitution by codons more tion efficiency. frequently found in host cells iii] Introduction of sequence coding for a iv) Insertion of a translble AT-rich syn removal aminoterminal extension in the thetic cistron upstream from the hor­ protein that include ATG (code for mone gene with termination codon methionine) as well as additional amino next to or overlapping the initiathri acid sequences that could facilitate codon of the gene. Such insertion pro­ preparative purification and analysis of vides efficient initiation. the gene product.

Gene Cloning Effect of Grovvth Hormone Injections

Firstly the DNA sequencing coding is Growth hormone is an important regu­ isolated from the appropriate tissue of target lator of skeletal growth. In vivo, growth hormone animal, following which either a c-DNA library deficiency results in decreased cartilage cell or genomic library is prepared (Maniatis, 1982). proliferation and matrix synthesis, impaired 104

osteoblastic function and a subsequent decline in did not observe any significant increase linear growth. Excess growth hormone produces in growth rate due to growth hormone an increase in linear growth of domestic animals injections in sheep and pigs. before the epiphysis are closed and an increase in bone formation after closure (Harris et al., vi) Only one report on growth hormone in 1972). Summers et al., (1978)reported increase jections in buffaloes (at NDRI, Karnal, in size of dwarf growing hens when daily injec­ India) indicates that buffaloes showed tions from 30-40 g/kg body wt is given to an increase of 19 .4 and 32.4 percent in young pullets commencing from 3 months of age milk yield when injected daily with 20 to sexual maturity. However daily injections had and 50 mg of b GH respectively. Fat resulted in decreased egg production of the hens content and S .N.F content of milk, and due to hormonal imbalance. feed and water intake did not change as a result of these injections. Su (1988) has reviewed recent results on galactopoietic resposes to GH as follows: Transfer of Cloned Genes in Germ Lines i) GH increases total milk production in dairy cattle by 10-40 percent. Comparison of different mammalian species with regard to the production of transgenic ii) During early lactation, the response is animals, a function of gene integration effi­ much less than that after the peak lac ta ciency, has been made earlier. Gene involved in tion. The possible explanation for this this transfer include GH. It is obvious from Table is that in early lactation period the cows 2 that integration frequency decreases with in­ are in considerable negative array ba creased size of species which is again due to lance and have high circulating levels more complexity in technical procedures re­ of endogenous GH. quired for larger group of species.

iii) Long term (188 days) administration Wagner andJochle (1985) gave a com­ showed similar effects (Bauman et al., prehensive survey on the potential forimproving 1985). growth in livestock by gene transfer. Most gene transfer in animals followed the mice example of iv) In short-term administration, milk fat Palmitter et al., (1982) in which recombinant and protein composition increased, but constructs utilise the mouse metallothienein no such effects were found in long-term promoter .to regulate the expression of a protein tests (Mercier, 1985). coding sequence. However in most cases, unlike in mice, increased levels of GH found in the v) Conflicting results on growth rate of transgenic pigs and sheep did not increase body pigs and sheep in response to GH injec weight. Brem et al., (1985) used MT-1-h GH tion have been reported. A daily injec construct to produce transgenic piglets but un­ tion done at 60 ug/kg gave optimized fortunately most of the transgenic animals were performance in pigs. Increasing the born dead or killed by their mother. Two of them maximum lean meat requires higher were reard but only one shows very little expres­ doses with some sacrificing in the opti­ sion of the transgene in serum. There were no mum weight gain and feed efficiency difference observed in growth rate between the (Su, 1988) (Personal communication transgenic and control animals. Hammer et al., to Su from scientists of Cornell Univer (1985) have produced 20 transgenic pigs and 11 sity). However Hammer et al., (1988) of them express the GH but do not show increase 105

in body weight. genes into the germ line of poultry. Shoffner (1985) indicated the target cells of importance In another experiment porcine GH was would be gonadal cells. Basically there are two transferred by microinjection into pigs. One approaches of effecting these gonadal cells. One transgenic pig showed accelerated growth rate is the direct micro injection ofrecom bi nan t DNA upto about 70 Kg. Further examination was as calcium phosphate precipitates and liposome. inturrupted by death of the animal because of The handling of large yolky fertile ova at the pneumonia (Viz et al., 1987). Therefore a series optimum stage of microinjection and of external of possible modifications have been proposed. manipulation and reinsertion of the altered ova into the female oviduct is cumbersome. There­ Seldon et al., (1985) showed that a gene fore attempts have been made to obtain 4-day old construct consisting of the human growth embryo from the incubator, place it horizonall y, hormone, structural gene and the glucocorticoid cut a small square portion of the shell using a receptor binding site, ligated to the mouse met­ dentist's drill, inject gene solution,place back the allathionein promoter sequences can be induced cut shell and reinserted into the incubator (Opitz, by glucocorticoids. The identification of the Guise and Shoffner - personal communication, primary structure and expression of a function 1988). However gene intergration was found to human glucocorticoid receptor c-DNA allows be of very low percentage when DNA h ybridiza­ an analysis of the molecular mechanism by tion technique was followed. which glucocorticoids regulate gene transcrip­ tion (Hollenberg et al., 1985). These results in­ An alternate method is employed by dicate that it might be possible to use modified Shuman (1984), Shuman and Shoffner (1986) binding sequences, modified receptors and syn­ and Crittenden and Salter (1985) in which they thetic glucocorticoid analogues as means of are using retroviruses as vectors for germ line external regulation of transgenes. insertion in the chickens. Retrovirus containing It has been shown that injections with the dis ired gene is injected into the blastoderm of genetically engineering b GH dramatically in"' embryos just before incubation and hatched creases milk production per lactation (Baumann chicks are tested for hybridization with match­ et al., 1985). But, it is disputable whether gene ing DNA probe. transfer experiments in cattle with bovine growth hormone structural gene in order to increase The chicken growth hormone gene milk yield are useful. Firstly drastic increase of which may be useful in the regulation of growth level of growth hormone will bring in chain has also been cloned. However, preliminary reactions for modified regulation and production studies showed thata somatically inserted growth of other hormone. Secondly, from genetic point hormone gene led to increased hormone produc­ of view, milk yield, growth and other quantita­ tion but had little influence on growth (Souza et tive characters must be controlled by more than al., 1984). one gene. Therefore search for other genes re­ sponsible for these characters are necessary in Discussion order to combine a quantitative and molecular approach for production of transgenics with One major point about expression of improved genetic merit. transgenes is that there is considerable variation in the level of activity in progeny of GH transgenic Chickens animals as shown in mice. One explanation of the variations in the expressed level of trans gene In chickens, most of the researchers are products may be due to differences of methyla­ seeking an easy method for inserting beneficial tion of the trans genes. On the other hand precise 106

tissue specific expression does not oceur (Brin­ administration on lactation in dairy cows. J. ster and Palmiter, 1984). Dairy Sci. 68: 1352-1362. Recently ·Sapienza et al., (1987) and Reik et al., (1987) have reported that the degree Brem, G., Brenig, B., Goodman, H.M., Seld0n, of methylation oftransgenes is dependent on the R.C., Graft, F., Kruff, B., Stringman, K., =~::m­ gamete of origin. Heritable molecular differ­ dele, J., Meyer, J., Winnacker, E.L., Krausslich, ences between maternally and paternally derive H.( 1985) Production of transgenic mice, rabbits transgenes complicate the application of gene and pigs by microinjection into pronuclei. Zuch­ transfer procedures in livestock. Thus, while it ts thygiene, 251-252. probabely possible to introduce any gene con­ struct as a transgene in animals, several charac­ Crittenden, L. B., and D.W. Salter. (1985).Ge­ teristics of expression have yet to be learned to netic engineering to improve resistance to viral achieve optimum gene regulation. disease of poultry. A model for application to livestock improvement. Can J. Anim Sci. 65: The efficiency of gene transfer tech­ 553-562. niques will increase in the future involving growth hormone gene and other genes, but still there are Goedde!, D., Heyneker, H., Hozumi, T., Ar­ many problems to be solved. Transgenic animals entzen, T., Itakura. K., Yansura. D., Ross. M., sometimes have problems with fertility and Miozzari. G., Crea. R., and Seeburg. P. (1979). health. Brem and Wanke ( 1987) reported in their Direct expression in Escherichia coli of a DNA transgenic h GH mice, glomerular lesions, cystic sequence coding for human growth hormone. tubular atrophy and hyaline casts appeared within Nature 281: 544-548. tubules. The hepatocytic nucleus were extremely pleomorphic and necrosis of liver cell groups Hammer,R. E.,Pursel, V. G.,RexroadJr. C. E., were seen. Wall, R. J., Bolt. D. J. Ebert. K. M., Palmiter, R. D., Brinster, R. L. (1985). Production of Inspite these problems, there is an over­ transgenic rabbits, sheep and pigs by microin­ all enthusiasm among the scientists that growth jection. Nature 315 : 680-683. of an animal could be enhanced by establishing transgenic lines where GH and other genes Harris W. H., Heany, J.P., Jowsex. J., Cocking. beneficial for growth had been inserted. J.,Atkins. C. Graham. J., Weinberg. E. H. (1972) Growth hormone - the effects in skeletal muscle. Literature Review Calcif. Tissue Res. 10: 1 - 13.

Brinster, R. L. and Palmiter, R. D. (1984). The Hollenberg, S. M., Weinberger, C., Ong, E. S., transfer of new genes into mice. Transfer and Carelli, G., Oro, A., Lebo, R., Thompson, E. B., expression of eucaryotic genes. Academic Press Rosenfield, M. G., and Evans, R. M. (1985) 135-140. Nature 318, 635-641.

Brem, G., andR. Wanke (1987). Phenotypic and Krausslich, H. (1986). Criteria for useful gene patho-morphological characteristics in a Half­ transfer. Proc. 3rd. World Cong. Applied to sib family of transgenic mice carrying foreign livestock Prod. XII. Biotechology pp. 22-31 (held in Lincoln, Nebraska, U.S.A. July 16-22). MT-h GH genes. 3rd. FELASA symposium, Armsterdam, 01-05 June, 1987. Maniatis, T., Fritsch E. F., and Sambrook. J. (1982). Molecular cloning- a laboratory manual Bauman, D .E., Eppard, P.J., De Geeter, M.J. and cold spring Harbor laboratory. pp 128-132. Lanza G.M. (1985). Long term effect of GH 107

Mercier, J.C. (1985). Exploiting new technolo­ Seldon, R. F., Wagner, T. E. Yun, J. S., Moore, gies in animal breeding. Proc. C. E. C. Seminar, D. P, and Goodman, H. (1985). Cited by Wag­ Edinburgh. 19-20th. June, 1985. ner, T. E and Jochle W. P. J. Buttery, Editor, Buttersworth. Press, London (In press, 1985). Palmiter, R. D., Brinster, R. L., Hammer, R. E., Trumbauer, M. E., Rosenfield, M. G., Birnberg, Summer, J. D. and Luke, E. P and Reinbart, B. N. C., and Evans, R. M. (1982)Dramatic growth S. (1978). Effect of different levels on growth of mice that develop from eggs microinjected hormones in growing hens. Poultry Sci. 38 (4): with metallothionein growth hormone fusion 72-76. genes. Nature. 300: 611-615. Su, T. Z. (1988). Manipulation of animal growth Reik; W,m Norris, M. L., Barton, S. C., and milk and fibre production through somatotro­ Surani, M. A. (1987). Genomic imprinting de­ phic hormonal and immunization techniques termines methylation of parental alleles in (Manuscript personally communicated). transgenic mice. Nature, 328: 248-251. Seeburg,P. H., Shine, J.,Martial. J. A., Ivarie,R. Sapienza, C.,Peterson, A. C.,Rossant, J., Balling, D., Morris, J. A., Ullrich A., Baxter J. D. and R. ( 1987) .Degree of meth yla ti on of trans genes is Goodman, H. M. (1978) Synthesis of growth dependent of gamete of origin. Nature 328: 251- hormone by bacteria. Nature 276 (21-28) 795- 254. 798.

Shoffner, R. N. (1985). Perspectives for mo­ Vize, P. D. (1987).Improving growth in lecular genetics research and application in transgenic farm animals. EMBO Workshop poultry. Paper No. 14729. Scientific Journal Germline Manipulation of Animals. Nethybr­ Series, Minnesota Agricultural Experiment Sta­ idge, Scotland. 23-25th. June, 1987. tion. Watson, J. W., Tooze, J andKurtzD. T., (1983). Shuman, R.M andR. N. Shoffner. (1986). Gene Recombinant DNA- a short course. W. H. Free­ transfer by avian retroviruses. Poultry Science man Coy., New York. p 236. 65: 1437-1444. Ward, K. A., Franklin, I. R., Murray, J. D., Shuman, R. M. (1984). The avian retrovirus as Nancarrow, C. D.,Raphael,K. A.,Rigby, N. W., potential vector for gene transfer in the chicken. Byrne, C. R., Wilson, B. W., and Hunt, C. L Ph.Dthesis, Univ.ofMinnesota,St.Paul, U.S.A. (1986). The direct transfer of DNA by microin­ jcction. Proc. 3rd. World Cong. AppliedtoLive­ Souza, L. M., Boone, T. C., Murdock, D., Lan­ stockProd. XII Biotechnology. pp6-21, (held in gley, K., Wypych, J., Fenton, D., Johnson, S., Lincoln, Nebraska, U.S.A. July 16-22). Lai,P. H.,Everett,R., Hsu,R. y.,Bosselman, R. (1984). Application of recombinant DNA tech­ nologices to studies on chicken growth. Journal of Experimental Zoology 232: 465-473. 108

Table 1 Somatothophic Hormone gene cloning and expression

Gene Source of DNA Expression System Reference pGH cDNA E.coli Seeburg et al., 1983 cDNA E.coli Vize et al., 1987 cDNA E.coli Xia et al., 1988 cDNA E.coli Su et al., 1988 Genomic library E.coli Vize et al., 1987 bGH cDNA E.coli Keshett et al., 1981 cDNA E.coli Seeburg et al., 1983 cDNA Streptomyces lividans Gray et al., 1984 cDNA E.coli Schaner et al., 1984 cDNA E.coli Buell et al., 1985 cDNA E.coli George et al., 1985 cDNA E.coli Winggfield et al., 1987 cDNA E.coli Kim et al., 1987 Genomic library 1 Voychik et al., 1985 Genomic library mammalian cells Ramabhadran et al., 1985 Genomic library mammalian cells Desrosiers et al., 1985 Genomic library mammalian cells Pasleau et al., 1985 Genomic library mammalian cells Prarr et al., 1986 Genomic library mammalian cells Kopchick et al., 1985 Genomic library mammalian cells Reitz et al., 1987 cGH cDNA E. coli /mammalian cells Souza et al., 1984 cDNA E.coli Burke et al., 1987 sGH cDNA E.coli Sekine et al., 1987 lGH cDNA E.coli Agellon et al., 1986

IGF - I/11 cDNA 1 Janson et al., 1983 cDNA 1 Bell et al., 1984 Genomic library 1 Bell et al., 1985 SM-C Synthetic 1 Li et al., 1985 Synthetic E.coli Buell et al., 1985 Synthetic E.coli Elsevier et al., 1985 · Synthetic E.coli Niwa et al., 1986 Synthetic E.coli Saito et al., 1987a, b

SRIF Synthetic' E.coli Itakura et al., 1977

GRF cDNA E.coli Gubler et al., 1983 cDNAE. coli Mayo et al., 1983 Genomic library E.coli Mayo et al., 1985 Synthetic E. coli Pages et al., 1987

PGH: Porcine growth hormone; bGH: Bovine growth hormone; cGh: Chicken growth hormone; sGH: Salmon growth hormone; tGH: Trout growth hormone; IGH - I/II: Insulin like growth Factor - I and II; SM-C: Somatomedin - C (-IGF - I); SRIF: Somatotropin release inhibiting factor; GRF: Growth hormone releasing factor.

(Compiled by Su, 1988) 109

Table 2 Comparison of different mammalian species with regard to the production of transgenic animals (Krausslich et al., 1986)

Mice Rabbit Pig Sheep Cattle

Ovulations per animal: i) Without 6-10 8-12 10-15 1-3 1 superovulation ii) with superovulation 15-30 20-30 30-40 4-8 4-7

Visualization of pronuclei +++ +++ ++ + + and injection

Survival rate (offspring/ injected egg) 10-15% 8-12% 5-10% 5-7% 3-5%(?)

Integration frequency (transgenies/offspring) 15-25% 10-15% 8-12% 2-5% ? 110

(+} (-)

GRF S RIF~-----....,

PL

RPL LI POLY SIS GH-- GLUCOSE. METABOLISM

INSULIN SEX HORMONES SM (IGF-1, IGF-11)

THYROID HORMONES

GR 0 WT H t------1 GLUCOCORTICOIDS

SKELETAL NON-SKELETAL GROWTH GROWTH

Figure 1 The hormone control of growth performance (Su, 1988) 111 SYNTHETIC OLIGONUCLEOTIDE FOR HIGH AMINO ACIDS 1-24 HUMAN PITUITARY

HGHmRNAt REVERSE TRANSCRIPTION t • cDNA OII1jlJJl!IJIJJlllll!JJlllill s4BP tHoe Ill lac PROMOTER lIOtllllllJlllllllllllj] RI / HGH AMINO ACIDS 25-191

HGH 25-19\

pHGH 107

TRANSFORM E.COLI

Figure 2 Human growth hormone produced(Watson et al.,1983) 112

Hind IH Pif I Hine.I 111 \ ------1 Hind JU ROH I

·- ttorn I

v F'11.t f. Com f l\lh ;\line rho~ph.1t:1~~C' \_

llOATr. TflAHSTO~M S.l l l CT. H I 0 H' Tr Ta

Hind Ill \

( (~ • -~. 'OC P1H ~ SJON'

Pl A ~MID

Figure 3 Construction of a hybrid gene between bacterial and mammalian DNA sequences (Seeburg et al., 1978) 113

TRANSLATlON---i...,

,,,.------·~ ~ • LACT AMAS.E .1 ... ') ') ~ - 2 ___ ._____ ,...... _ __ -.. ---., -23 ...... -:.>I 179 100 181 182

Th r Pro /,lo· ,, s p S e r Gin AC.C ,,.,,,. t ,.... " • s '___ CC T' GCA J G/,( T ( T t_ ' ~ \..I ~ .• - ~\

( T,... ITGC GGfA- ~cc r •V 1-\C.r\ ,..... 'c t \..; ' l I Psi I I I L-·------, r--·------·-----J

f\JA.\AP\ I ) ( 'ncu/\ HI

Ceo HI

Figure 4 Postulated nucleotide sequence aound the Pst 1 site in the hybrid gene. 114

- '

-- ,,

I

Figure 5 Gene construct using methallothionein promoter (Hammer et al., 1985) {a) .... a: ...... -'O 0 e:z a:J .!: w0 (/) (/) I

MT Promotor Growth SV40 ori Hor.mane cDNA ...,..______.,.,,_,..,.,_,,.,,._,,..... (2. 19k b) - ·- 'IP-

'b) .... a: ...... "O g e:z a:J c w ro en J:

I -.- ...\...... -~ ···w·N .'*',.,i;, .. -...... I , J J . - • . k e e . ' -~·=·~~;.: -=='". 1==:.: K.J».. •::•:.:-:-:-:- r I MT Prornotor Growth Hormone Gene S 0 ori

.... ~~~~~~~~~------~~~~-(5.26kb)~~~~~~~~~~~~~~~~~~~-lli> ...... Figure 6 Gene construct for injections in sheep (Ward et al., 1988) Vl Genetic Improvement of Livestock in Developing ountries Using the pen Nucleus reeding System

John. Hodges Senior Officer Animal Breeding & Genetic Resources FAO, Rome, Italy

A genetically superior nucleus flock or Summary herd is established under controlled conditions Thegeneticimprovementofruminants where testing and genetic selection are carried in developed countries has made much progress out. The test group is first established by screen­ in the last 30 years by the use of large-scale field ing the base population for outstanding females. data derived from many herds based upon the They are then recorded individually in the nu­ progeny testing of males. A new option is now cleus herd and the elite females among them are being discussed which is to use the new tech­ used by MOET with superior sires to contribute nique of Embryo Transfer with Multiple Ovula­ embryos which are carried by recipient cows tion (MOET) in an Open Nucleus Breeding from the base population. The resulting off­ System (ONBS). The genetic theory of the new spring are reared and recorded and the males option has been established (1,2,3) and in a few among them are evaluated genetically using the developed countries trials are being established performance of their sibs and paternal half-sibs to test the feasibility in practice. Other important and their own performance where they exhibit factors still needing investigation are the ability the trait. From these an elite group of males with to use MOET successfully as a routine in a large high breeding value is selected which is used in herd, to compare the costs of the new option the base population for genetic improvement by against the existing system and to calculate how natural service or A.I. The ONBS yields higher much of the theoretical gain is achieved in prac­ rates of genetic progress per annum than estab­ tice, especially when applied to commercial lished progeny testing systems mainly because conditions with a field population. This will all of the shorter generation interval. It is capable of take time. Meanwhile, the suggestion has been being adapted to different species in which MOET made that the Open Nucleus Breeding System is available. It also permits control of the mix of (ONBS) may be especially valuable for develop­ genes released to the base population. The 0 NBS ing countries (3 ,4) where the use offield progeny is flexible for the introduction of exotic testing and A.I. has largely been a failure due to germplasm from other populations, for example the need for an established infrastructure, both semen or embryos from temperate breeds and for field recording and A.I. This paper therefore also for the continuous addition of outstanding outlines the principles of ONBS and considers individuals screened from the base population. its possible use related to the needs, problems and opportunities of genetic improvement in This paper emphasizes that in theory, developing countries. the ONBS offers a method for the improvement of (i) purebred exotic breeds, (ii) purebred in­ recording system introduced temporarily in the digenous breeds and also (iii) stabilized crossbred field or by relying upon the owner's knowledge genotypes. The level of response is naturally of the animals. These exceptional animals are dependent upon the scale of the ONBS testing brought together in the nucleus flock or herd to programme, the size of the base population and form a test group under controlled management the selection intensity. Individual ONBS plans where recording of their performance traits can need to be designed for each situation and spe­ be carried out routinely and where embryo trans­ cies. The paper gives an example of an ONBS fer facilities can be located. The elite females plan for dairy cattle using a female adult group of from this test group are brought into multiple 250 milking animals plus the associated young ovulation and their embryos are transferred to stock.! the other females in the test group and unre­ corded females in the base population. The off­ spring, both males and females, are kept in the Introduction test group where their traits of interest are re­ corded. Then the male offspring are evaluated on Successful livestock improvement the basis of their siblings' performances and their needs changes in several components of produc­ own records where these are available for a trait tion. A genetic improvement strategy in devel­ recordable in both sexes. The best of the males oping countries is rarely successful unless ac­ are then used extensively in the base population companied by improved nutrition and herd health either by A.I. or natural service. together with better stockmanship and feed re­ sources management. In practice such changes The female offspring are next consid­ are best accompanied by genetic improvement ered as potential elite females to donate embryos programmes, either for the purebreed indige­ by MOET for the following cycle. This selection' nous breeds, by breed substitution or by cross­ involves appraising them against the elite cows breeding with exotics. already in the nucleus flock or herd which have already been used for MOET. The critical deci­ In many developed countries, genetic sion about which females to retain for MOET is improvement is achieved by the use of field a balance between those with the highest indi­ performance recording on a large scale. For vidual genetic merit combined with their suita­ example, the progeny testing of dairy bulls using bility for multiple ovulation, while seeking to daughters' records has been highly successful minimize the generation interval between each when combined with A.I.. However, in many cycle of MOET. developing country conditions, where field re­ cording and A.I. are not possible, genetic im­ The comparison between progeny and provement has been limited. The advent of sibling tests is illustrated in figures 1 and 2 for Multiple Ovulation Embryo Transfer (MOET) dairy cattle. Sibling test results are obtained in 4 may offer a new means of achieving genetic years compared with 7 for progeny testing. This progress by the use of Open Nucleus Breeding difference may be larger when, as often happens, Systems (ONBS), using the sibling test instead the age of the first calving of daughters in the of the daughter test. The genetic relationships of field progeny testing scheme is delayed. On the brother and sister are the same as those of father other hand the female siblings in the nucleus and daughter. The system makes use of full and herd under controlled conditions can be calved at half-sibs to estimate the breeding value of males younger ages. Although the sibling test is less under test. First, the unrecorded base population accurate due to there being fewer siblings for is screened to find apparently superior females. each male on test in an ONBS than there are This can be done either with a very simple offspring in a field progeny testing plan, the 118

Figure 1

PROGENY SIULING YEARS TEST TEST TEST BULLS I !-<2 SEMEN SISTERS PREGNANT

2~2 TEST~ DITTO I30RN CALVE

3

4 . DITTO 1st LACTATION COMPLETE TEST RESULTS

4 l~ -.PREGNANT 2 ~~ --.. 1st LACTATION . 6 !-::2 COMPLETE

7 ~TEST RESULTS

Figure 2

------PROGENY TEST------

30 - 50 DAUGHTERS 7 YEAR TEST

-----SIBLING TEST------4 .-8 SISTERS 16 - 20 HALF SISTERS 4 YEAR TEST LESS ACCU RA TE SHORTER GENERATION INTERVAL 119

shorter generation interval of the sibling test ures used in practice. Thus numbers used here provides higher genetic gains per year. must be regarded solely as an example. The actual size of any ONBS will depend upon a Open Nucleus Breeding System for variety of factors which will be discussed later in Dairy Cattle the paper. Figure 3 shows that in this example, it The ONBS is illustrated now for a dairy may be possible under developed country condi­ cattle ONBS in a developed country. (Figure 3). tions to produce 500 embryos annually from the It is assumed in this example that when the 32 elite cows, from which an estimated 260 ONBS is in full operation, the nucleus herd will calves are born. In developing country condi­ comprise 250 females. These will include the tions, where local conditions for MOET are less elite females subject to MOET, and their female favourable, it is sure to be considerably less. For offspring in first lactation and other cows screened ease ofillustration, it is now assumed that all 260 from the base population and on test. The younger animals remain alive, fertile and available for animals must also be kept in addition. From this selection. In practice an inevitable but variable herd of lactation cows on test, it is assumed for loss will occur from different causes such as the example that each year the 32 top perform­ failure of heifers to conceive or to complete a ance animals are chosen for MOET using semen pregnancy or lactation. There will also be some from 8 elite sires. This semen may be locally loss and infertility among males. These losses, produced from previously tested males in the though of importance in practice, are ignored scheme, or it may be introduced from other here to simplify the logistics which show ani­ improvement programmes elsewhere. In this mals moving through an ONBS for dairy cattle. way the Nucleus Breeding System is open. The 130 females calve at 2 years of age and are milked in the test group with full recording. (In It should be noted that the figures here the case of a scheme combining milk and growth chosen for illustration are arbitrary. The genetic selection then both the males and females would progres? per year will depend upon the selection be growth recorded). When the first lactation intensity which is determined by the actual fig- records are available, either complete or esti-

Figure 3

/".. EXAMPLE OF OPEN NUCLEUS DREEDING SYSTEM FOR-DAIRY CATTLE

250 cow NUCLEUS PLUS~~ HERD (FULL RECORDING SURPLUS ocf & SEMEN

HEIFERS ANNUAL SELECTION S BULLS CHOSEN CALVE AT TOP 32 00T .,- ON SISTERS 2 YEAR sod PERFORMANCE

130 :j? 500 130 cf CALVES CALVES 120 mated from partial lactations, they are used to on individual performance records and the elite calculate breeding values for the sibling males. cows among them are retained for MOET as long On average this will involve 4 full-sibs and 12 as they remain responsive to the technique. This half-sibs per bull evaluated. The summary aver­ means that annually about 130 lactation cows age logistics are given: will return to the base population leaving the best 120 cows in the test herd drawn from those who 32 elite cows for MO ET with 8 sires (4 have just completed a first lactation and from cows per sire) · older cows previously subjected to MOET. This 512 embryos recovered (16 per cow) permits the new crop of 130 replacement heifers 256 successful pregnancies (50% sue- to enter for their first lactation and thus maintain cess) the size at 250. (8 calves per cow -4 females and 4 males) Genetic Progress in ONBS (130 heifer and 130 bull calves in total) First, it is appropriate to define "Adult" 130 bulls to be evaluated by sibling test and "Juvenile" MOET. These alternatives are (4 full sisters, 12 half - sisters per applicable in principle to several species, but bull) illustratedfordairycattle(Figure4).AdultMOET means that embryo transfer takes place only after An elite group from these 130 test bulls the first lactation when the individual cow rec­ with breeding values is now chosen for extensive ords of performance are available. Juvenile use when A.I. is available. Where there is need or MOET means embryo transfer at about one year demand for natural service, then a higher propor­ of age when the heiferis sexually mature, but has tion of the above-average bulls is made available no lactation records on which to evaluate her for use in the base population. Annually the test genetically. Adult MOET with dairy cattle gives females whose records have been used for their a generation interval of about 3. 7 years, whereas brother's genetic evaluation, are then evaluated

Figure 4

(FROM SMITH (3) )

---...------EM l3 R YO TRANSFER------.

ADULT AFTER FIRST LACTATION GENERATION 3.7 YEARS

JUVENILE 1 YEAR OLD GENERATION 1.8 YEARS 121

Figure 5 (FROM SMITH (3) AFTER WOOLLIAMS AND SMITH (5) )

POSSIBLE RATES OF GENETIC CHANGE PER YEAR FOR MILK YIELD IN DAIRY CATTLE WITH DIFFERENT BREEDING SELECTION SYSTEMS

GENETIC RESPONSE (%PER YEAR)

--PROGENY TESTING SCHEMES RATES CURRENTLY ACHIEVED 0.2- 1.1 RATES POSSIBLE CURRENT SYSTEM 1.5 EFFICIENT SYSTEM 2.0 EFFICIENT SYST. WITH FEMALES SELECTED & BRED WITH ADULT MOET 2.0- 2.3 EFFICIENT SYST. WITH FEMALES SELECTED & BRED WITH JUVENILE MOET 2.1 - 2.3

--MOET NUCLEUS HERD SCHEMES ------""'"II RATES POSSIBLE: ADULT MOET NUCLEUS SCHEME 1.8 - 2.4 mVENILE MOET NUCLEUS SCHEME 2.6 - 3.5 (WITH EMBRYO SPLITTING x 2) 2.7 - 3.8 (WITH EMBRYO SPLITTING x 16) 2.9 - 3.9 ruVENILE MOET NUCLEUS SCHEME WITH INDICATOR TRAIT (Co-heritability= 0.25) 2.7 - 4.8

THE RANGE IN RESPONSE WITHIN A SYSTEM REFERS TO MODERATE AND HEGE MOET RA TES. MODERATE 4 MALES, 4 FEMALES PER FULL SIB SHIP AT SELECTION; 8 DONORS PERSIRE.HIGH8MALES, 8FEMALES PERFULLSIBSHIP AT SELECTION; 16DONORS PER SIRE. COEFFICIENT OF VARIATION= 0.15.

which may be used for ONBS. Juvenile MOETreduces this to 1.8 years. Figure 5 shows the theoretical rates of genetic change The scale of each ONBS needs to be per year for milk yield in dairy cattle using designed individually for actual conditions and different selection systems. These are potential species. It is not possible to give these in the changes. The actual gains will always depend abstract and expect that they will automatically upon the numbers of animals and the selection fit any or all circumstances. Some of the critical pressure. The theoretical gains show that ONBS issues are the size and distribution of the base using MOET may be a worthwhile alternative to population, the management system under which progeney testing. In developing countries the the base population is kept, the numbers of 0 NBS merits further research to test its practical animals which can be accommodated in the feasibility since field progeny testing is rarely nucleus herd facilities and the level of success possible, whereas governments often have live­ with MOET. Calculations to take account of stock station facilities already in their control animals being held while awaiting test must be 122

Figure 6 shows the annual rates of re ti cal grounds from use of ONB S for traits other genetic change which may be expected on theo- than milk in cattle and also traits in sheep and pigs.

Figure 6

(FROM SMITH (6) QUOTED IN SMITH (3))

------RATE OF ANNUAL GENETIC CHANGE(% MEAN) ------CATTLE SHEEP PIGS GROWTH 1.4 1.4 2.7 2.6 2.4* LEAN 0.5 0.9 1.6 .LQ .L.8.* L2. SEX LIMITED MILK LITTER LITTER TRAITS YIELD SIZE SIZE 1.5 2.1 4.7 2.0 M"' 5...5.

*JUVENILE EMBRYO -NORMAL REPRODUCTION TRANSFER EMBRYO TRANSFER made and cost-benefit evaluations are needed on purebred indigenous breed, as for example in an the advantages and disadvantages of alternative environment with highly specific adaptability strategies for age at first parturition and use of needs or can exotics be introduced as purebreds Juvenile or Adult MOET. Another important where the environment and management can be point is whether semen is to be used by A.I. from adapted to their needs? Very often in developing the elite proven sires or whether genes from the countries the. first option is not desirable and the elite proven animals are to be spread through the second is not possible. Therefore crossbreeding base population also or only by natural service. is often the prefered route. ONBS could be adapted for controlling the gene mix in a base A most important issue in developing population. The type of semen used for MOET countries relates to the genetic improvement on the elite indigenous females must be adjusted strategy (Figure 7). Is it designed to improve the subsequently as the base population becomes

Figure 7

OPEN NUCLEUS BREEDING SYSTEM PUREBRED IMPORTED BREEDS INDIGENOUS PUREBRED BREED INDIGENOUS FEMALES (ADAPTED) & IMPORTED MALES/SEMEN (HIGHER PERFORMANCE) CONTROL% GENE MIX 123

crossbred. When the exceptional females result- ONBS and MOET. The staff to be trainednatu­ ing from the sib test are crossbreds and are rally must include the station personnel who are themselves treated by MOET with semen from responsible for the livestock in the nucleus herd crossbred males, they maintain the desired gene or flock. The training of extension staff is vital as mix. they will be involved in sharing the details with the livestock owners in the area whose animals form part of the base population. The success of Figure 8 FROM SMITH (3)

ADVANTAGES AND DISADVANTAGES OF NUCLEUS BREEDING UNITS COMPARED WITH FIELD BREEDING SCHEMES -----ADVANTAGES ------+GENETIC LIFT IN ESTABLISHING THE UNIT +FASTER RATES OF GENETIC CHANGE +CONTROL OVER HUSBANDRY & TESTING +MORE SELECTION POSSIBLE FOR ECONOMIC MERIT +USE OF TRAITS DIFFICULT TO RECORD IN FIELD +CONCENTRATION OF BREEDING RESOURCE +POSSIBLE USE OF EXPENSIVE TECHNOLOGIES +ECONOMIC BENEFITS OBTAINED SOONER +LOW COSTS ON A NATIONAL SCALE +SEPARA TE NUCLEUS UNITS FOR DIFFERENT SETS OF BREEDING +OBJECTIVES/ENVIRONMENTS -----DISADVANTAGES------..----- + RISK OF DISEASE AND LOSS (avoid by dispersing age classes) +RISK FROM CONCENTRATION STOCK & RESOURCES IN ONE UNIT + POSSIBLE GENOTYPE x ENVIRONMENT INTERACTIONS IN COMMERCIAL PRODUCTION +NEW FUNDS NEEDED TO SET UP AND OPERATE +RE-EDUCATION OF PRODUCERS TO ACCEPT MOET BRED STOCKS

Conclusions for Developing Countries such a system is dependent upon the willing cooperation of the owners of the livestock. They, The benefits of ONBS combined with as always, need to be convinced of the value of MOET, compared with field breeding schemes this new scheme being proposed by the govern­ are summarized in Figure 8. The specific advan­ ment. Patience and skill in describing its opera­ tages and expected rates of annual genetic gain tion, benefits and possible risks must be em­ should be assessed in each case and must include ployed. Ideally the ONBS/MOETsystem should not only the genetic aspects, but also the logistic haveacooperativeaspectwhich brings the lead­ and economic factors. ers of the local livestock owners into the discus­ sions and decisions, especially concerning the Another important concern should evaluatidn of the animals and choice of which always be the training of the staff at all levels so males are to be used in the base population. that they fully understand the theoretical basis of 124

An old problem with trying to buy key techniques for channelling biotechnology outstanding animals from local owners for a into genetic improvement of livestock in the government programme, has been their natural future. reluctance to part with their best animals. It is understandable. Cooperation between the gov­ At present, without molecular genetics ernment who operate the nucleus flock or herd ONBS and MOET have theoretical advantages and the owners may be made more productive which if adapted successfully to the conditions and successful if the females screened from the of developing countries, could offer a new ap­ base population are loaned rather than bought. proach to livestock improvement. They also As borrowed females they then enter the test offer a vehicle which, if successfully estab­ flock or herd for a period of recording. When lished, will be suitable for conveying expected they return to the owner it should be possible to benefits of future biotechnology research and provide a genetically superior offspring to ac­ development in embryo manipulation and gene companying them as a return to the owner. transfer.

