RECENT ADVANCES IN LIVESTOCK PRODUCTION TECHNOLOGIES PROCEEDINGS OF A NATIONALWORKSHOP
N A
Editors
Dr. B. R. Mangurkar Dr. A. L. Joshi
I ARCHIV
101490 •0 F AIF Development Research Foundation a /Q/Lf/3
RECENT ADVANCES IN LIVESTOCK PRODUCTION TECHNOLOGIES
PROCEEDINGS OF A NATIONAL WORKSHOP
HELD MAY 21 - 22, 1993
Editors:
DR. B. R. MANGURKAR Research Programme Organiser BAIF Development Research Foundation Uruli-Kanchan, Pune 412 202
and
DR. A. L. JOSHI Research Programme Coordinator BAIF Development Research Foundation Uruli-Kanchan, Pune 412 202
BAIF Development Research Foundation Uruli-Kanchan, Pune 412 202 Maharashtra, India
I SPONSORS
Ministryof Agriculture, Government of India.
Indian Council of Agricuftural Research, New Delhi.
DRC International Development Research Centre, Canada.
HOSTORGANISATION
BAIF Development ResearchFoundation Uruli-Kanchan, Pune 412 202, India.
Printed : March 1994.
Price : Rs.50/-
Published by BAIF
Designed and Produced by Ashish Advertising, Pune. CONTENTS
Preface
Welcome Address 1 Dr. B.R. Mangurkar
Inaugural Address 2 Dr.V.Gnanaprakasam
Keynote Address : Gearing up for a Veterinary 5 Service in the 21st Century. Dr. C.M. Singh
Opening Remarks 15 Dr. Manibhai Desai
ANIMAL REPRODUCTION
Recent Advances In Biotechnology In Livestock 19 Production Dr. B.S. Prakash
Role of Hormone Assay In Monitoring Livestock 20 Reproduction Dr. V.M. Mehta
Recent Advances In Bovine Reproductive Technologies 23 K.L. Sahni
Experience In Embryo Transfer Technology During 34 Preliminary Trials Carried Out At P.K.V., Akola M.S. Kadu
Effect of Different Cooling Rates in Programmable 43 Freezer On The Quality Of Frozen Buffalo Semen M.R. Bhosrekar, S.P. Mokashi, J.R. Purohit, S.B. Gokhale and B.R. Mangurkar.
Recent Advances In Livestock Production Technology: 53 Gujarat's Experience Dr. B.K. Bhavsar and Mr. H.H. Chhaya ANIMAL NUTRITION
Constraints and Perspectives 57 In Dairy Animal Nutrition in India V.D. Mudgal
Role of Fodder Crops, Bushes and Trees 73 for Livestock Feeding L.L. ReIwan
Production and Utilization of Green Fodder, Bushes 83 and Trees for Livestock V.S. Upadhyay
Recent Advances in Approach Towards Development and 86 Research in Livestock Production D.V. Rangnekar
ANIMAL BREEDING
Breeding Strategies.tor Buffalo Improvement in India 97 S.C. Chopra
Field Recording and Sire Evaluation under Indian Conditions 104 S.B. Gokhale and B.R. Mangurkar
ANIMAL HEALTH
Strategies in Animal Health Programme 113 K.P. Mallick, P.N. Kaul and D.S. Baiain
Application of Biotechnology in Veterinary 122 Diagnostics and lmmunobiologicals M. R. Gaendragad and C. Natarajan
Biotechnological Approaches in Diagnosis and 131 Control of Pathogens Transmitted Through Semen S.N. Singh, P.K. Gupta, S. Kilari, R.S. Kataria, N. Pandita & M.N. Takale
Strategies in Animal Health Programme 135 Dr. V.A. Sapre
Group Discussions and Recommendations 141
List of Participants 145 PREFACE
Livestock forms an integral part of small holder mixed farming systems in developing countries. Cattle, buffaloes, sheep, goat etc. are maintained for various purposes like security, status, food, draft, fuel. Livestock component in mixed farming systems also serves to augment the farmer's income. The problems related to livestock production mainly consist of animal management (breeding, reproduction, health and nutrition), marketing and quality of the produce, and socio-economic constraints. Different technologies have been suggested to overcome some of these problems. It is however essential to examine their relevance to our situations, their adaptability, costeffectiveness, and probable impact. It is recognised that the pace of these innovations is much greater than their adoption by human minds, especially in the developing countries.
Our late founderPresident, Dr. Manibhai Desal was one of the few distinguished disciples of Mahatma Gandhi who endeavoured to translate Gandhiji's philosophy of rural development into action. He has shown that through the adoption of appropriate technologies, the degraded resources (like unproductive livestock, land, vegetation and water) can be transformed into valuable assets and further that the rural communities can be involved in the process of development. He advocated a synchronisation of the wheels of research and development for improving the quality of life of the rural masses. This requires constant exchange of information between the laboratories, farmers and administrators. The workshop has fulfilled this objective by bringing together researchers and extension workers involved in livestock development in the country for better understanding of the tasks ahead.
It is sincerely hoped that this proceedings will stimulate new interest on the research in relevant and practical technologies for developing countries.
We gratefully acknowledge the support received from Indian Council of Agricultural Research, New Delhi, International Development Research Centre, Canada. Ministry of Agriculture, Govt. of India and MIS. DAAPS Cryogenic, New Delhi., M/S 1BP New Delhi. We are thankful to the Director, NIBM, Pune., Director, MIDA and Director, Animal Husbandry, M.S. Pune. fortheir kind cooperation in organising the workshop. Our sincere thanks are due to Ms. Tinku Dhar for valuable editorial support and also to our colleagues at BAIF, for their cooperation.
B.R. Mangurkar A.L. Joshi WELCOME ADDRESS
DR. B.R. MANGURKAR
Respected Dr. Manibhaiji, President, BAIF, Dr. C.M. Singh, President, Veterinary Council of India, Dr. V. Gnanapraksam, Vice-Chancellor, Tamil Nadu Veterinary & Animal Sciences Uni'iersity, distinguished guests, Directors of State Animal Husbandry Departments, eminent scientists from the ICAR Institutes, and Universities, invitees, colleagues, ladies and gentlemen. It is my privilege to welcome you all on behalf of the organiser and sponsors.
The aim of this workshop is to provide a common platform for all those concerned with Research, Development and Extension in Livestock Production. The deliberations will highlight new horizons of technologies and their assessment in our situations. I am sure with your active participation, the workshop will conclude with fruitful recommendations which can be utilised for development of livestock in the country. Dr. C.M. Singh has kindly agreed to deliver the Keynote Address and Dr. V. Gnanaprakasm has consented to inaugurate this workshop. I request Dr. Manibhaiji to preside over the function.
I would like to express my sincere thanks to IDRC, Canada, Ministry of Agriculture, Govt. of India and othersfor their moral and financial supportwithout which this workshop would not have been realized. Once again, I am thankful to you for your attention and hope you will enjoy your stay.
1 INAUGURAL ADDRESS
DR. V. GNANAPRAKASAM Vice-Chancellor, Tamil Nadu Veterinary & Animal Sciences University, Madras
With the growing human population, optional and judicious utilisation of food resources of animal origin has become a subject of vital concern, to meet the increasing demands for high quality animal protein. A strategic approach to satisfy this demand lies in upgrading our conventional animal production systems by transforming from the existing extensive to semi-intensive and intensive production capabilities. Harnessing and maximising the use of available land and water-spread areas for animal and fish protein undoubtedly forms the basis for farming operations for intensive production systems.
The animal industry in India has shown a spectacular growth and it should be no exaggeration to claim that at present, no other branch of agriculture has attained such rapid strides in its development like milk, egg and meat production. The introduction of exotic germplasm in cattle and hybrids in poultry has paved the way for commercial production of milk and eggs. The improvement in genetic quality combined with better nutrition, health cover and overall management on scientific lines has given a boost to the commercial production. Of late, more and more research and development programmes are directed towards the application of biotechnology for animal improvement.
1. Nutritional Efficiencies And Feed Technology
India with nearly 275 million heads of cattle and buffaloes and another 125 million heads of sheep and goats is facing a situation where there is shortage of about 44% dry fodder and 38% green fodder. Considering the limitation on the land available for fodder production and pasture development, it is necessary to use fibrous crop residues as feed for ruminants. Cellulose is the most commonly and abundantly available organic compound in nature. According to an estimate, the net photosynthetic productivity of earth is about 155 billion tonnes dry matter per year, of which about 63 billion tonnes are the lignocellulosics including 6 billion tonnes of the crop plants. These lignocellulosic crop residues (cereal straws, stovers and sugarcane bagasse) contain 30 - 40% cellulose, 16 - 27% hemicellulose and 3 - 13% lignin. However, because of their low nutritive value, there is an imperative need to upgrade them for use as livestock feed.
2 2. Growth Promotion
We should obtain maximum growth in a specified periodto obtain more economic return from the animal production systems. In the recent past, various growth promoting agents like probiotics, hormones etc., were used to improve growth rate and maximise production in various livestock species.This being a new field, we have to exploit this area to the advantage of animal production systems.
3. Disease Diagnosis
Early diagnosis and identification of the causal agent form the most important criteria for adopting appropriate measures for the treatment, and control of animal diseases. The conventional methods used for this purpose are lacking in rapidity and accuracy. They also involve large quantities of biological reagents and do not have the needed sensitivity to detect clinical cases early. With modern methods, identification of microorganisms or their antigens is possible in vivo or in vitro using the more sensitive radio-immune assays. Similarly, the identification of such antigens is also possible through the method of tagging fluorescent dyesto corresponding antibodies and examine for specific fluorescence under uv light. More specific qualitative and quantitative assays for immune response for definite diagnosis have also been made possible through the use of enzyme linked immunosorbant assay (ELISA) system. The development of molecular biochemistry has provided rapid and sensitive methods for precise diagnosis of many animal diseases by detecting the specific protein subunits of the infectious micro-organisms as elicited by the gene sequences in the genetic material. The application of genetic engineering techniques in recent years has added precision to the diagnosis of diseases.