The theory of ONBS appears to be very References attractive. The system is already being tested in some developed countries in Europe for dairy 1. NICHOLAS, F.W. and SMITH, C. (1983). cattle. It now needs to be tested and practical Increased rates of genetic change in dairy cattle protocols designed for application in developing by embryo transfer and splitting . Animal Pro­ countries. Only then will it be possible to calcu­ duction. 36. 341 - 353. late how much of the theoretical genetic gain can be achieved in practice with defined species and 2. CHRISTENSEN, L.G. and LIBORIUSSEN, traits and whether the needed levels of success T. (1986). Embryo transfer in the genetic im­ with MOET can be achieved with indigenous provement of dairy cattle. In :Exploiting New animals. Technologies in Animal Breeding: Genetic De­ velopments (ed. C. Smith,J.W.B.King,andJ.C. Finally, it may be asked what all this McKay), pp 37 - 46 Clarendon Press, Oxford. has to do with biotechnology? The answeris that Embryo Transfer is an essential part of biotech­ 3. SMITH, C. (1988). Applications of embryo nology for mammals, since it is by the manipu­ transfer in animal breeding. Therio genolo gy 29 .1 lation of the embryos that molecular genetics is 203 - 212. applied to livestock. In the longer term future, biotechnology with ET is likely to open up new 4. SMITH, C. (1988). Genetic improvement of means of producing animals with the desired livestock in developing countries using nucleus mix of genes, whether as improved purebreds or breeding units. World Animal Review 65. 2- 10. by bringing together the desired high perform­ FAO,Rome. ance traits of one breed with the adaptive traits of an indigenous breed. 5. WOOLLIAMS, J.A. and SMITH C. (1988). The value of indicator traits in the genetic im­ While the molecular engineering tech­ provement of dairy cattle. Animal Production niques are still very imprecise and the results 46. 333 - 345. variable, intensive research is taking place in developed countries. Also the sexing and clon­ 6. SMITH, C. (1984). Rates of genetic change in ing of mammalian embryos is the focus of much farm livestock. Research and Development in research activity. ONBS andMOET appear to be Agriculture. 1. 79 - 85. Potential of Multiple Ovulation, ryo and Associated Technology for Livestock Improvement in Developing Countries of Asia

P.N. Bhat Director & Vice-Chancellor National Biotechnology Centre Indian Veterinary Research Institute Izatnagar - 243 122 [up] India

Introduction gonadotrophin (PMSG) was reported by then (Cole and Hart, 1930) and its action was tested In farm animals, the number of times a for folliculogenesis and ovulation. Although con­ female can become pregnant is limited. The siderable research on collection and transfer of number of offspring that a female can produce in bovine embryos was conducted during 1930s its life-time could however be increased by re­ and 1940s (Dowling, 1949; Pincus, 1949; Row­ peatedly allowing her to produce a number of son and Dowling, 1949; Willet et al., 1951; ova and transferring them to reproductive tracts Lamming and Rowson, 1953), the first calf of other females to complete the gestation. produced as a result of egg transfer was born at Advances in reproductive physiology during the Wisconsin in 1951 (Willet, 1951). Interest in last 50 years have greatly helped in understand­ embryo transfer, thereafter, increased as studies ing the control of estrus and ovulation, induction involving mammalian eggs began to appear in of superovulation, collection of ova/embryos the literature. These studies revealed that em­ nonsurgically and cryopreservation of embryos. bryo transfer in farm animals was not as simple During the past two decades these discoveries as that in laboratory animals and thus a simple have been developed into viable technologies technique similar to artificial insemination for and are now routinely used for livestock im­ embryo transfer was needed. Classical tech­ provement. nique for nonsurgical collection of bovine em­ bryos was made available by Newcomb in 1979 The first embryo transfer was done in and since then it has been in use. rabbitbyWalterHeapein 1891 to understand the embryo uterine interactions.Warwick and Berry It was in the early 1970s that great in 1932producedalamb by embryo transfer. No commercial interest in bovine embryo transfer one, however, thought about application of developed. European dual purpose breeds of embryo transfer technology (ETT) for animal cattle were imported to North America and improvement till superovulation technique in because of their small numbers these became cattle was reported by Casida et al. (1940) and extremely valuable. Breeders wanted a method (1943). Discovery of pregnant mares' serum to increase the reproductive rate of these females 126

for profitable sale of their offspring. This helped probably results in an overestimation of embryo the technology of embryo transfer in farm ani­ recovery. mals to become commercially viable. Over the past decade, several European and North Ameri­ Follicle Stimulating Hormone (FSH) can firms have been using this technology com­ mercially and several ET centres have been Pituitary glands of pigs contain large established. Governments in Wes tern Europe, amount of FSH and these, therefore, have been particularly in UK, France, Ireland, West Ger­ used for commercial FSH production. FSH-P is many, Canada, United States, Japan, and Austra­ closely related to hormone produced by cows lia have supported considerable research in and is responsible for stimulating growth of embryo transfer. ovarian follicles prior to ovulation. FSH has relatively short biological half-life and in order Embryo transplantation research in to obtain a superovulatory response, breeders bovines in developing countries, including In­ must inject the cow twice a day over a period of dia, is in the initial stages of experimentation. time. Informa{~on on superovulation response, recov­ ery rate of embryos, quality of embryos in our In order to maximise superovulatory animals under existing agroclimatic conditions response, FSH dose ranging from 32-50 mg have is still not available. Information, therefore, on been used (Belloues et al., 1969; Ha.~ler, 1978; various areas of embryo transfer technology, Schams et al. 1978; Seidel et al., 1978; Lubbadh including embryo sexing, splitting and gene et al., 1980; Barnes et al., 1982; Yadav et al., manipulation has been reviewed to know the 1986). However, there exists a strong ambiguity present status of this technology and its applica­ regarding the appropriate dose schedule for get­ tion in livestock improvement. ting optimum response. Nelson (1979) and Belevich et al. (1984) demonstrated thatFSH at Superovulation in cattle higher doses gave better response. Lerner et al. (1986) correlated the FSH dose with age of the Superovulation may be defined as the donor. They reported that while among the older development and the ovulation of more than the donors increasing dose of FSH was associated normal number of follicles (Avery et al., 1962 with increase in number of ova plus embryos ab). A number of papers and reviews on su­ recovered, among the younger donors increas­ perovulation using PMSG and FSH at different ing dose of FSH had a negative effect. It was dose levels have been published (Beehan and therefore, recommended that in order to maxi­ Sreenan, 1977; Elsden et al., 1978; Seidel et al., mise production of transferrable embryos, ad­ 1978;Newcombetal., 1979; HyttelandGreeve, justment in dose ofFSH should be made accord­ 1983; Beal et al., 1984). Pregnant mares serum ing to the age of the donor. As an explanation gonadotrophin (PMSG), follicle stimulating they assumed that increasing dose of FSH in the hormone (FSH) and human menopausal gona­ older donors resulted in the stimulation, recruit­ dotrophin (HMG) have been used extensively as ment and development of a greater proportion of superovulatory drugs. secondary or small antral follicles. In contrast, the lesser number of ova and embryos obtained Evaluation of the superovulatory re­ with increasing dose ofFSH in younger donors sponse by rectal palpation has also been widely could be due to over stimulation of ovaries. used. This assessment is gen~rally inaccurate Similar observations were reported by McGowan and often gross underestimates are recorded when (1983) when the dose ofHMG was increased in the number of corpora lutea on any one ovary beef heifers. exceeds nine (Guay and Bedoya, 1981). This 127

Halley and Rhodes (1979) observed Treatment regimes employed were constant dose an increase in number of CL and embryos re­ (Schams et al., 1979), decreasing dose (Elsden covered when animals were treated with FSH et al., 1978; Barnes et al., 1982) or increasing between 36 and 43 mg and decrease in response doses (Crister et al., 1980). Decreasing doses, between 43 and 48 mg. They concluded that however, gave better response (Chupin et al., neither a very high nor a very low dose yielded 1983; Daniella et al., 1984 a). The FSH treat­ good results. Elsden and Kessler (1983) reported ment both for five days (Seidel et al., 1978, that in Nellore cows 24 mg FSH was more Barnes et al., 1982; Lauria et al., 1983; Alcivar effective than 36 or 50 mg. Munro (1986) re­ et al., 1984; Beeker et al., 1986; Lerner et al., ported that the number of embryos recovered 1986; Lindsell et al., 1986) and four days was not significantly affected by dose of FSH (Lubbadeh et al., 1980, Chupin and Procureur administered to either B. Taurus or B. Indicus 1983; Donaldson, 1984 a; Walton and Stab­ cows. The number of embryos collected from B. bings, 1986; Yadav et al., 1986) have been used Taurus cows increased as the dose of FSH in­ with similar results. creased while the greater number of embryos were recovered from Brahman cows after ad­ The removal ofLH contamination from ministration of some intermediate dose (32 mg). commercial FSH preparation increased the re­ In Gircows, satisfactorynumberof embryos had· sponsiveness of the cow to FSH treatment been recovered after superovulation with 28 mg (Humphrey et al., 1984; Murphy et al., 1984). of FSH (Zan war, 1978). However, 36 to 40 mg LH in superovulatory regimes appear to be dele­ of FSH had been found to stimulate a better terious; it acts at several stages in the reproduc­ follicular growth in Haryana cows (Dutta and tive processes. TheFSH in pure form increased Taneja, 1987). the production of transferable embryos by in­ creasing the number of fertilized embryos FSH has been used both as single (Donaldson and Ward, 1986; Donaldson et al., (Lubbadeh et al., 1980; Hill et al., 1985; Becker 1986). The ratio of FSH; LH was crucial; 10: 1 and Pinherio, 1986) and double (Barnes et al.; ratio gave optimum superovulatory response 1982; Chupin and Procureur 1983) injection per (Chupin and Procureur 1984). day. Considering the short half- life of exogenous FSH, twice daily schedule of injection appeared Pregnant Mare' Serum Gonadotrom to be essential (Laster, 1971). However, Beeker phin (PMSG): and Pinherio (1986) did not find any statistical difference between fractionated or single injec­ PMSG is a glycoprotein found in high tions per day. concentration in blood of pregnant mares be­ tween 46 and 130 days of gestation (Cole and It is believed that gonadotrophin treat­ Hart, 1930) produced by the specialised cells of ment stimulates the growth ofnormal follicles in foetal origin. It is a highly potent gonad-stimu­ 1.7 - 2.0 mm range, and at the same time de­ lant and has been shown to possess both FS Hand creases the proportion of atretic antral follicles · LH activities (Monniaux et al., 1983). As com­ (Monniaux etal., 1983). Medium size non-atretic mercial preparation, PMSG is purified from the follicles were shown to be most abundant in the serum of pregnant mares. ovariesbetweenday0-5 and9-13 of the estrous cycle (Moore et al., 1984). As such FSH treat­ PMSG has been used as superovulatory ment was generally initiated between day 8 and agent in bovine for quite a long time (Hammond 12 of the cycle, although day 9 was found to be and Bhattacharya, 1944; Rowson, 1951; Brock the most suitable for a greater yield of embryos and Rowson, ~ '\2; Gordon et al., 1962; Hafez (Lindsell et al., 1985; Lindsell et al., 1986). et al., 1963a). lt increases the number of normal 128

preantral follicles and decreases the number of (Betteridge, 1977). An increasing interval from atretic follicles, particularly those at the size of administration of PMSG to observed estrus was antrum formation (Monniaux et al., 1984 ). Doses shown to coincide with a decreased ovulation of PMSG as 1000 - 3000 LU. (Dowling, 1949; rate (Barbella et al., 1979). But the use of Rowson, 1951; Hafez et al., 1963 a; Fulka et al. prostaglandins and its analogues have greatly 1976; Greve, 1980; Gupts et al., 1983; Riha et facilitated the degree to which the estrous cycle al., 1984; Almedia, 1987) have been used suc­ of the cow could be synchronised (Tervit et al., cessfully to induce superovulation in cows. 1973). Sreenan and Beeham (1976) reported that the ovarian response increased with the dose of Preliminary information on the use of PMSG. But higher doses of PMSG have been PMSG and PG in combination have been reported to yield more number of degenerated reported by Tervit et al. (1973) and Elsden et ova (Schilling et al., 1980). Superovulation in al., (1974). It is now a common practice to use Zebu taurus cross-bred cows with PMS G yielded PMSG alongwith PG either as a single injection two corpora lutea in each ovary (Ramakrishna et after 48 hours (Schilling et al., 1980; Gupta et al. 1987). Luteal phase stimulations with 1500- al., 1983 Boland et al., 1984; Gregory et al., 2000 i.u. ofPMS Gin Indian cows yielded greater 1986) or after 72 hours of initiation of the treat­ number ofovulations than follicular phase stimu­ ment (Parmigiani et al., 1984). Two injections lation (Rao and Ramakrishna, 1987).Superovu­ of PG 12-24 hours apart have also been used by lation attempted in cross-bred repeat breeding several workers (Dhondt et al., 1978; Sanchez cows using 1500 i.u. of PMSG gave moderate to Gareial et al., 1984; Parnigiani et al., (1984). poor response (Verma et al., 1987). Larger number of an ovulatory follicles were reported as Injection of PMSG during the mid­ the major problem of superovulatory response luteal phase of the estrous cycle of the cow led to with 2000 i.u. PMSG in Haryana cows (Dutta a drastic increase ofLH andFSH concentrations and Taneja, 1987). for several days due to long half-life of this glycoprotein. Consequently follicles continued There are apparently two waves of fol­ to grow and release high amount of oestradiol licular growth in cows one occuring at the begin­ into the circulation (Bevers andDieleman, 1987). ning of the estrous cycle (3-6 days) and another Higher concentration ofoestradiol may adverse! y between 12 and 14 days (Rajakoski, 1960; affect early embryonic development of the fer­ Mariana and Huy, 1973). It has been shown that tilized oocytes (Bouters et al. 1983). PMSG administered during mid-luteal phase (8- 12 days of the cycle) was more effective in terms Moyaert et al. (1985) found that in the of estrus and ovulatory response as compared to cows treated with 2 ml of monoclonal PMSG that given on earlier or later days (Mariana and antibody at the time of Ist insemination after Huy, 1973; Elsden et al., 1974; Phillippo and superovulatory estrus, concentrations of oestro­ Rowson, 1975; Elsden et al., 1978). Since it has gen declined to basal level within 24 hours of · a long half-life (Laster, 1972; Schams et al., treatment and remained low until embryos were 1978; Lauria et al., 1982) superovulation with recovered on day 7. These animals treated with PMSG requires only one injection. Subdivision anti PMSG showed shorter period of estrus than of the total dose into multiple injections does not the control group. Dhondt et al. (1978) reported improve the response (Hafez et al., 1963b). that 1 ml of PMSG antiserum at the time of 1st insemination resulted in more number of CL, Variation in time from PMSG treat­ greater number of recovered eggs, higher per­ ment to the onset of estrus was a major problem centage of fertilised eggs and lesser number of in using this drug as a superovulatory agent unovulated follicles. 129

Both PMSG and FSH were employed Lactating dairy cows had rather low in many early cattle superovulation studies. ovulatory response to gonadotrophins. But by Comparative trials revealed that FSH was more repeated use of the nonsurgical embryo recov­ effective than PMSG in terms of the yield of ery, sufficient number of embryos may be ob­ fertilized eggs (Dowling, 1949). Dzium et al. tained on a yearly basis to justify the use of the (1958) noted that FSH produced less variable procedure for improvement and dissemination ovulation rate than PMS G. Several other studies of dairy breeds (Brand et al., 1978b). indicated that FSH was better than PMSG in terms of number of CL produced and number of Temperature and photoperiod had been embryos recovered (Laster, 1972; Elsden et al., shown to affectreproductive parameters in cattle 1978; Lubbadeh, 1980; Danielle et al., 1983; (Tucker, 1982). Greater population of vesicular Bakhitov, 1984; Kim et al., 1986; Sergeev et follicle in winter and spring than in summer and al., 1986). autumn suggested that this variation had high effect on the outcome of superovulation (Rajak­ Effect of age, postE partum interval, oski, 1960). Superovulation response, embryo lactation and season on superovulaa recovery rate and quality of embryos were tion response, embryo recovery and maximum in winter and spring than in summer quality and autumn (Hasler et al., 1983; Sergreev et al., 1983; Shea et al., 1984; Lerner et al., 1986). Donors with characteristics that make them valuable are kept beyond the age at which Collection, Evaluation and Transfer most cows are culled. Age-related changes, there­ of Embryos fore, are of economic importance among these donors. As the age advances, the reproductive Initially embryos were collected surgi­ capacity in terms of population of oocytes or cally around day 4 after the estrus. The proce­ follicles seems to decrease (Yamauchi, 1963; dure used was cumbersome, expensive and there Erickson, 1966). Moore (1975) studied superovu­ was a danger of scar tissue and adhesions. The latory response with PMSG or HAP in cows mid-ventral laparotomy was originally used at (average age of 8 years) and heifers (14-18 Cambridge, England (Rowson et al., 1969). months) and reported that response in heifers Nevertheless, in cows, embryos are still col­ was greater than in cows. Several workers subse­ lected surgically for research purposes. quently demonstrated that younger animals yielded more number and higher percentage of With the success of nonsurgical transferable embryos (Brand et al., 1978a; Hasler collection techniques, it was possible to collect et al., 1981;Donaldson, 1984; Kim et al., 1985ab; embryos several times from a donor cow without Walton and Stabbings, 1986). damaging its reproductive organs and subse­ quentfertility. During early years, several work­ Time interval since last calving was ers described methods for nonsurgical collection considered by many workers as a factor affecting of embryos in cows (Rowson and Dowling, the superovulatory response. Sixty to one hundred 1949; Dziuk et al., 1958). The technique, how­ and twenty days post-partum was found to be ever, wasperfectedin 1970s (Sugie, 1970; Sugie most suitable where 81-86% of animals showed et al., 1972ab; Newcomb, 1979). Methods of superovulatory response (Hasler, 1983; Riha, collecting embryos have not changed since then. 1986). However, superovulatory response did Most nonsurgical recoveries were made on day not vary with time since calving, although fertili­ 6, 7 and 8 as bovine eqibryos descend into the sation rate did so. uterus around day 4.5 to 5. Embryos represent- 130

'ing 50-90% of ovulations were harvested non­ ferred into virgin heifers resulted in acceptable surgically in cattle (Betteridge, 1977). pregnancy rates. Embryos transferred to heifers resulted in higher pregnancy rates than those Cattle embryos were first described by transferred to repeat breeder recipients. While Hartman and his Coworkers in 1931. Since then synchrony of estrus between recipients and a lot of information on chronological develop­ donors was necessary to obtain optimal preg­ ment of bovine egg has been published (Hamil­ nancy rates, in practical situations this was diffi­ ton andLairg, 1946; Thibault, 1967). The overall cult to achieve because neither all superovulated diameter of the cow embryo is 150-190 mi­ (Elsden et al., 1974; Moore, 1975) nor nonsu­ crometer including the zona pellucida. The evalu­ perovulated (Johnson, 1966;Hurniketal., 1975) ation of ova and embryo is very subjective; even cows exhibit estrus all the time after synchron­ experienced technicians encounter embryos that isation treatment. are difficult to evaluate. Most commonly used criteria to evaluate embryo includes good over­ Ovulation time and insemination of all structure, uniform size of the cells and proper donors stage of development after breeding. Shea ( 1981) described the morphological evaluation of In order to determine the time of in­ embryos. Embryos after evaluation, are trans­ semination of donors, either fixed time after ferred to the recipient either surgically or non­ PGF2 alpha or careful observation for the onset surgically. Embryos of doubtful quality are of estrus was the common practice. On the as­ usually cultured for a few hours before transfer. sumption that in superovulated cows ovulation Surgically transferred embryos yield higher occurs over a prolonged period, multiple insemi­ pregnancy rates (60- 70%), than those transferred nations (3-4 times) were performed at 12 h nonsurgically (50-60%). The standing lapa­ interval during and immediately after estrus to rotomy in the paralumbar fossa perfomed with ensure good fertilisation (Mapletoft, 1981). local anaesthesia is the common method used for Others have used two doses of semen at each surgical embryo transfer in cows. insemination (Elsden, 1980).

Synchrony of donors with recipients Although there were no large differ­ ences in fertilisation rates using fresh or frozen Survival of the transferred embryo semen, a single insemination with fresh semen depends on a close alignment between the devel­ was demonstrated to be sufficient (Betteridge, opmental stage of the embryo and the stage of the 1977), perhaps because spermatozoa which have uterus of the recipient. Synchronisation is usu­ not been frozen and thawed survive longer in the ally ensured by using the onset of estrus as a gential tract of the cow (Gordon, 1983 b). reference point in matching the donor and recipi­ ent animal. Maximum pregnancy rates (91 %) It was not known untilrecentlywhether were achieved when recipients and donors were ovulations in superovulated animals occur si­ exactly synchronised at the onset of estrus multaneously in all the follicles which are stimu­ (Gordon, 1983). Recipients out of phase by 1 lated to grow or whether ovulations will be day had low pregnancy rates between 52 and extended over a number of hours. Maxwell et al. 56% (Rowson et al., 1972). ( 1978) reported that in cows superovulated with PMSG, 45% and 91 % of ovulations occurred Almeida et al. (1974) demonstrated that within a period of 24 and 48 hours after the onset uterine environment at transfer was of greater of estrus, respectively. No ovulations were ob­ importance than the origin of the embryos. served in the first 18 hours. In another study, Embryos from repeat breeder donors, when trans- Angel (1979) used faproscopy in cows treated 131

with PMSG and FSH and reported that ovula­ loss through the vagina due to embryo-uterine tions were spread over 24 hours or more. asynchrony(Chang, 1966; Chang, 1971)orfrom failure of follicles to shed their oocytes Schiewe et al. (1983) demonstrated that (Monniaux et al., 1983). a single insemination with two units of high quality semen (20 x 10 6 sperm/unit) resulted As a general rule, however, the number in 63% and 88% fertilisation rates when su­ of recovered embryos increased with ovulation perovulated (FSH) beef cows were inseminated rate (Angel et al., 1984) and the number of at either 12 or 24 h after the onset of estrus, transferrable embryos per collection was related respectively. Insemination with two units of (r = 0.64) with total embryos and ova in a semen four times each at 12 h intervals resulted collection (Donaldson, 1984 b). Others were of in a fertilisation rate of 74%. It was concluded the view that the fimbria may not pick up all the that a single insemination with two units of high oocytes because the ovaries were grossly en­ quality semen, 24 h after the onset of estrus, larged (Betteridge, 1980). should result in optimal conception rates. The same group (Schieweetal. 1985) using asimilar Present status of superovulation in protocol, obtained fertifi.sation rates of 87.2% buffaloes and 90.0% when'donors were inseminated 24 h after the onset of estrus with one or two units of Two types of gonadotrophins have been semen as compared to 26.1%and78% fertilisa­ used to superovulate donor buffaloes. These are, tion after insemination at 12 h using one or two pregnant mare serum gonadotrophin (PMSG) units of semen, respectively. Insemination with and follicle stimulating hormone (FSH), usually two units of high quality semen at 24 h after the administered during the mid luteal phase (9-14 onset of estrus would seem desirable because a days) of the estrous cycle. Among these two lower number of degenerating embryos ( 14 .3 %) glycoproteins PMSG has more sialic acid con­ were recorded in this group. tent which accounts for its longer half-life. For this reason PMSG is usually administered in a From these studies, it would appear that single dose of 3,000 - 5,000 IU whereas FSH is ovulation in superovulated (FSH) cows initiates injected twice per day followed 48-72 liours later approximately 24 h after the onset of estrus and by a luteolytic dose of prostaglandin F2 alpha. would not be expected to continue over a long time. The fertilisation rates would be reduced if PMSG has the advantages of being cows were inseminated only once. available in large quantities and at relatively low cost as compared to the gonadotrophins of It has been estimated that only 50-80% pituitary origin (FSH-P). Its use also reduces the of embryos are recovered (Angel et al., 1984) labour cost because only single injection is Shea et al. (1976) demonstrated that both fertili­ required to superovulate a buffalo. sation and embryo recovery rates tend to de­ crease as ovulation rates increase. Sreenan et al.' The most common method of supero­ (1974) recovered 32% and 50% of embryos from vulation involves a single dose of PMSG (3,000 animals with average ovulation rates of 23 .2 and IU) during the mid luteal phase of the estrous 13.6,respectively. Angel et al. (1984)recovered cycle followed 48 hours later by a leuteolytic only 14.8% embryos in heifers and 39.5% in dose of PG F2 alpha (500 microgram) (Vlahov, cows when uterine horns were flushed and cor­ 1985;Karaivanov, 1986; Nanda and Bhat, 1988). pora lutea were counted on the ovaries after More specifically injection of PMSG on day 14 slaughter. Such loss of embryos might have of estrous cycle results in higher ovulation than resulted from early embryonic death, lysis or the injection on day 12 of the cycle (Parnpai et al 132

., 1985). Of course there has been an indication Vlahov et al., 1985; Drost et al., 1986; Karaiva­ that PMSG when administered on day 15 of the nov, 1986), decreasing doses (Drost and Cripe, estrous cycle followed by PG 60 hours later 1985; Daraivanov, 1986; Motwani, 1987 Nanda results in higher ovulation rate (Motwani, 1987). and Bhat, 1988) doses over 5 days (Drost et al., As the sample size is very low in Parnpai's 1983; Drost and Cripe, 1985; Drost et al., 1986 experiment and as there is no experimental and Motwani, 1987) or doses over 4 days period evidence to support Motwani's statement no (Vlahov et al., 1985andKaravanov,1986Nanda conclusions can be drawn. Shariffudin and and Bhat, 1988). There was no difference in Jainudeen, 1984; Madan, 1987 got better result response between constant versus decreasing in terms of ovulation rate after administration of treatment regimes (Drost and Cripe, 1985). PMSG on day 10 of the induced cycle. Doses of PMSG varying from 2,500-3,000 IU (Sharifud­ Both PMSG and FSH produce similar din and Jainudeen, 1984; Parnpai et al., 1985; ovulatory responses but more follicles ovulated Vlahov et al., 1985; Karaivanov, 1986) have with FSH than PMSG (Sharifuddin and been used successfully to induce superovulation Jainudeen, 1984; Nanda and Bhat, 1988). No in buffaloes though many workers have pre­ substantial difference in the ovarian response ferred the upper limit. has, however, been established either for PMSG or FSH schedule. Percentage of the donors ex­ During superovulatory treatment, es­ hibiting estrus is high in both PMSG and FSH trus in donors is regulated by intramuscular group (Vlahov et al., 1985). The protaglandin - administration of PG. The normal dose of PG is standing heat intervals established in PMSG in the range of25 mg (Parnpai et al., 1985) or its treated group is significantly longer than in FSH analogue 500 to 1,000 microgram (S harifudding treated group. Percentage of the nonovulated and Jainudeen, 1984; Vlahov et al., 1985; follicles following the PMSG treatment is con­ Karaivanov, 1986) administered as one dose or siderably higher (Vlahov et al., 1985). Besides as two doses 12 hours apart (Drost et al., 1983; the equal initial follicular response, significantly Drost and Cripe, 1985; Motwani, 1987) either lower percentage of anovulatory follicles are after48 hours or after24 hours (Sharifuddin and obtained after giving GnRH in superovulatory Jainudeen, 1984) or after 60 hours (Motwani, regimen at the time of estrus (Sharifuddin and 1987) after initiation of superovulation treat­ Jainudden, 1984 and Karaivanov, 1986). Per­ ment. This induces luteolysis in donor cows. centage of animals responding with more than two CL in PMSG regimen is considerably lower Follicle stimulating hormone and than that of FSH schedule but the average num­ prostaglandins were used to superovulate the ber of good quality embryos per buffalo donor is buffaloes according to treatment regimes de­ not high in bothPMSG andFSH treatment(Kari­ veloped in cattle (Drost and Cripe, 1985). In vanov, 1986). order to maximize superovulatoryresponse, doses of FSH ranging from 40 to 70 mg have been used Superovulation often frustrates embryo (Drost et al., 1983; Drost and Cripe, 1985; Vla­ transfer practitioners because the donor buffalo hov et al., 1985; Karinov, 1986) as two injec­ response is inconsistent. There is a lot of indivi­ tions per day. Drost et al. (1986) have tried to dual variation while using either PMSG or FSH. stimulate a greater superovulatory response with Nonsurgical collection generally results in the an increased dose of FSH in buffalo. Too varied recovery of satisfactory number of embryos in a response and small sample size could not detect relation to the number of corpora lutea estimated any significant differences on response. Treat­ by the rectal palpation. Sometimes no embryos ment regimes employ either constant doses are recovered even though corpora lutea are (Drost et al., 1983; Drost and Cripe, 1985; present on ovaries (Nandy et al., 1984). 133

The evaluation of ovaries through rec­ tion involved in follicular growth is severely tal palpation in buffaloes is much more inaccu­ limited. Potentiality of a buffalo to be superovu­ rate than in cows and often the ovulatory re­ lated is very low in terms of number of primor­ sponse has been underestimated. It is difficult to dial follicles and growing follicles (Nanda and determine accurately the number ofcorpora lutea Bhat, 1988). In surti buffaloes the average number on the small ovaries of buffaloes, particularly of primordial follicles in the right and left ovary when it exceeds 5 or 6 or when they are ac­ is 4,600 (304 to 16,565) and 5,200 (420 to companied by several large tense follicles, 25,390) respectively (Danell and Settegen, 1983). leading to overestimation of the response and These numbers are much lower than those re­ poor recovery of embryos. The CL of Buba/us ported in Nilli-ravi heifers where the average bubalis is small, more deeply embedded, fused numberwas 19,000(SamadandNasseri, 1979). and generally has a less pronounced papilla than that ofBos taurus (Drost and Cripe, 1985; Nanda A study conducted by Nanda and Bhat, and Bhat, 1988). 1988 on Indian buffaloes indicated the meager population of growing secondary and tertiary Failure in recovery of embryos from follicles at different stages of the cycle, which superovulated buffaloes may be attributed to may plausibly explain the lower response to several factors. Overestimation of the ovaries superovulation in buffaloes. Danell (1987) has can lead to a failure of the fimbriae to envelope reported that the average number of primordial the ovary at the time of ovulation. There is a great follicles per pair of ovaries from surti buffaloes problem in fluid recovery (20 to 60% ). Sharifud­ is about 12,000 (Range: 1,300 to 40,000) indi­ din and Jainudeen support the problem of fluid cating alowerreproductive potential than cattle. nonrecovery by suggesting that it may be due to Though there is individual difference between the uterine wall adhering to the tip of the catheter the two ovaries, on an average there are about the and preventing backflow of the fluid. Evidence same number of primordial follicles in the left based on laparoscopy indicates that as the and right ovaries of the cycling surti animals, uterine horn distends, the uterotubal junction 6,234 (49.3%) and 6,402 (50.7%) respectively. opens and causes fluid to escape through the Buffalo matures later and lives longer than ovarian end of the fallopian tube. This may be a cattle which probably means a relatively slow valid reason for less fluid recovery and loss of decrease in follicle number compared to cattle. embryo into the peritoneum which escapes The cattle heifers ovulate 6-14 times, while the through the ovarian end of fallopian tube. This buffalo heifers have only 4.2 ovulations on the may be of antomical origin which need further average. Thus it seems that there is a real dif­ investigation. Drost and Cripe (1985) have ference in number of primordial follicles be­ reported that hatched blastocysts particularly tween cattle and buffaloes (Danell, 1987). This when collapsed, are very difficult to identify and is perhaps one of the reasons why buffaloes do may easily slip from microscopic vision when not show a positive response to superovuratory flushing is done beyond 5 days of superovula­ treatment. tory cycle. Multiple ovulation, Embryo Transfer Variability between individuals and (MOET) and Progeny Testing within the individuals has made progress of embryo transfer uncertain in buffaloes. MOET is considered mainly as a tool Different dose regimen and various treatment for production of superior breeding bulls. Theo­ schedule have less repeatable results when tried retical considerations indicate that it might be more and more to find out an optimum criteria. possible to increase the rate of genetic progress Part of the difficulty is that quantitative inform a- 134 in a dairy population by 10-15 percent in a the process is cumbersome and technically dif­ conventional breeding scheme by practising ficult since the Indian experience is that farms MOET on the very best cows. But in practical beyond 200 to 300 cows are unmanageable under performance ET will only have small effect on our conditions. the rate of genetic progress in a conventional breeding scheme. Traditional breeding schemes The European and American alterna­ must be changed in order to make better use of tive is to do the same under field conditions with the new technique. village cows. The information on such experi­ ence is not a happy one in our country. Besides Quality improvement is possible by the problem of different management regimes rigorous selection within indigenous breeds for being practised within the system which vary drought capacity. Selection of males can bring from zero input to marginal supplementation, about rapid improvement but it can only be done the reliability of the data is difficult to ascertain if the performance of their daughters and sons is and thus any expenditure on progeny testing is known so that estimates of breeding value are not of any value. Attempts made so far have not available for selection" yielded any worthwhile results. Therefore, a method has to be found which can produce This process involves breeding of a sire reliable data at low cost. MOET is a scheme to several females ( 100 cows) to produce records which can give the necessary reliable data and a on 30 progeny. If 10 males are bred to a 1,000 reduction on costs. females and data recorded on 30 females size for on lactation for each male for one year, cost per In a MOET project for testing one bull record comes to about Rs.50,000 and takes a based on 20 recorded daughters we would need total of 5 years for crossbreds and 7 years for 15 donorsand51 recipients. These recipients can indigenous animals. If such 10 bulls are put to be used from farmers' herds. Female calves within test, on an average two bulls would be one one month of birth from these nominated cows standard deviation above the mean, and only can then be transferred to the farm for testing. these two bulls would be proven at a cost of This would reduce the expenditure on recording, Rs.5 lakh each. Even if the money is available, breeding and follow up. It would produce con-

Population And Production Of Livestock

1982 1990 2000 Change due to ETT

Total population 263.0 299.0 355.0 Total bullocks 88.0 0.97.6 123.7 113.0 Milk production from indigenous cows 11.9 15.8 21.0 21.6 Crossbred cows 2.7 5.8 18.0 20.20 Buffaloes 17.0 24.2 27.5 28.30 Others 2.0 3.0 3.0 3.0 Total 33.6 47.8 69.5 73.36 135

temporary data reliable for a good genetic evalu­ If an additional 50,000 cows, i.e. top ation. In a milk shed for one million cows, if five 5% are also given embryos of high-producing such farms were established, 20 proven sires will cattle/buffaloes every year, this will contribute become available from them after the 4th year. an additional 0.3 million cows/buffaloes of high The situation would be as follows, if this pro­ genetic merit which will replace the entire 1 gramme involves only one quarter of the avail­ million within the next 10 years cutting down the able population. numbers by almost 20%. If embryo sexing is superimposed on this, the time interval is re­ duced by half, i.e. from 24 years to 12 years. Ifembryo sexing is perfected, the entire expenditure on nominated services could be There are other possibilities such as reduced by half. If embryo splitting was also selecting cows on progeny test. This is not so resorted to, it would reduce it to one fourth if only helpful as may be thought. Progeny testing of one split was standardised. bulls is so important because they cannot be evaluated on their own phenotype.But cows can Reduction in numbers, increase in milk be, and thus the value of a progeny test is much yield and draught animal power (DAP) can be smaller. The accuracy of selection of a cow on done only by embryo transfer and associated her first record is 0.5. If the mean of 5 daughters technologies. In iridigenous,crossbredcowsand is used as well, the combined accuracy is 0.632. buffaloes by subjecting them to breeding with By the time the progeny test records are avail­ proven sires milk production per cow can be able, the cow should have another 2 or 3 records, increased from 480 kg to 600 kg and eventually and the accuracy of her average production would to 1,200 kg in crossbreds from 1,500 to 2,500 kg be 0.645 or 0.674. Progeny test information and buffaloes from 9 50 to 1,200 kg and to 2,000 would increase this accuracy, but by less than kg eventually. This genetic improvement should with a cow's single record. Selection decisions make it possible to provide 74 million tonnes of would have to be delayed while progeny test milk in 2000 AD and bulk of this milk will come records are obtained. Thus raising the generation from 10 million crossbred cows. length progeny testing of cows would seem to have little application. If embryo transfer could All this is based on the presumption be applied in all breeding herds, then the rate of that proven sires of high genetic potential will improvement in a progeny testing programme become available for breeding the cows and could be increased by some 15%. large number of sons of Proven sires will also become available around the periphery of milk Associated Technologies sheds to produce better and improved indige­ nous cows. Cryopreservation of embryos Cryopreservation of embryo forms an For a cow/buffalo milk shed with popu­ important facet of embryo transfer for longterm lation of 1 million, 60 proven sires by 10th year conservation of germ plasm, easy and rapid trans­ would cover 0.6 million cows with proven sires port of potentially valuable strains or breeds of and 0 .4 million with sons of proven sires. By the livestock for future use. end of 18th year enough frozen semen will be available from 120 proven sires to bring about a Embryo during cryopreservation or quantum jump in milk yield and animals' power thawing may be damaged either by formation of which will allow 10% reduction beginning 18th large intracellular ice crystals or by increased year in cow and buffalo population. intracellular concentration of solutes and ac­ companying changes that results from dehydra- 136

tion of cells during freezing. Fast freezing of Wilmutet al. (1973) studied the effects embryo minimize damages from solution effect of cooling rates on survival of blastocyst frozen but it leads to formation of large ice crystals in presence of 2.0M DMSO or 0.3 M sucrose. which causes severe mechanical damage. On the Complete degeneration was observed in embryo other hand, slow freezing though it prevents cultured for 24 hours after being frozen and large ice crystal formation, it leads to increased thawed in the presence of sucrose but a propor­ damage from solution effect. The addition of tion developed after being frozen in presence of cryoprotectant such as glycerol or dimethyl sul­ DMSO. Willadsen et al. (1978) froze day7 cow foxide (DMSO) in freezing medium remove embryos in 1.5 M DMSO in PBS at 0.3°C minute dehydration effect of cells; embryo thus may be to -36°C and at the rate of 0.1°C/minute between cooled slowly enough to prevent formation of . -36°C and -60°C before being plunged directly large ice crystals. Rapid thawing at 28° - 37° C into liquid nitrogen. The embryos were subse­ prevents reformation of ice crystals. The cry­ quently thawed at room temperature. Maximum oprotectant is then removed at room tempera­ pregnancy rate (8/12) was obtained with surgical ture. transfer immediately after thawing and step wise dilution of DMSO. Campo and Akes son (1983) A wide variety of compounds have froze day 7 or 8 cattle embryos in straws con­ been demonstrated to protect cells against freez­ taining PBS+20% foetal calf serum and gly­ ing damage. These are divided into two catego­ cerol. Freezing was done from room temperature ries, those which freely permeate most of the to -7°C at the rate of 1°C/minute and then to cells and those which do not permeate. The -35°C at the rate of 0.3°C/minute. Thirteen former includes glycerol, dimethyl sulfoxide embryos were thawed and 10 of them transferred (DMSO) and ethylene glycol while the latter nonsurgically to 10 heifers, of which 2 con­ includes sucrose, polyvinylpyrolidone (PVP), ceived. hydroxyethyl starch (HES), dextrans and albu­ min. Massip et al. (1984) successfully froze day 6.5-8 cattle embryos in straws in 1.36 M Mainly two methods of embryo freez­ glycerol and 1.36 M glycerol + 0.25 M sucrose ing have been tried. The first method involved in PBS. Embryos were directly transferred into cooling of embryos slowly to sub zero tempera­ freezing chamber of -7°C to -25°C or -35°C the ture, storing them in liquid nitrogen at - 196°C cooling rate was 0.3°C/minute. Thawing was and then warming them slowly until it gets done by dipping straw in 20°C water bath. The thawed. The total time required for freezing and glycerol was removed by keeping embryos in 1 thawing was about 2 1/2 hours (Whittingham M sucrose solution for 15-20 minutes. The preg­ et al. (1977). In this the embryos were cooled nancy rate of embryo frozen in 1.36 M glycerol slowly to the temperature of-33°C thus reducing and 1.36 M glycerol+ 0.25 N sucrose was 38.5 the time required for cooling. The method was and 60 per cent, respectively. primarily developed for freezing of embryo of large animals such as cattle and sheep. It was The most commonly adopted medium demonstrated that early stages of cattle embryos for freezing cattle embryo was 1-1.5 M glycerol upto morula stage were sensitive to cooling and in PBS. The cooling rate from room temperature did not survive well after exposure to tempera­ to -7°C was not very important and could be ture around 0°C. Embryos at blastocyst stage, adjusted between 3° and 5°C/minute. Seeding however, tolerated cooling much better and were was done at -7°C and straws were kept at -7°C for therefore, more suitable for freezing (Trounson 5 minutes. Cooling from -7°C to -30°C or -35° et al., 1976 a,b). was done at the rate of 0.3°C to 0.6°C/minute. Straws were then plunged directly into liquid 137