4. Genetic Progress
The advent of biotechnology has brought in potential impact on animal breeding. The application of genetic engineering has opened up vistas for (1) production of new products (2) enhancing the efficiency of production of existing products; and (3) modification of present products to enhance or reduce certain components. Of these, improving the efficiency has probably been the greatest alteration since the efficiency in production of milk, meat, fibre etc. from animals is beneficial to the farmer as well as to the consumer. The technique of embryo transfer has made it possible to exploit the genetic potentiality of both sire and dam for rapid improvement in the genetic potential for milk production.
3 5. Post-harvest Technology
Millions of cattle, sheep, goat and poultry are slaughtered every year in our country. A number of slaughter house by-products like blood, viceral content and inedible offals, horn, hoof and bone are not properly processed for preparing various value added products for industry and human animal consumption. This field is a rich mine for exploiting the economic utility of these by-products.
6. Farm Management
Due to modernisation, animal and poultry production has been transformed in the last two decades. It is today commercially viable and an agro based industry contributing significantly to the gross national product. This development has been largely possible due to the combination of various factors such as improvement in breeding, feeding, management technology and also due to active participation of private entrepreneurs. Housing is one of the important components in the step towards disease management in intensive production. Houses are designed in such a way that the livestock are subjected to least stress and disturbance. Automatic feeders, lighting, delivery of feed in bulk quantities, automatic waterers, ventilation system and medication dispensers have brought down not only the labour requirements but also provide optimum comfort with least stress to the livestock.
Agrotorestry For Livestock Feeding
As per the National Forest Policy, the forest cover should be 33.3% of the total land area of the country. But in our country only around 15% is under forest cover. Further, improvement of forest area is not possible because man keeps on expanding his territory of activities for habitat and dwelling. In this situation, Agroforestry has a vital role to play in strengthening the forest policy. This will also help animal husbandry production systems like stall feeding of animals with tree fodder. It will also save grain feeding of animals which will be a costly proposition in the years to come.
Our country has 26 cattle breeds, 7 buffaloe breeds, 20 goat breeds, 40 sheep breeds, 6 horse breeds, 4 camel varieties and 18 poultry breeds. These valuable germplasm should be preserved for the existence of human race and to evolve efficient livestock production systems.
I have touched only a few areas of the modern technology available for efficient livestock production systems. In this context, we need land saving agriculture and grain saving animal husbandry to sustain modern livestock production systems.
4 KEYNOTE ADDRESS GEARING UP FOR A VETERINARY SERVICE IN THE 21ST CENTURY
DR. C.M. SINGH President, Indian Veterinaty Council
Manibhaiji, distinguished guests, officers, scientists and participants of this workshop on 'Recent Advances in Livestock Production Technologies'. I am indebted to the organisers for inviting me to addressthis important congregation of professionals. Being the President of the Veterinary Council of India, I would begin my words with the professional responsibilities that we owe our country and its great people and of course, to our animal folk who serve us uncomplainingly.
Veterinary services in India include animal health care, treatment of diseased animals, organisation and management (macro & micro management) of animal and avian farms, protection of public from diseases spread through animals, animal products and animal wastes. There is an additional responsibility of biodiversity and control of environment to the benefit of man and animals.
The field scenario in India has undergone drastic changes over the years. There is an increased awareness on the value of pressure food of animal orgin and other animal products. This has necessitated intensive management, resulting in pressure on the animal. This in turn has necessitated an assured protection from diseases, deficiencies and casual management.
Import of animals and transport of biomaterials from across the world has introduced newer diseases. Exotic animals are unfamiliar to Indian environment and our system of management, and are more prone to diseases in comparison to local animals. With the introduction of new methods of breeding and production, a veterinarian's responsibilities for hi-tech work has increased. Newer diseases call for quickerdiagnostic and therapeutic methods. Increased value of animals now warrants lengthy and expensive treatments which hitherto were not in demand. The compassion for life in India, makes slaughterof uneconomic and diseased animals impractical. This has added the responsibility for treating them even if there is no return from them. Deterioration of environment has put additional stress on animals and more responsibilities on veterinarians. Public health hazards due to diseases of animals, animal wastes and animal products have risen with increased use of animal products and closer animal-man contact.
5 The graduate veterinary education programme is neither meant nor expected to prepare a graduate on all aspects of specialised service requirements in veterinary science. For this purpose there are post-graduate and continuing education programmes. The time has come when a general veterinary practitioner is frequentlyfacing the need for specialised services to support his day to day practice in diagnosis, treatment, micromanagement etc. Disease surviellance and disease prediction are as important as emergency disease management, special diagnostics and special therapies or malor surgery.
Livestock management has more vistas to cover since commercially viable stud farms, poultry farms, hatcheries, animal product units, animal feed processing units, rabbitry etc. are coming up.
The entire responsibility will be difficult for the govt. to burden itself with, if not provided relief by private sector organisations by sharing some responsibilities.
Now, I would like to focus on our future responsibilities that are to be confronted by the turn of the next century. Health production and technology are the inseparable parts of our major responsibility as professional veterinarians.
CERTAIN EMERGING AND EXOTIC LIVESTOCK DISEASES
During the last four decades, a number of emerging diseases of livestock and poultry have been introduced, diagnosed and established in India e.g. :- blue tongue, infectious bovine rhinotracheitis, parainfluenza Ill, bovine leukosis, infectious bronchits, infectious laryngo-tracheitis, avian encephalomyelitis, marek's disease, eggdrop syndrome, infectious bursal disease, gumboro disease, avian mycoplasmosis, equine influenza, equine infectious anaemia, suspected cases of equine cerebral exanthema, etc.
There are diseases which are important to India like African Swine Fever, Vescicular Diseases of cattle and Swine. Aujeszky's Disease,, Porcine Parvo Virus, African strains (SAT-i SAT-2, SAT-3) of Foot and Mouth Disease (FMD), Bovine viral diarrheoal mucosal disease complex, Limpy skin disease, Louping ill, Bovine Spongyform Encephalopathies, Ibraski disease and Jembran disease in cattle, certain strains of Blue tongue virus infections, pest des petit-ruminants, Rift valley fever in sheep, Caprine arthritis/encephalitis (lentivirus infection) in goats; Contagious equine metritis; Fowl plague, Duck virus hepatitis, Reticuloendotheliosis in poultry and man, and others including haemorrhagic disease of rabbits. Many of these diseases are prevelant in other countries including neighbouring countries.
6 RETROVIRUS IMPOSES A BIOLOGICAL THREAT TO MAN AND ANIMALS
Retroviral infections in man have been recognised only from 1976 whereas animal retroviruses have been studied much earlier. However, with the emergence of Acquired Immuno Deficiency Syndrome (AIDS) caused by Human immunoefficiency virus (HIV) an unprecedented emergency has been created and global challenge has been made to mankind for undertaking urgent steps to tackle the problem at international, national and various other levels.
The microbial threat retroviruses in and lenteviruses in posed by " general particular, can be very well appreciated in the words we are facing a catastrophe unmatched since the Black Death of middle ages. If ever we needed an international effort it is now". (World Health, Nov. 1987).
WHO has started a drive for worldwide efforts on "EDUCATION FOR HEALTH" programme with a symbol and special pullout supplement in WHO magazine such as "World Health". During the year 1987, the continuing spread of AIDS has been placed in the fifth place amongst ten top information polled by Associated Press, New York.
It may also be worthwhile to mention that it is universally recognised that the knowledge already available on the studies conducted on animal lentiviruses, such as Equine Infectious Anaemia Virus (EIAV), VisnalMaedi Virus (W) and Caprine Arthritis Encephbalitis Virus (CAEV) have helped in understanding the nature of the primate lentiviruses e.g. Human Immunodeficiency Virus (HIV) and Simian Immunodeficiency Virus (SIV) in their close relatedness and developing animal models for augmenting research on lentivirus. The comparative knowledge on lentivirus studies, non availabilityof effective treatment or immunobiologicals and diagnostics, haterogenecity of strains, lack of facilities for molecular virology with containment laboratories for screening out prevalence of viruses and their variants in the populations particularly in developing countries have posed a serious problem to understand and contain the maladies induced by these group of viruses.
Animal lentiviruses are included in the livestock emergency diseases by FAO and require international regulations for prevention and control in the areas free from these diseases in the world. It is imperative to safeguard against the intrusion of animal lentiviruses such as CAEV and EIAV through the import of animals to developing countries from developed countries where the lentivirus infections are prevelant (FAO/RAPA). Gobal efforts are also required for taking precautions and strict measures and to educate all the concerned people including the scientists against the dissemination of diseases through frozen semen and embryo transfer. (Part III the proceeding of the 75th session, Indian Science Congress. 1988).
7 BIOTECHNOLOGY IN VETERINARY SCIENCE
It would be interesting to note that since the major break-through in molecular biology, in recent years, much greater strides have been made in the applied aspect of Biotechnology in biomedical sciences.
A number of discoveries relevant to veterinary science place this field in the forefront of such progress. The responsibilities of veterinarians are considerable because these discoveries ( be it medicine or vaccine) have first to be tested in animals whether in the form of products administered or feed given to them.
In the field of Biotechnology ONBS, ETT & MOET has brought in a breakthrough in Animal production. Other fields that benefitted are disease diagnosis (Hybrydoma, Monoclonal DNA probes, Probiotics, Rumen microbe manipulation), Pharmacology (Insulin from cell culture, target oriented drugs), Genetics (Restricted Fragment Linked Polymerisation) etc.
All these have a large component of training need for veterinarians leading to high degree of professional perfection and skill, demanding an entry into a realm of Superspeciality in veterinary science. Such procedures are seldom derived from a single discipline or a single discoverysince they result from simultaneous application of several of these discoveries for solving a particular problem.
It is imperative that an integrated approach involving the expertise of animal physiologist, veterinary biochemists, animal geneticists, animal breeder, animal gynecologist, animal health specialist, clinician, and surgeon would be the only scientific way to go about such a venture. Many such ambitious ventures where one has tried to be the 'know all' has proved to be either a non-starter or has provided no tangible results inspite tall claims. What one must understand is that in the initial stages all the animals involved are to be screened periodically, handled aseptically, minitored scientifically, examined precisely and reared efficiently. The scope is unlimited if biotechnology is given its due respect but hazardous if mis-handled. It is a stake that demands high monetary input, high tech laboratory and very careful handling.
OPEN NUCLEUS BREEDING SYSTEM (ONBS) WITH MULTIPLE OVULATION EMBRYO TRANSFER (MOET) The description, scope, advantages and disadvantages of Open Nucleus Breeding System with Multiple Ovulation and Embryo Transfer has recently been reviewed at a recent Regional Workshop on Biotechnology in Animal Production and Health held at Bangkok in 1988. Dr. Bhat, Director IVRI, Izatnagar also advocated the use of this strategy (ONBS and MOET) for cattle herd improvement in developing countries, at this seminar.
8 In view of the importance of research on buffaloes, an FAO Round Table meeting was held at New Delhi in 1988 in conjuction with the llnd World Buffalo Congress. The meeting also highlighted the importance and need for intercountry cooperation in buffalo research and development.
A very important conference on the Open Nucleus Breeding system was arranged by FAO at Biatalerzegi, Poland in June, 1989 in collaboration with the Polish Academy of Sciences. It is the committee's assessment that a faster growth in the pedigreed sector can be achieved through the open nucleus breeding system. Geneticimprovement of livestockin developing countries using the Open Nucleus Breeding System has been presented by Dr. John Hodges. El can be utilized as necessary adjunct and supplement to existing breeding practices for genetic improvement of cattle and buffaloes. In contrast to progenytesting of males, a new option is now available by using the new technology of Embryo Transferwith Multiple ovulation (MOET) in a Open Nucleus BreedingSystem and this is considered valuable for developing countries.
The advantages and disadvantages of nucleus breeding units compared with field breeding schemes are
Advantages
* Genetic lift in establishing the unit. * Faster rates of genetic change. * Control over husbandry and testing. * More selection possible for economic merit. * Use of traits difficult to record in field. * Connection of breeding resource. * Possible use of exp9nsive technologies. * Economic benefits obtained sooner. * Low costs on. a national scale. * Separate nucleus units for different sets of breeding objectives/environments.
Disadvantages * Risk of disease and loss * Risk from concentration of stock and resources in one unit. * Possible genotype x environment interactions in commercial production. * New funds needed to set up and operate. * Motivation of producers to accept MOET bred stocks.
9 HEALTH AND REGULATORY ASPECTS OF EMBRYO TRANSFER
According to the document on safety guidelines and procedures for embryo transfer in animal sciences prepared by the DBT, Ministry of Science and Technology, October 89, the recommendation in respect of safety aspects relate to a) Having standardised laboratory facilities for ET. b) Thorough screening of donor recipient animals. c) Health evaluation with particular reference to reproductive diseases among the recipient animals. d) A standardised procedure for handling of fresh and frozen embryoswith information on the following aspects (through standard forms).
i) Genetic profile of dam and sire and their health status.
ii) Time and place of collection
iii) Time and place of freezing and procedures used.
iv) The individual(s) responsible for collection and handling of embryos.
v) Strict identification of embryos including age, quality and processing procedures.
vi) Record of any micromanipulation, use of spare zona DNA recombination and cloning.
vii) Identify the health status of zona donors.
viii) Details of birth recorded through earlier transfer involving the above siredam and the health status of the new born calf.
ix) Health status of recipients. e) Development of data and sera bank in the country for immunological testing of transmissible diseases. f) Setting up an apex body of Veterinary/Animal Scientists to oversee the embryo transfer programme particularly, when embryo micromanipulation of DNAtechnology
10 is involved. Setting up of a statutary authority under the Govt. of India to regulate the import and export of genetic material. This would also involve document examination, certifIcation etc. g) Identification of research laboratories and institutions in the country capable of taking up research/development work in biotechnology of ET including disease investigations.
According to the manual of the International Embryo Transfer Society (lETS), the recommendations for sanitary handling of embryos have been based on the conclusions of the Research Sub-committee of the lETS Import/Export Committee reproduced from the Scientific and Technical Review of OlE Vol. 10, NO. 2 1991 as follows:
Category 1.
Diseases or disease agents for which sufficient evidence has accrued to show that the risk of transmission is negligible provided that the embryos are properly handled between collection and transfer
- Enzootic bovine leukosis. - Foot and mouth disease (cattle) - Brucella abortus (Cattle) - Infectious bovine rhinotracheitis (trypsin treatment required) - Pseudorabies (swine) (trypsin treatment required).
Category 2.
Diseases for which substantial evidence has accruedto show that the risk of transmission is negligible provided that the embryos are properly handled between collection and transfer, but for which additional transfers are required to verify existing data
- Blue tongue (cattle) - Hog cholera (classical swine fever).
Category 3.
Diseases or disease agent for which preliminary evidence indicates that the risk of transmission is negligible provided that the embryos are properly handled between collection and transfer, but for which additional in vitro and in vivo experimental data
11 are required to substantiate the preliminary findings
- Rinderpest (cattle) - Bovine virus diarrhoea - Blue tongue (sheep) - Campylobacter foetus (sheep) - Foot and mouth disease (swine, sheep and goats) - Swine vesicular disease - African swine fever - Scrapie (sheep) - Haemophilus somnus.
Category 4.
Diseases or disease agents on which preliminary work has been conducted or is in progress.
- Akabane (cattle) - Vesicular stomatitis (cattle and swine) - Chlamydia psittaci (cattle) - Ureaplasma - Mycoplasma spp, (cattle and goats) - Maedi/visna (sheep) - Pulmonary adenomatosis (sheep) - Scrapie (goats) - Bluetongue (goats) Caprine arthritis - encephalitis - Parvovirus (swine) - Enterovirus (cattle and swine) - Leptospirosis (swine) - Bovine harpes virus - 4 - Mycobacterium paratuberculosis (cattle) - Brucella ovis (sheep) - Brucella abortus (sheep) - Border disease (sheep) - Para influenza-3 virus (cattle) - Bovine spongiform encephalopathy agent.
It may be further added that health monitoring should be implemented in letter and spirit in the ET programme. Similarly the recommendations of the scientific and technical review of OlE Vol. 9 No 5 1990 in respect of Biotechnology and Veterinary science should be utilised for control and regulation of Biotechnological procedure in Veterinary Science. The safeguards implemented at research laboratories should guarantee
12 containment (premises of categories P1 to P4) and biological containment (classification of the risk of an escape from I to IV for live organisms manipulated in the laboratory). It is further emphasised that the safety regulations for India for recombinant DNA research based on the report of the Recombinant DNA Advisory Committee" of the DBT should be implemented.
In view of the international movement of frozen semen/embryos the set of requirements for import of semen/embryos as laid down by Govt. of India and FAQ recommendation for movement of embryos should be made obligatory to all the laboratories/institutions carrying out work on frozen semen and embryo transfer.
In contrast to the relatively lower risk of transmission of bacterial and viral diseases transmitted through embryo transfer technology, much greater potential of transmission of diseases is through the semen specially the frozen semen.
The committeetherefore reccomends that the pathobiological aspects of embryotransfer relevantto Indian conditions should be carried out at IVRI, while it should be mandatory for all frozen semen laboratories/centres to establish semen microbiology laboratory for screening against bacterial, protozoan and viral diseases at least on a level of identification of microorganisms. The reference laboratories must be made available at IVRI for further specification of the type of microorganisms involved. It would also be necessary to grant recognition of a network of laboratories on regional as well as institutional basis in view of the infrastructural facilities and expertise available in the country for carrying out the biotechnological diagnostics. To achieve this objective, a suitablecollaborative project should be funded jointly by DBT and ICAR with international agencies if considered necessary.
Utilisation And Control Of Biotechnological Procedures In The Field Of Animal Health. (Resolution of the OlE international committee, 58th gn session -1989).
Considering the progress made in biotechnological procedures during recent years, the possibility of applying such procedures to diagnosis and to prevention of animal diseases with the objective of improving animal production, the potential ethical implications of certain applications of biotechnology particularly genetic manipulation and due to the urgent nature of biotechnological developments, the committee recommends that
1. Member Countries bring to the attention of OlE, the results of their research for the application of biotechnological procedures for diagnosis, prevention and treatment of diseases of animals with details of methods used. These should include bioengineered vaccines, diagnostic tests utilising monoclonal antibodies and nucleic acid probes, proteins of medical importance, development of transgenic animals and molecular basis for disease resistance.
13 2. The development and implementation of these procedures at the different stages of their development, such as trials in the laboratory and confined field tests before environmental release remain under the control of the authorities both national and international.
3. The research be conducted, under safety containment guidelines established by competent authorities in each country. It is suggested that each country establish specific legislation for the registration of products or animals obtained by biotechnology processes.
4. The member countries provide OlE with their existing or pending legislation and regulations regarding the use of products or animals obtained by biotechnology processes.
5. The Director General calls on the services of a group of experts whose tasks shalt be, in consultation with the standards commission, to prepare guidelines or legislation concerning utilisation of products of biotechnology, which have fulfilled the requirements for environmental release in the country of origin and the country of use.