nitrogen for long term storage. Thawing was licular oocytes as near as possible to the time of done by direct transfer of straws into water bath ovulation and thus allows maximal physiologi­ 20°C or 37°C. Stepwise removal of glycerol with cal maturation of the oocytes within their fol­ 0.25-1 M sucrose in PBS was followed. It has licles and accounts for improvement in fertiliza­ recently been demonstrated that with the use of tion rate of bovine oocytes (Brackett and others, 1.36 M glycerol+ 0.25 M sucrose in PBS as 1984). cryoprotectant medium, the removal of glycerol after thawing can be avoided and embryos upon A major limitation to the use of in vitro thawing could directly be transferred into re­ matured oocytes is the number of oocytes ob­ ceipient. By this method, pregnancy rate of42.1 % tained per female. Even when cows were stimu­ against 30% with 1.36 M glycerol was obtained lated with FSH, the usual number of oocytes (Massip et al., 1987). recovered from a donor ranged from 5 to 10 (Lambert et al., 1986; Leibfried-Rutledge et al. In vitro Fertilization , 1986). Whenever large number of embryos are required, in vivo matured oocytes proved an An eventual alternative to superovula­ expensive supply. Collection of immature oocytes tion as source of bovine embryos, especially for from ovaries obtained at the abattoir would research purposes, may prove to be the in vitro provide a much cheaper source. maturation and fertilization of the follicular oocyte. Recent advances in understanding of The success of fertilisation in vitro re­ oocyte maturation, sperm capacitation and lies on completion ofboth oocyte maturation and embryonic development have accentuated in sperm capacitation. In the in vitro fertilisation vitro fertilisation (IVF) in domestic ruminants system, one of the prime reasons for failure of the (cattle, sheep and goats). The first ruminant resulting zygotes to develop was the occurrence offspring from IVF was a bull calf born in 1981 ofpolyspermy, which however, was not the case (Brackett et al., 1982). In vitro fertilisation re­ in vivo. Sperm concentration and time of sperm­ quires (1) a system for harvesting the oocyte oocyte interaction should, therefore, both be which is efficient and does not damage the oocyte controlled to minimise polyspermy. or donor, (2) nuclear cytoplasmic and cumulus cell maturation of the oocytes cumulus complex, The production of off-spring from in (3) non-damaging culture systems, (4) a system vitro fertilisation is considerably restricted by for capacitating the spermatozoa and (5) effi­ the absence of suitable in vitro culture systems. cient systems and conditions for accomplishing Cattle and sheep embryos resulting from in vitro fertilisation and development of the embryo from fertilisation have been developed to morula and fertilisation to a transferable stage (First and blastocyst stage. This was accomplished by di­ Parrish 1987). rect surgical transfer of zygotes or early embryos to the oviduct of the cow (Brackett et al., 1982; The quality of oocyte used for the in 1984). The oviducts of sheep in situ (Eyestone vitro fertilisation is critical. Ifit presents an ideal et al., 1985)andrabbits (Sirardetal., 1985)were morphology, i.e. an expanded and clear cumu­ · also used as temporary surrogate in vivo incuba­ lus, the probability that it will reach the two cell tors for development of cattle zygotes or early stage was about 50percent (SirardandLambert, embryos to a transferable morula or blastocyst 1985). However, oocytes recovered at the same stage. time, but without a cumulus or with· only a compact cumulus (corona), have lower chances Chang's experiment of in vivo capaci­ of developing normally (Sirardand Lambert, tation of semen and fertilisation in vitro laid 1985). Laparoscopy permits the recovery of fol- down the foundation ofIVF. Subsequent devel- 138 opment of high ionic strength medium capacita­ cattle embryos have shown that the embryos, tion and suitable culture medium by Whitting­ inspite of a great reduction in their cell number, ham rolled the cart of IVF onward for a better are able to develop through early cleavage application in domestic animal species. Produc­ (Willadsen andPolge, 1981) and upto blastocyst tion of calves from fertilisation in vitro of oocytes formation (Ozil et al., 1982)/ The simple bisec­ maturedin vivo (Bracket et al., 1982; 1984; Sirard tion of fully compacted late morula or early and Lambert; 1985; Sirard, et al., 1985) or in blastocyst allows the production of identical vitro (Critser et al., 1986) and lambs from oocytes twins in routine embryo transplantation proce­ matured in vitro (Chang et al., 1986) proved the dures in cattle. The twinning rate (66.6%) ob­ fact that IVF could be successfully done. The tained after cervical transfer of monozygotic efficiency of producing off-spring was less than half embryos showed that it was possible to that achieved by natural processess. The most produce identical twins after direct transfer of limiting step was the inefficiency of develop­ micromanipulated embryos. (Ozil et al. 1982). ment of embryos in vitro from the pronuclear to The pregnancy rate, 64.2 per cent, was similar to the blastocyst stage. Embryo developmental the rate (58.8 per cent) obtained after non-surgi­ competence could be acquired by immature cal transfer of non-manipulated embryos (Renard oocytes cocultured with abundant number of et al., 1981). This indicated that these embryos preovulatory granulosa or cumulus cells which had an ability to survive after having been bi­ receive stimulation from FSH, LH and estradiol sected. (Staigmiller and Moore, 1984; Crister et al., 1986). Immediate benefits of this procedure are two folds. Not only the potential number of Fertility of bull could be assessed by calves available from a donor was increased, but IVF in a short tenure and number of spermatozoa there was possibility of producing genetically per ovum required for fertilisation could be identical twins. The production of identical twins reduced. Commercial multiplication of iden­ in large numbers could facilitate sire testing, tiical embryos was expected to be dependent on reduce cost of semen production and enhance the use of abattoir obtained oocytes as recipients testing of maternal traits. Identical twins have for transfer of nuclei from valuable multicellular been useful in studying the genotype x environ­ stage embryos and on the ability to develop the ment and genotype x level of nutrition interac­ resulting embryos to a transferable stage. Par­ tions. ticularly in buffaloes, large drainage of germ plasm through slaughter can be retrieved by de - Sexing veloping embryos through fertilisation in vitro. Abundant number of embryos for research in The desire to control sex and to produce reproductive biology and gene microinjection offspring of a predetermined sex is one of the could be provided by much needed alternative - major goals of embryo transfer industry. For IVF than tubal recovery. Development, there­ this, one would require insemination of donor fore, of the biotechnologies for producing with a homogenous population of either X-bear­ transgenic off-spring was dependent on volume ing or Y-bearing spermatozoa, identificaiton of production of precisely staged embryos and on embryonic sex prior to transfer to a recipient or the ability to develop these unique embryos to a identification of embryonic or foetal sex early in state compatible with transfer to reciepients. pregnancy so that undesired pregnancies could be terminated. The genetic sex of zygote is fixed Splitting with the penetration of ovum by fertilizing sper­ matozoon. Those ova that are fertilised by sper­ Micromanipulation experiments on matozoa bearing a Y-chromosome become 139 genetic males, while those fertilised by X-bear­ The technique of chromosome analysis ing spermatozoa become genetic females. The on half embryos presents a feasible means of gonadal and phenotypic sex are dete1mined later, sexing embryos, which may then be stored or but rarely do these differ from genetic sex. transferred to recipients. The disadvantage is that one of the twin embryos has to be destroyed Techniques for separation of X- and Y­ for chromosomal analysis which might be miti­ bearing spermatozoa have not withstood testing gated by using a small proportion of the embryo in controlled experiments involving farm ani­ for analysis. mals (Amann and Seidel, 1983) and currently is not an acceptable option. Embryos before trans­ Other approaches to embryo sexing fer, therefore, seem to be a better candidate for include quantification of differences in meta­ sexing because ofpractical and economic disad­ bolic activity between male and female em­ vantages of procedures of identification of bryos. A colorimetric test for glucose-6-phos­ embryonic or foetal sex during pregnancy. phate dehydrogenase activity is able to correctly identify the sex of 64% of mouse embryos Embryos form cattle, sheep and rabbit (Williams, 1986). Attention has been given to have been successfully sexed before transfer by the possibility of differentiating male and female four cytological approaches i.e. (1) sex chroma­ embryos by assessment of the nuclear density of tin (Barr body) detection in day 6 rabbit blasto­ interphase cells of living embryos (Kim, 1986). cysts (Gardner and Edwards, 1968), (2) chromo­ Among the newer techniques under study is some analysis on trophoblast biopsies in day 12 DNA hybridization for identification of male to day 15 bovine embryos (Hare et al., 1976; embryos. DNA sequences that are specific to the Wintenberger et al., 1980), (3) chromosome Y-chromdsome are known to exist and probes analysis on biopsies from day 5 to day 6 bovine for such specific sequences are being tested for embryos (Moustafa et al., 1978; Singh et al., their use in embryo sexing. The isolation of 1980) and (4) chromosome analysis on half DNA sequences speecific to the human Y-chro­ embryos produced by micromanipulation mosomeanditspotential use in in situ hybridiza­ (Popescu et al., 1982; Picard et al., 1984). tion techniques for sexing (Bishop et al., 1983) may have application to bovine embryos, once Although identification of Barr bodies similar segments of the bovine genome have was highly accurate, only a small proportion of been isolated. embryos examined could be sexed. Furthermore, the granular nature of cytoplasm in embryos of A non invasive approach to sex selec­ domestic animals obscure nuclear material, thus tion is provided by immunological detection of making identification of the inactivated X-chro­ a factor that is specifically expressed as male but mosome difficult. not female embryos (Anderson, 1987). Ample evidence has been provided that H-Y antigen The limiting factors with trophoblastic was expressed on pre-implantation embryos of biopsy and karyotyping procedure were the rela­ various species. Furthermore, its detection with tively low number of cells in metaphase and its reasonable accuracy was to be used to identify quality. The techniques used were also quite the sex of an embryo. Current procedures for tedious, time consuming and relatively inaccu­ identification of embryonic sex by detection of rate making them impractical for routine com­ H-Y antigen remain largely experimental at this mercial use. In this method, two-thirds of the time. Wachtel (1984) suggested that H-Y anti embryos tested could be sexed with certainty gen present in early embryos of bovine and (Betteridge et al., 1981) but only one third of the serological reagents that identify H-Y antigen sexed embryos resulted in pregnancy. could be used to identify the sex of the embryos. 140

A method that could be used routinely at the field Cloning generally provokes the state of level, however, remains to be developed. nuclear transplantation which remains as a desir­ able goal for cloning non homozygous adults. Cloning Nucleus of higher genetic potential could be transferred into an egg that had previously been Difficulty in achievement of a desired enucleated. Bromhall (1975) was the first to phenotype is faced when the elements contribut­ show that, nuclear transplantation into mammal­ ing to that phenotype are distributed in a varied ian eggs was possible when he transplanted gene pool. Conservation of a required phenotype embryonic nuclei into rabbit eggs. New form of through sexual mode of reproduction was diffi­ nuclear transplantation in mice aims at removal cult. Development of new phenotypes by selec­ of nucleus by pinching it off in a small part of the tive breeding and subsequent conservation and egg cytoplasm and plasmamembrane with an propagation of the desired phenotyes by vegeta­ enucleated egg by the use of sendai vims to bring tive reproduction i.e. to produce clones of iden­ about fusion (Mc Garth, 1983). The procedure tical individuals may be economically advanta­ avoids the damage done by microsurgical inser­ geous. Markert (1984) hypothesized that mam­ tion of a pipette into the egg. However, normal malian eggs could easily be provoked to develop development from any advanced nucleus do not parthenogenetically but no mammalian parthe­ occur. This art is successful only in very early note had ever developed to term. However, the embryonic nuclei. Cloning by nuclear transplan­ parthenote cells could be rescued by aggregating tation using adult nuclei from desirable adult them with normal cells to make a chimera that phenotypes could be achieved by prolonged could reach adulthood and reproduce using the incubation in oocyte cytoplasm. parthenote cells. Major advantages of cloning comprise Cloning in mammals, a common oc­ of removal of genetic variation, copying small currence was expressed in the form of identical number of animals with outstanding genotypes twins, triplete, or quadruplets. It could be accom­ for breeding, rapid production oflines with special plished artificially in the laboratory by dividing traits, and automatic sex selection. Active reces­ early embryos. However, cloning embryos of sive gene perpetuation and increase in breeding unknown potential phenotypes was of no impor­ depression limits the use of cloning. tance. Major interest lied in cloning of adults with demonstrated phenotypes that were eco­ Trans genesis nomically valuable. In vitro multiplication of early embryo to have many copies for storage by Animals that have integrated foreign freezing could be a potential tool for practical DNA into their germ line as a consequence of application of cloning if few embryos were tested experimental introduction of DNA are termed as for their economic viability of desired pheno­ transgenic. The transgenic animals offer new type. opportunities for the genetic improvement of economic merit. Useful transgenes may come The future of embryo transfer industry from 1) known genes with large effects, 2) genes lies in production of identical homozygous indi­ with restrict metabolic pathways, reducing feed­ viduals through removal of one pronucleus and back factors or unwanted products, and 3) fusion by application of cytochalasin B that stops cy­ genes with exogenous regulatory sequences tokinesis and leads to nuclear division. Many which are out-with the normal feed back control such embryonic homozygotes were successfully system (Smith et al., 1987). transplanted into the uteri of recipient and car­ ried to term (Hoppe and Illmensee, 1977). Methods of producing transgenic ani- 141

mals comprise of microinjection of DNA, infec­ and the development of systems for an appropri­ tion of preimplantation embryos and cell injec­ ate regulation of transgene expression in tion into a host blastocyst that leads to develop­ transgenic animals are the most important pre­ ment of chimera. One of the most powerful suppositions for this realization. Production of embryological techniques is the aggregation of transgenic cattle (Kramer et al., 1985), rabbit cells from two or more preimplantation embryos sheep,pig (Hammer et al., 1985), goat(Fabricant to produce chimeras (Mintz, 1965) or the injec­ et al., 1987) and poultry (Donald et al., 1987) tion of cells from one embryo into the blastocoel has given hope for better future. Although, the of another embryo (Gardner 1978). The most potential benefits from gene transfer forincreas­ spectacular chimeras are the "geeps" produced ing the efficiency of food and fiber production by Fettilly et al. (1984) showing phenotypic are theoretically great, definition of the true characteristics of both parental genomes (Sheep value of such techniques awaits experimenta­ & Goat). tion. Kraublich (1986) suggested identification of following genes for transfer: Limitations for application of trans gen esis to domestic animals are as follows: 1) for­ 1. Cloning genetic sequences that re­ eign genes once integrated in the genome may gulate body growth, wool growth, lactation and not be expressed, 2) transmission of foreign gene reproduction. from transgenic germline may encounter varied situation, 3) expression of foreign genes may 2. Cloning genetic sequences which vary not only between lines of transgenic ani­ influence immune response and disease resis­ mals but also between the offsprings within one tance and given line and 4) fertility of transgenic animals may be severely lowered (Renard and Babiner, 3. Identification and cloning of single 1987). The application of gene transfer tech­ genes with major effect in farm animals like niques to domestic species has been impeded Booroola gene of sheep, halothane sensitivity - primarily by the inability to visualize egg nuclei. stress susceptibility gene in pigs. However, by using interference - contrast mi­ croscopy (I-C) for rabbit and sheep eggs Embryo Transfer in India (Hammer et al., 1985), and centrifugation and I-C microscopy for pig and cow eggs (Wall et The Government of India initiated a al., 1985), the nuclei become visible and can be National S & T project on 'Cattle Herd Improve­ micro injected. ment for Increased Productivity using Embryo Transfer Technology,' through its Department of Gene transfer offers a powerful new Biotechnology. This project is underimplemen­ tool in animal research. Transfer of genes into tation through a multiagency network on mis­ the bovine genome has been accomplished. sion mode. However, successful directed expression of these incorporated genes has not been achieved to Objectives of the S&T date. New combinations offusion genes may be an effective way of producing transgenic domes­ To introduce embryo transfer technol­ tic animals which show controlled expression of ogy as an effective tool for increasing the pro­ the desired genes. duction and productivity of cattle and buffaloes.

Gene transfer in farm animals will have To create institutional infrastructure for a considerable impact on the future of livestock research and development in E.T. production. Efficient production of transgenes 142

To create a skill pool of scientists, prac­ germplasm to the native recipients. By increas­ titioners and technicians. ing number of progenies from high genetic po­ tential females superior nucleus herds can be And above all, to create a seed stock of developed for better perpetuation of desired traits genetically superior animals for rapidly building in the population. up a national milch herd of highly productive cows and buffaloes. Artificial insemination coupled with progeny testing specially in cattle and buffaloes Future Prospects has made it possible to achieve genetic gains of 1 to 1.5 % per year in milk production and other Research on embryo transplantation in economic traits. Deep freezing of semen has cattle and buffaloes is fairly a recent develop­ made it possible to conserve semen for its later ment and is in the initial stages of experimenta­ use besides the transfer of superior germplasm tion in most of the developing countries includ­ across the world to achieve higher livestock ing India. Information with regard to superovu­ productivity in deficient areas. lation response, recovery rate ofembryos, hormo­ nal profile etc. is very meagre on cattle and Superovulation, embryo collection and buffaloes under the existing agroclimatic condi­ transfer has now become a reality and has made tions of Asia. The female reproductive prob­ it possible to multiply the superior female geno­ lems, associated with delayed puberty, long types. Multiple ovulation and embryo transfer partum anoestous intervals, ovum fertilisation (MOET) allows progeny testing of both males failure, interim infection, silent heat, seasonlity and females and can increase the genetic gain per and early embryonic mortality, are quite preva­ year, over that is possible by artifical insemina­ lent in tropical livestock. Embryo technology tion alone, through increased selection intensity coupled with uterine histological, biochemical for dams of bulls and dams of cows. Sire selec­ and immunological events may enhance the tion through progeny testing is the only way for understanding of embryo uterine interactions in making continous genetic improvement in milk order that one may significantly decrease the production - a trait with low heritability. Field incidence of early embryogenic mortality. recording in most of the developing countries is absent and sire testing is confined only to the Considering that embryo transfer tech­ organised farms. The number of sires tested are nology provides a potential tool for increasing far below the numbers required for breeding production from farm animals, the studies on purposes. There is no possibility for developing these aspects are important. This has special field recording system for progeny testing in importance in Indian conditions where milk near future. consumption needs to be increased (from 132 grams per day) to a minimum recommended The only option, therefore, is to use intake of 201 grams per day. MOET for multiplication of superior genotypes and production and progeny testing of superior Since little selection intensity has been males. It has been estimated that we would need placed upon the female buffalo, one would pos­ 8 donors and 27 recipients to progeny test a given tulate that greater progress in milk production male in shortest possible time. This could be yields could result from embryo transfer because done both at organised farms and by identifica­ of the more extreme variation in production tion of donors with the farmers. The progeny efficiency in this species as compared to dairy born out of planned matings could be brought to cattle. Disease resistant and environment toler­ the farms for performance recording. This sys­ ant animal can be produced by transfer of exotic tem would allow a larger number of bulls to be 143 tested with lesser number of fcmals. MOET a lm·ge number of markers at species level could scheme coupled with progeny testing would most readily be accomplished by utilising wide make it possible to achieve genetic gain up to 3 %. crosses between divergent breeds or species.

Superovulation, sexing, micromanipu­ Transgenic animals offer vast potential lation of the embryos and in vitro fertilisation to improve the yield targets. The most common are some of the other important areas for future method of producing transgenic animals is to research which would help in multiplication of inject DNA sequencing into fertilised ova. The the large number of quality males and females isolation, therefore, of desired gene sequences and thus increase genetic gains in the milk pro­ and the regulatory DNA sequences required for duction and other desired traits. Most obvious controlled expression in a genetically engineered benefit of embryo splitting is to obtain greater animal is of paramount importance for success­ number of offspring from a given superior fe­ ful application of this technique to livestock male, which is extremely important for the In­ improvement. The important task is to identify dian situation. Selection intensity for females superior genes for cloning and their dissemina­ could, therefore, be increased with halving or tion which shall result in dramatic initial in­ quartering of embryos. If three or four geneti­ crease in genetic value of production. Future cally identical males are produced by embryo potential areas of research for gene transfer are micromanipulation, each one could be placed in hormones and growth factors, modification of different environment and used as "reference structural proteins, identification of major genes sire" enabling among farm comparison of sires. and possibility of adding new metabolic path­ Splitting of embryos can also be used in estima­ ways and novel promoters through transfer of tion of animal's performance traits where indi­ genes across wide taxonomic boundaries. viduals are sacrificed. Recommendations In vitro fertilisation is another impor­ tant area which would be extremely useful in 1. The protocols for superovulation, embryo saving the superior genotypes especially the collection, evaluation and cryopreservation buffaloes, which in metropolitan cities go to of zebu and zebu taurus crossbreds have slaughter after the lactation. been standardised. These need to be tested on all breeds/crossbred in Asian region to Due to extensive crossbreeding in the perfect the methodology. No major prob­ country, the original zebu genome is likely to get lems are envisaged in this task. It should be lost or diluted and, therefore, two major research possible to collect embryos and get con­ thrusts need to be identified. One is to identify firmed pregnancies by transfer. the amount of genome which is introduced from the exotic parent primarily Friesian, Brown Swiss 2. R & D effort is needed for developing viable and Jersey and to investigate the data produced technologies of in vitro fertilisation, em­ from various crossbred groups in relation to their bryo culture and sexing so that the pro­ chromosomal components using molecular cy­ gramme can be made cheap and effective at togenetic techniques. For this it will be necessmy the level offarmers. that restriction fragment length polymorphism (RFLP) is investigated in these crossbred popu­ 3. The research should also be directed at lations. This technique will also be useful in sexing of sperm/embryo as this would result monitoring the embryos. These RFLP markers in developing single sex progeny for pro­ will open broad prospects for mapping and breed­ duction of meat or milk. ing manipulation of quantitative traits. Mapping 144

4. A new work project on MOET, involving hormonal profile of both riverine and swamp some of the important zebu breeds of dairy buffaloes. The hormonal profile has to be and meat type should be taken upasaninter­ done under varying field conditions. Con­ country net work. This would involve estab­ currently, the structure of buffalo ovary lishment of one, two or many MOET sta­ should be investigated in detail, in terms of tions where at least 10 bulls could be tested. primary, secondary oocytes in various In this way across the eight countries, 80 breeds. Since the number of primordial bulls could be tested annually. From 5th oocytes is very low in buffaloes, it is neces­ year onwards, 16 proven bulls of superior sary that an entirely different regime for quality could become available for a quan­ superovulation be introduced. Priming of tum jump in milk/meat production within these oocytes is necessary to become viable the next 10 years. Initially Sahiwal, Red follicles, before actually introducing a su­ Sindhi, Haryana, Ongole could be selected perovulatory regime utilizing either FSH/ as the major breeds of interest. LH.

5. It would be necessary to develop an embryo 8. Once the problem of superovulation in buf­ bank in each country for maintenance of faloes is sorted out, the situation in regard to embryos from superior females. These could other items of interest like embryo splitting, be exchanged between these countries. in vitro fertilization etc. can be undertaken. An inter-country net work on problems of 6. No studies have been taken up on disease buffalo embryo transfer need to be given the transmittance through embryos. It is neces­ highest priority and all the eight countries sary that we take up studies on disease trans­ must cooperate to produce the necessary mittance through embryos, so that the inter­ data within the next 2 years on (a) hormonal national safeguards for embryo exchange profile, (b) structure of the gonad and (c) can be implemented. possible ovulatory regimes which can be taken up for priming the follicles to be 7. In the case of buffaloes, a cooperative pro­ followed up by a superovulatory regime. ject should be taken up to study the normal Embryo Transfer Technology in Cattle: Experience in Pakistan

M. Anwar * & M. Anwar** *Director of Research (Animal Production) **Scientific Officer (E.T). Pakistan Agricultural Research Council Islamabad , Pakistan

Introduction litres per lactation but the average milk produc­ tion in Sahiwal Cattle, an important dairy breed of tropics, is about2,500 litters per lactation. The Genetic progress in large ruminants is good specimens of Sahiwal breed are limited to limited due to low reproduction rate of the fe­ some big farms. males. Reproductivity of selected females can be increased many fold by superovulation and In such a situation where a large num­ subsequent transfer of embryos to low value ber of low producing cows are available as mothers. First successful bovine embryo trans­ embryo reeipients, transfer of embryos from fer was performed in 1951. The technique is elite dairy cows will speed up the genetic pro­ being used in cattle in many parts of the world. gress. Similarly there is great genetic variation An experienced operator can now obtain an among buffalo population. Establishment of this average of about 10 live calves per year from technique in buffaloes will be of great help in each cow. The technique permits the rapid ex­ uplift of this species. pansion of elite rare breeds. Genetic progress is enhanced by increased selection intensity among Method females. Genetically superior dams are selected The standard embryo transfer proce­ as embryo donors. They should have regular dure consists of treating a donor female with calving record and should be normally cycling, gonadotropins which induce the maturation and with no signs of endometritis or vaginitis. The ovulation of large number of ova intstead of ideal recipient is a young, disease free, cow with . usual one. These ova after being fertilized are proven fertility and mothering ability. Recipient flushed out from the donor and transferred to - donor oestrous cycle synchrony is important embryo recipient cows (foster mothers) forges­ for successful embryo transfer. Prostaglandin F tation to term. alpha or cloprostenol is used for this purpose.

Need of Technology in Pakistan Superovulation in donor female is achieved by injecting pregnant mare's serum Majority of the animals in Pakistan is of gonadotropin (PMSG) or Follicle stimulating non-descript type with very low milk produc­ hormone (FSH). Example of superovulation and tion. Average milk production of cows is 870 oestrus synchronisation regimen is as following: 146

Day 0 : Donor comes into natural or induced Embryos can be kept viable at room oestrus. Inject all recipents with 500 temperature during the period between recovery microgram cloprostenol. from the donor and transfer to the reGipients in a micro-organism free medium having osmolality Day 10: Injectdonorwithgonadotropin(PMSG 260-310 mOsM and pH 7-7.4. The presence of 2,000 to 3,000). serum albumin acts as energy source. Many different kinds of media such as tissue culture Day 11 : Inject recipients with cloprostenol (5 to medium 199, Ham's FlO, modified Dulbecco's 7 recipients are prepared for each do phosphate buffered saline etc. are used for embryo nor). storage. PBS is most popular medium for this purpose. Day 12 : Inject donor with cloprostenol. The majority of the embryos recovered Day 14 : Estrus should occur in donors and re should be at the same stage of development. cipients. Breed donors 12 and 24 hours Embryos collected 7 days after oestrus are late afterJirst standing heat, us~ng 2 doses morulae or early blastocysts which are the opti­ of semen on each occasion. mum stages for transfer or cryopreservation. Diameter of embryo at this stage is 150 micron. En1bryo Recovery Accurate embryo evaluation based on morpho­ logical appearance of embryos is most practical Presently most of the embryo transfer one. Embryos are classified into 4 categories: units use nonsurgical embryo recovery tech­ excellent, good, fair, poor. Conception rates are nique. A 2 or 3 way foley catheter is introduced highest with excellent and good quality em­ into the uterus of donor female with minimum bryos. damage to the donor cow. Embryos are usually recovered 6-9 days after oestrus. Before this Transferable embryos are washed 3 time, many eggs are in the oviduct. The donor times in PBS + 10% serum. Each embryo is cow is placed in a crush and epidural anaesthesia picked up in a half cc.or quarter cc french straw is given with lidocaine 2% (5-6 cc). Vulvar area which is loaded in an insemination gun. The is washed and an 18 or 24 guage foley catheter embryo is deposited into uterine horn as deep as with a stiffening rod of stainless steel inside is in­ possible ipsilateral to ovary with CL. Care must serted into one of the horns at palpable bifurca­ be talcen to avoid trauma or infection. tion. Baloonofthecatheterisinflated with 15 ml of air. The uterus is flushed with a buffered An embryo donor cow can be superovu­ sodium chloride solution. AmodifiedDulbecco's lated after 2 months. An average of 5-7 embryos phosphate buffered saline plus 1 % bovine serum can be recovered per donor with 60-80 percent is a commonly used medium. At one time 50 to conception rate after transfer. 100 ml of fluid is introduced into the horn. A total Work Done at Animal Sciences Instim of 800-1,000 ml fluid is used to flush the uterus. Medium is recovered into a 1,000 ml cylinder. tute the cylinder is then placed at room temperature for 30 minutes. The fluid from cylinder is si­ Cattle phoned off leaving behind 200 ml of the me­ i) Superovulation: Different trials were dium. This 200 ml fluid is placed in scored petri conducted on cows (AIS x Sahiwal) to induce dishes and embryos are searched using a dissect­ superovulation with the help of pregnant mare ing microscope at 10-15 x magnification. serum gonadotropin (PMSG) and follicle stimu lating hormone (FSH). Luteolysis was brought 1

were 20 cows on an average of ranged from early to cyst stage. 68% the embryos were morphologically to be good ones. embryos recovered from individual donors ranged from 0 to 15. equipped embryo Eleven good quality embryos were has been developed. transferred to non-descript cows, cies proposed as source became pregnant a conception rate this regard. Provision been for percent. First calf through embryo transfer term trainings and equipment supply in the PC-1. Pakistan was born on June, 1987, at NARC.

embryos were recovered from 2 Sahiwal cows indicating a good response. Seven of these embryos were of good quality were L private enterprise Messers Uqab late or early blastocyst stage. Breeding Farms established a livestock 184 importedFriesian and Jersey cows near Hyderabad (Sind) in 1987. Purpose was to use L Superovulation response is variable these cows as embryo donors and sell embryos to and unpredictable cows. farmers as well as transfer the embryos to indige­ 2. Superovulation response is much nous cows maintained at the same farm. lower in buffaloes as compared to that cattle. American commercial "Granada Interna­ the gonadotropins i.e. PMSG and FSH tional" is extending technical cooperation to the gave similar results buffaloes. Uqab Breeding Farms Although they 3. While flushing embryos in case of produced & preserved Friesian and Jersey em- 1

bryos, they have not been able to sell these actual work will get underway. embryos. The approximate cost per embryo as advertised by them is Rs. 4,000/- which is too high for a farmer to pay. 2. Government of Punjab and Balu­ Seven lactating normally cycling Nili­ chistan have planned to establish embryo trans­ Ravi buffaloes were treated with PMSG (3,000 fer units at Pattoki (Bhuneki Research Station) IU) on 3 different days of the oestrous cycle as and Quetta respectively. Equipment for the units shown below: has been ordered but it will take sometime before

Day of Cycle Treatment I Treatment II Treatment

9 PMSG 11 Cloprostenol 12 PMSG 14 Cloprostenol PMSG 16 Cloprostenol

Uteri of the buffaloes were flushed with modefied Dulbecco's phosphate buffered saline 6 days after insemiantion.

Results:- Average number of corpora were 25% unovulated follicles present on the lutea, indicating the number of ovulations, was ovaries. Only 2 embryos were recovered from 2 highest in the animals treated on 12th day of the animals treated on day 12 of the cycle. oestrous cycle. Alongwith corpora lutea, there

Table: Superovulatory response in buffaloes

Hormone treatment at No.of No.of day of cycle No.of embryos Animals Gonadotropin Cloprostenol Ovulations recovered

3 9 11 3 2 12 14 4.5 2 2 14 16 LO

In another trial, 10 buffaloes were (n=4) of the cycle. Only five embryos could be treated with FSH (40 mg).Gonadotropin treat­ recovered from 3 animals treated on day 12 of the ment was initiated on day 9 (n=6) and day 12 cycle. The embryos were in early rnorula stage. Embryo Transfer Technology in Cattle : Experiences in Thailand

11 21 Samphan Singhajan , Vanda Sujarit , C.hamnean Satayapunt31, Samutra Sirivejapandu21, l/Ratchaburi A.I. Station center, Livestock Developmetn Department. 2/ Faculty of Veterinary Medicine, Kasetsart University. 3/ Animal Science Department, Kasetsart Uniersity. Bangkok, Thailand.

Introduction upgrading obtained faster, but an imported embryo transferred to the womb of local cow is believed to receive immunity from that mother at Thailand has long been known to be an birth. These advantages has been brought to the agricultural country. Livestock and Livestock attempt to develop the embryo transfer techno­ products contribute to approximately 15% of the logy in Thailand. total agricultural production. In 1988, cattle population are approximately 4 million heads. Embryo tran sfer technology in The number of dairy cattle is around 60,000. However, Thailand can only produce about 5% Thailand of its local demand for dairy products. Approxi­ mately 95% has to be imported in the form of Although both milk and meat produc­ powdered milk. The local demand is expected to tions are at the equivalent levels of importances, increase steadily over the next decades because embryo transfer in beef cattle is behind the dairy of the present population growth rate of 2.0- cattle. Embryo transfer technology in dairy cattle 2.2 % and because of extensive governmental has been developed in Thailand with two main campaign to promote milk consumption. The objectives: to increase the rate of milk produc­ current average milk production per cow in tion and to improve the dairy cattle breed by Thailand is approximately 2,400 kg over a 305 developing a type of superior genetic livestock day lactation period. This compares to milk that will yield maximum milk and meat produc­ production of over 7 ,000 kg for cows in the tion. Two approaches had been simultaneously western countries. Although, artifical insemina­ undertaken: importing the frozen embryo of tion has been practiced for 30 years in Thailand, superior stock and developing the techniques by conception rates and the genetic quality of se­ flushing embryo locally. men are low. This situation, coupled with the nutritional and management problems of a dairy Collaboration of researchers among industry in a tropical environment, accounts for Kasetsart University, Department of Livestock the relatively low level of milk production in Development and private sectors has established Thailand. firm project since 1984 to develop embryo trans­ fer technology in dairy cattle and utilize its Embryo transfer offers a better oppor­ application under Thailand condition. The pro­ tunity than artificial insemination for upgrading ject has been supported by the National Center of indigenous stock, not only are the effects of Biotechnology and Genetic Engineering 150

(NCBGE), Ministry of Science, Technology and In 1987, the project supported by Energy of the Royal Thai Government. At the NCBGE imported thirty-four holstein frozen present time, eighteen calves were born from this embryos from the United States. Twelve recipi­ project, and some are still pregnant. Neverthe­ ents were pregnant and ten calves were born, 4 less, several research efforts had been reported to males and 6 females. These calves are expected accomplish the technology in the country. All to be good donor in the future (Sujarit et al., attempts aimed at improving overall reproduc­ 1988b). tive efficiency in Thailand. Details are in the following section and Table 1. In 1987, Parnpai et al., reported 4 calves born from the transferring of fresh em­ The success of embryo transfer bryos to the worn bs of recipients. This work was conducted at the Dairy Promotion Organization, in cattle in Thailand Saraburi.

Although several attempts at transfer In the same year, Singhajan et al., re­ had met with minimal success in many year ported the utilization of FSH to enable the su­ period, the techniques had continued to develop perovulation. After the flushing and transfer­ without any distress. ring, two out of ten recipients were pregnant and two male calves were born from these transfer­ In 1984, Aimlamai et al., reported the ring of the fresh embryos. non-surgical flushing of 7 day embryos and resulted in no pregnancy. Eventhough anumberof embryos were born, the success rate are considered unsatisfied. In 1984, a private company in Thailand Various factors are during the processes of fur­ imported frozen embryos of the Genetic Carna­ ther investigations. Early in 1988, our group had tion Co. from the United States. Twelve embryos flushed the embryos from the pure breed Hol­ were surgically transferred to the recipients at stein. Two out of six recipients were pregnant. two private dairies. One male holstein calf was The calves are expected to be born in October. born in February, 1985 at Chok-chai farm, Nakhon Rachasima (personal communication). Future aspects of embryo trans­ In 1985, fourteen frozen embryos were fer in cattle in Thailand imported from the United States and transferred to the local cows. The first non-surgical trans­ The capabilities of conducting the em­ ferred, brown-swiss calf was born in September bryo transfer technology in cattle in Thailand are 1986 at Udom farm, Ratchaburi (Sujarit et al., enormous. The understanding of the basic meth­ 1986). odology of veterinarians and animal scientists is adequate. Successful embryo transfer clearly Because of the high cost of the U.S. demonstrated that biotechnology- embryo trans­ frozenembryos,embryosfromothersourcewere fer could be accomplished in Thailand. also research. The purpose was to investigate the condition of recipients. Therefore, the frozen With the necessary methodology of embryos at lower cost from New Zealand were embryo transfer, we aim to improve the dairy imported. We have 2 male calves (out of 24 cattle breed by the achievement of the modern transferring) born from this trial (Sujarit et al., reproductive biology to the basic technology. 1988a) in 1986. Recent advances in gene transfer, molecular genetics and recombinant DNA technology and 151 the expression of this genetic potential in our material in genetics or early embryos hold prom­ cattle. Superior breed of our embryo transfer ise of greatly increasing the rate of genetic im­ calves are expected to be the sources of genetic provement and making genetic combinations stock in the future. Freezing the embryo will not beyond those possible through natural fertiliza­ only be supplied to the demand locally but can tion processes. also be applied marketably to other tropical agricultural systems. Formation of embryo bank With the current technology success for long-term storage of the most productive rate, embryo transfer could lead to an increase in breed is promising. the rate of genetic improvement of from 10 percent to nearly 100 percent, depending princi­ Increasing of the fertility of the animals pally on current intensity of sire selection and on through the production of twins leads to a higher the trait in question. The potential impacts and intensity and better economics of animal pro­ long-range payoff for the research are enormous. duction. Selection of embryos through sex ratio Previous experiences and subsequent trainings is benefit. Genetic engineering, embryo manipu­ of embryo transfer in Thailand will achieve lation the exchange or replacement of genetic optimum output both in dairy and beef cattle.

Table 1. Summary of the Success of Embryo Transfer Technology in Cattle in Thailand

Year No.of No.of Trials transfer calves born

1984 12 1 Frozen embryos from Genetic Carnation Co., USA., at Chok-chai farm (Surgical Technique).

1985 14 1 Frozen embryos from USA., at Udom Dairy Farm, Banpong, Ratchaburi (Non Surgical Technique).

1986 24 2 Frozen embryos from New Zealand, at Udom Dairy farm, Ratchaburi (Non Surgical Technique).

1986 16 4 Fresh embryo transfer at the Dairy and Promotion Organization, Muak Lek, Saraburi, 1987 (Non Surgical Technique).

1987 34 10 Frozen embryos from USA., at Udom Dairy Farm, Racha-buri, (Non Surgical Technique).

1987 10 2 Fresh embryo transfer at Udom Dairy farm, Ratchaburi (Non Surgical Technique).

1988 6 2 Fresh embryo transfer at Nang-Po Dairy cooperative, Ratchaburi, (Non Surgical Technique) 152

References Sujarit, V.K.; Singhajan, S.; Sirivejapandu, S.; Satayapunt, C.; V angtal, U. and Sukpatarapirome, V. (1986) "Sai-fon": An Embryo Transfer calf. Aimlamai, S.; Sawangsri-buntearg, S. andLikit­ Proceedings of the 13th Annual Veterinary padungkit, C. (1984) Non-surgical Embryo Sciences Conference, TVMA. 2-4 December Transfer in Cattle. Case conference, Faculty of 1986, Bangkok, Thailand. Veterinary Science, Chulalongkorn University. Sujarit, V.K.; Singhajan, S.; Sirivejapandu, S.; Parnpai, R.; Sophon, S.; Kamonpatana, M.; Satayapunt, C.; Vangtal, U. andSukpatarapirome, Meebumrong, K.; Srisakwattana, K.; Intarachote, V. (1988a). Performance of New Zealand Hol­ P.; Suwankumjai, T.; Wattanodorn, P.; stein Fresian Calves Born by Frozen Embryo Kurdchuchurn, P. and Tantrakul, S. (1987) Transferring. The 26th Annual Conference Embryo Transfer of Dairy crossbred Cows in Kasetsart University. 3-5 February 1988, Thailand.Jn: IVF and Embryo Transfer. Chula­ Bang~ kok, Thailand. longkorn University Press. pp/ 273-288. Sujarit, V.K.; Singhajan, S.; Sirivejapandu, S.; Singhajan, S.; Sirivejapandu, S.; Sujarit, V.K.; Satayapunt,C.; Vangtal, U. andSukpatarapirom, Satayapunt, ~.; Vangtal, U.; Sukpatarapirome, V. (1988b). Proceedings of the VI World con­ V. and Bodh1puksa, P. (1986) Superovulation ference on Animal Production. 27 June - 1 July with FSH in Embryo Transfer Technology. Pro­ 1988, Helsinki, Finland. ceedings of the 13th Annual veterinary Sci­ ences Conference, TVMA. 2-4December1986, Bangkok, Thailand. Embryo Transfer Technology in Pigs: Experiences in Thailand1

Peerasak Chantaraprateep Department of Obstetrics Gynaecology and Reproduction Faculty of Veterinary Science, Chulalongkorn University Bangkok, Thailand.

Introduction (Singh, 1987, 1988). Thus, theoretically, the di­ sease transmission potential of embryos is li­ 1. Embryo transfer has been used successfully mited. Table 1 shows the different reasons for during the last 25 years both as an experimental introduction of swine embryos into recipient tool in various studies related to reproductive herds in the US in 1981. physiology and early pregnancy in the pig. Due to the high ovulation rate the pig is also a good 3. The objective of thepresentpaperis to under­ animal amongst the domestic species for the line some of the basic techniques we used, to provision of eggs and embryos for investigation describe the results achieved and to draw atten­ in vitro, the techniques used which have been tion to certain areas where more research and developed (Hancock and Hovel, 1962; Polge, development are needed. 1982) and for practical application in our condi­ tion are not complicated and effective (Kitrun­ 1. The basic techniques groj-Charoen et al., 1980; Chantaraprateep et al., 1985; Techakumphu et al., 1987; Chantara­ 1.1 Selection of donors and recipients prateep et al., 1987 a, b; Techakumphu et al., 1988). The most important goal for performing 4. Depends on the needs of the owner. However, commercial embryo transfer in this species is the only animals that have the genetic potential to introduction of new genetic material into herds improve the breed should be selected as donors which are closed for the purpose of disease and they should be in good health. Sows are control and in this repect it can be regarded as an preferable as the source of embryos. Sound health alternative to hysterectomy (Polge, 1982; Mar­ and normal cycling animals without having a tin 1983, 1986; Einarsson, 1985). As the disease good genetic background can be served as re­ transmission potential of embryos is much less cipients. than those of either the live animal or semen. 1.2 Oestrus synchronization 2. It may be used for achieving an increasing rate ofreproduction of superior genetic animals and 5. Several methods are commonly employed for there is also the possibility of export or import of controlling estrus and ovulation for embryo trans­ embryos between countries particularly iflong­ fer purposes. The first method involves weaning term storage of swine embryos becomes pos­ a group of sows on the same day, with estrus sible. Furthermore, cryopreservation has been occuring 4 to 10 days later (Martin, 1986). found to be effective in inactivating low levels of Another method is using PMSG and HCG dose many viruses that can be adhered to embryos ~.ate of 400/200 I. U. (PG 600®, Intervet) admini- 154

Table 1: Primary reasons for performing commercial embryo transfer in swine in the US in 1981 as well as the number of transfers and recipient herds involved (Martin, 1986).