To conclude, I must admit that the task before us by the turn of the century is herculean. But the fact remains that many countries have already braced themselves to the situation that await the world which is becoming smaller and smallerwith lightning transport and communication systems. We in India cannot afford to be conplacent or indifferent to the developments that are materialising around us. I wish and expect that a workshop of this nature would sow the seeds for a meticulous planning, that will ferry us comfortably along the 21st century and bring brighter colours to the profession and fruits of its benefit to the people.
JAI HIND
14 OPENING REMARKS
DR. MANIBHAI DESAI Founder President, BA/F Development Research Foundation, Pune
Friends,
We have very learned and experienced veterinarians here: Dr. Gnanprakasam, Vice Chancellor of the first Animal Science University in India and Dr. C. M. Singh, the doyen of veterinary profession. Unfortunately another senior scientist, Dr.P.N. Bhatt was not able to come. I was keenly listening to the views expressed by the earlier speakers and wish to make the following observations:
My first concern is that we must be very careful about adopting or imitating whatever is being done in the Western World. We are in a very adverse climate which cannot be compared with the Western World. Animal behaviour regarding growth, production and disease very much depends on the environment where the animal is living.
The rigorous environmental and social conditions limit the availability of facilities and resources to the farmer who is managing the animals. We must thereforebe very careful before transplanting exotic technologies from developed countries. If this factor is overlooked, the chances of rejection by the farmer would be high. Farmers will follow what they are able to manage. You are going for a very modern technology and producing an excellent animal. How will you protect this calf ? What do we do to feed this calf ? What is the health cover you can offer ? What is the management level you have ? So you have to also bear in mind that we walk on the soil, Indian Soil, and not just fly in the air. I don't want to discourage you to go for modern technology, but this is a very important factor which we cannot afford to overlook.
Second thing, is the cost aspect. In the inaugural speech at this conference, Dr. Gnanprakasam has given a very commercial approach which is very much needed, but we have to see what we can manage. Equally important, is the economics which farmers can manage. How livestock can provide self employment to the farmer and improve his economy ? It has great potential which we have demonstrated in India, but when you go ahead, there are dangers.
We had a very strong difference of opinion with few experts who were not for cross breeding. They advocated that you must bring pure exotic. Don't try for this 50%, 62.5% or 75% and they were quoting Israel. I said Israel is very fortunate in one thing. The Israelis eat beef, they cut the unwanted cow and put it on the table next day.
15 You cannot overlook that in this country as there is a ban on culling. You cannot get rid of the calves which may not be required. You cannot sell it off. You just have to throw it away. So what do you do with rejected animals ? When culling is not possible, improvement of cattle is extremely slow.
You starve the cow, and still worship her. You torture the cow but don't kill her. On the contrary, the well-wishers of the cow collect more money. When a cow is in a bad stage, people immediately give funds and save the cows. This is a very important hurdle in Indian conditions. These are the practical problems which you have to face.
In Indian conditions you can cull a goat, and a sheep and a bull and even a buffalo. Sometimes excellent buffaloes are unfortunately culled. Those which are cullable are not culled. But when an excellent buffalo (Murrah) coming from Punjab to Bombay, is sold for beef-buffalo meat, you are losing the germplasm.
I was working with a small herd of Gir breed of cattle and I was awarded the Gopal Ratna. But friends, our concept of improving a breed is also narrow. After fifteen years of experience, I can say that improving a breed is very important but you must have atleast 10,000 cows. These may be with the farmers. The farmer is willing to improve his Tharparkaror Sahiwal or Gir or Kankrej or Rathi but he cannot wait. He therefore, wants a cross-bred. ICAR is discussing indigenous cow projects since last seven years. In principle they say that it is a very good idea. But good ideas cannot come on the ground unless they are funded. There is one shortage and that is shortage of money. In this situation let us be very realistic, practical and take those steps which can be taken 10-15 years ahead. I am very much for innovations and new technology but not for readymade transferred technology. We must adopt new technologies to serve mankind and to make the whole industry commercially viable, not for us but for the farmer who is managing the livestock.
In Jaisalmer, we have Tharparkar and Rathi. Nothing to drink and nothing to eat, but there are cattle - and an excellent cattle. How has it survived ? Has anybody bothered to find out? Why such cattle tracks are developed in dry areas ? In Gujarat, in Dang in Kutch, (850 Sq.miles) there is nothing but cattle - excellent Kankrej. We don't know how they are managed. So these are the things, the indigenous and domestic wisdom and technology, which also should be studied properly along with the Western Modern Technology and choose what is relevant for India.
I think we have to learn a lot from the persons who are managing livestock. They know much better than what we have been taught. There should be a mechanism in the Universities to study their knowledge and wisdom. They are really illiterate but their knowledge is innovative. They have no information, but they have wisdom. We
16 have to respect the farmers, although they may be illiterate. We should try to learn something from them and also try to learn how they have lived.
I got into animal production activity accidentally and I thought livestock industry has a fantastic role to play. It has great potential for employing illiterate people wherever they are staying. Many landless families are with us today, managing the cow and buffalo. The nutrition is very crucial and so is health. So, breeders should also know what you are able to feed to the new calf. Whichever excellent animals you are producing are all very precious and we should maintain them properly. For this purpose you should have a single package. The package of breeding, health and nutrition is very crucial. I do hope that you would strive to bring about the economic package of livestock technologies for the benefit of Indian farmers.
The next issue is, how to transfer the package to the farmer? Our experience in laboratories, in institutions or in organised farms, is quite different from farmer conditions. Whether you will consider a bridge between university and farmer for exchange of information? This is very important. Even Government Departments can also use NGOs as a bridge. Looking to the weaknesses of the system itself, to make it more effective and complementary to each other, we can involve the people's organisations, wherever possible. This should also be considered.
As the Founder Trustee of BAIF, I am very happy that you all are here. I hope this workshop will be very fruitful. Thank you very much!
17 ANIMAL REPRODUCTION RECENT ADVANCES IN BIOTECHNOLOGY IN LIVESTOCK PRODUCTION
DR. B.S. PRAKASH National Dairy Research Institute, Karnal 132 001
The sensitive and humane implementation of biotechniques in the livestock industry should focus on economizing yield of animals and producing animal products with high nutritional quality for man. In this process, it is to be kept in mind that such overall increase in livestock productivity is not associated with any detrimental impact on animal reproduction and health and on the environment. Our current state of knowledge relating to animal reproduction in the aspects of regulation of folliculogenesis, oocyte maturation, corpus luteum function and of late, on molecular and cellular mechanisms controlling reproductive processes have triggered off a spate of biotechnological innovations for a quantum improvement in animal productivity. The biotechnology of embryo transfer which has made considerable impact in augmenting reproduction, has also brought in its wake the application of a whole gamut of new biotechnologies. The emerging technology of in-vitro fertilization and embryo production has also developed to a point of commercial relevance. The ability to transfer genes into the germline of livestock can help create qualitative modifications in many of the traits of commercial interest for improving the productivity of animals. The use of nuclear transfer procedures to produce large number of identical bovine offsprings (cloning) is of great potential value for the multiplication of genotypes of superior economic value. The recent technique of separation of x and y bearing sperms through flowcytometry has heralded a procedure for producing bovine offspring of desired sex.
In addition, the endocrine biotechniques of oestrus synchronisation, pregnancy diagnosis and confirmation, parturition induction, application of hormone analysis for augmenting fertility, lactation induction and enhancement of milk production through recombinant growth hormone administration are particularly important for improving productivity in the Indian context.
19 ROLE OF HORMONE ASSAY IN MONITORING LIVESTOCK REPRODUCTION
DR. V.M. MEHTA College of Veterinaty Science & Animal Husbandty Gujarat Agricultural University, Anand 388 110.
INTRODUCTION
Immuno assays are methods for utilizing immunological reactions, to measure substances of biological or clinical importance. Such assays have inherent exquisite specificity due to their immunological nature and by various procedures can have high sensitivity. The immuno assays used in the past were relatively simple but the increasing appreciation of their versatality has led to highly complex assays.
In the beginning, the tests were almost all done manually but now these are being rep!aced by an automated system often accepted for large scale batch processing of samples. At the same time the progress in electronics has led to vastly improved data processing capabilities on instruments allowing much better interpretation of the results of tests.
The immuno assays according to their sensitivity for detecting antigen are classified into (a) Low sensitivity assays (Radial diffusion, Double diffusion, Counter current electrophoresis and Immuno-electrophoresis), (b) Medium sensitivity assays (complement fixation), (c) High sensitivity assays (Direct agglutination, Haemagglutination inhibition, Passive haemagglutination, immunofluorescenceand immunoperoxidase) and (d) Extemely high sensitivity immuno assays (Radio Immuno assay, Enzyme Immuno assay and Fluoroimmuno assay).
The livestock reproduction is monitored by high sensitivity immuno assays involving either radioactive tracers (RIA) or enzymes (ELISA). Endocrinology in animal production involves scientific evaluation of the hormonal influences in (a) health and adaptation, (b) growth and fattening, (c) reproduction and lactation, (d) gynaecological disorders (e) environmental stress, (f) hormone and drug therapy and (g) biotechnology. A critical approval is necessary to evaluate whether or not the estimates obtained are casual, symptomatic or meaningful.
Since the epoch making discoveryof Radio Immuno assay by Vallow and Berson (1959), the radio isotopic and nonisotopic immuno assays have been explained - keeping the principle of competitive binding between labelled and unlabelled (sample)antigens with homologus antibody. The systems using radio labelled antigens are known as Radio Immuno Assay and those using enzyme labelled antigens are Enzyme Immuno Assay.
20 Both the systems are precise, replicable and accurate when homologus system of antigen and antibodies are used. In employing the immuno assay, the following points need consideration.
(a) Extraction of samples where hormone (Antigen) concentration is extremely low. (b) Storage of samples without losing the activity. (c) Specific nature of sample-plasma, milk, tissue etc. (d) Estimation of true hormone (antigen) versus damaged hormone. (e) Diurnal variation and episodes of release. (f) Seasonal variation in hormone level. (g) Effect of animal handling. (h) Correlation with function.
Attempts have been made to use hormone immunoassay in selection criteria in animal breeding and reproduction. Some metabolic hormones have generated keen interest because they are involved in growth and lactation. The growth hormone, insulin, thyroxine and triodothyronine are related to growth and lactation. There is current discussion on value of measuring the thyroxine degradation rate in bulls as parameter to predict the lactation per(ormance of their offspring. Similarly, significantly higher circulating level of growth hormone were found in lactating dairy cows as compared to lactating beef cows. The growth hormone level study need intensive sampling since the hormone level exhibit diurnal variation and effect of feeding.
The importance of testosterone for normal sex function in male produced the idea of using testosterone determination to estimate or even predict male fertility. Though there is a dose response curve between androgen and function of male accessory sex glands, there seems to be low threshold level which is sufficient to maintain normal sexual behaviour and spermatogenesis. Moreover, there are fluctuations which demand intensive blood sampling due to diurnal variation in matured animal and pronounced changes around puberty. Stimulation tests may be helpful to overcome the lack ot correlation between actual testosterone values during the trial period and production abilities. One special steroid however, might be suitable as a parameter, the C-19 steroid 5 x-androst-16-en-3-one which is synthesised in the testes of boars and causing the characteristic sex odour in boar carcasses.
The use of immuno assay for animal production depends on further knowledge about physiological pattern of hormone level. In two examples it can be pointed out how a situation can be presented like the kind of 'hormone score'.
21 (a) The hormonal events of estrous cycle which can be elucidated for several species, the endoscopic observations of cow ovary with FSH level in blood have been correlated.
(b) The endocrine profiles have been correlated with the time of parturition. The endocrine events between foetus and motherhave been correlated and induction of early parturition has been made successful.
On the basis of physiological knowledge, biotechnological measures to control the oestrus cycle and to induce parturition have been developed. The immuno assays have been applied to monitor and supervise animal reactions after the application of hormone drug such as synthetic gestagen, prostaglandin, GnRh etc. Several studies were performed to control the response of endocrine system to environmental influences such as sexual stimulation, feeding regimen, starvation, heat stress, transport stress, and vitamin A & E deficiencies.
For further biotechnical improvement, application of immuno assays is required to elucidate the endocrine status viz.
(a) Oestrus induction in post partum cows and buffaloes. (b) Initiation of cycle in seasonally anoestrus sheep. (c) Exact oestrus synchronisation between donor and recipient animal in embryo transfer. (d) Determination of neuroendocrine reactions in milking stimulus as a parameter for development of improved milking machines. (e) Influence of nutritional factors in endocrine and reproductive factors.
One of the hormones, progesterone was shown to be a sensitive parameter for fertility control. In bovine species, the quantitative estimation of this hormone is made for diagnosis of pregnancy and reproductive disorders. Further, the subjective evaluation of oestrus period towards insemination to follow can be made by immuno assay. The conception is biologically impossible if inseminations are performed during the phase where the progesterone level in milk is 2 mg/mI. The importance of the application of immuno assay for cattle A.l. breeding management can hardly be over estimated as the figures recently obtained indicate that under controlled conditions, poor inseminations were performed in 15% of the cases. However in practice this figure is more than 26%.
The continuation of pathogenic processes and hormonal changes by measuring progesterone in specific clinical case of gynaecology has been found to be correlated. Oestrogen determination was applied for diagnosis of dead foetus. With regard to role of phyto-estrogens in fertility failure in cattle, the immuno assay can be used. The serious metabolic disorders of cattle and buffalo (parturient paresis) can be diagnosed by these techniques. The hormone assay systems have practical utility in understanding the reproductive physiology and applied reproduction in livestock.
22 RECENT ADVANCES IN BOVINE REPRODUCTIVE TECHNOLOGIES
K.L. SAHNI Indian Veterinary Research Institute, Izatnagar (U.P)
ABSTRACT
Advanced reproductive technologies have been applied in securing rapid rate of genetic improvement of dairy animals during the last decade. Inspite of its application during last 40 years, the A.I. Technology is only moderately successful. The research done on frozen semen technology has resulted in increasing the efficiency of cryogenic storage of sperms under tropics. Due to production of thinner ejaculates, the efficiency of Bos taurus semen has become lower although freezability is excellent. The semen of crossbreds is comparatively poor. The sephadex filtration technique has been found to be valuable in improving the semen quality which is initially poor. The incorporation of lower level of Yolk, lower glycerol and milk (1.5% fat) have given good results in freezing of buffalo semen. The development of faster cooling rates, shorter equilibration and faster thawing rates have reduced the operational period considerably. The crenellation pattern techniques (CPT) is considered as a field method for assessing the semen quality. Methods for predicting the freezability of semen and use of liquid nitrogen vapours for cooling have been developed. The technology of storage and freezing of semen in highly concentrated form and its use in micro-insemination of cattle need further refinement.
The oestrus activity in tropical cows and buffaloes is not so intense which is partly responsible for obtaining lower fertility rates. Besides getting control over oestrus, the low efficiency of oocyte production is a major limiting factor for genetic improvement through female line (E.T.T)
To become effective, these technologies require intensive system of management which is difficult to obtain in general farming situation of the tropics. The use of hormones is becoming increasingly important in clinical cases of reproductive disorders. These technologies are expensive and research has to be conducted on adjusting them to local economic conditions particularly for improving the fertility of bovine species. INTRODUCTION
Fertility is a complex expression of outcome of both male and female reproduction. Its objective is to maximize the profitability of animal production through effective management.
23 During the last two decades, several techniques have been developed for the effective control of reproduction in dairy animals. For the male, the technology of artificial insemination with frozen semen has almost become a routine in practical breeding but some new techniques have also been developed for increasing the reproductive efficiency of the female mainly through control of oestrus, superovulation and embryo manipulation.
Because of economic pressure and over increasing emphasis on efficiency through production of high quality animals, it is certain that the farmer would search for newer technologies in animal reproduction. The research and development organisations cannot escape from the responsibility of simplifying some of these techniques so that these become operative as well as cost effective in the long-run underfarming/husbandry conditions in developing countries. It should be understood that most of these technologies are costly and applicable under intensive management system.
There is a world-wide interest in the research and application of E.T.T. and therefore a good deal of knowledge has accumulated over the last decade. We have tried to present that knowledge along with expected future developments in this paper. Because of its comparatively long standing, the interest in research for improving the frozen semen technology is limited. However, the problem of lower fertility generally encountered in the field through use of frozen semen and also dependence of Ex-situ conservation of genetic resources on technology of cryopreservation, call for research/modifications under tropics. Accordingly, we have been conducting research on semen technology during the last few years at l.V.R.l. Izatnagar with the main aim of making this technology more relevant and efficient under tropics.
SEMEN TECHNOLOGY
Exotic Bulls (Bos-taurus) Sperm concentration is low otherwise its semen is normal and quite freezable. However, sperm production efficiency is only 30-50% of what it could be in advanced countries. This calls for modification in rearing and management practices for rearing exotic bulls under tropics. Crossbred bulls
Ordinarily nearly 30-40% of synthetics produce semen with poor initial quality which is not freezable. In certain situations a fairly large number of bulls produce semen of Azoospermatic quality (Holstein x Sahiwal crosses) or quality is poor and only semen of 20% of bulls are freezable. This is a serious problem. Research investigations should be carried out to determine if the cause is located in Genome or these bulls should be reared under controlled climatic/feeding conditions to avoiddamage to spermatogenesis.
24 Our work at I.V.R.I shows that Shephadex filteration technique could help in improving the quality of such ejaculates as well as storage and freezability of semen. Buffalo Bulls
A very high incidence of occurrence of static eiaculates in areas of extreme hot and cold climate causes considerable difficulty in successful freezing of semen. However, direct collection in buffer, slight warming and dilution help in revival of 80% of ejaculates. However, the success of preservation of these ejaculates is only marginally lower. An optimum schedule of collection and good management could reduce the incidence but there is a lot of variation between bulls.
Assessment of Semen Quality through Crenellation Pattern Technique
Certain biological fluids when allowed to dry on a glass slide, settle down in a characteristic pattern. This pattern is termed as "Crenellation Pattern" (CPT) and is significantly correlated with otherseminal characteristics. This test is simple, inexpensive, rapid and reliable. The versatility of the test lies in the fact that the slides could be prepared even by unskilled workers under field conditions and examined at a later date in the laboratory. A permanent record of semen quality of a breeding bull could be obtained even from those places where microscope facilities or professional skills are not readily available.
Cryopreservation of Semen
With a view to reduce the operational time, we have been trying the freezing by mixing semen and diluent ( containing glycerol ) at room temperature ( 20°- 30° -C), filling the straws and then cooling them in optimum quantity of water in the freezing chamber of a refrigerator so as to ensure that cooling to 5°C takes place in 2 hours. The results have been equally satisfactory with Tris as well as Milk diluent. However, buffalo sperm could yield acceptable post-thaw motility even without equilibration and after faster cooling in 30-60 minutes.
An equilibration period of 1-2 hours has further improved the freezability. The incorporation of additives such as 0.1% cysteine hydrochloride and \ or 0.1% EDTA significantly improved the keeping quality, freezability and fertility. Faster thawing at higher temperatures significantly improved the post-thaw sperm recovery. Heterospermic semen significantly improved freezability and fertility.
Lower yolk levels (5%) were found to be more effective in cryopreservation of buffalo semen. Buffalo sperms could be frozen successfully with minimum quantities of yolk and glycerol. The beneficial effect of certain sugars have been noticed in freezing.
25 Modifications In Processing Procedures
A simplified kit for freezing of semen. in field for initial screening of bulls has been developed. Faster cooling rates and shorter equilibration periods have almost reduced the operational period by almost 50% which has increased the efficiency of semen banks considerably.