Group Reason No.of No.of No. recipients herds

1 Establish new herds from herds with Pseudorabies 113(43) 4

2 Make additions to specific pathogen free (SPF) herds 67(26) 13

3 Obtain boards for closed commercial herd 61(23) 11

4 Obtain more offspring from superior gilts and sows 19(7) 8

Total 260 36

stered subcutaneously, 90.5 percent (19/12) of follicular phase of the cycle, 15 or 16 days after treated animals came in heat within 5 days after the onset of estrus (Hunter, 1964) and the estrus the treatment (Techakumphu et al., 1987). The occurs 3.5-4 days later. HCG at the dose rate of most effective treatment for estrus synchroniza­ 500 I.U. can be used to enhance ovulatory re­ tion in mature gilts is by feeding ally I trenbolone sponses and is given 3-4 days after the treatment (Regumate® Roussel Uclaf) for 18 days.This ofPMSG. progestational agent blocks follicular matura­ 7. For optimum conception, donors should be tion during the time that it is administered and the bred every 12 h throughout estrus. The volume animals then rebound into a follicular phase of the inseminate should be 50 to 100 ml and following withdrawal. Results of the experi­ contain at least 4-5 x 106 live spermatozoa. ments with Regumater were 98.5 percent of 586 gilts came into estrus within 9 days after the end 1.4 Collection of embryos of treatment (Polge, 1982) and 80.7 percent of the heats were synchronized within a 2 day 8. Swine embryos are usually collected surgi­ period on the 5th and 6th days (Chantaraprateep cally 4 to 6 days after the onset of estrus. They are et al., 1986; Chantaraprateep et al., 1987 c). AI mostly at the 4 to 8 cell stage and expanded on the second day of estrus resulted in 90 percent unhatched blastocyst stage respectively. At 4 of the animals with fertilized eggs (Polge, 1982). days after the onset of estrus 4 to 8 cell embryos are easily identified and evaluated. The recovery 1.3 Superovulation and breeding rate of embryos were usually high 94-100 per­ cent (n=320 donor gilts) (Polge, 1982) 85-100 6. When superovulation of donor is required, kit percent (Chantaraprateep et al., 1987 a), 80-90 is usually achieved by one injection of 1,000- percent (Techakumphu et al., 1987). 1,500 I.U. PMSG at weaning or at the early 155

9. The techniques used is a mid-ventral lapa­ uterine horn only as they will migrate throughout rotom y under general anaesthesia and the genital the uterus (Dziuk et al., 1964; Dziuk, 1985). A tract is exposed through a small incision. Ovula­ minimum of 4 embryos is required in order to tion in pigs occurs around 36-40h after the onset avoid luteolysis around day 14-15 of the cycle of estrus and since the eggs remain in the ovi­ (Polge et al., 1966) gilt recipients seem to be ducts for less than 48 h following ovulation and better than those old age sows in term of farrow­ enter the uterus at the 4 cell stage, recovery from ing rate 36.4 percent (n=3) vs 11.8 percent(n=4) the uterinehornispracticed(Polge, 1982). Warm as shown by Techkumphu et al. (1987). flushing medium (PBS) at 37° C is introduced from the fim briated end of the oviduct or via 2. Factors affecting results oviduct with a blunt 12-14 gauge needle near the conjunction of each horn. The fluid about 20-50 14. The pregnancy rate is reduced when transfer ml is gently massaged along the horn and col­ are made to recipients which come in heat before lected through a canula or a Foley catheter No. the donors. There was no drop in pregnancy rate 12 inserted into the lumen via a small longitudi­ following transfers to recipients which come on nal incision. Embryos can be collected from the heat 1-2 days after the donors (Polge, 1982). This proximal one third of each horn. It is necessary phenomenon indicates that a greater degree of to avoid bleeding during the operation in order to asynchrony between donor and recipients can be reduce adhesion and allows donor animals to be flexible in the pig than in cattle or sheep (Row son re-used again. et al., 1969).

10. Donor can be re-used as shown by Chantara­ 3. Certain areas to be more inm prateep et a/. (1987) 3 operationsduring7 months period. vestigated

1.5 Handling embryos Preservation of embryos

11. The flushings are examined for embryos with 15. Simple media (PBS + 20 percent sheep a stereomicroscope at 10 x magnification and serum) will support early cleavage at 37° C from evaluation of embryos stereomicroscope at 10 x single cell fertilized eggs to the 4 cell stage. Viability of embryos cultures for 1 day is good magnification anQ. evaluation of embryos at 40 x. As embryos are located they are transferred to while 48 h culture results in poor survival. culture plates. After several rinses, they are stored 16. Pig embryos are sensitive to cooling and few until transfered to the recipient. The embryos embryos survive following a reduction of tem­ should not be chilled below 15° C. perature to below 15° C. 12. Short-term storage of embryos can be made 17. Investigation on culture of pig embryos and through flushing medium added with 15 percent fertilization transfering them to recipi­ bovine serum albumin (Whittingham, 1971) for in vitro ents as well as embryo manipulation and preser­ several hours as shown by Cameron et al. ( 1986) vation are required in order to extend the possi­ and Techakumphu et al. (1988) for conducting bilities of practical application of embryo trans­ embryo transfer of pig between different farms. fer. 1.6 Embryo transfer

13. Embryos is transferred surgically in a small volume of fluid to the anterior one fourth of one 156 References Einarsson, S. 1985. Embryo transfer in swine. Proc. F AO/SIDA Local Seminar on Pig Repro­ Cameron, R.D.A. Durack, M. and Fogarty, R. duction, P. Chantaraprateep (ed.) Whale Hotel, 1986. The use of embryo transfer to establish Nakhon-Pathom. 30-34. new breeding lines in a commercial S.P.F. pig­ gery. Proc. 9th. IPVS, Barcelona Spain: 45. Hancock, J.L. and Hovel, G.J.R. 1962. Egg trans­ fer in the sow J. Reprod. Fert 4, 195-201. Chantaraprateep, P. Kunavongkrit, A. Bodhipaksha, P. Lohachit, C. and Virakul, P. Hunter, R.H.F. 1964. Superovulation and fertil­ 1985. Feasibility study of potential and demand ity in the pig. Anim. Prod. 6, 189-194. of research and development of technology of embryo transferin pig in Thailand. Submitted to Kitrungrojcharoen, T., Wong, C., Bodhipaksha, National Centre of Genetic Engineering and P., Chantaraprateep, P. Lohachit, C. and Vira­ Biotechnology, Ministry of Science, Techno­ kul, P. 1980. Embryo Transfer in Pigs. Senior logy and Energy, 46 pages (in Thai). Project. Faculty of Veterinary Science, Chula­ longkorn University. 13 pages (in Thai). Chantaraprateep, P., Prateep, P ., Poomsuwan, P. and Kunavongkrit, A. 1986. Estrous synchroni­ Martin,P.A.1983. Commercialembryotransfer zation of gilts with Altrenogest. Songklanakarin in swine : who is interested in it and why. J. Sci. Technol. 8(2) 161-165. Theriogenology. 19(1) 43-48.

Chantaraprateep,P.,Kunavongkrit,A.,Lohachit, Martin, P.A. 1986. Embryo Transfer in Swine. C., Virakul, P., Bodhipaksha, N., Minmanee, S. In : Current Therapy in Theriogenology. D .A. and Bodhipaksha, P. 1987 a. Embryo trnsfer in Morrow (ed). W.B. Saunders Company. pp. 66- pigs. Thai. J. Vet. Med. 17(2) 166-177. 69.

Chantaraprateep, P., Lohachit, C., Kunavongk­ Polge, C. 1982. Embryo transplantation in the rit, A., Virakul, P. and Prateep, P.1987 b. Em­ pig. 2nd Internat Congress. Annecy, 20-22 Sep­ bryo transfer in swine : using repeated donor. tember,Ch. MerieuxandM. Bonneau (eds)235- The 14th Annual Veterinary Sciences Confer­ 242. ences, Asia Hotel, Bangkok. November 25-27. p. 76. Polge, C. Rowson, L.E.A. and Chang, M.C. 1966. The effect of reducing the number of Chantaraprateep,P.,Prateep,P.,Poomsuwan,P. embryos during early stages of gestation on the and Kunavongkrit, A. 1987 c. Altrenogest for maintenance of pregnancy in the pig. J. Reprod .. control of estrus in gilts. Thai J Hl th Resch 1(2) Pert. 12. 395-397. 1-7. Rowson, L.E.A., Moor ,R.M. andLawson,R.A.S. Dziuk, P. 1985, Effect of migration, distribution 1969. Fertility following egg transfer in the cow, and spacing of pig embryos on pregnancy and effect of method, medium and synchronization fetal survival. J. Reprod. Fert. Suppl. 33 : 56-63 of oestrus. J. Reprod. Fert. 18 : 517-523.

Dziuk, P .J., Polge, C. and Rowson, G.J .R. 1964. Singh, Elizabeth, L. 1987. The Disease Control Intra-uterine migration and mixing of embryos PotentialofEmbryos. Theriogenology.1: 9-20. in swine following egg transfer. J. Anim. Sci. 23, 37-42. Singh, Elizabeth, L. 1988. Determining the 157 Disease Transmission Potential pf Embryos and Techakumphu, M. Rujtikumporn, B., Rakari­ Semen. 3rd World Congress Sheep and Cattle yatham, N., Maicharoen, A., Horcharoen, A., breeding. Paris. June 19-23, 659-672. Punchai, K. and intarauthok, P. 1988. Pig em­ bryo transfer between farms. Thai. J. Vet. Med. Techakumphu, M., Horcharoen, A., Rujtikum­ 18(2) 173-180. porn, B. Rukariyathum, N. Jungthanacharoen­ lert, S. and Puangsilpa, V. 1987. Preliminary Whittingham, D.G. 1971. Survival of mouse Study on Pig Embryo Transfer in Farm. Thai. J. embryos after freezing and thawing. Nature, Vet. Med. 17(3) 227-242. London. 233 : 125-126. Superovulation and Embryo Transfer in Goats : The Malaysian Experience

W. Sharifuddin, Y. Rosnina and M.R. Jainudeen Department of Veterinary Clinical Studies, Universiti Pertanian Malaysia, UPM43400 Serdang, Selangor, Malaysia.

Goat rearing in Malaysia is restricted to small­ established either with 2 injections of 125 mcg holder farmers owning between 4-10 goats of cloprostenol (Coopers-England) or fitted with a non-descript breeds producing approximately 8- vaginal sponge impregnated with medroxypro­ 10 kilos of meat per animal. Genetic improve­ gesteron acetate (MAP) (Repromap-Upjohn) for ment through AI has not been encouraging. Poor 18 days. Following the second injection of conception rates associated with low semen cloprostenol or sponge removal, the goats were survival rate and poor estrous detection tech­ tested for estrus using a vasectomised buck fitted niques under smallholder conditions all contrib­ with a sire-sign harness. The number of animals ute to the slow rate of progress. Importation of in heat after treatment was higher in the clopros­ live animals for cross-breeding pro grammes has tenol treated group (Table I). The time taken for its own limitation. Adaptation, enviromental the animals to be in estrus was spread until 72 stress and diseases greatly affect the perform­ hours in the cloprostenol group where as in the ance of these imported animals. repromap group 92% animals were already in estrus by 48 hours post-treatment. The duration Embryo transfer technology is now an of estrus was shorter in the repromap group. established breeding tool for farm animals. This technique involves in making greater use of ova Superovulation in the ovaries of the genetically superior animals. Embryo transfer is often viewed as the female Two types of gonadotropins were used counterpart of artificial insemination (AI) where to induce multiple ovulations. The does either females of superior genetic merit may be su­ received follicle stimulating hormone (FSH­ perovulated and embryos collected and trans­ Sigma) injected twice daily in decreasing doses fered to recipients. over a five day period or a single injection of Pregnant Mare Serum Gonadotropin injection, This paper reports the research activi­ the does were treated with 250 mcg cloprostenol. ties on embryo transfer in goats carried out by All does in heat were mated naturally to a buck. Universiti Pertanian Malaysia, the application and possible micromanipulation of embryos in The superovulatory response to treat­ progress and future plans of our department. ment with FSH and PMSG is shown in Table II. The incidence of unovulated follicles was high­ est with 1500 iu of PMSG while the best su­ Establish1nent of Estrus Cycle perovulatory response was obtained with 20 mg ofFSH (Table II). Cycling females goats of the indegenous breed, Kambing Kacang had their estrus cycle 159

Table I. Onset of estrus following treatment with Cloprostenol and Repromap

Cloprostenol injection Intravaginal sponge (125 mcg) (60mgMAP)

No: of animals 48 43

No: of animals in estrus(%) 39 (81) 26 (60)

End of treatment to estrus 12 - 48 h 29 (79) 24 (92)

Duration of estrus 12 h 23 (59) 22 (85) 12 - 24 h 16 (41) 3 (12)

Embryo Collection and Transm incision was made and the uterus exteriorised. A fer 2-way foley catheter (gauge 8-Euromedic) was inserted into the lumen of the uterine horn and the balloon cuff inflated. The collection media On day 4-5 of the superovulatory cycle (PBS+ 2% fetal calf serum) was infused slowly eggs were recovered using a surgical technique. through a needle inserted close to the utero-tubal Anaesthisia was induced with an intravenous junction. The flushings were collected through injection of alphaxolone (5mg/Kg body weight) the catheter in a flat bottom flask. The same and maintained on halothane. A ventral mid-line procedure was repeated for the other horn. After

Table H. Superovulatory response in goats treated with FSH and PMSG

FSH_(mg) __ _ __ PMSG_(iu) ___

10 15 20 500 1000 1500

No: of animals 11 9 8 14 18 10

Total no: of CL 79 67 83 19 106 55

Mean no: of CL 7.2+2.9 7.4+2.8 10.1+3.8 1.4+0.4 5.8+1.3 5.5+1.9

Mean no: of follicle unruptured 1.3+0.9 3.1+2.2 0.3+0.3 0.8+0.4 2.9+1.4 5.1+1.8 160 flushing, the linea alba and peritoneum were Future Developments sutured followed by the sub-cutaneous layer and the skin. The flushings were examined under a i. Non-surgical embryo transfer stereomicroscope (Wild) at 18x magnification for identification of eggs. Work is in progress to develop a non­ surgical technique of embryo transfer in sheep The eggs recovered were either unfer­ and goats. The technique involves the use of a tilised (25 %) or in the morula or early blastocyst laparoscope for visual guidance to deposit the stage. The mean embryo recovery was better in embryo into the uterus via a cannula punctured theFSH treated group as compared to thePMSG through the abdomen. (47%) group.

Embryos were transfered surgically into ii. Rapid freezing and thawing of the ipsilateral horn of the recipients using a glass embryos pipette. Experiments are being conducted for a Cryopreservation of E1nbryos one step equilibration in cryoprotectan t and freez­ ing without the use of sophisticated freezing machines. In addition, we are experimenting on Cryopreservation of embryos offers a a one step thawing technique where by the cry­ practical solution for long term storage and fa­ oprotectant would be removed from the embryo cilitates greater use of embryo transfer. One without having to remove the embryo from the major advantage of frozen embryos is that syn­ straw. This would make transfer of frozen em­ chronization ofrecipiepts is eliminated and trans­ bryo under field conditions without the use of fer can be done when the recipient is in the expensive microscopes a reality. appropriate stage of the cycle. Also, goats can now be cheaply and rapidly transported from one country to another. iii. Micro manipulation of em- bryos The embryos to be frozen were allowed to equilibrate in 1.5 m glycerol/PBS (Cryopro­ Chimeras tectant) in a stepwise manner. The embryos were then loaded in 0.25 plastic straws in between 2 A chimera can be produced by fusing cleavage­ air bubbles and was gradually cooled until -30° C stage embryos divested of the zona pellucida. in a freezing machine (Minicool, IMV) followed Thus by mixing appropriate embryos, chimeras by rapid cooling before being immersed in liquid with two, three, four or more parents may be' nitrogen. produced. Chimeras can also be produced by The frozen embryos in the straws were injecting embryonic cell into the early blasto­ thawed by immersion in a water bath at 37° C and cyst. The freemartin is a naturally occuring chi­ the cryoprotectant removed gradually in a step­ mera. wise manner from 1.5 M glycerol/PBS solution. The thawed embryos are transfered to recipients Cloning surgically. The word is derived from the Greek "klon" meaning twig or cutting and has been used to describe a number of vegetative or asexual re­ production in animals. Several different proce­ dures all have the end result of producing a 161

number of identical young which are known as not nuclear division, doubling the remaining clones. haploid DNA; the two identical haploid nuclei form the first cleavage spindle and a homozy­ The simplest procedure is to bisect an embryo gous diploid egg begins development, with nor­ and obtain two "identical offspring". This has mal cleavage and development thereafter. Only been recenly achieved in cattle, sheep and goat. females can be produced by this method. The A further method of producing clones would be value of producing homozygotes that can sur­ the transfer of the nucleus a cell of the embryo to vive to term and be used in various schemes for a previously enucleated egg. Work in amphibi­ breeding desirable strains of animals is enormous ans have shown that it is possible to produce but to date no live offspring have been born. clones by nuclear transplantation. However, successful cloning in mammals has been slow to Fertilization perfect because of many technical difficulties, particularly their small size. Recently, success­ By microsurgery it is possible to fertilize eggs by ful cloning in mice has been achieved by insert­ injecting sperm into them instead of relying on ing the nucleus of a somatic cell into a fertilized normal fertilization procedures. It is also pos­ one cell egg and then removing the original sible to inject an additional sperm from a highly genetic complement (male and female pronu­ desirable male used to fertilize the egg, then to clei) of the egg. Nuclei from only two embryonic remove the female pronucleus from the fertil­ tissues, the ectoderm and the proximal endo­ ized egg. The resulting embryo would carry derm of the embryo retain the potential to pro­ genes derived from a single male. Thus embryos, duce whole mice. both male and female, produced entirely from the genetic makeup of a prize bull might develop If we can devise procedures to restore totipo­ into valuable breeding stock. tency to adult nuclei, cloning mammals by nu­ clear transplantation will become a basic part of Gene tr an sfer animal breeding. Even now, it is possible to transplant the nuclei of mammalian embryos, It is now possible to inject genes into the pronu­ but to be useful in animal breeding, transplanted cleus of a fertilized mouse egg with a reasonable nuclei must be derived from a desirable adult expectation that these genes will be incorporated whose phenotype has already been tested. But in the genome. This gene transfer technolohy at the low heritability of many commercial traits present resembles a shothun approach to im­ may lead to disappointment when clones fail to proving the genotype, but we are at the very live up to the reputation of their "parent". beginning.

Clones are uniquely suited for research into iv. In vitro fertilization diseases and their treatment. It would be ex­ tremely useful to be able to apply a variety of As the mammalian ovary contains thou­ treatments to clones which had all been given the sands of primordial follicles, it is clearly an ·same infection. important objective to seek ways of promoting oocyte maturation and fertilization in vitro in Production of homozygous embryos order to obtain a large number of young from selected females. Production of young from Microsurgical removal of one pronucleus from a oocytes fertilized in vitro has been achieved so fertilized mouse is technically feasible. This far in only four mammalian species: rabbit, then results in a haploid egg containing either the mouse, rat and man but not in cattle and sheep. In nucleus of the sperm or egg. When treated with those species in which it is successful, fertiliza­ cytochalasin B, cell division is suspended but tion depends upon incubation of the spermato- 162 zoa in salt solutions which usually contain Y - bearing sperm appears difficult, one method macromolecules which are believed to play a of selecting the sex of an embryo is to discover crucial role in inducing capacitation of the sper­ the sex and select those which are of the desired matozoa. The search in other species is for suit­ sex for transfer to foster mothers. Since chromo­ able salt solutions and macromolecules. somal analysis of embryos is tedious, attention has now been focused in detecting the gene v.Sexing of Embryos product of the Y chromosome, ie. the H-Y anti­ gen. Already the sex of mouse embryos has been Modern approaches to controlling the identified by the H-Y antibody. Whether or not sex ratio at birth have centred on separating X­ the H-Y antigen is present on embryos of farm and Y- bearing sperm. As separation of X- and species remains to be ascertained. Genetically Engineered Vaccines for Animal Diseases-Present Status

Kazu.ya Yamanou.chi Institute of Medical Science, University of Tokyo Japan

1. Development of smallpox vaccine in late 18th occuring variant of microbes with attenuated century has resulted in the global eradication of properly and live microbes that are attenuated smallpox in 1980. It is rather surprising that the artificially by passages in cell cultures or in production method of smallpox vaccine using laboratory animals. calflymph which was developed in 184 2 in Italy had remained unchanged except for minor 4. The application of recombinant DNA tech­ modifications and contributed to the smallpox nique has led to development of essentially new eradication. Meantime, the substrates for the type of vaccines for many infectious diseases production of other vaccines have been changed including virus diseases, bacterial diseases, and from the whole animals to the embryonating parasitic diseases. Hereafter, I wish to mention chicken embryos and then to in vitro cultured the current situation of virus vaccines which are cells. All these methods of vaccine production the major subjects for development. are based on the propagation of virus or bacteria in those substrates. However, this situation has 5. Genetically engineered virus vaccines are been rapidly changing by the introduction of classified into three types, i.e. subunit vaccine, recombinant DNA technique for the develop­ synthetic vaccine, and vector vaccine. The first ment of genetically engineered vaccines. type, subunit vaccine, consists of viral protein produced in prokaryotes such as Escherichia 2. In this paper, I wish to briefly summarize first coli or eukaryotes such as yeast and insect cells. the current situation of genetically engineered Outer proteins of virus particles which are enve­ vaccines in general, and then wish to introduce lope proteins of enveloped virus or capsid pro­ ourresults in developing new heat-stablerinder­ teins of non-enveloped virus serve as protective pest vaccines. antigens. Therefore, the genes of these protec­ tive antigen proteins are integrated in these (1) General features of genetically prokaryotes or eukaryotes. engineered vaccines. 6. The second type, synthetic vaccine, is pro­ 3. Current vaccines which have been produced duced on essentially the same concept as the byclassicalmethodsareclassifiedinto two types, subunit vaccine, except chemical synthesis in­ i.e. inactivated vaccine and live vaccine. The stead of production in bacteria or cells. Anti­ inactivated vaccine is further classified into the genic regions which are effective in protection vaccine which uses the whole microbes and the are limited within the s_hortamino acid sequences vaccine which uses only antigenic portion of the of the protective antigens. For instance, pol­ microbes. The live vaccine consists of naturally ypeptides consisting of only about 20 amino 164

acids are responsible for the protection in case of 9. As to the application in the veterinary field, capsid protein, VPI, of foot and mouth disease cattle is highly susceptible to vaccinia virus as virus. Such small polypeptides can be synthe­ shown by the fact that cattle had been used for the sized chemically, and are expected to be the most production of smallpox vaccine. Thus, vaccinia pure form of subunit vaccine. The subunit vac­ virus is particularly expected for the develop­ cine and synthetic vaccine have disadvantages in ment of vector vaccine for cattle use. One of the their low immunogenicity, so that the use of major controversies about the use is an acciden­ adjuvant is essential. For this purpose, many tal infection of humans from the vaccinated attempts in developing effective adjuvant are animals. In this aspect, the use of an attenuated being made. smallpox vaccine will be useful to solve this problem. Dr. Hashizume and his colleagues in 7. The third type, vector vaccine, consists of Japan had developed attenuated vaccinia virus, vector virus in which the gene of protective 16m0, and then LC16m8, from the Lister strain antigen is inserted. As a vector virus, vaccinia that was widely used in global smallpox eradica­ virus which is a smallpox vaccine itself has been tion program. The LC16m8 was a plaque puri­ most widely used. Besides vaccinia virus, use of fied clone from the LC16m0. Side reactions of several other attenuated viruses, most of which the LC 16m0 and LC 16m8 in humans were much are used as live vaccines, is also considered. milder than the Lister strain, the latter being They include sheep pox virus, swine pox virus, slightly more attenuated than the former. In bovine enterovirus, varicella virus, Marek's dis­ addition, neurovirulence of these two viruses in ease virus, and fowlpox virus. monkeys and rabbits as examined by the intrac­ erebral inoculation was markedly decreased than (2) Vaccinia virus as a vector for re­ the Lister strain. Based on these findings;-:the combinant vaccine. LC 168 was licensed as an attenuated vaccine in 1975 in Japan. Although theLC16m8 is slightly 8. Vaccinia virus has several advantages as a more attenuated than theLC16m0, infectivity of vector. The first is its safety in humans. This is the former in rabbits is about 100 times lower clearly shown by the fact that the global vaccina­ than the latter. The;efore, the LC 16m0 is consid­ tion program by WHO had been conducted except ered to be suita~le as a candidate vector for for rare cases of complications. The second is its animal use because of its attenuated property in genetic stability. Thus, the essentially same type humans and its effectiveness in animals. of smallpox vaccine had been successfully used for nearly 200 years since Jenner's time. The (3) Development of recombinant rin­ third is its high immunogenicity conferring both derpest vaccine. humoral and cell-mediated immunities without using adjuvant. The fourth is its high degree of 10.Rinderpestvirus(RV)isamemberofmorbil­ heat stability, and this is the most remarkable livirus subgroup of family paramyxoviridae. In property of the smallpox vaccine which can be this subgroup, measles virus (MV), canine dis­ stored for weeks under tropic climate without temper virus (CDV) and pests de petits ruminant cold chain system. All of these advantages are virus (PPRV) are also included. Except PPRV, expected for the vector vaccine. In addition, such these viruses are well known to show similar big size of the vaccinia virus genome DNA as biological, pathological and serological charac­ 185 kb makes the insertion of multiple genes of teristics. Recently, their similarity was also re­ protective antigens possible, so that polyvalent vealed on the gene structures. We have been vaccine can be developed. Because of these ad­ interested in the pathogenicity ofMV, CDV, and vantages, vaccinia vector vaccine is becoming a RV from comparative aspect by using animal most promising new vaccine. models infected with these viruses. Among these 165

models, rabbits infected with the Nakumara III­ vector vaccine was investigated. The stock of the L strain of RV which had been adapted to rabbits recombinant virus was prepared in PK13 cells, has especially attracted us because of its high and was lyophilized with 5 percent polypeptone virulence in rabbits. In this model, severe clini­ as a stabilizer and examined for its heat stability. cal signs such as high fever and diarrhea are After the incubation at 37° C or 45° C for one experimentally induced. Histologically thses month, decrease in infectivity titers of the re­ clinical course was shown to be associated with combinant virus were only 0.2-0.4 log plaque characteristic lesions mainly consisting of giant forming units at similar degree with that of the cell formation and necrosis in the lymphoid parental LC16m0 strain. This is a marked con­ tissues. Immune capacity such as the mitogen­ trast to the current rinderpest vaccine which can induced proliferative response of the peripheral be stored at 45° C for only a few days at most. The blood lymphocytes, delayed type hypersensitiv­ result indicates that the insertion of H gene did ity to tuberculin and antibody response to sheep not affect the heat stability of the parental virus, red blood cells was also found to be markedly and that this recombinant virus can be safety suppressed in RV-infected rabbits. Moreover, used in tropic area without cold chain. the virus infection induces development of two types of autoantibodies, i.e. anti-nuclear anti­ 13. The immunogenicity of the recombinant body reacting with DNA and his tone and hemag­ virus was examined in rabbits. Seven rabbits glutinating antibody reacting with rabbit red were immunized with the recombinant virus blood cells. Thus, RV-infected rabbits provide intradermally on the back and anti-RV-H anti­ an excellent model for the study on pathogenesis body was examined every week by the virus of morbillivirus. neutralization test and by immunoprecipitation. Anti-RV-H antibody was detected by im­ 11. Recent epidemics of rinderpest in Africa and munoprecipitation in all rabbits inoculated with South Asia have prompted us to develop recom­ the recombinant virus from 1 week after immu­ binant rinderpest vaccine based on our long nization. Virus neutralizing antibody was pro­ experience with RV-infection model of rabbits duced at mean titers of 2 in 3 to 4 weeks after · and on our results of gene analysis of RV. Be­ immunization. Four weeks after immunization, cause of the reasons discussed above, we se­ all rabbits were challenged with the virulent L lected the LC16m0 as a vector. The hemag­ strain of RV. Control groups consisting of rab­ glutinin (H) gene of RV was cloned from the L bits immunized with the parental LC16m0 or strain of RV. The H protein which is a product of those unimmunized were also challenged with H gene serves as the receptor-binding protein the L strain. None of the seven rabbits immu­ and is responsible for virus neutralization, so that nized with the recombinant virus showed any it is the most important protective antigen of RV. clinical signs and immunosuppression. One to 2 The RV-H gene was integrated downstream of weeks after the challenge, anti-RV-H antibldy promoter, p75K, within the HA gene of the was markedly elevated. In addition, antibodies LC116m0 strain. The recombinant virus was against NP, Mand F which are the other viral cloned as hemagglutination-negative mutant. proteins were also transiently produced but at This recombinant virus produced in mammalian low degree. These results indicate that the rab­ cells the glycoprotein with the same molecular bits are successfully protected from the disease weight as the natural RV-H protein suggesting in spite oflocal growth of the challenged virus at that glycosylation occurred properly as authen­ low degree. In contrast, all rabbits of control tic RV-H protein in those cells. groups developed typical clinical signs includ­ ing high fever, severe weight loss, immunosup­ 12. Since the recombinant virus was found to pression and lymphopenia. Three out of five produce RV-H protein, possibility to use it as a rabbits inoculated with the parental LC16m0 166 died 4 days after challenge. Upon autopsy, these virus will hopefully be further confirmed in rabbits had coagulopathy and showed haemor­ cattle. Since F protein, another envelope protein rhagic lesions in the lung, urinary bladder and of RV, may act also as a protective antigen, the colon. These results clearly indicate the recom­ construction of a divalent recombinant vaccine binant virus can be used as a highly heat-stable with Hand F protein genes of RV, in which even vaccine even in the absence of a cold chain higher immunogenicity is expected, is in prog­ system. The immunogenicity of the recombinant ress in parallel work. iotechnology In Animal Disease Diagnosis Developing Countries: New Types of Diagnostic Methods

B. W. J. Mahy, N. J. Knowles and S. Pedley Pirbright Laboratory, AFRC Institute for Animal Health, Pirbright, Woking, Surrey GU24 ONF, U.K.

Introduction in the 1970's (Engvall and Perlmann, 1971 a,b; Voller et al., 1977). Numerous modifications 1. Within the field of animal health, recent ad­ and improvements of the original method have vances in genetic engineering and biotechnol­ been described, and the application of monoclo­ ogy offer the prospect of improvements is three nal antibodies combined with ELISA can further principle areas: 1) better disease diagnosis, 2) improve the specificity of diagnosis. Tests are better and safer vaccines for disease prevention, now available which combine rapidity and accu­ and 3) genetic manipulation of the germ line of racy with high specificity. To illustrate these, economically important livestock so as to im­ some example of ELIS A in use at Pirbright will prove specific disease resistance. Applications be presented here. of biotechnology to the first of these areas is now well advanced, and the principles and examples 3. the World Reference Laboratory for Foot­ of these new methods will be outlined in this and-Mouth Disease atPirbright undertakes diag­ paper. Three methods will be considered: ELISA nosis and typing of foot-and-mouth disease virus combined with monoclonal antibodies or ex­ (FMDV) and swine vesicular disease virus pressed antigens, nucleic acid hybridization, and (SVDV) in field tissues taken from cases, and the nucleic acid sequence analysis. serodiagnosis of FMD of SVD by detecting specific antibodies in animal sera by an indirect Applications of ELISA double antibody sandwich ELISA (Roeder and LeBlancSmith, 1987:FerrisandDawson, 1988). 2. Besides the clinical and epidemiological pic­ The virus typing test uses high titre rabbit antis­ tures, traditional methods for disease diagnosis era to each of the seven serotypes ofFMDV and have mostly relied upon isolation of causative SVDV coated onto polystyrene immunoplates organisms and typing by neutralisation with as 'capture antibody'. The test virus preparation specific antisera, and the converse of examining is then added. Trapped virus antigen is detected sera from affected animals for the presence of by addition of type-specific guinea pig antisera, specific antibodies by tests based upon neutrali­ followed by rabbit anti-guinea pig gammaglob­ sation, immunoprecipitation, immunodiffusion, ulin serum conjugated to horse radish peroxi­ or haemagglutination. Considerable improve­ dase, with orthophenylenediamine as substrate ments in the ease and accuracy of serological for colour detection. The potency of the antisera tests were introduced by the development of the is such that dilutions of 1 in 5000 of rabbit sera enzyme-linked immunosorbent assay (ELISA) or 1 in 1,000 of guinea pig sera can be used. This 168 ELISA is more sensitive and specific than the ELISA kits original complement fixation test for typing FMDV. 6. At present, countries which lack facilities for laboratory identification of FMD or SVD send Monoclonal Antibodies field samples to a regional reference laboratory such as PANAFTOSA in Rio de Janeiro or to the 4. A second approach, which is likely to be of World Reference Laboratory in Pirbright. Re­ increasing importance, is the typing of FMDV cently we have found that ELISA plates can be using panels of monoclonal antibodies ofknown precoated with specific rabbitantisera and stored specificity as the second antibody. In such a test, for long periods at 4 °C or - 200 C with no loss of a 'monoclonal antibody profile' can be obtained reactivity (Ferris et al., 1988). This approach, which is characteristic of the subtype ofFMDV combined with other necessary reagents in (Crowther and Samuel, 1987). This can provide freeze-dried form, should enable the develop­ valuable information on the precise relationship ment of simple 'kits' for antigen detection in less between virus strains (Figure 1). However this developedcountries. TestsforantibodytoFMDV method requires the availability of suitable in animal sera have also been developed at Pir­ monoclonal antibodies. bright (Hamblin et al., 1986a,b) and these have a similiar potential for development in kit form. 5. The production of suitable monoclonal anti­ bodies for use in diagnostic tests requires basic 7. Where it is necessary to reduce costs, it is facilities which are available in any laboratory possible to avoid microtitre plates for the en­ which is capable of maintaining cell cultures. zyme immunoassay by carrying out 'dot-ELIS A' However, the procedure is labour intensive and assays on a sheet of nitrocellulose paper. All calls for a high level of technical skills. The basic reagent addition and washings can then be made technique, which has hardly changed since its in a single container. A combined dot-ELISA first description by Kohler and Milstein (1975) capable of assaying simultaneously for both requires fusing lymphocytes from an immunised adenovirus and rotavirus in faeces has been mouse with a mouse myeloma cell line, selecting described which uses this technique (Gouvea et resultant hybridomas by growth in selection al., 1987). media, then clonal expansion. Antibody can be purified later in tissue culture or in mice. Al­ Co1npetitive ELISA though screening of the hybridoma cell lines for specific antibody production is usually easy, 8. Where a monoclonal antibody of appropriate characterisation of the nature and specificity of specificity is available, the ELISA can be adapted individual monoclonals is time consuming and to exploit its diagnostic potential. One example may involve sophisticated technology. In addi­ is a group-specific monoclonal antibody against tion, only a few cell lines, perhaps one to ten bluetonque virus, which reacts with all 24 sero­ percent of those screened, may yield a monoclo­ types ofbluetongue, but not with other orbi viruses nal antibody of interest. For these reasons it is such as African horse sickness or epizootic preferable for a diagnostic laboratory to obtain haemorrhagic disease of deer viruses (Ander­ appropriate monoclonal antibodies for use in son, 1987). A competitive ELISA developed ELISA from a major centre, if this is possible. using this monoclonal antibody (Anderson, 1984) The sharing of monoclonal antibodies between has been found reliable and unequivocal in tests laboratories is often prevented by commercial of large numbers of animal sera (Afshar et al., interests, but it should fonn a part of the technol­ 1987a). A simple dot-ELISA procedure has been ogy transfer process in assisting developing adapted from this as a rapid, inexpensive test for countries with disease diagnosis. 169 bluetongueantibodies (Afshar et al., 1987b).Itis Nucleic Acid Hybridization of interest that of 24 monoclonal antibody cell lines (from more than 200 produced) which were 11. Analysis of the nucleic acid genomes of found to be bluetongue virus group-specific, disease-producing organisms has advanced only one was suitable for use in competition with considerably in recent years. In particular, many positive sera for diagnosis of bluetongue infec­ virus nucleic acids have been cloned in bacterial tion. This illustrates the value of such a reagent plasmids, so that large amounts of DNA copies once it is obt.ained, but argues against the use of of RNA or DNA sequences are available. These valuable resources by less developed countries clones can be adapted for use in specific hybridi­ in attempting to obt.ain similar monoclonal anti­ zation assays for diagnostic purposes. bodies. General Principles Expressed antigens 12. The potential value of nucleic acid hybridisa­ 9. Most types of ELISA require a source of virus tion for diagnosis was inst.antly recognised but at antigen, which means thatinfectious virus, which first the techniques used to measure the forma­ may be hazardous, must be grown in quantity in tion of hybrids were costly and time consuming cell cultures or, in some cases, in animals. The and thus an obstacle to the general use of the availability of cloned copied of virus genes al­ methods. Simple methods for separating the lows the possibility to express a single protein hybrid molecules from the remaining single antigen in vitro, using an expression vector, thus strands have now been developed. For most eliminating the need for whole virus. applications this involves binding the target nucleic acid to a membrane support. 10. A recent application of this approach em­ ploys the nucleoprotein ofrabies virus, produced 13. The earliest and most widely used membrane from arecombinantDNAclone (Reid-Sanden et support are prepared from nitrocellulose. Chemi­ al., 1988). The rabies virus nucleoprotein gene cally modified cellulose papers (Alwine et al., was cloned, and the DNA inserted by recombi­ 1979) bind nucleic acids, in particular RNA, but nation into the baculovirus Autographica cali­ the papers are expensive and require activation f ornica in association with strong late promoter immediately prior to use. More recently nylon from thepolyhedringene(Matsuuraetal., 1987). membrane filters have been introduced. The Growth of the recombinant baculovirus in high capacity of these filters for nucleic acids, Spodoptera frugiperda(Sf9) insect cells pro­ their robust strncture and ease of handling has duced milligram quantities of rabies nucleopro­ contributed to their widespread use. tein which was fully reactive with monoclonal antibodies in the ELISA, and could also be used 14. Radioactivity is widely used for labelling in immunofluorescence assays. One consider­ nucleic acid probes. It is relatively simple to use able adv ant.age of this expression system for de­ and can be detected at very low levels. Further­ veloping countries is the ease of growing the Sf9 more, the probe nucleic acid and associated label insect cells, which grow optimally at 28° C, but can be washed off the target nucleic acid leaving can survive between 22° C and 30° C, and do not the filter, or other solid support, ready for reuse. require CO during growth. Virus stocks will The disadvantages are the short half life of the survive up t~ 2 years at 4° C with little change in commonly used isotopes and the hazards associ­ titre(SummersandSmith, 1987). The use of this ated with the handling of radioactivity. expression system with other antigens for which cloned DNA genes are available seems entirely 15. The use of nucleic acid hybridisation in feasible. routine disease diagnosis in developing coun- 170

tries will require non-radioactive lables. Biotin, 19. Biotin labelled probes can be detected by a water soluble vitamin, is the most widely used antibodies to biotin followed by reaction with a of the non-radioactive lables. The biotin mole­ second antibody linked to an enzyme or fluores­ cule is linked to the pyrimidine ring of a nucleo­ cent marker. Alternatively, it is possible to ex­ tide triphosphate through an allylamine linker ploit the strong affinity of the biotin molecule for arm (Langer et al., 1981). The labelled nucleo­ avidin, a protein constituent of egg white, or for tide can then be introduced into the probe by any streptavidin, a similar protein from fungi. The of the polymerase based labelling methods. Biotin avidin or streptavidin can be conjugated to en­ molecules linked to a photoreactive arm (photo­ zyme or fluorescent markers. The commonly biotin) have been developed and can be linked used enzyme labels are horse-radish peroxidase directly to the probe nucleic acid by exposure to and alkaline phosphatase and there are several strong light. commercial systems for measuring their pres­ ence. The end product of the reactions is a water 16. Several methods are available for in vitro insoluble coloured precipitate. Chemolumines­ labelling of the probe nucleic acid. The majority cence and fluorescence markers have been de­ of methods can be used to introduce both radio­ scribed (Dahlen, 1987) but the equipment neces­ active and non-radioactive labels although some sary for the detection of the photon emissions is methods are specific for particular labels. Nick­ expensive and not easily available to less devel­ translation (Rigby et al., 1977) has now been oped countries. largely replaced by random primer extension with Klenow nucleic acid polymerase to label Specific Techniques the probe nucleic acid (Feinberg and Vogelstein, 1983). (a) Extraction of the Nucleic Acid 17 .Renz and Kurz (1984) described a method by which enzymes can be linked directly to the 20. For most applications the target nucleic acid nucleic acid probe by a polyethy leneimine bridge. must be separated from other cellular compo­ The enzyme retains its ·activity during the la­ nents which may affect the hybridisation or the belling reactions and may be stored for several binding of the nucleic acid to the solid support. weeks linked to the polyethyleneimine. Since Phenol extraction is still the method of choice for different nucleic acid probes can be linked to purifying nucleic acids and many, if not all, different enzymes it is possible to add several procedures use phenol at some stage of the probes to the hybridisation assay and to differen­ extraction. tiate the probes by the enzyme labels. No other labelling method offers such a simple and con­ 21. The successful extraction of RNA presents venient method of detecting more than one type greater problems than the extraction of DNA. Cells contain high levels of the enzyme RNase, of nucleic acid with a single hybridisation. The an extremely stable and active enzyme which disadvantage of the direct label is the restriction upon the hybridisation conditions which result digests RNA. Rapid methods for the extraction of RNA have been published (Chomczynski and from maintaining the activity of the enzymes. Sacchi,1987). 18. Probes labelled with radioactivity. are de­ tected by exposure of the washed filter to X-ray (b) Transfer and Binding to a S glid Sup­ film. Alternatively, it is simplest of the detection port methods, but exposures of several days or weeks are sometimes required if the hybridisation is 22. Several methods are available for transfering weak. nucleic acids to membranes. The simplest 171

method, and one widely used for detecting the or cells. genome nucleic acid of viruses, is to apply the extracted anddenatured target nucleic acid di­ (c) Hybridisation rectly to the filter. Thomas (1980) developed a method whereby nucleic acid is applied too the 26. Areas of the filter or solid support which have filter in a small volume, approoximately Sul, as not bound nucleic acid are first blocked by the a dot. Large numbers of samples can be analysed addition of an excess of a non-specific nucleic simultaneously by this technique which has been acid- sonicated, denatured calf thymus or salmon termed "dot-blots" sperm DNA - and a mixture of synthetic poly­ mers and bovine serum albumin (Denhardt's · 23. Dot-blotting is a simple and rapid method for solution; Denhardt, 1966). Theprehybridisation detecting the presence of specific nucleic acid solution also contains salts and organic solvents, sequences in large numbers of samples. How­ which are used in the hybridisation step, to ever, information which can be gained from this equilibrate the filter. The labelled probe is dena­ method is limited. More involved techniques are tured by boiling and added to the filter in hybr­ needed if specific characteristics of the target idisation solution containing salts, blocking nucleic acid, for example the size, are required. melting temperature of the nucleic acid hybrid. Southern (1975) described a method for trans­ The temperature of incubation during hybridisa­ ferring DNA fragments from an agarose gel to a tion is determined by investigation but is nor­ nitrocellulose membrane by passive diffusion in mally set at 25° C below the melting temperature a flow of high salf buffer. The fragments were of the hybrid. At this temperature the rate of then analysed by hybridisation to probe nucleic hybridisation is highest. acid. Methods were developed subsequently for transferring RNA from gels to membranes by 27. Excess probe is removed from the filter by diffusion or more rapidly in an electric field or repeated washings in salt solutions at controlled vacuum. By using a combination of nucleic acid temperatures. The presence of hybridisation is fractionation techniques and nucleic acid hy­ determined by detection of the label on the probe bridisation it is possible to obtain a great deal of nucleic acid. These methods have been dis­ information about the target nucleic acid. cussed earlier.