A Protocol for Freezing of Buffalo Semen
Out of several protocols developed for dilution and freezing of semen, the one which is found to be most efficient is based on milk plus 5% yolk. The fat in milk is reduced to 1.5% after boiling, storage and filtration. This protocol could eliminate the import and use of costly chemicals such as Tris etc. Another advantage is that the level of glycerol could be reduced to 3% from the conventional level of 6-7%. Post-thaw "Holding Time" For Buffalo Semen When straws are allowed to remain at 37°C for 5-10 minutes after thawing there is an improvement of 10-15% in revivability of sperms. Although response to holding time is quite variable between bulls, the waiting period can save many straws from rejection.
Cryopreservation and Storage of "Concentrated Semen"
A really good advantage (20-30%) in storage as well as in freezing is if semen is diluted so as to ensure a sperm concentration 100 x 106!ml. or 100 million/mI. In practical terms it may be stated that the dilution rate may be kept below 1:10. This advantage of preservation of concentrated semen is retained when it is rediluted to a single dose of 10 million sperms. Another alternative could be to develop straws of micro sizes (0.1 ml) and then there is no need for re-dilution immediately before actual insemination.
Cryopreservation of Epidydimal Semen
The epididymal semen of buffalo was found to be an excellent source of Germplasm for storage and cryopreservation. Even one time sperm collection from epididymus of slaughtered bull could produce 400-500 doses of frozen semen. Perhaps surgical cannulation may be clinically necessary for males with poor sex libido. Once the potential for continuous supply of epididymal semen through surgical intervention is established, it would be necessary to test the fertilizing ability of epididymal semen both in vivo and or in vitro for making various aspects of semen technology more efficient. This technology has wider implications for ex-situconservation of the other breeds of livestock and could form the base for cryogenic preservation of animal biodiversity including wild life and endangered species.
26 Predicting the Freezability of Semfn
We have recently developed certain "Test freezing methods" which can give quick post- thaw results within a period of 45 minutes. A very fast cooling to 5°C is done in 15 minutes and equilibration period of 30 minutes is given. A highly significant correlation value ( r= 0.7) was obtained between the post-thaw results of test freezingand standard freezing methods.
Efficiency of Cryogenics under Tropics
Those imported cryogenics with a static holding time of six months work only to the extent of 50% under tropics consuming nearly double the quantity of liquid nitrogen. However, those with a static holding time of one year could function satisfactorily for a period of 9 months, thus requiring filling with liquid nitrogen after every 3 months. Because of high evaporation rate of liquid nitrogen under hotter climate, there is an urgent need to make efficient cryogenics indigenously. Otherwise the long-termstorage of semen will become cost prohibitive.
Fertility of Frozen Semen
The results have been varying in different situations. Generally the conception rates varied from 30-50%. A comparative study on use of fresh and frozen semen from the same bulls gave higher conception with fresh semen. Heterospermic insemination and use of certain additive have given comparatively higherconception rates. A wide variation among individual bulls in buffaloes have been noticed. The CR varied from 14 to 56 percent among 10 buffalo bulls. Thawing at higher temperatures gave better conception rates as compared to thawing in tap water.
In our assessment, much of the variation in fertility observed in different places could be accounted for by differences in husbandry and reproductive management, rather than semen quality alone.
FEMALE REPRODUCTION
Control of Oestrus Cycle
Ovarian cycles of females must be controlled and ovulation induced when needed. Progestogens and/or prostaglandins are in wide use in domestic animals for oestrus cycle induction and/or synchronization. A major limitation in the use of prostaglandin F2 alpha is that it cound be used in cyclic females having an active corpus luteum. Gonadotropins (PMSG or HCG) or gonadotropin releasing hormones are also used widely for the induction of ovulation.
27 Superovulation
The ovulation rate could be increased moderately to improve natural prolificacy or it could be stimulated significantly i.e. superovulation to increase the numberof high quality oocytes for embryo transfer programme. Various treatments with FSH preparations containing a controlled level of LIH, GnRH analogues, or bovine follicular fluid have been tested to determine those having the least effect on the normal endocrine profile of the animals. PMSG have limited effects on superovulation of cattle. However when a specific anti-PMSG antibody is used to limit the duration of action of the PMSG, the number of viable embryo per treated cow is similar to that obtained with FSH. Superovulation could be repeated every 6-10 weeks for increasing the total production of ova or embryos per donor female. However, an important individual variability in response to hormonal treatments remains and, with repeated treatments, there is a tendency for the ovulation rate to decrease possibly due to the formation of antibodies against injected hormones. In Vitro Fertilization
This technique is now being increasingly used for studying the biological mechanisms of fertilization and early mammalian development. Its applied uses include the multiplication of lines of superior animals, the production of transgenic offsprings and the prediction of male fertility. Successful in vitro fertilization has been obtainedin almost all domestic animals.
Oocytes must be collected by either surgical or laparoscopic routes, or at slaughter. They can be collected at the time of ovulation after in vivo maturation or punctured from ovarian medium sized follicles before ovulation and matured in vitro in an appropriate medium.
Semen must be collected from males producing spermatozoa with high fertilising ability. In vitro sperm capacitation has been a major problem in domestic animals and many media and technical conditions have been used to achieve this process. Efficient media for sperm capacitation must preserve high motility, facilitate membrane maturation and increase in percentage of sperm undergoing spontaneous acrosome reaction. High concentrations of spermatozoa are required to achieve in vitro capacitation which takes nearly 6 hours but once completed it takes a few minutes for spermatozoa to penetrate the ovum.
Transfer
Embryo transfer is performed with 6-8 day old embryos either surgically by laparatomy or non-surgically through the vagina and the cervix in cows/buffaloes. The success of implantation of transferred embryos might be increased by co-transfer of trophoblastic vesicles and the embryo.
28 Freezing
Freezing 6-8 day old embryos has been done routinely in cattle. However, freezing of younger embryos has not been achieved successfully but it is possible to obtain in vitro development of fertilized eggs to normal morula or blastocyst stages by co- culture on tubal or uterine epithelial cell monolayers. These blastocysts could be frozen, but this has not yet been reported.
Vitrification of Embryos -
The technique of bovine embryos freezing is likely to change enormously in the next few years. Vitrification of the embryos is rapid cooling under protection of high glycerol solution to -170°C at the rate of 200°/mm. This is about three times faster as compared to the conventional method. Similarily a new thawing technique has been developed where it is possible to thaw the straw containing vitrified embryos 'behind the cow' in approximately 6 minutes before the actual transfer is performed.
Splitting Embryos
All the cells derived from the first segmentations after fertilization, until the morula stage, are equipotents to develop into normal embryos. When pairs of bisected embryoswere transferred to each recipient, the overall yield of offspring increased and under best conditions could even exceed 100%.
Sexing
With the survival of split embryos, it is possible to sample a few cells for sexing a future off-spring. Any breeder will certainly appreciate the possibility now available to transfer embryos of known sex. Out of several methods used, the most promising is that using DNA- probes specific to the Y chromosomes. The most recent method is to allow the use of a small number of embryonic cells to which is applied the Po!ymerase Chain Reaction (PCR) technique for amplification of DNA specificto the V chromosome. This specific sequence gives a strong hybridization signal with the probe only for male embryos. This method is accurate, reliable and rapid. It is expected that this method will be used extensively in the near future.
Sexing techniques based on separation of X and Y spermatozoa have not proved their utility in practice so far. However, a recent technique based on Sperm flow sorting developed for rabbit is likely to be adapted for farm animals.
29 Cloning
Recent developments in basic knowledge of embryology suggest new techniques that would allow extensive replication resulting in rapid dissemination of genetic progress. It has been shown that nucleii of cells from the first egg cleavages or even those from the inner cell mass of the early embryo have the capacity to develop into normal embryo when transplanted into a recently ovulated and incubated oocyte. The techniques of micromanipulation such as cellular enucleation and electrofusion require extensive improvement and cloning is expected to increase rapid genetic change in the population. However, one must be highly efficient in selecting 'Unique' animals to be cloned, otherwise there is a risk of diffusing lead genes.
Transgenic Animals
The recombinant DNA technology has made it possible to insert genes into genome of farm animals and thus alter their genetic potential. This is only an optimistic view since success is very low (about 1/100). To be economically efficient, genes need to be correctly targetted and their expression controlled to obtain expected results. At present, this is extremely difficult and molecular gene engineering must be developed further before significant success can be obtained in the form of changing animal performance.
In-vitro Production of Embryos
The application of multiple ovulation and Embryo Transfer(MOET) have maximally given 25 to 30 embryos per cow per year. Much research has been done recently to enlarge the number of progeny of top cows. With a view to gain as much as possible from the genetic potential of top cows, in vitro production of embryos is a new technique in this area which includes i. The collection of oocytes from cows. ii. The maturation of oocytes (IVM). iii. In-vitro fertilization of oocytes (IVF). iv. In-vitro-culture of the fertilized oocytes to transferable embryo (IVC).
A new technique (Ovum pick up) has been developed for collection of oocytes from the follicles in the ovary. Follicles are punctured with the aid of ultrasound scanning. This technique does not require the application of hormones and is harmless and can be multiplied several times on the same cow. Nearly 500 oocytes have been collected per cow in one year. The collected oocytes are matured first for one day before fertilization. After fertilization with semen, they are grown in an in vitro culture for a week till they reach the transferable stage.
30 The results of in-vitro production of embryos are encouraging but efficiency is still low. Only 10-20% of the oocytes collected develop into a transferable embryo. Therefore, more fundamental research is needed on various steps involved in the process. The creation of same environment (in-vivo) of uterus in laboratory is not an easy task. However, expectations are that in-vitro production of embryos can replace E.T. in future. Even now 50-100 embryos can be produced per cow per year as compared to 25- 30 embryos through E.T. method. This will enable intensive selection of potential bull dams resulting in greater genetic progress. Because of in-vitro fertilization, the oocytes can be fertilized with semen of several different top sires resulting in different combinations in breeding programmes.