24. After being transferred to a filter the nucleic 28. Each of the hybridisation methods so far acid must be fixed. Nucleic acids are fixed to described requires the target nucleic acid to be nitrocellulose filters by incubation at a tempera­ bound to a solid support. These methods present ture of 80° C for approximately two hours in a problems when designing diagnostic kits. A vacuum oven, or to nylon membranes by a short modification of the basic hybridisation, called exposure to U.V. irradiation. However, the re­ "sandwich hybridisation" (Dunn and Hassell, tention of some nucleic acids or nylon mem­ 1977), has been described and will be valuable branes can be improved by including an incuba­ for the design of diagnostic reagents. Two nu­ tion at 80° C (Mertens et al., 1987). cleic acid probes are synthesised to neighbour­ ing, but not overlapping, regions of the virus 25. Nucleic acids to be analysed by in situ genome. One of the probes is fixed to the solid hybridisation are fixed to the glass slide by on of support and used to capture the target nucleic the standard histological fixatives, for example acid. The free tail of the target nucleic acid, 60% acetone, ethanol acetic acid mixture or which has not reacted to the filter bound probe, formaldehyde. Retention of the tissue and thus is detected with the labelled second probe. The the nucleic acid is improved by including a advantage of this method over the basic hybr­ coating on the slide before applying the sections idisation techniques is that the solid support can 172

be supplied with the probe attached, eliminating 32. Two important observations were made from the need for the diagnostician to bind the target these analyses. First, the genome of the probe nucleic acid to the support. virus hybridised strongly to all segments of the homologous, target virus and to 8 of the 10 Applications segments of the other viruses. Two segments, segments 2 and 5, dig not cross-hybridise be­ 29. In common with all diagnostic procedures, tween viruses of different serotype but hybr­ adequate controls must be included with each idised between viruses of the same serotype. hybridisation assay. McClure and Perrault (1986) This result shows that the genetic information have shown that the genome RNA of some from two virus segments is involved in the con­ viruses will cross-hybridise with the genomes of trol of the virus serotype and has implications for unrelated viruses nd certain cellular RNA's under generation of new serotypes by the reassortment conditions which permit the hybridisation of · of these segments between viruses. Second, the nucleic acids with high sequence homology. relative strengths of the hybridisation between These results emphasise the need for including viruses of the Australia group (1 Australia and appropriate controls with each experiment. 20) and the South Africa/America group sug­ 30. There are several examples of the use of gests that these two virus groups are genetically nucleic acid hybridisation for animal disease distinct and have been isolated long enough for diagnosis. These fall under two headings; epi­ the genome sequences to diverge. In the latter demiology and diagnosis. example nucleic acid hybridsation provides valuable information about the evolution of the (a) Epidemiology bluetongue viruses which would not be revealed by serological methods. 31. Nucleic acid hybridisation can generate valu­ able data for the understanding of the epidemiol­ (b) Diagnosis ogy of certain virus groups, in particular the viruses with segmented genomes. The epidemi­ 33. A recent publication by Rossi et al. (1988) ology of bluetongue viruses has. been studied, describes the use of a cDNA clone from the using these methods, atPirbright (Mertens et al., polymerase region of the 01/Campos/Brazil/58 1987). Viruses of different serotypes and viruses strain of foot-and-mouth disease virus (FMDV) of the same serotype but from different geo­ for the detection ofFMDV sequences in samples graphical locations were cross-compared using of bovine oesophageal-pharyngeal fluids. The nucleic acid hybridisation. The genomic double probe detected viruses of the A,O and C groups - stranded RNA (dsRNA) segments of the vi­ with equivalent sensitivity and was capable of ruses were separated on a polyacrylamide gel detecting virus sequences from cells which would and transferred to a membrane. Nucleic acid not yield virus in tissue culture. The method will from each of the viruses was radioactively la­ be valuable for the screening of animals sus­ belled and used to probe the blots. Figure 2 pected of a persistent virus 9ainfection which shows an example of the results of this analysis. may be over-looked by virus isolation or sero­ The lanes A are control lanes showing the full logical detection methods. genome profile of the viruses. Lanes B were loaded with unlabelled virus nucleic acid and a 34. Nucleic acid clones from specific regions of positive hybridisation is indicated by a band on the virus genome can, by careful selection, be the autoradiogram. In this example the genome used to differentiate viruses which are closely of bluetongue virus serotype 1 from South Af­ related and difficult to distinguish by other rica was used as the probe. methods. At Pirbright we have isolated a clone 173 from the N-gene of the rinderpest virus which hybridisedtoFMDVRNAandFMDVcDNAin can be used to differentiate the closely related the individual wells of the plate. Following the rinderpest and PPR virus (Barrett, 1987; Diallo washing steps the presence of hybridisation was et al., 1988). Theprobewillhybridiseonlytothe detected by incubation with streptavidin/horse­ genome nucleic acid of the rinderpest virus under radish peroxidase (HRP) and measuring the conditions which permit the formation of hy­ presence of the HRP with orthophenylenedi­ brids between nucleic acids which share high amine (OPD) and hydrogen peroxide. The colour sequence homologies (high stringency). Figure was read on a Flow multichannel plate reader. 3 shows the probe hybridising to the nucleic acid from all strains of rinderpest virus but only 38. The nucleic acid bound to the plates has been weakly, if at all, to the PPR virus nucleic acid. shown to be stable for several months. More studies are required but it would appear that the Recent Developments microtitre plates are an ideal solid support if nucleic acid hybridisation is to be used in diag­ nostic laboratories. (a) Use of alternative solid supports (b) Use of synthetic probes 35. For nucleic acid hybridisation to be used as a routine diagnostic method it will be essential 39. Where the nucleic acid sequence is known, for as many as possible of the manipulative steps it may be possible to use synthetic oligonucleo­ to be automated. The membrane filters currently tides of about 15 nucleotides for specific diag­ used for binding the target nucleic acid do not nosis. Sano et al. , (1988) have described the readily lend themselves to automation. Plastic use of such probes with plant viroids, and it microtitre plates would appear to be an ideal may prove possible to adapt the techniques for support for nucleic acid hybridisation. They are animal viruses. extremely robust and many of the items of equip­ ment required for handling large numbers of (c) Sequence amplification by the samples are already available and equally appli­ "Polymerase Chain Reaction" cable to the techniques of nucleic acid hybridisa­ tion. 40. Sequence amplification by the "polymerase chain reaction" (PCR) is a simple system 36. A technique for binding high levels of nu­ whereby a sequence of interest is copied many cleic acid to plastic surfaces, in particular the times by repeated short reactions, (Ou et al., wells of microtitre plates, has been developed at 1985; Saiki et al., 1988). The method can only the Pirbright Laboratory (National Research & be applied to nucleic acids for which nucleotide Development Corporation and Pedley, 1988). sequence information is available. A region of The treated plastic surfaces bind significantly the nucleic acid is chosen and oligonucleotide higher levels of nucleic acid than untreated sur­ primers are synthesised to two sequences sepa­ faces and 50 to 100 fold higher levels than other rated by a short distance (usual! y less than 1.5 published binding methods. kilobases). The primers must have opposite polarities such that the priming 3'-ends of the 37. Several types of nucleic acid have been nucleic acid are directed towards one another. assayed using the micro titre plate system. These The analyte nucleic acid is denatured and include FMDV RNA, bluetongue virus RNA allowed to hybridise to the oligonucleotides. andcapripox virus DNA. Table 1 shows the type DNA polymerase then synthesises a comple­ of result which is obtained with this system. The mentary DNA strand using the oligonucleo­ probenucleic acid was labelled with biotin and tides as primers. Two copies of the original 174

sequence now exist. The denaturing, hybr­ copy of the molecule using deoxynucleoside idisation and polymerase steps are repeated triphosphates, a specific synthetic oligonucleo­ many times - 25 to 30 rounds - each time poten­ tide primer and a DNA polymerase. However, tially doubling the amount of the sequence one relied upon base-specific chemical cleav­ between the primers. As shown in Figure 4 it ages to ascertain the sequence of the resulting is the sequence which is flanked by the DNA either directly or after insertion into a oligonucleotide probes that is preferentially plasmid vector (Maxam and Gilbert, 1977, 1980), amplified and thus the size of the final product while the other incorporated chain-terminating can be predicted with great accuracy. The reac­ inhibitors (dideoxynucleoside triphosphates) and tion product can be analysed on an agarose gel thus was able to determine the sequence directly and detected by staining with ethidium bro­ from the cDNA fragments generated (Sanger et mide. Alternatively nucleic acid hybridisation al., 1977). Both techniques used polyacrylamide can be used to detect the product. gels for the analysis of the cDNA fragments, and were later modified to use reverse transcriptase, 41. Figure 5 shows an example of the use of this enabling RNA genomes to be copied and se­ method for the detection of FMDV RNA. The quenced (Zimmern andKaesberg, 1978; Biggin result was obtained in four hours, compared to a et al., 1983). minimum of 24 hours for standard nucleic acid hybridisation. Furthermore, the sensitivety of 44. Both chemical cleavage and dideoxy-se­ the technique means that nucleic acids which are quencing and currently employed to determine present in amounts below that which can be foot-and-mouth disease virus (FMDV) nucleo­ detected by hybridisation alone can be rapidly tide sequences. The former technique, while amplified to levels which can be measured. There giving generally a less ambiguous sequence, can be no doubt that this technique will be used suffers from a greater complexity and is time widely and companies are now producing ma­ consuming to perform. The latter techniques is chines to automate the cycle of incubation steps. much more rapid and easier to perform but PCR has enormous potential for the diagnosis of problems may be encountered when analysing medical and veterinary diseases. the cDNA on polyacrylamide gels; firstly pre­ mature polymerase pausing (a "strong stop") is Nucleic Acid Sequence Analysis often encountered presumably due to secondary structures in the RNA template. Secondly com­ 4 2. This technique is in its infancy as a diagnostic pressions may be seen in the DNA ladder due to tool, but has great potential for extremely spe­ base pairing in the cDNA fragments generated. cific characterisation of viruses. Although in The first may be reduced by using terminal many cases such precise analysis is not neces­ deoxynucleotidyl transferase and dNTPS to sary, accurate discrimination may be required in randomly extend unblocked3' DNA ends thus certain circumstances, and is beginning to re­ revealing the correctly terminated base (De­ place the former laborious T, mapping technique Borde et al., 1986). The second can be resolved (Rico-Hesse et al., 1987). We have developed by the use of 2'-deoxy-inosine-5'-triphosphate RNA sequence analysis at Pirbright as a routine (dITP) in place of 2'-deoxy-guanosine-5'­ procedure in the accurate subtyping of FMD triphosphate(dGTP)in thesynthesisofthecDNA. virus. dITP is a precursor of dGTP and dATP, how­ ever, it is recognised as dGTP but does not base 43. In 1977 two techniques, which enabled the pair as readily. · nucleotide sequence of DNA molecules to be determined, were published. Both were based on 45. A number ofowrkers have compared nucleo­ constructing a complementary DNA (cDNA) tide and amino acid sequences of the FMD V 175

capsid protein lD (VPl) to ascertain possible capsid where monoclonal antibodies (MAb) react. regions involved in serotype and subtype speci­ By sequencing virus mutants which escaped ficities (Makoff et al., 1982; Beck et al., 1983; neutralization with a particular MAb, amino acid Cheung et al., 1983; Robertson et al., 1983; changes, presumably involved in or close to an Villanueva et al., 1983; Cheung et al., 1984; antigenic site, have been identified for serotype Weddell et al., 1985). Although variable and O(Xieetal., 1987;Pfaffetal., 1988; Stave et al., conserved intra-and intertypic regions were 1988) and serotype A (Thomas et al., 1988; identified this data alone was unable to define the Bolwell et al., in prep.) viruses. exact basis for serotype and subtype specifici­ ties. 48. The dideoxy-sequencing method relies on having a number of specific oligonucleotide 46. In epidemiological studies of FMDV, nu­ primers spaced thoughout the region to be stud­ cleotide sequencing has begun to replace con­ ied. To this end the World Reference Laboratory ventional serological methods because of its at Pirbright has been designing and making such unequivocal determination of genetic relation­ primers and is building a comprehensive library ships between viruses. It has been used for the so that at least one representative region of any identification of vaccine virus-related outbreaks FMDV can be quickly analysed and compared and determining the relationship between geo­ with known sequences which are also held on a graphically distinct disease outbreaks. Most computer database. studies have concentrated on sequencing the gene coding for the most variable capsid protein Conclusions and recommenda­ lD (VPl). Usingthechemicalcleavagesequenc­ ing method, Beck and Strohmaier (1987) con­ tions cluded that most of the recent outbreaks ofFMD serotypes 0 and A in Western Europe were 49. Several new types of diagnostic methods probably caused by incompletely inactivated which exploit recent developments in biotech­ vaccines of from laboratory escapes of vaccine nology are currently available for animal disease virus strains. Sobrino et al. (1986) and Martinez diagnosis. They provide considerable opportu­ et al. (1988) used dideoxy-sequencing to study nities to developing countries in terms of greater the evolution of closely related serotype C vi­ accuracy and specificity. The simplest of these ruses in Spain. Piccone et al. (1988) using both combine the ELISA test with monoclonal anti­ methods demonstrated two distinct genetic line­ bodies or expressed antigens. Transfer of such ages of the C3 subtype in South America. Re­ technologies to developing countries is already cently an outbreak of Sero type A in Iran in cattle underway, but will require free exchange of imported from the Federal Republic of Germany reagents such as monoclonal antibodies or ex­ was shown to neither originate from the export­ pressed antigens if full use is to be made of the ing country nor to be associated with the A22 potential of ELISA. Iranian vaccine virus strain (Ahl and Marquardt, 1987; Samuel et al., 1988; Figure 6). Another 50. Nucleic acid hybridisation techniques (probe study rapidly identified outbreaks of serotype 0 analyses) can be transferred fairly easily once in the Federal Republic of Germany in 1987-88 applications using non-radioactive labels have as being very closely related to the vaccine virus been developed. However, the free exchange of strain, 01/Kaufbeuren/FRG/65 (Knowles et al., cloned DNAs for use in such tests will be essen­ 1988). tial if all countries are to benefit. It would seem pointless for developing countries to have_ to 4 7. Nucleotide sequencing has also been of prime acquire sophisticated technologies in order to importance in identifying the areas on the virus clone DNAs which are already available else- 176

where. ALWINE, J.C., KEMP, D.J., PARKER, B.A., REISER, J., RENART, J., STARK, G.R. and 51. Finally, the application of nucleic acid se­ WAHL,G.M. (1979)Detectionof specificRNA's quencing is in its infancy, but has great potential or specific fragments of DNA by fractionation in for use in reference centres, where accurate gels and transfer to diazobenzyloxy-methy 1paper. diagnosis is required, e.g. for strain identifica­ Meth. Enzym. 68, 220-242. tion. It seems unlikely that this technique, which required high sophisticated apparatus and spe­ ANDERSON, J. (1984) Use of a monoclonal cialised reagents, will be readily transferred to antibody in a blocking ELISA to detect group all countries for diagnostic purposes. specific antibodies to bluetongue virus. J. Imm. Meth. 74, 139-149. Acknowledgments ANDERSON, J. (1987) Investigations on blue­ 52. We are grateful to Tom Barrett, Peter Mertens tongue with monoclonal antibodies. PhD thesis. and Alan Samuel for providing figures. Council for National Academic Awards.

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Table 1 Figures represent the mean optical density measured at 492 nm, of duplicate wells. Well number and amount of analyte DNA/RNA per well

l 2 3 4 5 6 7 8 9 10 11 12 250 pg 200 pg 150 pg 100 pg 60 pg 50pg 40pg 30pg 20pg lOpg 5pg Blank

A-B 0.611 0.721 0.586 0.800 0.713 0.690 0.515 0.370 0.279 0.271 0.272 0.280 C-D 0.572 0.560 0.463 0.365 0.331 0.350 0.289 0.260 0.245 0.241 0.239 0.256 E-F 0.268 0.267 0.267 0.267 0.277 0.282 0.281 0.279 0.274 0.271 0.270 0.274 G-H 0.243 0.231 0.243 0.239 0.244 0.250 0.248 0.248 0.242 0.237 0.238 0.255

Key Row A-B : Cloned dsDNA of FMDV RNA type AlO strain 61. Row C-D: Virus RNA of FMDV type AlO strain 61. Row E-F: As for A-B but without treatment. Rwo G-H: As for C-D but without treatment. Draft F·AO/UNDP Project entitled 'Biotechnology Development Network for Animal Production and Health in Asia

Inter-country project of Governments of China, India, Indonesia, Malaysia, Pakistan,Philippines, Republic of Korea and Thailand PROJECT DOCUMENT

Number and title: RAS/88/.... Biotechnology Development Network for Animal Production and Health in Asia

Duration: Three years

Project site:

ACC/UNDP sector and subsector: Agriculture and Forestry Livestock

Government sector and subsector: Agriculture and Livestock

Government implementing agency: Ministries responsible for Animal Production and Health

Executing agency: Food and Agriculture Organization

Estimated starting date: July 1989

Government inputs: In kind equivalent to US$ 1,175,000

UNDP and Cost Sharing Financing

UNDP IPF (Regional) $ 1,350,000 Other (specify) Nil Govt. or third party cost sharing (specify) Nil UNDP & cost sharing

Total: $1,350,000 183

Brief Description:

The major function of the project is to establish a network to share the responsibility of research, training and information exchange essential for the development of biotechnology in animal production and health in eight participating countries of Asia. The project will also provide assistance to ensure continued operation of the network on a permanent basis for an information exchange programme and the publication of the "Biotechnology Newsletter". A self supporting mechanism operated within the framework of TCDC with the responsibility ofJts management and operation being gradually assumed by the participating governments will be established. It is­ expected that in the developing countries where performance records of the herds are not properly maintained, the implementation of embryo transfer technology will have immediate application in a selection programme of genetically superior livestock through the organization of an Open Nucleus Breeding System.

Signed ...... on behalf of each participating country

Signed...... Signed ...... on behalf ofFAOon behalf ofUNDP

Table of Contents coting agency 6. Special considerations Cover page 7. Co-ordination arrangements 8. Counterpart support capacity A. Context C. Development objective 1. Description of subsector 2. Strategy of participating countries for D. Immediate objective(s), outputs, and acm the subsector tivi ties 3. Prior or ongoing assistance 1. Immediate objective 1 4. Institutional framework for development 1.1Output1 efforts in the subsector Activities 1.1.1 activity 1 B. Project justification 1.1.2 activity 2 1. Problem to be addressed; the present 1.1.3 activity 3 situation 1.1.4 activity 4 2. Expected end of project situation 1.1.5 activity 5 3. Target beneficiaries 4. Project strategy and institutional arrange 1.2 Output2 men ts Activities 5. Reasons for assistance from UNDP/exe 184

1.2.1 activity 1 A. Context 1.2.2 activity 2 1.2.3 activity 3 1. Description of the Subsector. 1.2.4 activity 4 1.2.5 activity 5 Livestock has become a priority sector for development in Asia, not only because of its 1.3 Output 3 importance in food production but also due to the Activities employment potential it generates for the small 1.3.1activity1 and landless farmers in the rural areas. The poor 1.3.2 activity 2 genetic potential of local animals, shortage of 1.3.3 activity 3 feed and fodder along with the prevalence of contagious diseases, have resulted in low pro­ 2. Immediate objective 2 ductivity of local livestock in the developing 2.1Output1 countries of Asia. Activities 2.1.1activity1 2. Strategy of participating countries for the 2.1.2 activity 2 sub sector 2.1.3 activity 3 2.1.4 activity 4 The major objective oflivestock devel­ opment of the participating countries is to in­ 3. Immediate objective 3 crease domestic production, thereby stimulating 3.1Output1 rural employment and reducing dependency upon Activities imports. The strategy for this includes the devel­ 3.1.1activity1 opment of new but effective technologies to 3.1.2 activity 2 improve the productivity of their livestock. 3.1.3 activity 3 Biotechnology is a relatively new science but the 3.1.4 activity 4 techniques related to it have already been iden­ 3.1.5 activity 5 tified as a potential means for improving the productivity of the livestock. 4. Immediate objective 4 4.1Output1 3. Prior or ongoing assistance Activities 4.1.1activity1 UNDPprojects in China (CPR/85/038)­ 4.1.2 activity 2 animal growth stimulants) and India (IND/85/ 4.1.3 activity 3 020)- embryo transfer and vaccine production) are supporting the development of biotechnol­ E.Inputs ogy in these countries. Different donor agencies are also assisting India, Indonesia, Pakistan and F. Risks Thailand in thefieldofbiotechnology. The Inter­ national Center for Genetic Engineering and G. Prior obligations and prerequisites Biotechnology (ICGEB) ofUNIDO has started to establish its component in New Delhi, India. · H. Project reviews, reporting and evaluation It is hoped that facilities developed at this unit will, in due course, provide assistance to the I. Legal Context countries in the region in their effort to utilize biotechnology in the improvement of animal J. UNDP Budget productivity. FAO Animal Production and Health 185 Division organized in 1986 organizing a global tivity of their livestock. The mission also identi­ Expert Consultation on Application of Biotech­ fied the institutes, equipment, research program­ nology to Animal Production and Health. Later mes, and human resources being directed for this in 1988, FAO organized a regional Workshop purpose. using its regular programme funds for the iden­ tification of regional research priorities in bio­ All the eight countries have made a technology applied to animal production and beginning in the field of biotechnology by initi­ health. The Workshop was attended by experts ating research in embryo transfer. In these coun­ from all the eight countries participating in the tries performance records of the herds are not project and recommended fields for training and properly maintained and it is difficult to carry out mechanism of the establishment of a Biotech­ progeny testing of dairy animals and perform­ nology Development Network for Animal Pro­ ance tests of meat animals. The disease diagnosis duction and Health in Asia. FAO has also pro­ facilities are also limited and it takes a long time vided assistance from its Technical Cooperation to obtain results. There is a shortage of properly Programme (TCP) funds to some countries of trained manpower, most of the laboratories are the region to initiate research and development without an adequate supply of chemicals and work in biotechnology for the improvement of reagents required and except for few institutions, animal production and health. do not have the equipment essential for the research and development work in the field of 4. Institutional framework for development ef­ biotechnology. forts in the subsector 2. Expected end of project situation In individual countries, the responsi­ bility for development of livestock lies with the It is expected that in the countries par­ respective Ministries of Agriculture. ticipating in the project there will be a sufficient number of trained scientists and technicians at The research and development activi­ the end of the proposed project to undertake ties in difference fields of biotechnology are research and development activities concerning carried out by the universities and research insti­ new techniques in biotechnology. A network tutes/centers in the participating countries. Some will be established to coordinate research and countries have established nodal agencies, such development, to avoid as far as possible duplica­ as the National Center for Biotechnology and tion, to engage in training in the spirit of techni­ Development in China and the Department of cal cooperation among developing countries Biotechnology in India for overall development (TCDC), to provide interfacing with other scien­ of biotechnology in all areas. tists working in developed countries, and to fa­ cilitate research and development targets with B. Project justification regional implications to be tackled cooperatively. It is also expected that laboratories engaged in the research and development work will be bet­ 1. Problem to be addressed; the present situation ter equipped and have adequate supplies of chemicals and reagents required for their work. A recently completed FAO Animal Production and Health Division fact finding The implementation of embryo trans­ mission to China, India, Indonesia, Malaysia, fer technology will have immediate application Pakistan, Philippines, Republic of Korea and in progeny testing in order to select genetically Thailand noted that there is definite interest in superior dairy and meat animals through the these countries in developing techniques in bio­ organization of an open nucleus breeding sys- technology for solving problems of low produc- · tern. 186

Another area of biotechnology which developed countries and to facilitate research will have immediate application in participating and development targets with regional implica­ countries is the development of tests such as tions to be tackled cooperatively. enzyme-linked immunosorbent assay (ELISA) for rapid and early diagnosis of animal diseases. Implementation arrangements consist of a Regional Coordinator to be based on one of The Project Coordination Committee the National Centers to be designated as the constituted during the operation of the project Coordinating Center. The subject matter spe will develop a self supporting mechanism to cialists will be based on specific Centers and will ensure continued operation of the network on a support the research and development activities. permanent basis for information exchange and The national professionals will assist the re­ the publication of the Biotechnology Newslet­ gional coordinator and subject matter special­ ter. The network then will be operated within the ists. framework of TCDC with the responsibility of its management and operation being gradually The Regional Network Coordinator will assumed by participating governments them­ be responsible for the overall operation, timely selves. implementation and coordination of the project. A Project Coordination Committee will be con­ 3. Target beneficiaries stituted to review the technical programme of the network. It will meet once a year and each of the The institutions which would benefit in participating countries will designate one of their the first instance would be selected national scientists to serve on this Committee. The centers in the eight countries. These centers will Committee in due course will develop a self have better trained personnel and suitable infra­ supporting mechanism to ensure continued structure to conduct research i_n one or more of operation of the Network on a permanent basis the specialized fields of biotechnology. A large for information exchange and publication of the number of women scientists and technicians will "Biotechnology Newsletter". The Network then be trained and this will improve their status as will be operated within the framework of TCDC scientists/technicians. The target beneficiaries with the responsibility of its management and will be the livestock farmer who, within the operation being gradually assumed by partici­ duration of the project, can obtain genetically pating governments themselves. superior livestock by embryo transfer technol­ ogy and facilities for early, as well as rapid 5. Reason for assistance from UNDP/executing disease diagnosis based on tests like ELISA. In agency due course there will be better vaccines by clon­ ing of specific genes and better utilization of The harnessing of biotechnology in animal feeds and fodder by manipulation of order to solve the problems of animal production rumen fermentation etc. and health in developing countries can only be done with the assistance of an international 4. Project strategy and institutional arrangements development agency like UNDP. The Animal Production and Health :Qivision of FAO con­ The strategy of the project is to estab­ ducted in 1986 an in-house review of the poten­ lish the network to coordinate research and tial of biotechnology and came to the conclusion development, to avoid duplication, to engage in that a networking arrangement was the most training in the spirit of technical cooperation cost-effective solution for developing such a among developing countries (TCDC), to pro­ programme in Asia. It therefore follows logi­ vide interfacing with other scientists working in cally that implementation of the activities origi- 187 nallyadvocatedbyFAOshouldbewiththesame C. Development objective agency. All the countries in the region have 6. Special considerations development programmes to improve the pro­ ductivity of their livestock and the objective of The biotechnology network will oper­ the projected network is to support the countries ate in the spirit of TCDC. The training, exchange in this effort. The project will develop and sup­ of information and utilization of technology port a rational and coordinated approach to ani­ developed in any one of the participating coun­ mal production and health in Asia and the Pacific tries by the other participating countries will lead region for training, research exchange of infor­ to regional cooperation. As a large number of mation and institution building. women scientists and technicians will be trained, they would be able to play an important role in D. Immediate objectives, outputs and the development of technologies suited to local activities conditions, thus resulting in integration of women in development. Immediate objective one

7. Coordination arrangements Establishment of a network of biotech­ nology centers in eight countries to share the Implementation arrangements consist responsibility ofresearch, training, and informa­ of a Regional Coordinator to be based on one of tion exchange essential to biotechnology devel­ the National Centers to be designated as the opment in the region. Coordinating Center. The subject matter spe­ 1.1Output1 cialists will be based on specific National Cen­ Activities ters. The expatriate staff will be based on spe­ 1.1.1 Confirm the suitability of the biotech- cific National Centers. The expatriate staff will nology national center in ...... support the research and development activities to serve as Regional Coordination of the national staff working in the centers par­ Center. ticipating in the biotechnology network being 1.1.2 Obtain, furnish and equip offices. established under this project. There will be 1.1.3 Recruit administrative support staff. substantial in-kind assistance from the host 1.1.4 Publicise the establishment of the governments along with technical and adminis­ Coordination Center to all interested par- trative back-stopping of FAO/UNDP. ties. 1.1.5 Establish links with institutes/centers 8. Counterpart support capacity working in the field of biotechnology.

Participating countries are interested in 1.2 Output2 developing techniques in biotechnology to solve Operational data and information processing the problems of low productivity of their live­ system. stock. A fact finding mission of FAO to the Activities participating countries noted that there are a 1.2.1 Recruit consultant sufficient number of scientists and technicians in 1.2.2 Establishment at the Regional Coordi these countries who, if trained under the project, nation Unit, a system for the evaluation, storage will be able to carry out different activities listed (data bank) and analysis of information to be under D Immediate objectives, outputs and provided by the national centers. activities of this project document. 1.2.3 Obtain and install the necessary equipment. 188

1.2.4 Train staff in operation of information 2.1.2 Locate suitable institutions abroad and processing system. place the trainees there for training. 1.2.5 Production of a regular "Biotechno - 2.1.3 Organize training for 40 other scien­ logy Newsletter" for circulation to all participat­ tists in the new techniques in biotechnology in ing countries. one of the participating countries in the spirit of TCDC. 1.3 Output 3 2.1.4 Organize inservice training of 48 tech­ Establishment and operational project coordina­ nicians in the new techniques in biotechnology tion committee. in one of the participating countries in the spirit Activities ofTCDC. 1.3.1 Formally establish Project Coordina­ tion Committee composed of designated scien­ Immediate objective three tists from all the participating centers. 1.3.2 Convene annual meetings of the Com­ Establish pilot projects in order to mittee. strengthen the research capabilities in one or 1.3.3 Circulate the proceedings of the meet­ more of the specialized subjects of the selected ings and take necessary follow-up action. national centers in each of the eight countries.

Immediate objective two 3.1Output1 Establish eight pilot projects resulting in strength­ To develop a core of suitably trained ened national center in each of the eight coun­ personnel at various levels for research and tries. development in new techniques in biotechnol­ Activities ogy in eight countries. 3 .1.1 Identify one selected center in each of eight countries. 2.1Output1 3 .1.2 Formulate eight pilot projects in order Sixty-four suitably trained scientists and 48 to strengthen research in one or more of the fol­ technicians for research and development in new lowing specialized fields for each center: techniques in biotechnology in eight countries. i) embryo culture Activity 1 ii) production of monoclonal antibod- 2.1.1 Select suitable candidate, 24 in num­ ies ber for training abroad in following specializa- . iii) gene mapping and recombinant tions in biotechnology DNA techniques iv) manipulation and control of rumen i) embryo culture and transfer fermentation ii) production of monoclonal antibod- v) cloning of specific genes for vaccine ies production iii) gene mapping and recombinant vi) cloning of specific genes for pro­ DNA techniques duction-related hormones iv) manipulation and control for vac­ vii) in-vitro treatment of feeds by cine production manipulated microorganisms v) cloning of specific genes for vaccine 3 .1.3 Review equipment and capability. production 3.1.4 Order and install additional equip­ vi) cloning of specific genes for pro­ ment as required. duction related hormones 3 .1.5 Arrange for staff training as required. vii) in-vitro treatment of feeds by manipulated microorganisms 189 Immediate objective four and other facilities for it will also be provided by the host country. To prepare at least one manual on each of the following subjects: The participating governments will i) use of recombinant DNA techniques provide appropriate national counterparts to work in animal improvement with the project staff and consultants during ii) embryo transfer technology assignments in the country. These countries will iii) biotechnology and animal disease also provide temporary office space, secretarial diagnosis assistance, meeting rooms and facilities in the iv) biotechnology and development of laboratories and in the field to carry out the vaccines activities listed under output 3.1 of the project v) biotechnology and modification of document. rumen micorbial ecosystem vi) biotechnology and production-re­ The part1c1pating governments will lated hormones designate a scientist involved in the biotechnol­ vii) biotechnology and improvement of ogy research and development to serve on the feeds Project Coordination Committee. The same person will liaise with the project on a continuing 4.1Output1 basis. Seven manuals in the areas of biotechnology dealing with embryo transfer, disease diagnosis, The participating governments will development of vaccines, modification of rumen nominate candidates for training under the pro­ fermentation, production related hormones, ject. The Governments will maintain the salaries improvement of feeds and use of recombinant and allowances of trainees during their period of DNA techniques in animal improvement. training. Activities 4.1.1 Identify the experts who will be mainly The participating governments will responsible for the preparation of each of the provide qualified instructors,if available, to manuals. supplement the work of the project staff in con­ 4 .1.2 Prepare and publish the manuals nection with training programmes. 4.1.3 Distribute the manuals to the partici­ pating countries and others interested in the 2. UNDP inputs subject. Regional Network Coordinator E. Inputs Will be responsible for overall opera­ tion, timely implementation and coordination of 1. Inputs of the participating countries the project. The Regional Coordinating Center will Consultants be located at the biotechnology center in and thus may be considered the host country for the Will be provided to advise and assist project. The Government of will therefore pro­ the Regional Network Coordinator and the par­ vide rent-free furnished office facilities for the ticipating national centers in different fields of project. The data and information processing specialization identified during the course of the unit of the project will also be located at the Co­ project. ordinating Centre. Necessary accommodation 190

National Professionals avoided by providing sufficient time for such recruitment. A similar precaution has been taken National experts from the region will to avoid risk of a delay in the selection and be provided to assist the Regional Network placement of suitable candidates for training Coordinator or consultants in different fields of abroad. specialization identified during the course of the project. G. Prior obligations and prerequisites

Official Travel Continued agreement of the host coun­ To cover the costs of travel by the try to allow the establishment of the Regional Regional Network Coordinator, the consultants, Coordination Center and the provision of office national professionals and members of the Proj­ facilities required for the establishment of data ect Coordination Committee. All costs of the and information processing unit of the network. review and evaluation missions. The project document will be signed by Training UNDP and UNDP assistance to the project will be provided only if the obligations stimulated To cover the cost of study tours and above are satisfactory to UNDP. participants to the group training. H. Project reviews, reporting and Equipment evaluation

For information processing and pro­ a) The project will be subject to tripar­ duction of newsletter at the coordination unit. tite review (joint review by representatives of the Governments, executing agency and UNDP) at Provision of urgently required and least once every 12 months of the start of full essential equipment, chemicals and glassware ~uch meeting to be held within the first 12 for the participating centers. months, the first such meeting to be held within the first 12 months of the start of full implemen­ A list of equipment etc. will be pre­ tation. The Regional Network Coordinator and/ pared for each center during the course of the or senior project officer of FAO shall prepare project. and submit to each tripartite review meeting a Project Performance Evaluation Report (PPER). F. Risks b) A project terminal report will be A risk that there may a delay in the prepared for consideration at the terminal tripar­ participation of a sufficient number of govern­ tite review meeting. It shall be prepared in draft ments to the project operation is minimized to a sufficiently in advance to allow review and tech­ great extent by the interest shown by the Govern­ nical clearance by FAO at least four months prior ments of the region in the establishment of a to the terminal tripartite review. biotechnology network. c) The project shall be subject to evalu­ A risk that there may be a delay in the ation 18 months after the start of full implemen­ recruitment of sufficiently qualified and mutu­ tation. The organization, terms of reference and ally agreed upon experts/consultants has been timing will be decided after consultation be- 191 tween the parties to the project document, plus ta1 Provisions. any associated United Nations agency. The following types of revisions may I. Legal Context made to this project document with the signature of the UNDP resident representative only, pro­ This project document shall be the in­ vided he or she is assured that the other signato­ strument referred to as such in Article 1 of the ries of the project document have no objections standard Basic Assistance Agreement between to the proposed changes; the Governments of those participating coun­ tries which sign such Agreement and the United a) Revisions in, or addition of, any of Nations Development Programme. The host the annexes of the project document. country implementing agency shall, for the pur­ pose of the Standard Basic Assistance Agree­ b) Revisions which do not involve sig­ ment, refer to the government cooperating agency nificant change in the immediate objectives, described in that Agreement. outputs or activities of a project, but are caused by the rearrangement of inputs already agreed to This project document shall be the in­ or by cost increases due to inflation; and strument envisaged in the Supplemental Provi­ sions to the Project Document, attached here to. c) Mandatory annual revisions which The host country implementing agency shall, for rephrase the delivery of agreed project inputs or the purpose of the Supplemental Provisions to increased expert or other costs due to inflation or the Project Document, refer to the government take into account agency expenditure flexibility. cooperating agency described in the Supplemen- UNDPBUDGET ProjectRAS/88/ Biotechnology Development Network for Animal Production and Health in Asia

Total 1989 1990 1991 1992 m/m $ m/m $ m/m $ m/m $ m/m $

10 Project Personnel 11.01 Regional Network Coordinator 36 315,600 6 51,000 12 104,160 12 106,200 6 54,240 11.02 Consultants 16 135,000 3 22,600 6 47,300 4 41,600 3 23,500 11.99 Experts Total 52 440,600 73 600 151,460 147,800 77,740 13 Administrative Support 102,700 15,700 33,000 35,500 18,500 15 Duty Travel 135,000 26,000 41,000 43,000 25,000 16 Mission Costs 10,000 10,000 17 National Professionals 40 60,000 8 12,000 12 18,000 16 24,000 4 6,000 19 Component Total 758,300 127,300 243,460 250,300 137,240 30 Training 31 Study Tours 45,000 5,000 15,000 20,000 5,000 32 GrouQ Training 255,000 60,000 95,000 75,000 25,000 39 Component Total 300,000 65,000 110,000 95,000 30,000 40 Equipment 41 Field Purchases 190,000 55,000 80,000 35,000 20,000 49 Com2onent Total 190,000 55,000 80,000 35,000 20,000 50 Miscellaneous 51 Operation and Maintenance 14,200 2,000 5,000 6,000 1,200 52 Reporting Costs 9,500 - 2,600 2,500 4,400 53 Sundries 62,000 12,000 15,000 25,000 10,000 59 Comronent Total 85,700 14,000 22,600 33,500 15,600 82 Pre2aration of Manuals 16,000 - 6,000 5,000 5,000

Grand Total 1,350,000 216,300 462,060 418,000 207,840

N 0\ .....-( AnnexD

Country Papers Presented

The People's of China : iotechnology Applied to Animal Production and Health

Wang Ruixiang Institute of Animal Science Chinese Academy of Agricultural Science Beijing, China

1. Biotechnology or bioengineering is Friesian embryos imported from W. Germany one important area of the Fourth Industrial was 40 percent (4/10). During 1983-1984 in 11 Revolution. In the last few years many break­ organizations and institutions transfer, a total of through in biotechnology applied to animal 329 cows were superovulated. The number of production and health have been made in the collected, transferable, and transferable embryos world. Although biotechnology in its narrow per donor were 952, 484and1.5 respectively .The meaning was developed much later than in de­ successful rate offresh, and frozen embryo trans­ veloped countries, Our Government has paid fer was 23.8 percent (38/160) and 20 percent and is paying great attention to it. The Chinese (5/25). The best results from small experiments Development Centre of Bioengineering was es­ were 46.6 percent (7 /15) and 33.3 percent (2/6) tablished in the National Committee of Science. for the fresh, and frozen embryos respectively. There is an Office of Biotechnology, which is in Sixteen calves were born from 32 recipients charge of research and development of biotech­ transferred by using frozen embryos in Institute nology applied to agriculture, animal production of Animal Science, CAAS in 1986 (Zhu et al, and health and fishery, in the Ministry of Agri­ 1986). In Jilin Provincial Academy of Agricul­ culture of China. There are either centres of tural Science, 4 85 embryos were recovered from biotechnology or centres of embryo transfer in 70 superovulated donor cows in 1986; Transfer­ most provinces as well as in Chinese Academy able embryos per donor was 3.8; 197 yellow of Agricultural Sciences. Numerous outstanding cattle recipients were transferred; Successful scientists are now working hard in this area in transfers were 50.3 percent (99/197); 93 calves universities and institutions. Some encouraging were born; 20 embryos were collected from progresses have been achieved. donor cow 185# in a superovulation; After trans­ fer 10 recipients became pregnant, 9 calves were 2. Embryo transfer. Research for the born (Jin et al., 1987). The successful rate of development of embryo transfer in China was embryo transfer in cattle increased from 17. 7 started in 1975. After nonsurgical collection and percent (3/17) in 1983 to 55.1 percent (27 /49) in transfer of embryos in cattle were successful, the 1986 in HebeiProvincial Centre ofembryo trans­ first successful deep-frozen embryo transfer in fer (Sang et al., 1987). During 1985-1986 in sheep in was made 1980. Afterwards, calves dairy farm (Chengdu,Sichuan Province), a total from frozen embryo transfer were born in 1982 of 387 embryos were recovered from 47 and 1983. Transfer of fresh and frozen embryos cows.Transferable embryos per donor were 4.9, imported from West Germany was also success­ 134 embryos were transferred to 93 recipients, ful. The rate of gestation of frozen Holsein - 43 cows became pregnant. and successful rate 196 was 46.7 percent(Chen et al., 1987). Institute of with aid of micromanipulator under stereoscope. Animal Science, JASS 352 embryos were col­ 14 (6 monozygotic pairs) demi-embryos without lected from 50 donor cows, 5 transferable em­ zonae pellucidae were transferred to 7 dairy bryos per donor, 200 embryos were transferred heifers (one pair into each recipient), 2 heifers to recipients (1 embryo to each), and pregnant became pregnant; One of them produced a pair rate was 40 percent (1987). In the North of China of male monozygotic twins at full term, The a total of 412 cows of Yellow Cattle were trans­ other gave birth prematurely a pair of female ferred in 1987. monozygotic twins (Tan et al., 1988).

3. Method of DAPI fluorescence for 6. In vitro fertilization system for the identification of embryo motality was esta­ mice was established and 11 'test-tube' mice blished in Institute of Animal Science, CAAS were obtained (Yin and Chen, 1987). In rabbits, (Ma et al., 1985). The accuracy of identification 72 oocytes were recovered from 8 superovulated for vital embryos was 94.7 percent, which was rabbits (from oviducts). Matured oocytes were significant higher than of morphological method. inseminated in vitro with sperm capacitated in rabbit genital tracts. After incubation, 56 eggs .4. Study on embryo transfer in sheep developed into 2-8 cell stage. Fertility rate was and pigs is in progress, but it is not as intensive 77 .8 percent (Zhu et al., 1987). In cattle, 166 as in cattle. Successful pig embryo transfers oocytes collected from ovanes of slaughtered were made in Beijing and Nanjing in 1987. yelolow cattle were incubated in TCM 199 complemented with FSH and LH,the fertili ty 5. Bisection of embryos. Study on rate of in vitro matured oocytes was 72 percent production of monozygotic twins, triplets, and (Qin et al., 1987). In pigs, preliminary experi­ multiplets started in our country only couple of ment indicated that oocytes collected from fol­ years ago. In the sheep, 17 previously softened licles and matured in vitro were inseminated embryos were bisected with microglass needles with fertility rate of 20 percent (3/15). under steromicroscope by hand, 10 pairs of demi­ embryos without zonae pellucidae were trans­ 7. Studies on sexing of embryos using ferred to 10 recipients, 4 of them became preg­ H-Y antigen and DNA probes, sexing of sperms nant and 5 lambs were born, including 1 pair of using gradient centrifugation in percol, and female monozygotic twins (Jiang et al., 1987), bioengineering applied to pig breeding are in 19 goat embryos were bisected in Northwestern progress. Agricultural University. 19 pairs of demi-em­ bryos were transferred into 18 recipients, 12 " 8. To sum up, it is concluded that became pregnant, 9 of them went to term produc­ although some progresses in biotechnology ing 11 half embryo kids (Zhang et al., 1988). applied to animal production have,been made In cattle, 8 late morulae and blastocytes pre­ during short period in China, it has a long way to viously softened in 0.5 percent pronase - PBS go to catch up with advanced countries. solution were bisected with microglass needles India: Biotechnology Applied to Animal Production and Health

P.N.Bhat National Biotechnology Centre Indian Veterinary Research Instttute Izatnagar - 243 122 (UP) India

1. Life Sciences are experiencing an excitement haps the greatest achievement of modem comparable to what physics experienced in science. It is the result of the efforts of 24 the early years of this century. There is an agricultural universities and 45 research in­ effort to synthesize various branches of life stitutes of the Indian Council of Agricultural sciences (Molecular Biology, Molecular Research and others which were established Biophysics, Genetic Engineering etc.) into in all major states in the country on the one integrated whole to explain all life pro­ pattern of US land grant colleges. Besides cesses on the basis of the behaviour of mole­ ICAR, projects in sectors allied to agriculture cules. This attempt has proved to be highly are also being supported by the Department successful and has brought about remarkable of Science and Technology (DST), Council developments which have provided deeper of Scientific and Industrial Research (CSIR), insights into the life processes. More impor­ Indian Institute of Technology (IITs) and tantly the knowledge acquired through these other universities. studies has been put to application in indus­ try, medicine, agriculture, animal husbandry 4. A good deal of attention has been paid to and veterinary science. enhance the genetic potential for rapid growth and increased milk production in livestock. 2. Modem research in veterinary science and This has been obtained by crossbreeding of animal husbandry in India started with estab­ animals that are adapted to tropical environ­ lishment of the Indian Veterinary Research ment and have greater resistance to diseases Institute (IVRI) at Izatnagar in 1889. This and parasites with improved breeds of Eu­ was expanded, when the Imperial Council of rope and the United States. A quantum jump Agricultural Research was set up in 1929. in the improvement of genetic potential by There was fast growth thereafter and today crossbreeding of indigenous animals has been there are 24 veterinary colleges, 12 research obtained such that 8 million crossbreds pro­ institutes and one veterinary university ser­ ducing about 15 million metric ton of milk a vicing research in veterinary sciences and year are available today. animal husbandry in India. 5. Health care in the tropics, both preventive and 3. In the middle of 1950's India produced less curative, is vital for the success of any animal than 50 million tons of food grains, whereas improvement programme. Making available in 1983-84 it produced more than 150 million suitable .vaccines and drugs at economical tons. This increased food production is per- prices within the reach of the small and mar- 198

ginal farmer is one of the biggest challenges 9. To promote activities in the multidisciplinary to veterinary scientists in all tropical coun aspects of biotechnology, the Government of tries. IndiasetuptheNationalBiotechnologyBoard in1982. Subsequently, in February 1986, the 6. Three major problems of the developing world, Board has been replaced by a separate De­ as far as animal production is concerned are: partment of Biotechnology in the Ministry of Science & Technology. It has been done in (a) How to make health protection recognition of the need for a focal point in the cheap, efficient and long lasting. administrative structure of the Government for planning, promotion and coordination of (b) Augmentation of fertility of farm biotechnological programmes. livestock so as to improve production of meat, milk, wool and pelt. The main responsibilities of this Department are: (c) Augumentation of feed resources by converting large quantities of avail­ i. To evolve integrated plans and pro­ able straws to protein and energy rich grammes in biotechnology; feeds to improve animal production in tropics. ii. To identify specific R&D programes in biotechnology and biotechnolo­ All these problems can be solved gy related manufacturing; through use of biotechnological tools now be­ coming available. iii.Establishment of infrastructural support at the national level; 7. Biotechnology has emerged as a frontier area of science as it has opened up possibilities of iv. To act as an agent of the Govern­ development of a variety of useful products ment for import of new recombi­ to combat the above mentioned problems in nant DNA based biotechnological production and health programme. The cur­ processes, products and technology; rent developments in biotechnology, particu­ larly in genetic engineering, are being di­ v. To evolve bio-safety guidelines for rected towards producing safe and cheap laboratory research, production health products for humans and animals. application;

8. The Government of India is committed to the vi. To initiate scientific and technical development of better livestock wealth to efforts related to biotechnology; provide high quality of animal products for human consumption. This involves the pro­ vii.Establishment of International duction of livestock with better productivity Centre for Genetic Engineering and and their maintenance through effective di­ Biotechnology. sease control. The advent of recombinant DNA technology has resulted in the produc­ 10. Programmes in biotechnology applications tion of new generation of vaccines against in agriculture and allied sciences are funded diseases, health products and newer methods by ICAR within its own institutes and agri­ of disease diagnosis, which need to be ex­ cultural universities besides the funding ploited to the maximum. For this we have provided by DBT. chosen to leap frog in technology. 199 11. Infrastructure: 12. Manpower Development:

11.1 The Department established a network 12.1 Department of Biotechnology had of infrastructure facilities as needed for drawn up a long term plan in biotech­ R&D, teaching and industrial activi­ nology. For successful implementation ties. The facilities include germplasm of this programme, it was felt that spe­ banks for plants, animals, algae and cialists capable of introducing modern microbes, animal house, oligonucleo­ genetic, biochemical and recombinant tide synthesis, production, import and DNA methods would be absolutely distribution of enzymes, reagents and essential. This has necessitated intro­ radio-labelled compounds, bio-process duction of changes in our existing optimisation and pilot plant and infor­ manpower development programmes. mation system. Most of the facilities The Department of Biotechnology re­ have become operational and consider­ cognizing these requirements and with able progress has been made in con­ a view to introduce teaching and train­ struction of buildings, procurement of ing programmes to produce manpower equipment, recruitment of personnel equipped in this multidisciplinary area etc. The details are given in Annexure. to meet future challenges, decided to evolve model systems of post graduate 11.2 The International Centre for Genetic teaching in biotechnology. This has Engineering and Biotechnology been done in cooperation with the Uni­ (ICGEB) has been established with two versity Grants Commission, Ministry components one in New Delhi and the ofHuman Resources and Development, other in Trieste, Italy, with the objec­ ICAR and Indian Council of Medical tive of bringing the fruits of modern Research. biotechnology to the dev~loping coun­ tries. In addition to the two compo­ 12.2 In the first phase for the initial 5 years, nents, the ICGEB would have a net­ 13 universities/institutions initiated PG/ work of national, regional and interna­ Post Doctoral Programme in multidis­ tional R&D centres which would en­ ciplinary sciences providing opportu­ deavour to promote an active pro­ nities for intake of top students with gramme of research and development background in basic and applied bio­ towards fulfilling the stated objectives. logy, chemistry,physics, engineering, The New Delhi component of the agriculture, veterinary and medicine and ICGEB will mainly focus its research to give them an intensive and up to-date efforts in the areas of agriculture (agro­ training in fast moving areas broadly biology-seed proteins,herbicide resis­ recognized as biotechnology-covering tance, cytoplasmic male sterility, tissue cell biology, molecular biology, gene­ specific expression of gene, and tran­ tics, immunology, tissue culture, en­ scription of genes of agricultural im­ zyme engineering, mathematical and portance and chloroplast DNA as trans­ computer modelling of molecular struc­ forming vector), human health (parasi­ tures and interactions and biochemical tology-antigenic variation in Giardia process engineering. lambia, Plasmodium,falciparum, and hepatitis) and animal productivity. 12.3 The action initiated by DBT by esta­ blishing 13 model PG institutions will undoubtedly stimulate other agencies 200

and universities to establish more such 12. 7 Visiting Scientists Programme : The ofinstitutional facilities during the VII th programme has been introduced this VIII th plan and together such a national year for organizing and supporting visits effort will produce a num her of trained of experts from abroad. The visiting people absolutely essential for achiev­ scientists depending on their areas of ing the objectives and goals in biotech­ specialization and interest will work in nology at the national level. selected institutes and associate them­ s~lves with the on-going research pro­ 12.4 Three task forces have been set up by jects, formulation of collaborative pro­ the Department to identify institutions/ grammes and training of young scien­ universities in the area of marine bio­ tists. technology, agricultural biotechnology and veterinary biotechnology where 13. Special Projects post graduate teaching can be imparted. At present, there is no institute giving 13 .1 Cattle Herd Improvement forlncreased post-graduate degree in marine bio­ Productivity using Embryo Transfer technology and there is only one each Technology. in the area of agricultural biotechno­ logy, and also one in veterinary bio­ 13 .1.1 Cattle and buffaloes are used for technology. a variety of purposes in the agricultural practices and, 12.5 Short-Term Training Courses in Bio­ therefore, forms an important technology: To reorient the existing element in India's rural eco­ manpower within a short period of time nomy. India has one of the larg­ to meet the immediate requirements of est population of cattle and the midcareer scientists, short-term buffaloes, but the average esti­ training courses are supported by the mated milk production in India Department. So far, 52 training courses is one of the lowest. Therefore, have been conducted and another 4 it has been our constant en­ have been identified. deavour to achieve herd im­ provement through fast multi­ 12.6 Biotechnology Associateship (Over­ plication of selected superior seas & National) : The scheme envis­ breeds. ages strengthening the research capa­ bilities of scientists both at junior and 13.1.2 In cattle and buffaloes, genetic senior levels through training under the improvement is limited by the overseas and national associateship low rate of female reproduc­ programme. So far, 35 associateships tion. Normally, a cow or a (31 overseas and 4 national) have been buffalo produces one calf in 13 awarded. Another batch of 17 scientists to 18 months. Increasing the has been selected (12 long-term and 5 number of offsprings from the short-term) during the year 1987-88for selected females by suitable overseas associateship. Under the methods should contribute to modified national associateship herd improvement by allowing scheme, the selection of candidates for higher selection intensities.By 1987-88 awards have been made. multiple ovulations and embryo transfer a superior female do- 201

nor can produce as many as 25 13.1.4 Achievements offsprings per year. During the last two years, the 13 .1.3 The Department of Biotechno­ project has made considerable logy has taken up an S &T Pro­ progress and established the ject on, "Cattle herd improve­ necessary infrastructure. Re­ mentforimproved productivity search and development facili­ through Embryo Transfer Tech­ ties in this speciality at the nology (ETT)", to be imple­ various participating/collabo­ mentedinamissionmode. The rating centres·have been estab­ organizational structure of the lished. While the construction project is as follows: of the main embryo transfer laboratories are in progress, i. Department of Biotechno­ interim facilities for embryo logy-Nodal Agency. transfer work ar.e already operational at these centres. A · ii. National Dairy Development donor/recipient herd of over Board (NDDB), Anand - Lead 700 animals has been esta­ implementing agency. blished under this project. Re­ search and development activi­ iii. Indian Veterinary Research ties on superovulation of do­ Institute (IVRI), Izatnagar - nors, estrus synchronization of R&D collaborating agency. recipients, flushing and embryo recovery has made much pro­ iv. National Dairy Research gress that the technique has Institute (NDRI),Kamal-R&D become routine. The nonsurgi­ collaborating agency. cal methods of successful embryo transfer in Indian cows v. National Institute of Immu­ have been standardized and as nology(NII),NewDelhi-R&D a result, the first birth of a calf collaborating agency. through ETT was reported by the NII during January 1987. The main objectives of the Project are: So far over 600 embryos have been collected and rough! y 270 i. To introduce embryo transfer embryo transfers were done in technology in the country in cows. This has resulted in the cattle and buffaloes. birth of 9 cow calves. About 28 more calves are expected soon. ii. To create institutional infra­ Over 43 more pregnancies in structure and a skill pool of cows have been confirmed and scientists, practitioners and several more are awaiting con­ technicians. firmation.

iii. To create a "Seed stock" of Research and development genetically superior animals. work on embryo transfer tech­ nology (ETT) in Indian buffa­ loes has also been making 202

steady progress under this Pro­ enhance germplasm stocks of ject. Experiments on superovu­ superior breeds at four differ­ lation of buffaloes using vary­ ent regions in the country. ing doses of gonadotrophins indicated response only in 50 It is also proposed to set up 25 per cent of animals. SimiJarly, ET sub-centres spread all over poor recovery of embryos also the country. These sub-centres remains a major problem would closely link their activi­ amongst superovulated buffalo ties with the Artificial Insemi­ donors. It is also noted that there nation Programme so that the is a proportion of buffaloes benefits of embryo transfer which are delayed ovulators. technology at the field level Concerted research and deve­ could be achieved, ultimately. lopment efforts are in progress During the year, an Embryo at the various participating Transfer sub-centre at Erode agencies to tackle the above­ became operational. Work on mentioned problems. Employ­ the establishment of ETT sub­ ing surgical methods of em­ centres at Patna,Bangalore and bryo transfer, the National Trivandrum are in progress. Dairy Development Board has reported 3 pregnancies among Under this Science & Tech­ buffaloes. A buffalo calf had nology Project, NDDB, has also already been born through ET undertaken a massive milk at the main Embryo Transfer recording programme among Laboratory at Bidaj. Pregnan­ the buffaloes in Bombay to cies in buffaloes have also been facilitate building up an elite reported now using nonsurgi­ donor/recipient herd. Already cal methods of embryo transfer 2,000 buffaloes have been en­ under this Project. rolled under this scheme. During 1987-88, five training Considerable progress has courses were held under this been reported in R&D on va­ project; four at the Indian Vet­ rious other aspects of embryo erinary Research Institute, Izat­ transfer technology by the par­ nagar, and one at the National ticipating institutions. The Dairy Research Institute, Kar­ National Institute of Immuno­ na!. A total of 48 practitioners logy (NII) has reported suc­ were trained putting the num­ cessful pregnancy in cows us­ ber of total trained personnel at ing cryopreserved embryos. 38 since inception of the pro­ Further work on cryopreserva­ ject. Two consultants from tionand developmentof a 'Seed abroad, have visited India and, Stock' of selected germplasm overseas training of scientists is in progress at the participat­ will begin during the year, under ing institutions. the NII also re­ this project. It is planned to set ported a break-through in up four regional embryo trans­ achieving successful transfer of fer centres under this Project to split embryos in cows leading ·create necessary expertise and to pregnancy. R&D on the 203 development of gene probes division is the birth of a calf also serological tests for sexing through embryo transfer in of embryos as well as early de­ August 1988, the first time in tection of pregnancy in cattle is the institute. in progress at NII. The NDRI has reported major efforts The total approved budget for towards early detection ofpreg­ the project for five years will be nancy and also development Rs. 16.85 crores. and evaluation of ET equip­ ments. IVRI, Izatnagar, is put­ 14. DBT has constituted a task force on veteri­ ting up major efforts towards nary biotechnology including animal hus­ production and purification of bandry and other allied sciences with the FSH and oocyte and ova cul­ following terms of reference: ture as well as in vitro fertiliza­ tion. 1. To identify already developed bio-tech­ nologies which have immediate rele­ IVRI's achievements in ET vance to the improvement of animal Research production.

The past two years saw the 2. To identify various bio-technologies successful culmination of se­ which have potential but need to be veral research centres at the ET researched on in the Indian context to research centre at IVRI. Scien­ become practical within few years. tists and researchers here stan­ dardized the technique of su­ 3. To identify institutions/universities work­ perovulation, collection and ing in these areas and to assess their transfer of embryos in cross­ infrastructural facilities and expertise breed, Haryana and Zebu tau­ available. rus crossbred cows and also 4. To identify specific projects with objec­ provided insights into how the tives/goals, funding level and time­ problems of annovulatory fol­ schedule, i.e., five or seven year time in locles during superovulation parallel with 8th or 9th plan in a mission could be avoided through the mode. use of GnRH in cattle. 5. To advise on successful mechanism of Scientists also purified the monitoring and implementation of the buffalo pituitary FSH for which programme. satisfactory biological assay has been obtained. It was also found 6. To hold workshops/seminars. that superovulation in Indian buffaloes can be satisfactory Recommendations of Task Force with the use of both FSH and PMSG. Attempts for in vitro 1. That a national facility on animal tissue fertilization of bubaline ova culture be established for the mainte­ were successful and is growing nance and supply of primary and estab­ upto two-cell stage in culture. lished cell lines organ cultures specific The latest achievement of this to veterinary science. This facility is to 204

be located in different institutions and lation to a large volume of information and will be linked to the National Facility for scientific knowledge. It has become essen­ Animal Tissue Culture located at Pune tialforconductingresearchindifferentareas for exchange of information.material etc. of biotechnology and new manufacturing programmes to keep abreast with the latest 2. Establishment of a centralized repository developments and advances that are taking of microbial agents on the line of Na­ place. In order to have access to the large tional Type Culture Collection Facility volume of information and to fulfill the of CDC, Atlanta. This facility will be growing need for timely availability of in­ created with the Coordinating Unit at formation, the Department of Biotechno­ IVRI, Mukteswar, and other participat­ logy has set up National Biotechnology In­ ing Institutes as the Regional Type Cul­ formation System (BTIS) with the twin ture Collection Centre. objectives of increasing the intensity of research and for realising commercial po­ 3. Application ofETT and other associated tential of the new technologies in a short technologies including hormone profiles span of time. Computer based information and growth promoting factors in live­ facility is established under the BTIS in a stock species. distributed manner having a strong compo­ nent of research and infrastructure. An apex 4. Development of immunodiagnostics, centre is being located in the Department to diagnostic kits, and other associated coordinate the activity of all the information procedures for making available the centres and to establish external linkage. diagnostic facilities at field level. The principal objective of DIC is not 5. Development of facilities for reagents, only to function as an information base and consumables and other biologicals make available the information to interested needed in the biotechnological proce­ scientists and biotechnology industries, but dures. also to develop software to analyse and assess the information thereon. The scien­ 6. Development of immunological tech­ tists can have ready access to computer niques through the application of assess­ based information through the National ment of Interleukin II in suitable cell Network System employing dedicated system for assessment of infection level communication lines or satellite facility. in chronic diseases, with special refer­ ence to tuberculosis andJohne's diseases. 16. Icar Programme On Biotechnology:

7. Application of microcarrier technology 16.1 National Biotechnology Centre atIVRI: for production of Rinderpest vaccine. The Government of India sanctioned a 8. Production of FMD vaccine through National Biotechnology Centre atIVRI rDN A and synthetic peptide techniques. in 1985 along with centres at IARI and NDRI; with research emphasis at IVRI 15. National Biotechnology Information Sys­ on two major areas genetic engineering tem (BTIS) in bacteria, viruses and protozoa using recombinant DNA technology and The recent developments in the field of embryo transfer and associated tech­ biotechnology have resulted in the accumu- nologies. 205

To attain this basic objective, follow­ iv) Haemoprotesta group of parasites unit: ing interdisciplinary units have been established at the Institute to undertake This unit will be responsible for deve­ research and teaching in certain basic lopment of competency in areas of im­ and applied aspects of biotechnology. munodiagnosis and immunoprophylaxis, especially for haemoprotozoa of animal i) Recombinant DNA unit: importance usingrecombinantDNA and hybridoma technique. This unit has been established to de­ velop competence in recombinant DNA v) Reproductive immunobiology unit: technology with bacteria, viruses, yeasts to be followed by applied microbial Development of research capability in genetic and genetic engineering work areas of immunobiology of reproduc­ involving bacteria, viruses and parasites tion in relation to hormone receptor of animal health importance, with a view systems to increase/decrease reproduc­ to develop better vaccines using genetic tion efficiency of animals leading to engineering techniques and gene clon­ fertility regulation will be tackled at this ing. Some of the selected vectors are unit. being propagated and maintained. Prepa­ ration and purification of plasmid DNA vi) Oncology unit: and its characterization have also been initiated. Some of the microbial antigens Development of immuno-biological have been identified, purified, labelled techniques for early diagnosis of cancer and used for immunodiagnosis. in animals and birds, identification of transformed and pretransformed host ii) Hybridoma technology unit: cells and their prevention using trans­ formed cell surface antigen as immuno­ This unit has the capacity to develop prophylaxis through use of monoclonal immunodiagnostic reagents against vi­ antibody and recombinant DNA tech­ ruses, bacteria, protozoan and other nique are the main work areas of this parasites of animal health importance. unit. iii) Embryo transfer technology and engi­ The National Biotechnology Centre is sup­ neering unit: ported by the following projects:

After successful in vitro fertilization of 1. UNDPProjectonimmunobiotechnology. goat embryos and long-term freezing, the techniques of synchronization of 2. Indo-USAID Project on Embryo Transfer estrus and superovulation have been Technology in Livestock. standardized and methods for nonsurgi­ cal collection and transfer of embryos 3. US-AID Project on Intracellular blood have been adopted in cattle and buffa­ protista with particular reference to loes at this unit. Work on long term Immunoprophylaxis and control. preservation of embryos, in vitro fertili­ zation, embryo culture, embryo sexing 4. S&T Mission Project on cattle herd im­ and embryo freezing for large animals is provement for increased productivity being taken up in a phased manner. using Embryo Transfer Technology. 206

5. Post graduate education and teaching of ering the prevalance of communicable diseases DBT. in the country and also because of the new opportunities available now in vaccine and 6. ODA Collaborative projects considera­ immunodiagnostic development through breal<­ tion: throughs in molecular biology, genetic engi­ neering, cell-fusion, cell-culture, protein engi­ (a) Establishment of Recombinant DNA neering, synthetic chemistry, etc. Under the V AP, Technology Centre for Research in Novel a unique opportunity is being envisaged for Molecular Vaccine Production and Im­ leading Indian scientists and institutions to join provement of Animal Production. hands with some of the best scientific groups in the USA for development of new vaccines and (b) Monitoring, surveillance and forecast­ immunodiagnostics for major communicable ing of important animal diseases. diseases in India.

( c) Feed standards for growth maintenance, U.S.S.R: production and work using direct and indirect calorimetry. A Memorandum of Understanding between the Govemmentoflndia and USSR on scientific and 17. International R & D Collaboration : technical collaboration/cooperation was signed by theHon'blePrimeMinister, Mr. Rajiv Gandhi During 1987-88 the following significant deve­ and the Hon'bleGeneral Secretary, Mr. Mikhail lopments have taken place in the field of coo­ Gorbachev in July 1987. The MOU inter alia laid peration between India and other countries in the special emphasis on pursuing collaborative R & areas of biotechnology. D projects in various frontier areas of biotech­ nology. Several exchange of visits between Indian U.S.A: and Soviet scientists have already taken place. Discussions are continuing with the Soviet side Indo-US Vaccine Action Programme: to identify new areas/projects for Indo-USSR collaboration. The Department of Biotechnology has also ini­ tiated several steps towards the development of China: new or improved vaccines through indigenous R&D. As part of this strong inhouse components An Indian delegation visited China during the for R&D in the new manufacturing units are .year to study the progress made by the Chinese being contemplated. In addition, the DBT is in the field of aquaculture, and antifertility drugs. encouraging the leading biomedical institutions A Chinese delegation is likely to visit India in the country to take up focussed projects in this during 1988 when areas of cooperation would be speciality. The department has also initiated a identified. new programme called, "Indo-US Vaccine Ac­ tionProgramme" (VAP) and signed a Memoran­ Czechoslovakia: dum of Understanding with the United States to promote R&D for the development of vaccines The Department had fruitful discussions on and immunodiagnostics for tackling communi­ immobilization technique applied to enzymes cable diseases that are causing major public with the Chief Research Worker of the Czech health problems in the country. Academy of Sciences, when the latter visited the Department during the year. Further discussions Indo-US V AP is of special significance consid- are on to identify areas of scientific cooperation 207

between India and Czechoslovakia. R&D work in the areas of bioprocessing and engineering, research in medicinal and economic Indonesi_a: plants and application oriented immunology. Subsequently, proposals from various Indian A three-member Indonesian delegation visited institutes were received, which are under exami­ the Department during the year. Discussion nation. between the delegation and the Department offi­ cials produced appreciable interactions and ef­ Vietnam: forts are on to promote exchange of scientists and identification of specific areas for develop­ A three-member Vietnamese delegation visited ing joint R & D collaboration. the Department of Biotechnology in October 1987. After discussions it was felt that the two Switzerland: countries could investigate possibilities of co­ operation in tissue culture on rice and medicinal Projects have been prepared for collaboration plants, embryo transfer techniques, use of bio­ between India and Switzerland. logical effluents/wastes for the production of spirulina and other nitrogen fixing bacteria. Italy: Netherlands: The Indo-Italy Joint Committee has been con­ sidering possibilities of identifying projects for TheDepartmentreceivedscientistsfrom theNe­ Italian assistance. Some proposals have already therlands and possibilities of cooperation were been received and are under consideration. discussed. Several institutes in India have been given the responsibilities for preparing specific Federal Republic of Germany (F.R.G.): research proposals for lndo-Netherlands co­ operation. Specific proposals are being prepared Through two workshops held in 1985 and 1986 and are likely to be finalized soon. in Delhi and Tuebingen it was agreed to start 208 Annexure

National Infrastructural Facilities

FACILITIES LOCATION

1. BLUE GREEN ALGAL COLLECTION Indian Agricultural Research Institute, New Delhi.

2. PLANT TISSUE CULTURE REPOSITORY National Bureau ofPlant Gene tic Resources, New Delhi.

3. MICROBIAL CULTURE COLLECTION Institute of Microbial Technology, Chandi­ AND GENE BANK garh.

4. ANIMAL TISSUE/CELL CULTURE University of Poona, Pune.

5. ANIMAL HOUSE i. National Institute of Nutrition Hyderabad ii. Central Drug Research Institute Lucknow iii. I.I.Sc. Bangalore

6. BIOCHEMICAL ENGINEERING PILOT Institute of Microbial Technology Chandi­ PLANT garh

7.0LIGONUCLEOTIDESYNTHESIS i. I.I.Sc. Bangalore ii. CSIR Centre for Biochemicals Delhi iii. Centre for Cellular & Molecular Biology, Hyderabad

8. PRODUCTION IMPORT AND CSIR Centre for Biochemicals Delhi. DISTRIBUTION OF ENZYMES AND BIOCHEMICALS

9. GENETIC ENGINEERING i. Indian Institute of Science, Bangalore ii. Jawaharlal Nehru University, New Delhi. iii. Madurai Kamraj University, Madurai.

10. LIPOSOME TECHNOLOGY Delhi University(South Campus) New Delhi. i. Indian Institute of Science, Bangalore. ii. Madurai Kamraj University, Madurai. iii. Bose Institute, Calcutta. iv. Jawaharlal Nehru University, New Delhi.

11. BIONFORMATICS v. Poona University, Pune. vi. Centre for Cellular and Molecular Bio­ logy, Hyderabad. vii. Indian Agricultural Research Institute 209

New Delhi. viii. National Institute oflmmunology, New Delhi. ix. Institute of Microbial Technology, Chan digarh. x. Indian Veterinary Research Ins ti tu te, Izat nagar.

12. POST GRADUATE EDUCATION i. Madurai Kamraj University, Madurai. IN BIOTECHNOLOGY ii. Poona University, Pune iii. Banaras Hindu University, Varanasi. iv. Jadavpur University, Calcutta. v. MS University of Baroda, Baroda. vi. Indian Institute of Science, Bangalore. vii. Indian Agricultural Research Institute, New Delhi. viii. All Indian Institute of Medical Sciences, New Delhi. ix. Indian Veterinary Research Institute, Izatnagar. x. Indian Institute of Technology, Bombay, Delhi and Kharagpur xi. G.B. Pant University of Agriculture & Technology. Indonesia: iotechnology Applied to Animal Production Health *)

B. Gunawan Balai Penelitian Temak P.O. Box 123, Bogor Indonesia

Introduction The aim of the present paper is to re­ view the present status and the roles of biotech­ nology applied to livestock production in Indo­ In Indonesia, development in the sector nesia. of animal husbandry achieved successful re­ sults. A decline in livestock population, espe­ cially ruminant, occured in 1970 but has now Current Status and the Roles of been overcome and the population is showing a Biotechnology trend of increase (Table 1). The demand for meat, eggs and milk has been steadily increasing The roles and impacts of biotechnology as a result of increase in human population and have been widely reported, such as in medicine, an increase in per capita income due to the agriculture and animal husbandry. In animal successful national development programme. husbandry, the technology has been used to Nevertheless the supply within the country is not produce monoclonal antibodies, vaccines, growth sufficient and imports of meat, eggs and milk hormones and others. In developed countries, the therefore cannot be avoided (Table 2). The Table products of biotechnology have been marketed shows that the animal protein consumption is commercially in a large scale. For example in the more or less equivalent to 2.55 gram/capita/day USA, the vaccine of foot and mouth disease which is far below the targeted minimal require­ reached the sale of about $300-$500 million per ment of about 4 gram protein/capita/ day which year. The sale oflivestock growth hormones has is equivalent to consumption of 6 kg of meat, 4 been estimated to be about $500 million per year kg of eggs and 4 kg of milk per capita per year. (Yoxen, 1983). The immunological modulation Thus stress the importance of dramatically in­ of livestock performance has been successfully creasing livestock production through better developed to increase reproductive rate in sheep management, nutrition, better breeding pro­ (ScaramuzziandHoskinson, 1984). The concept grams and disease control. Toward the year of immunization has been extended and has been 2000, biotechnology could play the major roles used for the improvement of carcass quality in accelerating animal genetic improvement, through a reduction of the proportion of fat by growth rate, maximising the utilization of unex­ immunization against fat cell membranes (Land, pensive agro by-products and the production of 1987). The technique of Multiple Ovulation and better vaccines. Embryo Transfer (MOET) has been applied for

•>A country report presented at a Workshop on Biotechnology Network for Animal Production and Health in Asia. Bangkok, Thailand, 17-21October1988. 211

the acceleration of the genetic improvement of Institute for Animal Production (RIAP) started livestock. For example the advantages of 50% or to study embryo transfer in sheep and cattle. The more can arise from the application of MOET to results are presented in Table 5, 6, 7 and 8 the genetic improvement of beef cattle (Land Research activities are still underway to improve and Hill, 1975). the technique of embryo transfer. A plan is being made to use MOET (Multiple Ovulation and In Indonesia, the use of biotechnology Embryo Transfer) in a local sheep breeding applied to livestock production has just reached experiment. It is expected that the technique the embryonic stage. Ata poultry breeding farm, could accelerate the genetic improvement of the a program of blood typing for the "B-complex" local sheep (Table 9). types has begun. It has been reported that "B­ complex" caused chickens to be resistant (over At the Research Institute for Animal 98% resistance) to Mareks disease while other Production (RIAP) a long term sheep breeding "B-complex" types caused extreme (over 98% program was started in 1980 in a collaboration susceptibility) susceptibility. Because different with "Small Ruminant Collaborative Research "B-complex" types can determine the disease Support Program" (SR-CRSP) from the USA. resistance of a chicken, it is important to be able The breeding objective is to increase the repro­ to identify different "B-complex" types in poten­ ductive performance (litter size) of the Javanese tial breeders. Blood typing provides an accurate Thin Tail sheep (JTT). Ovulation rate has been method to determine the "B-complex"type of used as the selection criteria by counting the each chicken. Blood typing requires only a small "Corpora Lu tea" in the ovary of the female sheep amount of blood. The test itself can be performed using "laporoscopy". The experiment was out­ within three hours, however the detection solu­ lined by Gunawan (1988b). Research is still in tions require much time for preparation. By progress and the current results show that the using the information from these tests, they will multiple birth is suspected to be controlled by a be able to select breeding stock with known few genes of large effect ("major genes"). The disease resistance characteristics and therefore segregation of the litter size from dams of high they can design matings to result in the healthi­ and low litter size is presented in Table 10. The est progeny possible. They are also testing a experiment will be followed by a progeny testing blood analysis procedure to monitor for elevated of the "carrier" rams. If the hypothesis can be stress levels in the breeders to ensure optimum proved, bfotechnology could play the major roles immunefuction. By detecting and the control­ in the genetic improvement of the reproductive ling unacceptable levels of stress in chickens performance in sheep. The major genes of large will be able to protect the immunity to disease effect affecting litter size can then be isolated that they poss. and transferred from the "carrier" to the "non carrier" sheep by the "recombinant DNA tech­ The technique of embryo transfer was nology". The method of gene transfer by micro­ just introduced to Indonesia in 1984 when a injection is outlined by Pinkert (1987) and has governmental company imported both frozen been successfully reported in mouse and live­ embryos and expertise from the USA. A total of stock (Pinkert, 1987; Franklin, 1985). However 477 frozen embryos (234 Frisian and 243 Brah­ we are not yet ready with this technology, in the man embryos) were bought and transferred to near future some of the scientists will be sent to local cattle by surgical technique. The results are the USA or Australia for a special training in this presented in Table 3. In 1985, the Directorate area. General ofLivestock Service started the Embryo Transfer Pilot Project on Bali cattle and the A long term breeding program to pro­ results are given in Table 4. In 1986, Research duce anew breed oflndonesian egg laying ducks 212 is also investigated atRIAP. The experiment was There could be possibly many other outlined by Gunawan (1988a). The current re­ research activities in biotechnology as they are sults indicate that the local Alabio duck has the scattered among various universities, research capability of producing "2 eggs per day". The institutes within the Governmental Departments genetic and reproductive mechanism has not yet and non Departments and private companies. been clear. We still continue investigating this The prospect of biotechnology in the develop­ unique phenomenon. Table 11 shows that they mentof agriculture, animal production and human are large genetic differences of the frequency of welfare was discussed by Gunawan (1987). "2 eggs per day" between flocks of Alabio ducks maintained under exactly the same management Conclusion system. This trait could behave like the fertility traits in Javanese Thin Tail sheep as described in previous paragraphs. Ifthe trait is also controlled Toward the year 2000 biotechnology by a few "major genes" then the biotechnology will play the major roles in boosting animal will play its maximum role in the production of production in Indonesia. The technique of em­ "transgenic super layer ducks". We are now in bryo transfer is being developed and continue to the stage of investigating the clutching pattern, be improved and eventually will be used in the frequency of 2 eggs per day and their correla­ animal genetic improvement program. The re­ tions with yearly egg production and the anat­ combinant DNA technology will also be used in omy of the reproductive organ in the female the near future in the production of transgenic ducks. The experiment has not yet completed sheep and ducks with higher reproductive traits. and the results are not presented. Specific ability of certain strains of yeasts and mushrooms will be used to maximize the utiliza­ Microbiologists and biochemists at tion ofunexpensiveagro by-products for animal RIAP are investigating bioconversion of starchy feeds. Efforts are also being pursued by a private materials into protein rich products by using company to use biotechnology in the production amylolytic yeasts in a solid subtract fermenta­ of a strain of chicken to be resistant to Marek tion system. Yeasts used were Candida ingeniosa disease and others. The use of biotechnology in , Candida ingeniosa plus Candida utilis and animal production and animal health will hope­ Endomycosis plus Candida utilis. The highest fully accelerate the achievement of the targeted protein content in the final product is 35% which minimum animal protein consumption for Indo­ is comparable to that of soybean. During the nesian people which is about 4 gram/capita/ day. fermentation process, 85% of starch in the cas­ sava tuber was consumed by yeasts and the yield References coefficient product/subtract (Yp/s) is 0.37 (Darma, unpublished). They are also investiga­ Bradford, G.E., Subandriyo and Iniguez, L.C. ting the ability of oyster mushrooms (P leurotus 1986. Breeding strategies for small ruminants in spp.) to degrade lignin as well as to produce integrated crop-livestock production systems. edible mushrooms for a dual purpose pretreat­ Proceedings of a Workshop on Small Ruminant ment of rice straw. Mushrooms yield (Yp/s) is Production Systems in South and Southeast Asia. 0.94 and the residual subtract contained 5%, Bogor, Indonesia, 6-10 October 1986, pp. 318- 22%, 4% and 4% hemicellulose, cellulose, lig­ 331. nin and protein respectively. Digestibility ofrice straw increased from 39 to 45% and 39 to 54% Dharsana, R. Embryo Transfer in Indonesia. for IDMD (In vitro Dry Matter Digestibility) (unpublished). and IOMD (In vitro Organic Matter Digestibi­ lity) respectively (Darma, unpublished). Franklin, I.R. 1985. Genetic Engineering and 213

Livestock Improvement. Proceedings of the Fifth Land, R.B. 1987. New Technologies for animal conference of the Australian Association of improvement and development countries. Tech­ Animal Breeding and Genetics. University of nical Committee Meeting SR-CRSP, Raleigh, New South Wales, Sydney,NSW, 26-28 August North Carolina, 13-15 January 1987. 1985. Land, R.B. and Hill, W.G. 1975. The possible Gunawan,B.1987.Prospectofbiotechnologyin use of superovulaiton and embryo transfer in the development of agriculture, animal produc­ cattle to increase responses to selection. Animal tion and human welfare in Indonesia. Proceed­ Production 21: 1-12. ings Seminar B ioteknologi Pertanian, Pusat An tar Universitas (PAU) Bioteknologi, Ins ti tut Per­ Pinkert, C.A. 1987. Gene transfer and the pro­ tanian Bogor, 21December1987. duction of transgenic livestock. Proceedings U.S. Animal Health Association 91: 129-141. Gunawan, B. 1988a. Teknologi Pemuliabiakan !tile petelur Indonesia. Seminar National Peter­ Scaramuzzi, R.J. and Hoskinson, R.M. 1984. nakan & Forum Peternak "unggas dan Aneka Active immunization against steroid hormones Ternak" II, BalaiPenelitian Ternak, Ciawi-Bogor, for increasing fecundity. In : Immunological 18-20 July 1988. Aspects ofReproduction in Mammals (Crighton, D.B., ed.). Butterworths, London., pp 445-474. Gunawan, B. 1988b. Teknologi Pemuliabiakan Domba Lokal Indonesia. Proceedings Seminar Yoxen, E. 1983 The Gene Business. Pan Books, ExporTernakPotong,HotelHorizon,Jakarta,2- London and Sydney, pp. 122-136. 3 September 1988.