A drawbackof in-vitro technology is that such embryos are not very suitable for freezing. Currently more research is being done to improve the results of pregnancy.
Zona Drilling and Sperm Injection
Zona drilling involves chemical dissolution of a hole in the Zona and sperm injection involves inserting a single sperm into the ooplasm of activated oocytes. In the bovine exposure of sperm injected ova to a Calcium ionophore greatly enhanced activation of the ova. Even killed sperm by repeated freezing and thawing can fertilize ova when injected into ooplasm. Four pregnancies resulted from E.T. of embryos fertilized by injection of dead sperm. Another variation of sperm injection, involving placement of a single sperm into perivitelline space resulted in low (10%) fertilization rates. Zona drilling and sperm injection are techniques that might benefit from the availability of semen that was sex selected by flow cytometry. Use of Hormones
The increasing understanding of the physiological mechanism controlling reproduction has led to procedures which may now be employed in many commercial situations in advanced countries. Because of uncertainty of existing level of hormones in Zebu and buffaloes and the cost involved, these packages are not being used in general farming situations in the country. One practical outcome of development in the area is the use of milk progesterone assay which can be employed in detecting cattle at 3 weeks after breeding those animals which fail to become pregnant. Prostaglandins are currently available in Synthetic form and PGF2 alpha is effectively employed to manipulate the oestrus cycle and ovarian function. Progestagens such as meaoxyprogesterone acetate (MAP) or fluorogestone acetate (FAG) have been extensively used for controlling oestrus and ovulation through the development of hormone delivery systems. The use of hormones is becoming increasingly useful in treatment of many reproductive disorders but these should not be used indiscriminately.
31 The Future
Advanced reproductive technology has already been applied in increasing the rate of reproduction/genetic improvement of dairy animals. Inspite of its application based over a period of 40 years, the A.l. technology is only moderately efficient (CR after single inseminati9n is around 40%). Undoubtedly there is a need to make this technology more efficient but the natural mating will certainly remain the most common method of animal reproduction in rural areas. There is an urgent need to reduce the number of spermatozoa required for obtaining optimum fertility. It is likely that the farmer may ask for the sexed spermatozoa. Any developments in early predictive assessment of fertility of male/ejaculates can be of considerable importance. Ultimately the success with injection of single sperm into ovum and use of lyophilyzed semen are the challenges for the future although indications of success are not encouraging in these areas. Attempts can be made to educe the mortality rate of spermatozoa during freezing. Currently 40-50% loss of sperms is common with most of the protocols around the world. The technology of freezing of concentrated semen and its use in micro- insemination of bovine needs further refinement.
T.he oestrus activity in tropical cows/buffaloes is not so intense and this is an important factor adversely affecting the conception rates through artificial insemination. There is a need to develop some simple methods of heat detection so that inseminations at the right stage of oestrus and ovulation can ensure optimum conception rates.
A more recent tool which is still at the stage of development is the measurement of the activity of the cows. Cows in heat are two to four times more active when in oestrus. Pedometers strapped to the legs of cows register the cow's activity which can be electronically monitored. Nearly 97% of heat periods have been detected. Obviously this efficient system requires a computerized herd management system.
Besides getting control over oestrus, the low efficiency of oocyte production is a major limiting factor for genetic improvement through the female line. As a matter of fact the progress of research in ovarian biology (Ovarian follicles and oocytes) will decide the extent and efficiency of best females which can contribute in genetic improvement through manipulation of embryos.
Besides conducting research on the possibility of successfully freezingthe oocytes, the embryos will be transferred with additional trophoblastic vesicles for increasing the chances of implantation in the recipient mothers. Alternatively, for increasing the pregnancy rates, injections of interferon recombinant (trophoblastic protein analogoues) can be tried on mothers. Mothers can also be treated to stimulate early production of pregnancy factors so as to reduce embryonic mortality. The freezing and storage
32 of clones can further facilitate the performance testing from their twins and then only, the test of the scope and challenges of research which are limitless in these areas of female reproduction.
CONCLUSION
India cannot afford to wait for the application of some of the technologies reviewed in this paper. The efficiency of semen banks and cryostorage facilitiescan be improved considerably without involving additional costs. Similarly the best females can be utilized under best conditions for the production of elite bulls through embryo manipulation. Most of the reproductive technologies require an intensive system of management and are not suitable for ordinary farmers. These techniques are rather expensive and research will have to be conducted on adjus(ing them to local economic conditions so as to enable the farmer to earn more money from animals as well as support the availabilityof much needed nutrition for the growing human population in the developing countries.
33 EXPERIENCE IN EMBRYO TRANSFER TECHNOLOGY DURING PRELIMINARY TRIALS CARRIED OUT AT P.K.V., AKOLA
M.S. KADU Punjabrao Krishi Vidyapeeth, Akola.
With rapid development of Embryo TransferTechnology, new vistasfor greater utilization of genetically superior female component has been opened to make rapid strides in milk production. Nevertheless, the potential benefits from the use of superovulation and ET are more likely to be realized only after taking due care at every step of the technology, which limits the superovulatory response, harvest of the viable embryos and the pregnancy rate thereafter.
Literature in respect of superovulation, recovery and transfer of embryos in cattle is scanty under Indian agroclimatic conditions. Introduction of the technology and experiences derived through initial trials in the eastern part of Maharashtra may interest the workers in the area.
Present trials were undertaken to study
1. The superovulatory response with regimens of FSH-P and PMSG + HCG.
2. The recovery of normal embryos and pregnancy on ET.
First Trial : Observations on preliminary trials for El in cross-bred cows
MATERIALS AND METHODS
Three cross-bred (Two Jersey x Sahiwal and one Holstein Friesian x Hariana) cows were selected as donors and ten cross-bred cows as recipients in this trial. The donor animals were given treatment with FSH-P as detailed in Table - 1.
Recipients were treated with PGF 2 alpha (Dinofertin) 24 hours earlier than donor cows in order to achieve exact oestrus synchronization. All the donors were inseminated with frozen semen, four times, at 12 hrs. interval after oestrus was detected. Non-surgical embryo collection was attempted on 7th day after first insemination. Epidural anaesthesia was given. The -ovaries were palpated and superovulatory response was recorded.
The uterine lumen was flushed with Dulbaco Phospate Buffer Saline (DPBS) by intermittent gravity flow. Each uterine horn was filled with 50-60 ml. of medium which was then allowed to flow into the collection vessel whilethe uterus was gently massaged.
34 This was repeated until 400 to 500 ml. of medium was used. The foley's catheter was then inserted into the other uterine horn and the process repeated.
The fluid along with embryos was collected through filter and transferred from embryo filter into a petridish. Embryos were isolated and examined underZoom Streo-Microscope at 10 x magnification. As soon as embryos were located under the microscope, they were transferred into a small dish containing fresh holding medium. The formation of holding medium was same as that of flushing medium. Except for a concentration of 20 per cent heat treated foetal calf serum. Morphological study of embryos was undertaken and only good blastocyst stage embryoswere transferred to the recipients.
RESULTS AND DISCUSSION
Three donors and ten recipients treated with 2 injections of PGF 2 alpha (Dinofertin) came in oestrus between 60 and 72 hrs. of treatment.
The estimated number of corpora lutea and embryo recovery is given in Table - 2. The mean number of corpora lutea was 12.0±0. The mean percentage of recovery of medium was 94.53±0.22.
Superovulatory response was better than that reported by other workers (Totey et a! 1987, 1988). However, the yield of embryos in this trial was very low when compared to results obtained in cattle by various workers (Totey et al 1988).
The number of unfertilized ova was 23 and only two embryos were obtained. These two Blastocysts were recovered from only one donor (No. 384) and transferred to two recipients (No. 427 and 249). One recipient (No. 427) diagnosed as pregnant on day 60 was found empty on subsequent examination.
The percentage of unfertilized ova was more in this trial. This may be due to suboptimal sperm transport. Ovuation over a period of time or other causes, fertilisation rate of ova from superovulated donors are usually considered below the rates for ova from untreateddonors (Elsden et all 976). The high incidence of unfertilized ova might have been due to 32 mg dose of FSH used for superovulation which may be high for crosses of Bos indicus x bos taurus cows. Higher dose of FSH may frequentlycause continued follicle stimulation after ovulation, resulting in persistent large follicle accompanied by high oestrogen level. Abnormal high level of oestrogen produces detrimental effect on sperm transport and embryo survival (Cahil et a! 1976).
35 Table 1. Treatment Regimens for Superovulator Employed in Crossbred Cows.
Day Treatment
0 day of oestrus Synchronised with PGF2 alpha
I. Superovulation with FSH-P
Morning Evening
11 Day FSH - P 1.1 ml (5.5 mg) l/M FSH -P 1.1 ml (5.5 mg)l/M 12 Day FSH- P 0.9 ml (4.5 mg) l/M FSH -P 0.9 ml (4.5 mg)IIM 13 Day FSH -P 0.7 ml (3.5 mg) l/M FSH -P 0.7 ml (3.5 mg)l/M + PGF2 alpha (Dinofertin) 5 ml ( 25 mg) l/M 14 Day FSH -P 0.5 ml (2.5 mg) tM FSH -P 0.5 ml (2.5 mg)l/M 15 Day Heat check and A.l A. I. 16 Day Al. A. I. 22 Day Embryo recovery and collection
Il. Superovulation with PMSG + HCG
0 Day Oestrus (Synchronised with PGF2 alpha Dinofertin 11 Day 2500 l.V. PMSG (Folligon) l/M — 13 Day 30 mg PGF2 (Dinofertin) tM 20 mg PGF2 X (Dinofertin)l/M 15 Day Head check and A.l. A. I. + H.C.G. 1200 lU 16 Day A.l. A. I. 22 Day Embryo recovery and transfer A. I.