Gunawan, B. 1988c. Frequency of2 eggs per day in Alabio ducks. (will be published in Majalah Ilmu dan Peternakan). 214

Table 1. Poultry and livestock population from 1978-1984 in Indonesian (x 1000 head)

Commodity 1978 1980 1982 1984

Dairy cattle 93 103 140 173 Beef cattle 6,330 6,440 6,594 6,741 Buffalo 2,312 2,457 2,513 2,724 Goat 8,051 7,691 7,891 8,210 Sheep 3,611 4,124 4,231 4,402 Pig 2,902 3,155 3,587 3,854 Horse 615 616 658 672 Native chicken 108,916 126,316 139,787 157,064 Layer 6,071 22,940 26,312 31,947 Broiler 0 25,462 31,033 37,548 Duck 17,541 21,078 23,861 27,144

Table 2. Production, imports and meat consumption

Commodity 1978 1980 1982 1984

Meat (xlOOO tons) Domestic production 474.6 570.8 628.6 685.6 Imports 1.7 1.6 2.6 2.2

Eggs (x 1000 tons) Domestic production 122.7 207.7 242.9 282.4 Imports 0 0.1 0.2 1.5

Milk (x 1000 tons) Domestic production 54.2 68.6 102.l 462.3 Imports 440.3 594.3 536.0 622.8

Consumption (kg/capita/year) Meat 3.4 3.9 4.1 4.3 Eggs 0.9 1.4 1.6 1.8 Milk 4.2 4.4 4.2 3.9 215

Table 3. Results of imported frozen embryos

No. of Imported - No. of recipients Rejection after thawing Pregnancy rate (%) Embryos

234 (FH) 222Cows. 10% 35%

2/3.(FH) 1/3 (Ongole)

243 (Brahman) 146 Ongole Cows 24% 12%

Source: Dharsana (unpublished)

Table 4. Results of embryo transfer in Bali cattle

Number of Donors flushed 24 Average embryo recovery 12 Average number of transferable embryos per donor 8.87 Pregnancy rate 62.5%

Source: Dharsana (unpublished).

Table 5. Ovarian respone to different doses of PMSG

Dose No. No. Corpora lutea/ewe No. Large persistent PMSG Ewes follicles (IU) Mean Range

300 5 1.40 1 - 2 None 500 6 7.67 1 - 17 Few 500 6 7.83 2- 10 Few 1000 5 0.80 0 - 1 Many

Source: Dharsana (unpublished). 216

Table 6. Embryo recovery 3 or 4 days after oestrus

Days after No. Total No. Eggs recovered Fertilized eggs oestrus ewes CL No. % of CL No. % of eggs recovered

3 6 46 34 74% 31 91% 4 6 47 27 57% 26 96%

Source: Dharsana (unpublished).

Table 7. Results of superovulation and embryo transfer in recipient ewes.

Recipient No. of CL No. embryos transfer Pregnancy diagnosis No.of ewes Ovary Comu 6 weeks after transfer lamb n=6 born Right Left Right Left

1 2 2 not pregnant 2 3 2 + 1 3 5 1 2 2 + 1 4 3 4 2 2 + 1 5 1 1 2 2 + 1 6 2 3 2 2 + 2

Source: Dharsana (unpublished).

Table 8. Results of non-surgical embryo transfer in Ongole cows.

Donors No. Corpora No.Eggs Transferred to Pregnancy lutea/cow recovered 5 recipients at 60 days

No. 451 6 1 No. 455 (1 embryo score A) + No. 4281 2 0 No. 4299 12 6 No. 4136 (1 embryo score B) - No. 448 (1 embryo score B) No. 449 (1 embryo score C) No. 450 (1 embryo score C)

+A healthy calf weighing 23 kg was born on June 13th, 1987.

Source: Dharsana (unpublished). 217

Table 9. Percentage of egg production and frequency of 2 eggs per day at 30 weeks of age in Alabio ducks.

Flocks % production frequency of 2 eggs/day

HR 91% 8% RA 96% 4% HS 91% 14% JO 104% 25%

Source: Gunawan (1988c).

Table 10. Segregation of litter size in Javanese sheep.

High dams low dams (1 or more records~ 3) (all records= 1or2)

No. No. records x No. No. records x

Dams 23 74 2.74 37 103 1.48 All daughters 39 95 1.65 42 86 1.38 High daughters 18 48 2.31 2 6 2.83 Low daughters 21 47 1.38 40 80 1.28

Source: Bradford et al. (1986).

Table 11. Genetic improvement of body weight in Javanese Thin Tail sheep with and without embryo transfer (ET).

Trait Without ET MOET"'

Number oflambs/ewe joined 1 4 Body weight gain/year - (kg) 0.8kg 1.7kg -% 2.4% 5.07%

* Multiple Ovulation and Embryo Transfer. Source : Gunawan (1987). Kyung Soon Im Seoul National University Korea

Ae Embryo transfer highest pregnancy rate and litter size than em­ bryos which were recovered at 3.5 days and 1. Study on embryo transfer technol­ transferred at 3 .5 days, and embryos which were ogy in Korea had initiated with rabbit and there­ recoveredat4.5 days and transferred at4.5 days. after extended to goat and cattle, and com­ menced recently to porcine. Oh et al. (1973') 2. In Korea, study on embryo transfer transferred 160 rabbit embryos at 2-6 cell stage for goat was initiated on 1975 at National Live­ or 16-morula stage to oviduct and uterus of 20 stock Experiment Station. Goat were intrave­ recipients. Eight embryos were transferred to nously injected with 1,000 IU HCG followed by each doe. Eleven recipients were pregnant and mating. Embryos were recovered by flushing farrowed 32 kids. A doe-farrowed 2.9 litter. oviduct or uterus with goat serum at 72 h after Good results were obtained when 16 - morula mating. One to eight cell embryos were trans­ stage embryo was transferred into uterus. No ferred to oviduct and 16-morula embryos were research work on embryo transfer of rabbit had transferred to utems. There was no difference in been observed for 10 years since 1983. Chi et conception rate between oviduct and uterus. al. (1984) transferred 64 embryos which were Nineteen embryos were transferred to 20 recipi­ recovered on 24-72 h after mating to recipients ents. Conception rate and kidding rate were 60 among which three were pregnant. Highest and 31.6 percent. recovery rate was observed when embryo was collected at 24 h after mating. Lian et al. re­ 3. Ten years later, Song et al. (1975) ported that 22.1 embryos per donor were recov­ induced estrus from goat by injection of 3 mg ered at first superovulation treatment, and 7.5 PGF2a on day 5-12 of estrus cycle. Eighty three and 5.5 embryos were recovered at second and to 100percentofthegoatshowedanestrusat38- third superovulation treatment with 17 days 56 h after treatment. interval between superovulations. Also 13.4 and 6.0 embryos were recovered at second and 4. National Breeding Station carried third superovulation treatments with 30 days ou~ porcine embryo transfer trial and obtained interval between superovulations in rabbit. Choi that 69.1 percent of embryos was one-cell em­ et al. (1987) reported that pregnancy rate and bryo when they were recovered from oviducts at number of litter per recipient were 62.5 percent 37-41 h after mating. Sixty seven embryos were and 3.1 head when embryos recovered from transferred to 5 recipients and the four farrowed donor at 2.5 days after mating were transferred 18 pigs. to recipient at 2.5 days after HCG injection. The embryos which were recovered from donor at 5. Study on embryo transfer of bovine 2.5 days after mating and transferred to recipi­ in Korea carried out actively from 1980. When ent at 2.5 days after HCG injection showed PMSG 2,000-3,000 IU was injected, 3.7-7.8 219 corpus lutea per head were formed, and when and 8-cell mouse embryos by elimination of zona FSH or FSH/LH 30 mg was administered, 6.6 - pellucida and fused physically. Some fused blas­ 7. 7 corpus lutea per donor were formed and 1.7- tomere were developed to morula stages. Ahn et 4.6 embryos were recovered. Nonsurgical re­ al. (1986) transferred fused morula and blasto­ covery and transfer techniques are preferably cyst, and produced 12.2 and 9 .2 percent chimera used and in case of imported frozen embryos, kids from fused morola and blastocyst in mouse. surgical transfer or nonsurgical transfer has been used. Suaket al. (1984) reported that conception D. Cryopreservation of embryos rate was 55.6 percent in fresh embryo and 26.2 percent in frozen embryo. Chung et al. (1987) 8. In Korea, for production of genetic reported that conception rate was 37.5 percent in superior dairy calf, imported frozen e'm bryo and frozen embryo. National Breeding Station and interior frozen embryo are practically used. Study National Livestock Experiment Station are on cryopreservation of embryos has been carried applying embryo transfer technique for produc­ out mainly in mouse. You and Lee (1984) re­ tion of young sire and heifer. Some dairy farms ported that livability of frozen-thawed 2-cell are applying embryo technique for production mouse embryo was higher in freezing rate 0.8- of breeding stock. A private company is doing 1.00 C/min. than in 3° C/min. and there was no business work of embryo transfer to the dairy difference between cryoprotectants DMSO and farmer. ethylene glycol. YunandChung(1984) reported that livability of frozen-thawed mouse embryo Be Embryo bisecting (cloning) was higher in 8-cell than in 1-4-cell, in slow freezing than in fast freezing,and higher in slow 6. In Korea, an attempt of bisecting addition and slow elimination of DMSO. Jin et embryo had been done by Oh et al. in 1983. Oh al. (1986) reported that implantation rate was et al. had bisected mouse 2,4 and 8 cell embryos 12.2 percent when frozen thawed mouse em­ by hand with thin glass needle (15 m diameter) bryos were transferred to recipients. Kim et al. where zona pellucida of embryo was softened or (1985) froze bovine morula and blastocysts in eliminated by exposure for 3-10 min. in the PBS containing 10 percent of glycerol. Embryos medium containing 0.5 percent of papaya pro­ were frozen at rate of 1° C per min. to -7° C and tease. Thereafter Yang et al. (1985) had cul­ seeded. Five minutes after seeding, embryos tured bisected embryos and confirmed that de­ were frozen atrate of0.3° C per min. to-38° C and veloping rate of zona softened and eliminated plunged into LN. Twenty (29 .4 percent) among was 46.7 and 38.5 percent in 2-cell embryo and 68 thawed embryos were developed normally in 10.8 and 6.9 percent in 4-cell embryo. Kim et al. culture. Several labs have automatic freezing (1986) confirmed that 54.3 percent of demi­ machine and doing research work with mouse, embryo was developed normal in mouse. Na­ rat, rabbit, porcine and bovine embryos. tional Livestock Experiment Station had trans­ ferred bisected cow morula and confirmed their E .. Sexing gamate conception. 9. Study on sexing of spermatozoa was Ce Chimera production carried out in 1980 in Korea with porcine and bovine spermatozoa. However evident results 7. As bisecting embryo has been suc­ were not obtained. cessfully done, fusioning of bisected blastomere for chimera production was carried out by sev­ 10. In recent year using mono- orpoly­ eral college laboratories in Korea since 1984. clonal H-Y antibody, several experiments were Lee et al. (1984) obtained blastomere from 2, 4 carried out for sexing sperm and embryo. Ko et 220

al. (1986) reported that when mouse embryos but none of Holstein oocyte developed up to 8 were treated withH-Yantibodyandcultured,45 cells. The medium with FMB showed higher percent was degenerated. Han et al. (1986) also cleavage rate (22.0 percent) than that with BSA reported when mouse embryos were treated (14.8 percent) in Korean cattle oocytes. with H-Y antibody, developed embryo has xx chromosome in their inspection of chromosome G. Nuclear transplantation in type. Ko et al. (1986) has reported that H-Y antibody from mouse inhibited development of embryo not only mouse embryo but also rabbit embryo. Chi et al. (1986) reported that when mouse 12. In Korea, study on nuclear trans­ embryo not degenerated by treatment of H-Y plantation in embryo is recently initiated. Chung antibody was transferred, 81.3 percent was born et al. (1988) reported that when transplanted to as female. Study on segregation and multiple enucleated mouse embryo by micro manupla­ production of H-Y antibody is done by Dr. Kim tion and fusion of pronuclei to the cytoplasm of in Keun Kuk University (1988). B Lympho­ embryo was mediated by Sendai Virus, 64.9 cytes of mouse producing antibodies to H-Y percent was fused to the cytoplasm of recipient antigen were fused with SP2/0-Ag 14 myeloma embryos and among those embryos, 69 .8 per­ cells and distributed to 384 wells. Eighty hybr­ cent was developed to blastocysts. Chung et al. idoma colonies (20.8 percent) were formed. (1988) transplanted pronuclei from C3H mouse Twelve wells were confirmed to produced embryos into enucleated ICR mouse embryo and monoclonal antibodies to H-Y by indirect im­ obtained 83 percent of fusion rate. Among the munofluorescence test. fused embryos, 80 percent was developed into blastocyst. In the near future study on nuclear transplantation with goat and bovine embryo F. In vitro fertilization will be initiated.

11. Recently in Korea, study on in vitro 13. In order to produce transgenic ani­ fertilization of follicular oocytes has been done mals, studies on expression of hGH gene in actively by several laboratories in college and mammalian cells and expression of human growth National Institute. Chung et al. (1986) reported hormone gene in transgenic mice were done at that when rabbit follicular oocytes were fertil­ Genetic Engineering Center. ized by capacit.ated spermatozoa in the medium containing 0.1-10 g/ml of FSH and cultured H. Animal health for 18 h in Ham's F12, 61.5 percent and 27 percent were developed in FSH 0.1 g/ml andin 14. At present monoclonal antibodies FSH 1 g/ml respectively. Park et al. (1987) against several viruses have been produced and reported that when bovine follicular oocytes characterized in Korea. These are antibodies were matured in mKRB and fertilized in rabbit specific to infectious bovine rhinotracheitis vi­ oviduct by capacitated sperm maturation rate rus (IBRV), bovine rot.a.virus (BRV), transmis­ was 57 .1 percent and fertility rate was 12.5-17 .6 sible gastroenteritis virus (TGEV), Japanese en­ percent in the medium with FCS. Chung and Im cephalitis virus (JEV), porcine paravovirus (1988) reported that when Korean cattle and (PPV), pseudorabies virus (PRV), Newcastle Holstein follicular oocytes were matured and disease virus (ND V), infectious larynotracheitis fertilized in Ham's FlO with FMS or BSA, virus (LTV), Marek's disease virus (MDV) and cleavage rate was 22.0 percent in Korean cattle herpesvirus of turkeys (HVT). Using these mono­ and 4 .4 percent in Holstein. Thirteen percent of clonal antibodies, rapid and sensitive diagnostic Korean cattle oocyte developed to morula stage, tests are being undertaken. Malaysia : Biotechnology Applied To · Animal Production and Health

T.K. Mukherjee Institute for Advanced Studies University of Malaya Kuala Lumpur, Malaysia

Biotechnology research in animal Malaysian Agricultural Research and production and health has neither been an en­ Development Institute (MARDI) is at present tirely coordinated effort nor a directed develop­ engaged with research relevant to country's ment in the past. Individual researchers in vari­ immediate needs. However the Institute has made ous institutions have developed some aspects of large scale plans for embryo transfer to increase biotechnology research to merge with their the progeny of genetically superior females in existing techniques. However, starting from this different nucleus .herd of cattle, and to multiply year, Malasian Government, through its sub­ exotic breeds of cattle. stantial R & D funds for 1988-1992 will pro­ The Institute for Advanced Studies, mote and coordinate biotechnology research in University of Malaya is a post-graduate institute agriculture, animal production and health, bi­ where large emphasis has been placed on high omedical sciences and health sciences. technology research for industrial development. The Universiti Pertanian Malaysia's Its biotechnology research and an M.Sc. course Faculty of Veterinary Medicine and Animal programme for teaching encompasses: Production and Faculty of Food science and a) Agriculture, Technology are the leading departments in the b) Animal production, and teaching, research and extension of biotechnol­ c) Biomedical sciences. ogy in animal production and health. A Bache­ In the area of animal science, germ lor of Science course in biotechnology is being plasm conservation through cryopreservation, run in the Faculty of Food Science and Technol­ embryo transfer in goats, fermentation, algal ogy with help from other relevant faculties. biomass as animal feed, genetic engineering, Teaching of Biotechnology in Animal Produc­ utilization of lignocellulosic materials as feed tion and Health forms a significant component and rumen bacteriology have received priority of the course work. These two faculties also status. The biotechnology programme of veteri­ offer M.Sc. and Ph.D. programmes in biotech­ nary Department of Ministry of Agriculture nology related to animal production and health includes abatement of pollution from piggery and food technology. In order to strengthen the waste obtained from approximately 1.5 million teaching programmes, various researches re­ heads from about an estimated 5873 farms. Some lated to animal biotechnology are in progress. small scale embryo transfer programmes have Some of these have been discussed in following also been planned by the department at the Central pages. Animal Institute at Kluang.

+ Paper presented at the Workshop on Biotechnology Network for Animal Production and Health in Asia, Bangkok, Thailand, 17-21October,1988. 222

Current Research in Progress Catheter but with the IMV Catheter, the cervix is dilated easily as the rigid tube could be manipu­ and Future Plans lated through the cervix.

Embryo Transfer The authors also pointed out that the location of eggs in the crevices of endometricum Universiti Pertanian Malaysia where flushing fluid could not reach to dislodge the eggs was the main reasons for failure to Embryo transfer and related research collect eggs. The authors suggested that a com­ has been carried out in buffalo, cattle and goat by bined surgical and nosurgical technique has to be Universiti Pertanian Malaysia, Serdang during developed for collection of ova or embryo in the last few years. buffalo. Similar opinion was also expressed by Professor Madan at NDRI, Kamal, India. In cattle the main objective had been to develop technique for future transfer of im­ Yussof, Jainudeen and Sharifuddin ported embryos into local recipient dams, and for (1987) while working on superovulation and the production of twins and chimeras through embryo transfer in goats found better ovarian microsurgery of embryos (Sharifuddin et al. responses to gonadotrophin treatment in goats. 1987). Lactating Sahiwal x Friesien donors, Using PMSG and different doses of FSH, the super-ovulated with PMSG or FSH and oestrus­ authors found percentage of does in oestrus to be induced with a single intramuscular injection of better with lower doses of FSH treatment than 1,000 mcg Cloprostenol, a PGF-2a analogue PMSG (Table 1) although percent does ovulat­ (Estrumate, Coopers) showed mean ovulation ing was better with PMSG and the highest dose ratesof7.0 2.5and15.6 5.5forPMSG(n=5) ofFS H given. Mean num her of follicles ruptured and FSH (n=3) respectively. were found to be higher with PMSG treatment.

Five out of six donors inseminated with Of the 16 recipients selected, 6 were frozen semen yielded 16 ova comprising one diagnosed pregnant (progesterone level> 0.7 ng/ unfertilised ova, three degenerated, three 4 - cell ml),and of these 6, one gave birth to triplets and embryos, six morula or early blastocyst stage one aborted at 3 months. when transferred to six recipients of some breed, yielded two pregnancies. Malaysian Agricultural Research and Development Institute (MARDI) has planned The above workers also developed embryo transfer work with following objectives: techniques for collection of embryos in buffalo (SharifuddinandJainudeen, 1987). A totalof77 a) To employ the embryo transfer work in : multiparous swamp buffaloes was superovu­ i) Sahiwal-Friesien cows, with milk lated with either PMSG or FSH, of which eight yield record above 4000 kg/lactation as animals wereslaughteredondays3,4,5 and 7 of donors and inferior Sahiwal-Friesien the superovulatary oestrus. Attempts to recover as recipients (for dairy) eggs surgically in two buffalo cows via a mid­ line incision was unsuccessful. Non-surgical ii) Sahiwal cows (donated by Pakistan recovery of eggs was more successful with IMV Government) with milk yield record embryo collection catheter than Foley Catheter, more than 3500 kg/lactation will be because in buffaloes, cervix had to be suffi­ chosen as donors and local inferior ciently dilated to allow the passage of the Foley Sahiwal-Friesienasrecipient(fordairy) 223

Table 1 : Ovarian responses to gonadotrophin treatment in goats.

PMSG(I.U.) FSH(mg) 1000 10 15 20

No of does 10 3 5 5 % of does in oestrus 60 100 100 40 % does ovulation 100 33.3 60 100 Mean no. of follicles ruptured a) All does 5.7 1.3 5.6 3.8 b) Ovulating does 9.5 4.0 9.3 9.5 Mean no. of large follicles 3.9 4.7 1.4 0.4

iii) Hereford or Kedah Kelantan (K.K) Studies (IPT) ). cows with body weight above 140 kg and 120 kg at 12 months respectively Professor Latif A. Ibrahim, Dean, Fac­ as donors, and inferior K.K's as recipi­ ulty of Veterinary Medicine, UPM is the leader ents (for beef) of the team that is establish recombinant DNA technology research for disease control and iv) Imported Limousine embryos from vaccine production. Research on molecular char­ West Germany as donors and K.K or acterization of New Castle Disease (NCD) virus K.K crossbreds as recipients. is already in progress. More and more virus and bacteria will be studied later in order to develop b) To supply embryos through embryo nuclear probes for detecting pre- sence of types splitting techniques. of different virus and bactelia.

c) To develop long term storage of embryo. Studies on rumen microbes and fungi are already in progress. Baseline data on diver­ d) To clone or produce identical copies of sity and performance of the rumen microbes are outstanding animals. being obtained to recognise and establish the dynamic interactions between rumen microbes University of Malaya's programme and feedstuff (normal and agro-industlial waste involved transfer of embryo from superior based rations). A major part of this study con­ females in the nucleus females in the nucleus cerns with molecular biological aspects con­ herd of goats to local goats. Research on in-vitro cerned with study of the genomic and plasmid fertilization and embryo freezing has been DNA in selected microbes and fungi. Molecular planned. cloning of a xylanase gene for Bacteroides suc­ cinogenes, using E.coli HB 101 as the host, has Molecular Biology Research been done by Dr. Abdullah Sipat, U.P.M, while he was working at University of Guelph, Can­ Major work on genetic engineering in ada. Xylan is a major polymelic component of animal production and health has been under­ hemicellulose, therefore characterization of taken by Veterinary Faculty of Universiti Per­ highly active xylanotic enzymes of ruminant tanian Malaysia (UPM) and two departments at microorganisms will contribute to an improved University of Malaya (Department of Genetics understanding of the enzymatic degradation of and Cellular Biology; Institute of Advanced hemicellulose in the rumen. Facilities for gene 224

mapping and finger printing and recombinant cloned DNA. Gene maps of these chicks would DNA techniques of Xylanase and other en­ be compared to control chicks to establish zymes found in microbes are available. whether the GHG has been inserted into chicken germ line or not. Dr. Gan Yik Yuen of department of Biology, UPM is using mitochondrial/nuclear Animal Feeding Biotechnology RFLP as a diagnostic markers for the indentifi­ cation of different breeds of local farm animals, and Microbiology Research and to study their genetic relationship based on DNA sequence. Dr. Gan also intends to study Intensive research and development plasmid DNA of certain bacteria that cause both work has undertaken by various institutes to human and animal infections. In her study DNA utilise large quantities of byproducts in the palm sequences will be compared from human and oil, rubber, coconut and other factories as ani­ animals to establish whether animals act as mal feed. Jalaludin (1986) mentioned that the intermediate hosts to human bacterial diseases. possibility of utilising agriculture byproducts, particularly from the palm oil milling industry, At the University of Malaya, Dr. Koh offers the best oppourtunity for improving live­ Chong Lek and his associates (1986) reported' stock production by establishing feed lots where the results of a study which relate transformable animals can be fed with palm kernel cake, fi­ antibiotic resistance traits in E. coli strains of brous residues (e.g. palm press fibre (PPF), animal origin (avian, bovine and porcine sources) empty fruit bunches (EPF) and palm oil mill with R plasmids, which are extrachromosomal effluent (POME). At present, most of these genetic elements coding the resistance markers, residues are not used to best effect as ruminant and are suspected to be responsible for drug feeds largely because of some intrinsic values of resistance. Among 15 independentE. coli strains the materials such as low digestibility and poor tested by them 8 were found to be antibiotic nutrient contents to support animal production. resistant, among which 37.5% weremonoresis­ tant and three or more multi-resistant i.e resis­ In order to know whether these defi­ tant to chlorampenicol (Cm), Kanamycin (Km) ciencies can be overcome by manipulation of and Nalidixic acid (Nx) (Koh and Kok, 1984). microbial organisms, research has been initi­ ated at Universiti Pertanian Malaysia. Three of the above antibiotic resistant strains were able to transfer all or part of their Ho et al. (1987) while doing a study resistance to an E.coli K 12 recipient by conju­ on the anaerobic fungi on 3 types of cellulolosic gation. Analysis of the plasmid profiles of all feed materials (guinea grass, rice straws and three donors and their respective transconju­ palm pressed fibres) in the rumen of cattle and gants after agarose gel electrophoresis provided swamp buffalo showed that attachment of fungi conclusive evidence that the transferable resis­ with feed material occurs 15-20 minutes after tance traits were plasmid mediated. the onset of the feeding by host animals. After 24 hours, the fungi could colonize the plant In the same University (UM), Mukher­ fragments extensively, especially vascular tis­ jee and Khadijah plans to introduce growth sues. In the guinea grass and rice straw, the hormone gene, both human and bovine origin epidermis and mesophyll were extensively near the developing embryo of unincubated degraded and many of the sclerenchyma and fertile eggs. Chicks hatched from these eggs vascular tissues were disrupted by the fungal would be analysed for their DNA's for restric­ rhizoids. Sclerenchyma tissues in the palm tion enzyme fragments that hybridize with GHG pressed fibres were also heavily colonized and 225

in many instances disruption and separation of bacteria and fungi (a part of it has been earlier the tissues occurred. mentioned in the genetic engineering section).

Besides this, rumen fungi have been Atthe UniversityofMalayaDr. Shaiful reported to digest plant structured carbohydrates Azni of Institute of Advanced Studies, and his such as cellulose, hemicellulose and xylan and associates are working with microbial distribu­ could digest up to 45% dry weight of plant tion and dynamics in rumen of goats fed with i) tissues. This indic"'ted that the anaerobic fungi grass and ii) grass plus concentrate containing are well equipped enzymatically to contribute to 50% palm oil mill effluent. Initial observations fibre digestion and rumen fermentation. There­ indicate a favorable distribution of microbes in fore the rumen fungi can play an active role in goats fed with grass compared to the other group the digestion of fibres in ruminants. Based on fed withgrassplusconcentrate(Manan, 1987; ). these facts, biotechnological processes in vitro Microbial growth, protein and energy contents can be developed for greater utilization of cellu­ of concentrate (based on palm oil mill effluent) losic materials especially those from agricul­ have also been studied, when concentrates were tural byproducts as feed resources forruminants stored for certain period in the form of pellets in Malaysia. Rohani et al. (1988) in another (Shaiful and Mukherjee, 1987). work on degradation of cellulosic agriculture waste by Aspergillus tefreus SUK 1 showed the Dr. Phang Siew Moi, also of Institute usefulness of this fungi in the degradation of of Advanced Studies, is presently involved in various cellulolytic wastes (cocoa seed testa, the utilization of agricultural wastes for the sugar cane residue). production of algae which may be used as a direct feed (in the culture of fish) or as a protein The UniversitiPertanian Malaysia has and vitamin supplement for other animals. appointed Dr. Mohamed Ali Rajion as coordi­ Wastes such as digested palm oil mill effluent nator to develop a multidisciplinary project for and animal manure are being used to generate manipulation of rumen microbes with the aim of algal biomass. The growth of fish (Tilapia and improving the utilization of locally available silver Catfish) on the algae, without supplemen­ feeds and agricultural byproducts.Research areas tary feeding, has been compared with growth on in this programme include: commercial feeding. Future work includes screening of algae for nutritional value (protein, 1. Microbial studies: isolation, char­ vitamin etc.), formulation and testing of feed acterization and screening for desirable charac­ (for poultry, goats, fish) incorporating algae, teristics e.g. cellulose, xylanase, protease,urease and upgrading nutritional value of waste mate­ and lipase production. rials (paddy straw, tapioca etc.) which are used as feed, by fermentation with algae. 2. Biochemical aspects: Studies on enzyme production by therumen wallandrumen One of the first industrial fermentation microbes. tower was made by Dunlop (M) Sdn. Bhd (Collier and Chick, 1979), a multinational company. 3. Morphological aspects: Studies on There POME has been used as a substrate for the host tissues and feed interactions with the ru­ growth of microorganisms. Filamento.us fungi men microbes. which were easily harvested and had short generation time of three hours were selected 4. Molecular biological aspects: (Collier and Chick, 1979). The effluent was Study on genomic and plasmid DNA in selected pumped into a fermentation tower from the 226

lower end where micronutrients and compressed The above faculty has done major re­ air were added and mixed. The fermentation search work in the production ofan oral Newcastle took place continuously as the effluent flowed disease (NCD) vaccine (Latif, 1986), which has through slowly. now been developed as a suitable protectant against NCD in village chickens. Here the chick­ The fermented effluent was removed ens are fed with optimum dosage of V 4 UPM slowly from the higher end of the tower. The virus in pellets to protect them against NCD. fibre fraction was reduced by 75% using a Two doses of vaccine were required for adequate vibratory screen. The remaining slurry had only immunity and a vaccine dose of about 105EID 7% solid and was dewatered by filter press and per chickens seemed adequate. Chickens placed dehydrated on a hot plate. The dry product was in contact with vaccinated chickens also devel­ called PROLIMA which contained crude pro­ oped protective immunity. tein levels of 45 .6 % compared to 13. 3 % for raw POME (Yeong, 1981). The waste water from Extensive field testing to establish this the process was channeled into anaerobic pond oral vaccine as a major protectant against NCD for further reduction -of BOD as it still carried is being done by Professor Latif and Dr. Aini at about 30% of BOD of the raw effluent. present.

PROLIMA was not found to be very The above faculty has also received a cost effective, which prompted the company to large grant from Malaysian Government for close down its plant. However feeding trial with biotechnology research in relation to production PROLIMA in broiler rations (Yeong, 1981) of monoclonal antibodies against diseases and showed that approximately 50% of soybeen and production of nuclear probe for detection of palm kernel cake could be substituted by PRO­ certain diseases. Laboratories to conduct above LIMA. research are being established.

The Veterinary Department of the At Universiti Kebangsaan Malaysia, Ministry of Agriculture and Malaysian Agricul­ Bangi, Selangor, Dr. Md. Annuar Osman and his tural Research and Development Institute, Ser­ associates at the Department of microbiology dang, have also made plans for biotechnology are attempting to developing monoclonal anti­ research in the area of agriculture waste utiliza­ bodies against known virulent factors ofPseudo­ tion. monas pseudomallei, particularly its lethal ex­ otoxin, haemolysin and pilli, and producing a Immunogens and Vaccines vaccine by way of utilising the purified antigen of P. pseudomallei in the control of melioidosis in domestic animals. (Personal communication). Major activities on the development of ELISA for rapid diagnosis of Newcastle dis­ In this regard the above researchers ease, Infectious bronchitis, Leptospirosis, Haem­ have isolated and purified the exotoxin, which orrhagic septicaemia and Brucella and detec­ accounts for lethal toxicity in mice. The availa­ tion of progresterone level in bovines are in bility of this relatively pure material enabled the progress at Universiti Pertanian Malaysia, ac­ development of a specific monoclonal antibody cording to Professor Latif A. Ibrahim, Dean, for use in the ELIS Aw hich itself would be of use Faculty of Veterinary Science. Efforts to de­ for the detection and determination of the ex­ velop monoclonal antibodies against Newcastle otoxin. disease are being made by Dr. Zubaida as a part of her Ph.D thesis. 227

Stu dies in the above laboratory demon­ Manan, A. (1987). Anaerobic cellulose diges­ strate the presence of pili on the surface of P. tion of palm oil mill effluents by rumen micror­ pseudomallei in culture and also the ability of P. ganisms. M.Sc thesis, University of Malaya, pseudomallei to adhere to human buccal epithe­ Kuala Lumpur, Malaysia. pp. 214. lial cells was mediated through pili (Nonya et al , 1987). Work is underway to purify and charac­ Razak, N., Y. Samah, R. Mohamad, N. Embi, terize the pili of P. pseudomallei towards prepa­ 0. Omar and G. Ismail. (1987). Pili of Pseudo­ ration of monoclonal antibody for use in the monas pseudomalleic and its haemaglutinating ELISA for the detection of pili. and adherence properties: Proc. 10th. Malay­ sian Microb Conf. (Kuala Lumpur, Malaysia) The Veterinary Service Division of the pp.10. Ministry of Agriculture will undertake ELISA for rapid diagnosis of diseases. One officer has Rohani A. K., 0. Olham and A. A. A. Shaiful been sent to USA under the Cochrane Training (1988). Degradation of cellulosic agriculture Programme. waste by Aspergillus terreus SUK-1. Proc. 11th. Ann. Conf. MSAP. pp. 84 - 90. References Shaiful, A. A. A. and T. K. Mukherjee. (1987) Ho, Y. W., N. Abdullah and S. Jalaludin (1987). Microbial growth protein and energy contents Contribution of rumen anaerobic fungi in the of stored goat feed pellets. Biological Wastes. utilization of cellulosic materials for the rumi­ nants. Proc. Colloq. An.Prod. &Health, Univer­ Sharifuddin, W., and M. R. Jainudeen. (1984) siti Pertanian Malaysia, Serdang. P. 75 - 77. Superovulation and non-surgical collection of ova in the water buffalo (B ubalus bubalis) Proc. Koh, C.L., andC.H.Kok(1984). Antimicrobial 10th. Int. cong. Anim. Reprod. & A.I., Univ. resistance and conjugative R plasmids in Esch­ Illionis, Urbana-Champaign, pp. 240 - 242. erichia coli strains isolated from animals in peninsular Malaysia. South East Asian Jour. Yeong, S. W. (1981). Biological utilization of Trop. Med. Pub. Health. 15(1); 37 - 43. palm oil byproducts by chickens. Ph.D. thesis, University of Malaya, Kuala Lumpur, Malay­ Latif, A. I. (1986) Vaccination of free range sia. pp. 168. chickens against New Castle Disease. 3 5th. Proc. West Poultry Diseases. Peutro Vallartta, Mex­ ico. pp. 85 - 86. List of Institutes and Engaged in Biotechnology Research

COUNTRY: Malaysia NAME OF INSTITUTE: Fae. of Biological Sciences, Univ. Kebangsaan Malaysia, P.O. Bangi Selangor. DATE VISITED: 5.11.1987 HEAD'S NAME: Prof. Zakri Hamid, Dean

Name Designation General Current Research/Development Future Research/Development work in Biotechnology work planned

Prof. Zakri Dean Plant breeding Plant biotechnology Plant biotechnology. Hamid

Dr.Anuwar Lecturer Veterinary Monoclonal antibodies against Continuation of present work. OsmanDVM Microbiology Meliodiosis (Pseudonwnas sp). M.Sc.

Dr. Ismail Lecturer Veterinary Monoclonal antibodies to T3 Continuation of present work. AhmadDVM Microbiology and T4 hormones M.Sc.

Dr. Abdul Lecturer Genetics and Gene transfer in mice Majeed Mohd. Breeding

00 N N 0\ COUNTRY: Malaysia N N NA.l\1E OF INSTITUTE: Faculty of Veterinary & Animal Sciences, Univ. Pertanian Malaysia, Serdang Selangor. DATE VISITED: 6.11.1987 HEAD'S NA.l\1E: Prof. :Latif A. Ibrahim, Dean

Name Designation General Current Research/Development Future Research/Development work in Biotechnology work planned

Syed Jalaludin Prof. &Dy. Animal Agro-waste utilization Animal Continuation of present work. b.Syed Salim V. Chancellor nutrition nutrition, Feed biotechnology

Prof .Latif A. Prof. &Dean Viral Vadccines 1. Production of a pelleted Gene mapping for NCD. Ibrahim Genetic Eng. vaccine against New Castle Produc.of Disease. monoclonal 2. Monoclonal antibodies antibodies 3. Cloning for viral vaccines.

Dr.W. Lee. Anirn. Animal Embryo transfer in goats and Nuclear probe development Sharifuddin Reprod Reproduction buffaloes. forNCD. Prof .Clinical

Prof Professor, Animal Embryo transfer in goats and Perfection of E.T M.R. Jainudeen Clinical Reproduction buffaloes techniques. Embryo Science freezing & solitting Chimera production.

Dr. Gan Yik Lecturer Genetics RELP in farm animals and C-DNA of New Castle Yuen Biology mitochondrial DNA Disease virus. Faculty

Dr. Abdullah Lecturer, Biochemistry Genetic engineering Cloning of enzyme genes Sipat Biology for manipulation microbes. Faculty COUNTRY: Malaysia NAME OF INSTITUTE: Faculty of Veterinary & Animal Sciences, Univ. Pertanian Malaysia, Serdang Selangor. DA'IE VISI'IED: 6.11.1987 HEAD'S NAME: Prof. :Latif A. Ibrahim, Dean

Name Designation General Current Research/Development Future Research/Development work in Biotechnology work planned

Dr.Abdul Lecturer, Pharmacology Brachiaria decumbens Microbial manipulation of SalamAbd. Biology {f oxicology t oxicity in livestock. plant poisons. Faculty

Dr.Mohamed Lec.Leadof Ruminant Essential fatty acids in Rumen metabolism of lipids. AliRajion Bfotech. Physiology ruminant nutrition. (Nut.group) Nutrition

Dr. Ho Yin Wan Lecturer , Plant pathology Anaerobic rumen fungi and To study interactions mycology their role in the between microbes, f eedstuff degradation o_ffibrous and animals. feed matertials in ruminant.

Ms.Norhani Lecturer Rumen function Enzymic studies os rumen fungi Continuation of present work. Abdullah andN2 and bacteria. metaboligsm

Dr. Baharuddin Lecturer Saccharificati Celluase of bacteria; cloning Cloning of other cellulase Ghani on of cellulose of b - glucosia-dase gene genes; Characterization of enzyme.

Dr.ARahim Lecturer, Vet. Vet Isolation and characterization To use these usilates mainly 0 Mutalib Bacteriology Bacteriology of beneficial intestinal in prevention of neonatal ~ N bacteria darrhoea in farm animals. """"4 COUNTRY: Malaysia ~ NAME OF INSTITUTE: Faculty of Veterinary & Animal Sciences, Univ. Pertanian Malaysia, Serdang Selangor. DATE VISITED:6.ll.1987 HEAD'S NAME: Prof. :Latif A. Ibrahim, Dean

Name Designation General Current Research/Development· Future Research/Development work in Biotechnology work planned

Rohani Lecturer, Agro-waste Degradation of cellular Agro-waste research Ab.Kadir Biochemistry utilization agricultural waste by Aspergillus terreus SUK-I. COUNTRY: Malaysia NA:rv.t:EOF INSTITITE: Dept. of Veterinary Services, Ministry of Agriculture Jln. Sweetenham, Kuala Lumpur. DATE VISITED: 6.11.1987 HEAD'S NAME: Dr. Mustafa Hj. Babjee, Director, General

Name Designation General Current Research/Development Future Research/Development work in Biotechnology work planned

Dr. Hadi Senior Vety. Animal Hashim Officer Production

Dr. Mustapa Vety. Officer Health Jalil

Dr. Chee.yee Vety. Officer Pig & Poultry Pig waste treatment. eng production

Dr. Raymond Vety. Officer Animal waste Animal Waste Management Biological probiotics in animal Choo Pow Yoon management and animal feed and biological pollution tools in waste mangement. control

Dr. Vincent Ng. Vety. Officer Animal Artificial Insemination and Embryo transfer in cattle. Production (work not started yet)

Dr. Gularn Shah Vety. Officer Animal Embryo transfer in cattle. Production

N ("(') N M M COUNTRY: Malaysia N NAME OF INSTITUTE: Institute of Advanced Studies, University of Malaya, Kuala Lumpur DATE VISITED: 7.11.1987 HEAD'S NAME: Prof. B. C. Tan

Name Designation General Current Research/Development Future Research/Development work in Biotechnology work planned

Prof. T. K Prof. & Chair Animal Genetic Cloning of pituitary growth Gene mapping and gene Mukherjee Nat.Res. and Breeding hormone gene using retroviruses expressions in chickens. Studies. as vectors.

Ms. Khadijah Lecturer Animal Genetic Cloning of pituitary growth Gene mapping and gene Embong and Breeding hormone gene using retroviruses expressions in chickens. as vectors.

Dr.Ramli Associate Animal Embryo transfer in a nucleus In-vitro fertilization in mice Abdullah Professor Reproduction herd of goats for genetic (Dr. Ramli) Embryo transfer improvement. work-perfection of technique.

Ds. S.Sivaraj Research Animal Embryo transfer in a nucleus In-vitro fertilization in mice Associate Reproduction herd of goats for genetic (Dr. Ramli) Embryo transfer improvement. work-transfer work-perfection of technique.

Dr. A.K. Associate Microbiology Production of fungal biomass Continuation of work. Kuthubuteen Professor using fermenter.

Dr. Shaiful Associate Microbiology Rumen microbiology Rumen microbes-animal feed Azni Professor interaction. COUNTRY: Malaysia NAME OF INSTITUTE: Institute of Advanced Studies, University of Malaya, Kuala Lumpur DATE VISITED: 7.11.1987 HEAD'S NAME: Prof. B. c. Tan

Name Designation General Current Research/Development Future Research/Development work in Biotechnology work planned

Dr. Masitah Lee. Biochem. Fermentation Cellulose degradation, Consolidation of present work. Hassan Engineering. technology computer application.

Dr.Phang Lee Natural Algal Single cell protein production Consolidation of present work. SiewMoi Resources production for feed and extraction of fine chemicals.

Dr. John Lee. Natural Microbiology Inoculation of fungi in empty Consolidation of present work. Tharnbirajah Resources palm fruit branches for growing mushroom and feeding of empty bunches after harvesting mushrooms.

Dr. A. A Associate Crop Science Agro-waste utilization in crop Composting of agric. waste for Ravoof Professor and animal production animal feeding.

Dr. Mohd Ali Associate Biochemical Bioreactor design, separation Solid state fermentation. Hashim Professor Engineering, process. Environmental Engineering.

'tj­ ('-r') N Pakistan: Biotechnology Applied to Animal Production and Health

M. Ashfaque Department of Veterinary Microbiology Faculty of Veterinary Science University of Agriculture Faisalabad, Pakistan

The main problem that we are facing is To achieve the above objectives, em­ the provision of enough food for a rapidly in­ bryo transfer technology has been tried at creasing human population from the earth which Government organizations as well as on private has a finite area and therefore, a finite capacity to level. produce food. This can be made available to some extent if modem technology is adopted in A successful embryo transfer in exotic the various fields of science. cattle was done at Animal Science Institute lo­ cated in National Agricultural Research In Pakistan, the application of biotech­ Centre,Islamabad. Further work is in progress. nology to animal production andhealth started in More technical experts are under training in eighties and most of its programmes are in the USA and UK. A second nucleous of embryo initial research stages and are briefly reviewed as transfer technology is being set up in a newly under: established Livestock Production and Research Complex, Rakh Bhunaky, with a division of Embryo Transfer Technology embryo transfer and micromanipulation which is in its early stage of procurement of equip­ There are numerous biological limita­ ments. tions and economic constraints in which animals fail to reach their production performance. In In the private sector this technology is livestock, the reproductive efficiency of male being practiced on small scale at Uquab Breed­ has been dramatically improved by artificial ing Farms, Kotri in collaboration with a foriegn insemination. The efficiency of females can be agency. TheratesfixedareRs.6,000.00forsingle­ further improved by producing twinning or to service and Rs.12,000.00 for a sure conception transfer eggs from top quality donors to inferior which is too costly. females. As we know, the generation interval is Nutrition fixed for a species, therefore the only mean of increasing the tum-over generation is by de­ To overcome the nutritional constraints creasing the time taken to reach sexual maturity. on the expression of milk yield/fattening per­ This could be done by breeding from immature ticularly animals or more likely the removal of immature ova from heifers and transplanting them into i) the heavy cost of protein mainte­ cows. nance of dairy cows superimposed with protein 236

used by their offsprings. - The restriction enzyme group has isolated a new enzyme from a locally isolated ii) to remove certain obvious disadvan­ bacterium Pseudomonas ovalis. The enzyme is tages ofruminent digestion such as loss of meth­ called Pov 1recognizes5'TGATCA 3'. ane from rumen and the inherent difficulty of as­ certaining amino acids requirements for milk - A study on the locally isolated bacte­ synthesis. ria revealed that some of these bacteria contain plasmids with uptoeightantibiotic markers.Two iii) to raise the overall feed conversion of the local strains have been identified as oil efficiency. In this context, experiments were emulsifire, three degraders oflong chain hydro­ conducted at the University of Agriculture,Fai­ carbon residues and five efficient nitrogen salabad in which low quality forage and agro­ fixer.Genes of interest are being identified and industrial wastes were processed with the help of cloned onto multicopy plasmids. fermentation technology to form a biomass. Rice straw, ricepolishing and husk were used as sub­ -The production group has purified strate for the growth of Trichoderma(a locally restriction endonuclease enzymes, a polynucleo­ isolated fungus) under predetermined optimum tide. ligase and an exonuclease. conditions. A good quality protein was obtained ranging from 11 to 20 percent in rice-polish­ In the long term plan the above centre fungal-biomass. The results of feeding trails in has the objective of preparing synthetic vaccine, rats are encouraging. The equipment used was till that time the animal scientists may adopt this locally designed (Hashmi, 1987). Further work is technology. Laboratory space and cell culture under way to try other microorgnaisms for more facilities are available in the department of recovery of protein. Veterinary Microbiology, University of Agri­ culture, Faisalabad to meet the basic require­ In another experiment on urea feeding ments in hy bridoma technology for the produc­ as a protein source in ruminants .with the objec­ tion of monoclonal antibodies. tive to use microbes urease (from dung) to de­ compose urea under anaerobic conditions with The construction of another institute wheat straw. The resultant product improved has started this year namely "National Institute with enhanced digestibility (Athar and for Genetic Engineering and Biotechnology" at Gilani,1988 personal communication). Similar Fai- salabad. experiments were made with com cobs and sugar cane tops improve the digestibility of the crop References residues (Mahmood and Gilani,1988.personal communication). Third five year report,Fifteen year of NIAB.Nuclear Institute for Agriculture and Recombinant DNA Technology Biology ,Faisalabad.1972-87. Annual report. Centre for Advanced Molecular The application of this technology is Biology,University of the Punjab,Lahore.1986. under consideration by the animal scientists for the production of molecular vaccine and mono­ Annual report.University of Agriculture,Fai­ clonal antibodies. At present there is one re­ salabad.1987-88. cently established centre"Center for Advanced Molecular Biology" at University of the Punjab, Lahore. The salient results obtained by various groups of scientists at this centre are: to al and

1 2 Chamnean Satayapunt , Vanda Sujarit , Samphan Singhajan3 I Animal Science Department Kasetsart University, 2Faculty of Veterinary Medicine Kasetsart University, 3Ratchaburi A.I. Station Center, Livestock Development Department. Thailand

Introduction : Implications of a decade now. The status and impact of the Biotechnology for Development technology have been the subject of several analyses (6-8). Although the infrastructure for biotechnology in Thailand is relatively strong Biotechnology makes many promises among developing countries, it still has .a ver;; for the future. These promises carry deep impli­ small number of qualified manpower m this cation, not only concerning economic benefits specific area. Thailand has some 60,000 scien­ for those who own the technology, but also tists and engineer at degree level, or 11 per concerning the livehood and development of 10,000 population. However it can be estimated people who will be affected by the new products that there are fewer than 500 with master degree and processes. For the developing world, ?~o­ and up, who are active in biotechnology research technology offers both threats and opportumties and most of these are in the universities. Current (1-3). The threats are posed by, among other degree programs produce some 40 bachelor things, substitution of new products for the tra­ degree graduates per year in biotechnology, and ditional ones which have been the sources of another 60 per year in food science and techno­ income for the developing countries. An depend logy. The combined production capacity for for export income, of the new sweeteners pro­ agriculture, medicine and other program related duced by biotechnology (4). The opportunities to biotechnology is about 1,700 bachelors per are offered by the fact that many developing year. The production capacity for master degree count:Iies are situated in the tropical belt rich in in biotechnology is about 30 per year, and the resources which biotechnology can turn to high combined production capacity for allied sci­ value-added products. These opportunities can ences is about 330 per year. The doctoral degree be met if they can increase their capability in the programs in life sciences presently ex.ist in ~ree technology (5). Good policies and strategies for Universities, with thehigheststrengthmMah1dol development and utilization of biotechnology University which has up to now produced some can therefore have a very significant impact on 50 graduates during the past 20 years. There is the future of developing countries. virtually no post doctoral system in Thailand at present. Status and Impact of Biotechnology in Thailand Research and development in biotech­ nology, as in other areas, are mostly done ~ the The importance of biotechnology to universities. Mahidol University has, various Thailand has been increasingly realized for over active research groups, with emphasis on mo- 238 lecular biology and genetic engineering of vari­ Future Directions and Strategies for ous biomedical areas, especially those concer­ Research and Development on Bio~ ning tropical diseases, and industrial micro­ technology applied to Animal prom biology. Kasetsart University is active in vari­ ous areas of agriculture biotechnology, includ­ ductions and Health. ing tissue culture for improvement and propaga­ tion ofvarious plants, embryo transfer technol­ Bearing the above considerations in ogy and fermentation technology. Chulalong­ mind, we can proceed to look at future direction kom University is well known for research on of research and development especially with re­ aquaculture of shrimps, rice tissue culture and spect to animal production and health. The main processing technologies. King Mongkut Insti­ direction should be a change from "adaptive" tute of technology Thonburi has various activi­ type of research toward more breadth and depth ties on bioengineering and biomass utilization. and from low-level trial and error type of re­ All of these institutes are located in the Bangkok search towards more systematic application of area. Among the provincial universities, Chiang biological knowledge. The change may be gra­ Mai University is the most active, with works on dual and many conventional technologies may tissue culture of various crops, including or­ still prove very useful over a long period in chids, teak and potato, and post-harvest biotech­ future. Conventional animal breeding, for ex­ nology. ample, should continue to be a very useful tool for development of animal breed and varieties as Among the government agencies, new technologies are being employed. In the Department of Agriculture has a very large re­ near future, improvement of domestic animal for search program, much of which is related to production of food and fiber is poised to undergo biotechnology. An AID supported project en­ a revolution by the utilization of recent break­ abled the production of Rhizobium on a large throughs and advances in molecular genetics, scale. Specialized research institute in these embryo manipulation, and gene transfer system. departments deal with rice, rubber and other Utilization of these techniques will have a wide crops. Department ofLivestockDevelopment do impact on animal agriculture by improvement of active research in genetic improvement of live­ production efficiency via manipulation and stock, Semen bank and ittechnology, Vaccine control of many physiological systems. The end production/or livestock, Disease diagnoses, pre­ result will be to decrease production costs, in­ vention, control and Eradication program of crease food production and quality, and lower livestock diseases and parasites, and demon­ food costs.Health and well being of domestic stration and transfer technology to the farmers. and other animals will be improved as a result of Other departments which have research with new methods of disease diagnosis, vaccine pro­ biotechnology components include Department duction, and disease prevention practices. Bio­ of Fishery and Forestry. The Thailand Institute technology and Genetic engineering also offers for Scientific and Technological research, a the possibility of utilizing animals for the devel­ government funded institute operating as public opment of pharmaceutical products to benefit enterprise, also has many activities in the bio­ society. Research progress will be enhanced via technology areas, mostly concerning industrial manipulation of the gene pool. microbiology. In the current status of animal bioengi­ The private sector in Thailand, to which neering and to realistically assess the potential agriculture and agroindustries are very impor­ applications of current and future genetic tech­ tant, already use biotechnology substantially for nologies for production of food and fiber to meet their products and processes. the needs of our hungry world, and to provide 239 animal scientists who may wish to utilize bioen­ realizable potential of solving some of the most gineering in cun-ent background information difficult problems facing agriculture today. regarding concepts, applications and method­ Thailand has the potential to do it in time on the ologies, it is important for Thailand to learn to near future. master these new technologies because of cer­ tain dominance they will assume in the future. Opportunities and Needs.

Genetic engineering is the field of · Thailand and other countries in south­ manipulating the DNA of a cell or of an animal east Asia constituting the Association of South­ in order to alter the genetic information con­ east Asian Nation (ASEAN) are middle-stage tained within the organism's genome (9). The developing countries with moderate to fast standard techniques of recombinant DNA are growth. The A SEAN region covers a land area of used. A number of excellent textbooks are now about three million sq.km., and has a total popu­ available that explain in clear fashion how to lation of 309 million, with an annual growth rate work with DNA to cloned, to alter known gene of about2%. The total GNP (1986) was US$ 205 and so on. OneconvenientaspectofDNAis that billion, and the average GNP per capita was the same techniques can be used whether the about US$ 675. The general socio-economic source of material is from human,bacteria, yeast, status of the A SEAN countries is given in Table plants cattle or chicken. Essentially, any DNA 1 (10). For over two decades now, the ASEAN can be recombined with any other DNA. Genetic countries generally enjoyed a good socioeco­ engineering technology is the major scientific nomic development record and, although still revolution of the century. Rapid development is relatively weak compared with the developed being made,especially in veterinary medicine. countries, are poised to become important num­ Genetically engineered vaccines and monoclo­ bers of the Pacific Rim in the year 2000 and nal antibodies are already in the marketplace for beyond. veterinary use. Many other animal health care products are being tested. In agriculture, genetic The ASEAN region is rich in natural engineering is focusing on manipulation of or­ resources, being the only region in the world ganisms to produce animal vaccine, hormones, which is both a net exporter of food (from Thai­ amino-acids, chemicals and drugs. These tech­ land) and of energy (from Brunei, Indonesia and nologies will have the greatest impact on im­ Malaysia). Agriculture plays a very important proved livestock production by : (a) reducing role in the A SEAN economy and will continue to animal losses through prevention of infectious be important despite recent trends towards in­ diseases, using effective genetically engineered dustrialization. The advent of new biotechnol­ vaccines and antitoxins ; (b) increasing produc­ ogy will likely enhance the role of agriculture, tion of meat and milk through the use of growth and help in its linkage with industries, especially promotants; (c) improving the nutritional values those concerning food processing. This has of animal feed. substantial implications not only for ASEAN countries themselves, but also for industrially The industrialization of recombinant developed countries in joint venture and tech­ DNA technology will lead to many useful pro­ nology transfer for local production both for ducts and processes. The underlying science of ASEAN and world markets. molecular biology and molecular genetics is dynamic ; it is reasonable to assume that new Apart from the advantage of natural opportunities will be created as the depth of resources, the main opportunities in develop­ understanding increases. Genetic engineering ment of biotechnology in the ASEAN region are technology is not a panacea, but it carries the offered at relatively low cost for skilled or semi- 240

skilled manpower. A few examples from Thai­ crop diversification for the developed countries, land can illustrate tJ1is point. Plant tissue culture with adverse effects on the developing ASEAN technology in Thailand has notably flourished countries. In fue long term still, lesser known commercially. Another example is fue remark­ plants of potential use in agriculture (11) can be able success of aquaculture technology. In Thai­ explored , although fuere are many hurdles to be land, com para ti ve advantage also exists for such overcome in introducing new crops to the mar­ downstream technologies as production of amino ket. With respect to increase in value-added of acids and antitoxins by fermentation processes. the products, the cassava starch provides a good However, lack of highly qualified manpower example whereby various technologies, most still largely precludes tJ1e exploitation of such well established in the developed countries, such high technologies as genetic engineering of as enzyme technology, carbohydrate modifica­ hybridoma technology in the production sector tion technology and fermentation technology, at this stage, although many universities and can be utilized following successful technology research institutes are now using them for re­ transfer. In other areas, joint research and devel­ search purposes. opment are needed such as the conversion of palm oil to more valuable products through trans­ The need for development of biotech­ esterification and other procedures. nology in Thailand and oilier ASEAN countries arises from the general decline and fluctuations National Policies Programs in commodity prices. These trends force the producers to lower the cost of production, to Thailand and a few other southeast diversify and to increase the value-added of the Asian countries have specific policies and meas­ products all of which require new approaches ures to promote the development and application through biotechnology. For example, in order to ofbiotechnology. Singapore has various schemes reduce fue cost of rubber production, iliere is a to help finance research and development acti vi­ need to predict fue potential yield ofrubber at the ties and commercialization in the private sector, young plant stage through enzyme typing or including the Research and Development Assis­ other techniques so that high-yield varieties can tance Scheme, the Science Park was recently be chosen for planting which will require some established near the National University of Sin­ five years to reach the production stage. Crop gapore, and the Institute of Cell and Molecular diversification can be achieved in the short term Biology has been set up to do research at the by focusing on import substitutes, for which the leading edge. The Philippines, Malaysia and technologies are well established. In Thailand, Indonesia also have biotechnology plans and for example, the rapidly increasing demand for programs at the national level. The six ASEAN potato seeds can be met by plant tissue culture for countries have recently formulated regional production of mini or microtubers. In the longer programs under the auspices of the Committee term, however, strategies for promotion of tropi­ on Science and Technology. cal fruits,medicinal herbs and other plants for which the ASEAN region is famous will bring a Biotechnology development programs higher return, not only from local but also from started in Thailand in the late seventies, first wiili export markets. The technologies for these plants the formation of centers in important universi­ are not well developed at present, and there is a ties. In 1983, the government set up the National need for Thailand and other A SEAN countries to Center for Genetic Engineering and Biotechnol­ collaborate with more developed countries in ogy to be the main policy, support and co­ research efforts. The need for indigenous tech­ ordination center for biotechnology research and nology development is very well demonstrated development projects, and to form linkage with here, because otherwise the result could well be the private sector. The establishment of the 241 National Center marked for the first time, sig­ Conclusion@ nificant local funding from the government towards development of a specific technology. National policies and programs on Support for the technology is, moreover, pro­ biotechnology as existing in Thailand demon­ vided as a complete package with provision of strate the current awareness and optimism in the funding, information, training, links to the in­ southeast Asian countries on the benefits of the dustries and international links. The National new technology. While these policies and pro­ Center has 4 affiliated laboratories, including grams still need to be developed further, .they pilot plants, and over 30 projects in 9 institutions provide ample basis for international co-opera­ in the network. The emphasis is on development, tion in various forms. Agriculture is the most transfer and utilization ofbiotechnology, includ­ important component of the national programs ing genetic engineering in the following areas: and projects, especially on the biotechnology industrial applications, agricultural applications, and genetic engineering applied to animal pro­ public health, energy and environmental appli­ duction and health, reflecting its importance in cations, and strengthening of basic infrastruc­ the economy. The private sector of both partners ture in genetic engineering and biotechnology. in the collaboration has a large role to play in the Under these guidelines, the National Center development of biotechnology as a whole, and of commission various studies on the status of agricultural biotechnology in particular. A few specific technologies and industries to assess the problems remain to be overcome regarding tech­ economic and social importance of the specific nology transfer, sharing of benefits from intel­ areas and to pinpoint the research, development lectual and genetic resources, and regulations of and technology transfer needs to be fulfilled. concerning biosafety. These problems are being Designated research projects are then formu­ tackled in Thailand through the National Center lated by researchers, often from more than one for Genetic Engineering and Biotechnology, the institution working together, and are funded Science and Technology Development Board, after proper peer review. Table 2 gives a sum­ and other relevant agencies. The needs for and mary description of the various projects sup­ opportunities in development of biotechnology ported by the National Center, and Table 3 gives in Thailand axe so substantial that the problems a summary of works undertaken at the 4 affili­ seem small by comparison. ated laboratories. The Science and Technology for De­ Reference velopment Program (12), established in 1985 with cooperation of the US Agency for Interna­ 1 UNESCO (1987) The Courier, Mar 1987 tional Development (AID), is another major supporter of biotechnology research and devel­ 2Ahmed, I (1988) The bio-revolutioninagricul­ opment in Thailand. The Science and Technol­ ture : Key to poverty alleviation in the Third ogy Development Board (STDB), which runs World. Int.Lab.Rev. 127, 53-72 this program, has a wide interest covering bios­ cience and biotechnology, material technology 3 Dembo, D. and Morehouse, W. (1987) Trends and applied electronics and computer technol­ in Biotechnology Development and Transfer. 1 ogy, areas which were determined to be of high PCT 32 UNIDO, Vienna, pp.96 priority by previous studies (13). Other biotech­ nology research are supported by the National 4 VandenDoel ,K. andJunne,A. (1986)Product Research Council and by grants from the univer­ substitution through biotechnology : Impact on sities. International funding is also significant, the Third World. Trends Biotechnol., Apr.,88- especially for biomedical and life sciences proj­ 90 ects. 242

5. McConnell, D., Riazuddin,S. and Zilinskas, 10. Far Eastern Economic Review (1988). Asia R.A. (1986) Capability building in biotechnol­ Yearbook 1988. ogy and genetic engineering in developing coun­ tries. IS 608. UNIDO, Vienna, pp.114 11. Vietmeyer,N.D. (1986)Lesserknownplants of potential use in agriculture and forestry. Sci­ 6. Yuthavong, Y., Bhumiratana,A. and Suwana­ ence 232, 1379-1384. adth,M. (1984) The Status and future of biotech­ nology in Thailand. In: Proceedings of ASEAN­ 12. USAID (1985) Science and Technology .for EEC Seminar on Biotechnology : The Chal­ Development. Project paper (493-0340) US AID/ lenges Ahead. Singapore, Nov.1983, Science Thailand, Bangkok. Council of Singapore, pp.29-39 13. Yuthavong, Y., Sripaipan,C., Kirtikara,K., 7. Yuthavong, Y. (1987) The impact of biotech­ Alankwandee, A. and Trakuku, K. (1985) Key nology and genetic engineering on development problems in Science and Technology in Thai­ in Thailand. Jr.Sci.Soc. Thailand 13, 1-8 land. Science 227, 1007-1011.

8. Suwana-adth, M. (1987) Thailand's position 14. Yuthavong, Y., and Bhumiratana, S. Nation in relation to biotechnology and bioindustrial (1988) Programs in Biotechnology for Thailand developmentln: Proceedings of the Conference and other southeast Asia Countries: Need and "First ASEAN Science and Technology week", Opportunities. Conference on "Strengthening April 1986, Kuala lumpur, vol.ii pp.239-246 Collaboration in Biotechnology: International Agriculture Research and the Private Sector". 9. Evans J.W. and Hollaender A. (1986) Genetic Rosslyn, Virginia, April 18-21 Engineering of Animals, Plenum Press, N.Y. pp.7-13 243

Table 1 Basic Socio-Economic Data of ASEAN Countries, 1986-7

Country Brunei Indonesia Malaysia Philippines Singapore Thailand

AREA, 1000 sq.km. 5.8 1,919 329.3 300.4 2.6 514 POPULATION Size (million),1987 0.2 174.9 16.1 61.5 0.6 53.6 % Average Annual 2.6 2.1 2.4 2.8 1.1 2.1 Growth, 1979-86 Projected Year 2000 0.3 219.8 20.2 85.5 2.9 65.5 Death Rate per 1000 4 10 7 7 5 8 Birth Rate per 1000 30 31 31 35 17 29 Life Expectancy 62 58 67 65 71 63

ECONOMIC DATA Workforce (million) 0.1 63.8 5.6 20.9 1.2 27.9 GNP/GDP at market 3.42 86.47 26.34 30.13 17.97 10.18 prices US$ b,1986 Real GNP/GDP -10 3.2 2.1 1.5 -1.8 3.4 % Growth, 1986 Average real -4.8 3.5 3.2 -1.1 7.1 4.1 growth (1982-6) Per Capita 15,556 530 1,636 515 6,431 612 GNP(US$)

.Table 2 Summary Description of Research Projects supported by the National Center for Genetic Engineering and Biotechnology

Project Description

I. Introduction Applications 1.1 Research and Development The major objectives is to develop local ca­ Development for the Industrial pacity in industrial production of glucoamylase and - Production of Glucoamylase and - amylase.Two fermentation procedures were carried Amylase. B-amylase out for the glucoamylase production. They are the pilot (Mahidol University)a scale solid substrate fermentation utilizing A niger MUCA-8 and the laboratory scale submerge culture fermentation utilizing A niger KUB-1. Two strains of Basillus sp KUB-B3 are being investigated at labora­ tory scale level for the production of -amylase.

8Parentheses indicate the principal institution carrying out the research. 244

Project Description

1.2 Production of Pure Glucose for Phar The objective is to develop technology for the maceutical Purpose. production of injectable grade glucose which are cur­ (Chulalongkorn University) rently imported at a very high value annually. The production of injectable grade anhydrous glucose with universal standard specifications was achieved at a bench scale. Equipment for pilot scale production were designed, fabricated and tested. Pilot scale production is being carried out to obtain data for industrial scale production.

1.3 Production ofG-Amino-penicillanic The activities include strain improvement for Acid by Biotechnological processes. increased enzyme production , optimization of the (Mahidol University) enzyme production process,scarching for appropriate methods for enzyme and 6 AP A purification, immobi­ lization of enzyme and cells and bioreactor design. Cloned PAC genes ofB. megaterium UNI andE. coli 194-3 were studied which included the restriction mapping and subcloning of both genes. Attempts to join strong promoter to the PAC genes to increase the enzyme production are being madd. Immobilization of the E. coli is studied using alginate, cellulose acetate, and cotton cloth.A low cost medium for enzyme pro­ duction has been formulated.

Thailand's local citric acid production using solid substrate fermentation satisfied about 50% of 1.4 Citric Acid Production in Sub­ total local demand. This project aims at developing a merged Culture. technology of efficient citric acid production method (Kasetsart University) using submerged culture and a technique for purifica­ tion of citric acid for medical usage. The target is to obtain a commercially viable concentration of 9-12 % . The system developed within this project has produced 13-15% citric acid from cassava starch in a laboratory scale fermenter. Scale up study is underway for a stirred tank and an air-lift fermenter. 245

Project Description

1.5 Development of Wine Production This recently developed project aims to study from the Grapes. the wine production process and to test the suitability (Kasetsart University) of wine grapes grown in Thailand for wine production.

1.6 Improvement of Yeast Strains for The project aims to select and improve yeast Production of Food Yeast and Yeast strains for the production of yeast and yeast autolysate Autolysate. utilizing cassava starch as raw material. Standard strain (Chiang Mai University) selection methods were carried out. Attempts will be made to transform amylase gene into C.utilis

1.7 Production and Utilization of High High test molasses is superior to black strap Test Molasses in Fermentation Indus­ molasses as raw material for fermentation and can be try. produced in current sugar mills with only minor (Thailand Institute of Scientific and adjustment. Technology to produce high test molasses Technological Research) from sugarcane was developed. It was also shown by the project that it is feasible to replace black strap molasses by high test molasses as raw material for ethanol production. Market study on the production of high test molasses by the existing sugar miller were carried out.

1.8 Design of a Pilot Plant to Produce A pilot plant to produce feed grade SCP from SCP from Cassava. cassava were designed. The pilot plant was designed as (King's Mongkut Institute of Techno - an attachment' to the existing 1,500 litre/day alcohol logy, Thonburi, and Thailand Institute pilot plant of TISTR. The design was based on results of Scientific and Technological Re­ of a 370-litre air lift fermenter using S. Cerevisiae search, TISTR) 281. Preliminary economic study showed that the major cost of production was the price of cassava roots.

1.9 Antibiotic Production for Use in The major aims of the project are to select Animal Feed. microorganism which can produce antibiotic substances (Kasetsart University) which are effective against a wide range of diseases and to develop technology for the production of the antibiotic. Two isolates have been obtained and are currently being investigated. 246

Project Description

II. Agriculture Applications

2.1 The DevelopmentofEmbryo Trans­ Embryo transfer technology has been deve­ fer Technology in Dairy Cattle and its loped in Thailand with two main objectives : to increase Application under Thailand Condition. the rate of milk production and to improve dairy cattle (Kasetsart University) breed by developing genetically superior livestock that will yield maximum milk and meat production under Thailand's condition. Many calves were produced from frozen imported embryos using nonsurgical transfer technique into native recipients. Other calves were born from fresh embryos produce under Thailand's condition. The adaptabilities and performances of off­ springs born from frozen embryos of exotic breeds will be determined.

2.2 Steroid Immunization to Recover Since swamp buffalo commonly shows ir­ Fertility in Swamp Buffaloes at Village regular cycling and subnormal fertility, a method for Level. improving ovarian activity and fecundity is needed. (Chulalongkorn University) Hormone Immuno Neutralization (IDN) using steroid immunization was investigated as a possible mean of stimulating ovarian function in the swamp buffaloes. A strategy and a system of HIN in swamp buffaloes at small farm level was established and is being assessed by thepercentageof increased fertility. A totalof3,758 females were studied. It appears that HIN will become perhaps the most important tool to efficiently regulate the endocrine system of the female and will provide a basis for more successful artificial insemination and embryo transfer.

2.3 Strain Selection and Cultivation of In Thailand, terrestrial snails are plentiful and Terrestrial Snails. of high economic value as export items. Among the (Mahidol University) many species ofland snails,Achatinafulica, has been the most popular export item. However the dark flesh of the species has not been popular among the Thais. The aim of the project is to systematically cultivate the snails in order to select strain of albinosnails through cross-breeding and to increase the growth and the re­ production rate of the snails by identification,isolation and priming of the hormonal factors. These factors and the study of the snails reproductive physiology are being conducted. 247

Project Description

2.4 Development of Technology for The project's major aim is to develop techno­ Commercial Production of Disease-free logy for commercial production disease-free tomato Potato Seeds. seed in an attempt to replace the nearly 200 tons current (Kasetsart University) annual import of the seeds. Virus free potato plant material was successfully produced by the apical mer­ istem techniques. Disease-free mini-and micro-tubers were also successfully produced. The tubers are poten­ tially suitable for large commercial scale production. Field trials are being carried out.

2.5 Technological Development of The major aims of the project are to apply Industrial Horticulture Crop Produc­ tissue culture technology for the development produce tion. disease free ginger seed for growers. Mass propagation (Kasetsart University) by tissue culture technique was canied out for several varieties of temperate cut flower eg. chrysanthemum and lily. Gerbera hybrids between Thai and European strains were produced and the selected plants tech­ nique. New gamma-induced varieties of chrysanthe­ mum were introduced.

2.6 Conservation Technology of Unique The major aim is to develop technology for Characteristic - Plants in vitro. conserving plants of importance to Thai culture. Tissue . (Kasetsart University) culture technique was applied to several selected plants which includedArthocarpus heterophyllus (Jack fruit) and Ervatania coronaris.

2.7 In vitro Propagation and Screening The major aim is to select varieties for high oflndigeneous Costus lacerus for High production of diosgenin. Chemically screened Costus Diosgenin Production Cultivars. laccrus were mass propagated by in vitro techniques. (Mahidol University) Multishoot was obtained by culturing in a nodal seg- . ment in Murashige and Skoog's medium supplemented with benzyladenine. Vigorous plantlets obtained were transplanted.Rates of diosgenin synthesis and accumu­ lation therefore are being compared between the ma­ ture rhizomes of tissue culture-derived and conven­ tional propagated plant. 248

Project Description

2.8 Plant Tissue Culture Biotechnol­ The network was set up for coordinating the ogy Network. research and development among any institutes active (The National Center) in the field of tissue culture. Several technical work­ shops were organized cooperatively. Person el and Plant Tissue Culture Data Bas es have been set up. "Plant Bio­ technology" newsletter has been published quarterly.

2.9 Research and Development on The major aim of this project is to study Rattan Production. factors affecting growth and development of rattan in (Kasetsart University) an attempt to speed up the propagation of rattan. Tissue culture technology has been applied and systematic study of rattan pest and disease are studied.

2.10 Application of Tissue Culture The unavailability of good quality oil palm Technology for the Improvement and seedling has inhibited the development of Thai palm oil Propagation of Oil Palm._ industry. The project aims to apply tissue culture tech­ (Prince of Songkla University) nology to mass propagate selected oil palm and subse­ quently to improve upon the quality of the palm.

2.11 Association between Rice ( Oryza An ability to reduce fertilizer requirement on sativa L.) on Nitrogen Fixation Bacte rice production can have a major impact on Thai eco­ ria. nomy and the earning ability of rural farmers. The (Chulalongkom University) project aims to study the association between rice and the nitrogen fixation bacteria. Chemotaxonomy of the associative bacteria attaching to the root surface and rhizosheric soil were studied. Initial results have indi­ cated that rice lactin may play a role as associative factor.

2.12 Research and Development on Mi More than one hundred strains of cellulolytic croorganism for Compost Production microorganisms were isolated. Their effectiveness in in Thailand. the production of compost from plant residue under the (Kasetsart University) field conditions are being investigated in comparison with commercially available strains. Searching for an appropriate techniques for testing microorganisms to assist consumer in evaluating the effectiveness of the commercially available strains is one of the aims of this project. 249

Project Description

2.13 Research and Development on the Vesicular-arbuscular(VA) mycorrhizafungus Utilization of Vesicular-arbuscular my­ is known to thrive well in symbiotic association with corhiza and Their Combined Effects the plant roots and to increase plant productivity by with Nitrogen Fixing Bacteria in Leg­ improving phosphate up-take (reducing fertilizer re­ umes. quirement). Several strains of VA-mycorrhiza have (Thailand Institute of Scientific and been isolated from legume planting areas. Preliminary Technological Research) assessment on the efficiency and the development of strains associated with the plant (Sorghum bicolor) has indicated the strains from Glomus spp. to be superior to other strain tested.

2.14 Screening and Improving Genetic The developmental objective of this project is Potential ofNf'ixing Blue Green Algal to improve the productivity of rice production and to Strains for Use as Bio-fertilizer to Im­ reduce the cost associate with fertilizer requirement. prove Rice Yield. Nearly 1,000 paddy soil samples from all over Thailand

(Thailand Institute of Scientific and were collected and the occurrence of N2-fixing blue Technological Research) green algae were investigated. Eighty two strains of the algae were isolated and tested for growth and N2fixing ability. Among other factors studied were the effect of salt and pesticide (Propanil) on the growing of the algae. Field experiments on the effects of the bioferti­ lizer on rice yield will also be carried out.

2.15 Biotechnology Technique for The main objective is to develop a reliable Selection and Breeding of High Yield technique for yield evaluation of rubber tree at the ing Rubber Clones. seedling stage. A good yielding tree will result in (Prince of Songkla University) substantial additional output over the average tapping period of more than 30 years. It was found that the activity of the hydro-methyl-glutaryl Coenzyme A reductase in latex tapped from mature trees correlates well with rubber yield. The same correlation was found in leaves detached from 2 different clones of rubber seedlings, whereas the activity level of mevalonic acid kinase showed no correaltion with rubber yield. 250

Project Description

2.16 Development of Biotechnology Thailand imports a large amount of shiitake for Cultivation of Shiitake Mushroom mushroom annually. Until recently the mushroom can (Lentinus edodes) only.be cultivated on the highland in Northern Thailand at the expense of precious oak tree. The project aims to develop the cultivation of the mushroom using agricul­ tural compost. Substrates for the cultivation was suc­ cessfully developed from para-rubber tree saw-dust, rice bran and sawdust. Factors influencing bag produc­ tion of the mushroom were studied. Several strains with desirable characteristics were isolated and physiologi­ cal studies were conducted.

III. Public Health, Energy and Environ­ mental Application.

3.1 Field Trials of Mosquito Control Field trials of mosquito control using spore Using Spore-forming Bcateria in Thai forming bacteria has been carried out in an area in land. Bangkok as well as in rural areas where cases of malaria (Mahidol University) were found. The study has indicated that the B. sphaer­ icus 1593 gave a good control effect on A. minimus faIVae for approximately three months. While the B. thuringiensis H-14 was effective against A. albopictus. Current research is concentrated on the minimizing of the cost of production of the microorganism and search­ ing for effective means for dispensing the microorgan­ isms.

3.2ConversionofSolid WastetoMeth­ Solid waste from fresh market were digested ane by Two Stages Anaerobic Diges­ to produce biogas utilizing a two stage anaerobic diges­ tion. tion technology. Effects of operating parameters were (Chulalongkorn University) stUdied.

3.3 Utilization and Treatment of Tapi­ The major aim of the project was to produce oca Starch Factory Effluent. valuable products from tapioca starch factory effluent. (King Mongkut's Institute of Technol Bench scale experiments have led to two pilot produc­ ogy, Thonburi) tion systems for the production ofbiogas for use as fuel in the starch factory and the production of Spirulina for animal feed. Estimated internal rate of return for a full scale biogas plant was shorter than five years in most cases. Commercial scale production of spirulina would depend on marketability of the product. 251

Project Description

3.4 Preparation of Opisthorchis viver­ The major aims to produce the specific anti­ rini Antigents by Genetic Engineering gen for 0. viverrini by rDNA Technology using E. Coli Techniques for Use in Immunodiag­ as host organism. It was found at this early stage of the nosis. project that it was possible to prepare RNA and con­ (Mahidol University) structed cD NAwithout the requirement for poly(A) tail of the mRNA.

IV. Strengthening of Basic Infrastruc­ ture.

4.1 The Preparation of Genetic Engi This project aims to stimulate the develop­ neering Materials as a Service. ment of genetic engineering in Thailand through the (Mahidol University) preparation and distribution of some basic genetic engineering materials as a service. The following ma­ terials have been prepared : restriction endonu­ cleases(EcoRI,PstI andBamHI),plasmid vectors(pBR 322,pBR 325 and pUN 121) DNA marker (pCBl-6), and T4 DNA ligase.

4.2 Research and Development of Cell The major objectives are to develop a mam­ Technology for Production, Collection, malian cell culture depository to serve the increased and Dissemination of Mammalian Cell demand in Thailand and to provide training in tissue and Tissue Cultures. and cell culture handling and manipulation. Thirty four (Mahidol University) cell lines are currently available. Techniques for pri­ mary culture of a number of mammalian tissues were developed.

4.3 Microbial Culture Collection and The main purposes of the program are to Services Program. collect and preserve microorganisms, to supply cul­ (Thailand Institute of Scientific and tures on demand and to help and assist researchers Technological Research) seeking for strains which are not available in Thailand. The methods of preservation used are lyophilization storage in liquid nitrogen.

4.4/n vitro Germplasm Collection and The programme aims to carry out research and Exchange of Economic Plants. development on tissue culture technology as applied to (Kasetsart University) the development of plant seeds and to use cryopreser­ vation technology in preserving germplasm. Germplasm of species of sugarcane,papaya,soy bean,tomato,fruit trees, and other cash crops are to be available by 1990. 252

Project Description

4.5 Development on the Production of Glucose and Fructose from Sucrose by Chromatographic Methods. (Kasetsart University) Project 4.5-4. 7 are small projects aim­ 4.6 Study of Methods for Agarose Pro ing at fostering the development of manpower in duction from Gracilaria spp. in Thai biotechnology. The project may later develop land into a larger programme under the three areas of (Srinakarinwirote University) application. 4. 7 Technological Development for the Commercial Production of Viral Insec ticide. (Kasetsart University)

Table 3 Summary of Works Undertaken at the Affiliated Laboratories.

Affiliated Laboratories Activities

1. Plant Genetic Engineering Unit at Organized in 1985, the unit is active in thearea Kasetsart University of development of plants of economic importance to Thailand resistant to diseases and adverse conditions, and the development of pest protection techniques. Current activities include development of papaya resis­ tance to the ring spot virus and tomato resistance to the yellow leaf curl virus. In addition, the unit regularly or­ ganizes seminars and workshops on various aspects of plant biotechnology.

2. Microbial Genetic Engineering Labo Organized in 1985, the unit is active in many ratory at Mahidol University areas of genetic engineering. Current activities include the success in cloning and expression of mosquitolarvi­ cidal -endotoxin gene of B. thuringiensis in E. coli and the development of DNA probe for the detection of microorganismswhich cause fish disease. The unit has also produced highly specific and sensitive DNA probes for detection and classification of mosquitoes and material parasites. National seminars on genetic engi­ neering and DNA probe technology are organized regularly.