Table 2. Results of Superovulator and Embryo Recovery.
Donor No of Recovery of Embryo Recovery ci medium infertile Blastocyst (Recovered/ infused)ml S x J 124 13 940/1000 9 — S x J 130 12 900/950 8 —
H.F.x h 384 11 930/980 6 2
36 Second Trial : Study of Superovulatory Response in Cross-bred Cows and Heifers Although superovulation is possible by several treatment schemes, the variability in response and quantity of embryo recovered remains one of the most limiting factors in development and planning of the Embryo Transfer Programme (Lerner, et a! 1986). PMSG, possessing both LH and FSH like properties, has been extensively used as a stimulator to follicle development, but presence of PMSG in the blood circulationafter the time of ovulation, might have an adverse effect particularly on quality of developing embryos. Hence, alternatively pituitary Gonadotrophin was used for superovulation. Some workers have reported relatively poor response as well as wider individual variability with PMSG as compared to FSH (Monniay et a! 1983, Elsden et a! 1978), while Christer et al (1980) found no difference between the response of the two gonadotropins. This experiment was designed to study oestrus behaviour, ovulatory response with FSH-P and PMSG+HCG regimen, in pubertal and parous cross-bred cows. MATERIALS AND METHODS In the second trial, fourteen cross-bred animals (Jersey Sahiwal), showing regular cyclic activity, with active corpus luteum (CL) and no signs of ovarian or uterine pathology were selected and randomly divided into two groups of 4 cows each and 3 heifers and were superovulated with FSH-P (Group 1) and PMSG+HCG (Group 2)regimens as detailed in Table - 1. The animals were closely observed for oestrus by parading vasectomised bull in the byre at 6 hourly interval and studied for duration required for onset, length, behavioural symptoms and intensity of estrus. The superovulatory (SOV) response was studied from the number of corpora lutea palpable per rectum on day 7 of the treatment. RESULTS AND DISCUSSION Oestrus Behaviour All the experimental animals (100%) exhibited oestrus on SDV treatment. The duration for onset of oestrus in FSH+P treated group was 43.00+3.00 hrs. in cows and 46.01 ± 1.50 hrs. in heifers. The duration in Group 1 and Group 2 was 44.28 ± 1.7 and 47.14 ± 1.22 hrs. respectively (Table 3). However, the difference was not significant. These observations are in agreement. (Scully et al 1982 and Yadav et a! 1986). The duration in heifers (Pooled) was slightly longer than cows, with non significant difference. The length of oestrus with FSH-P regimen was 35.01 ± 2.38,26.00 ± 2.0 hrs. in cows and heifers, respectively with an average of 31.14 ± 2.34 hrs. Whereas, the duration in PMSG ± HCG treated group was 36.0 ± 2.16 and 32.66 ± 4.37 hrs. in cows and heifers, respectively, with an average of 34.57 ± 0.80 hrs. The length of oestrus in cows (Pooled) was more as compared to heifers, although the difference was not significant. In both groups oestrus symptoms were intense.
37 Superovulatory (SOV) Response
The overall SOV response in terms of number of CL and anovulatory follicles was 10.57 ± 1.90 and 2.70 ± 0.42 in FSH-P treatment group. These findings are in agreement with those of Christer et a! (1980). The SOy response observed with FSH-P treatment was slightly better than reported by Totey et a! (1987) and (1988), but comparatively less than Chupin (1985). The SOV response observed in PMSG+HCG group was in agreement with Bhattacharya el a! (1987) and betterthan (4.4 ± 4.9) reported by Lastler (1973), but poorer than Scully et a! (1982). The overall number of C.L. and anovulatory follicles observed in cows was 14.12 ± 1.46 and 3.25 ± 0.45 as compared to 5.66 ± 1.02 and 3.50 ± in heifers. The SOy response in cows was significantly (P > 0.01) higher than in heifers. Present findings do not corroborate with those of Moore (1975), wo reported equal response in heifers and cows.
It is ooncluded that the SOV with FSH-P and PMSG regimen showed similar response in terms of duration for induction, length and intensity of oestrus. However, number of C.L. and anovulatory follicles palpated on day 7 of treatment, was significantlyhigher in cross-bred cows than that in heifers. All the super-ovulated animals exhibited intense oestrus behaviour.
Table 3. Comparative Superovulatory Response with FSH-P and PMSG Regimes Treatment.
FSH -P PMSG Treatment Heifer Cow Total Heifer Cow Total
No. of Donor 3 4 7 3 4 7 No. of oestrus 3 4 7 3 4 7 Duration for 46.0 ± 1.50 43.0 ± 3.0 44.28 ± 1.7 48.66 ± 0.66 46.0 ± 2.0 47.14 ± 1.22 onset of oestrus (Hrs)
Length of 26.0 ± 2.0 35.0 ± 2.38 31.14 ± 2.34 32.66 ± 4.37 36.0 ± 2.16 34.57 ± 0.80 oestrus (Hrs)
Average No. 7.0 ± 1.73 13.25± 2.49 10.57 ± 1.9 4.33 ± 0.6 15.0 ± 1.80 . 10.42 ± 2.34 of C.L.
Average No.of 3.0 ± 0.57 2.5 ± 0.6 2.70 ± 0.42 4.33 ± 0.66 4.0 ± 0.40 4.00 ± 0.30 A.F.
38 Table 4. Ovarian Response to Superovulatory Treatment in Crossbred Animals.
Sr. Treat- Class Treated Ovarian Response Total No ment No Left ovaryRight ovary
C.L. A.F C.L. A.F. C.L. A.F.
1. FSH-P Cow 4 6.50 1.0 6.75 1.5 13.25 2.5 ± 1.32 ±0.40 ±1.54 ±0.64 ± 2.49 ±0.6
2. FSH-P Heifer 3 2.66 1.66 4.33 1.33 7.00 3.01 ± 0.88 ±0.33 ±1.39 ±0.66 ± 1.73 ±0.57
3. PMSG Cow 4 6.75 1.75 8.25 2.28 15.00 4.0 ± 1.10 ±0.25 ±0.94 ±0.47 ± 1.82 ±0.40
4. PMSG Heifer 3 2.0 1.66 2.33 2.33 4.33 4.0 ± 0.0 ±0.33 ±0.66 ±0.33 ± 0.6 ±0.57
- Intense, M - Moderate W- Weak C.L. Corpus Luteum, A.F. Anovulatory Follicles.
Third Trial : Embryo Transfer and Pregnancy Rate
MATERIALS AND METHODS
The trial was undertaken on four cross-bred donors and seven cross-bred recipient cows belonging to CBF, Akola and Borgaon. The superovulatory treatment, study of superovulatory response and recovery of embryos were carried out as detailed earlier.
Embryos
Embryos were morphologically studied and only good blastocyst stage embryos were transferred to recipients. Epidural anaesthesia was given to recipients to prevent defeacation and to minimise straining. The blastocyst was aspiratedinto a 0.25 ml french straw between two air pockets and two columns of holding medium. The straw was loaded in A.l. gun. The embryos were trasferred non-surgically in a manner similar to artificial insemination in uterine horn ipsilateral to corpus luteum.
RESULTS AND DISCUSSION
The SOV response in PMSG treated cows was better (17.5) as compared to (12.0) in FSH-P treated cows (Table - 5). (However, the left horn of donor no. 264 could not be sufficiently flushed because of blocking of filter due to pus flakes). Present
39 observations are contrary to the previous reports where better SOy response with FSH over PMSG is reported. The overall SOV response (14.75) observed in the present study was better than that reported by Totey et a! (1987, 1988) indicating that the ovaries of these cross-breds respond well to exogenous gonadotrophins. The SOV response was similar to that reported by Chupin et a! (1985). Out of the total 59 (AV 14.75) ovulations 37 (AV 9.25) embryos were recovered. However, after the morphological examination only 14 (AV 3.50) embryos were found to be viable. The greater number of degenerated embryos observed in donor No. 124 and 157 was attributed to the low grade endometritis at flushing in these donors. Donor No. 128 which was free from such infection yielded highest 12 transferable embryos. These results indicate the vital importance of selection of the donor and strict aseptic measures required at all stages in the technique.
Table 5. Superovulatory Response in Cows treated with FSH-P and PMSG.
Sr. Donor Treatment No.of C.L. Total Aanovul- Total Medium Reco/ No. cow atory Rt Lt Rt Lt Infused
1. 124 FSH-P 7 10 17 7 5 12 980/1000
2. 264 FSH-P 4 3 7 6 7 13 500/700 Total (Mean)1 1(5.5) 13(6.5)24(12.0)13(6.5)12(6.0)25(12.5) 3. 128 PMSG 8 10 18 6 6 12 600/700 4. 157 PMSG 10 7 17 8 7 15 890/1000
Total (Mean)1 8(9.0) 17(8.5)35(17.5)14(7.0)13(6.5)27(13.5) Rt. = Right Lt. = Left C.L. = Corpus Lutium.
Table 6. Details of Embryo Recovery in Crossbred Cows.
Sr. Donor Treatment No.of Embryo Recovery Total Viable No. Cl UF DEG E.Blast Blast Exp.B
1. 124* FSH-P 17 2 7 — — 1 10 1 2. 264** FSH-P 7 3 — — — — 3—
3. 128 PMSG 18 — 3 5 4 3 15 12
4. 157" PMSG 17 — 8 — 1 — 91
Total (Mean) 59 5 18 5 5 4 37 14 UF= Unfertilized, Deg. = Degnerated E. Blast = Early Blastocyst Blast = Blastocyst Exp. Blast = * Expanded Blastocyst. Embryos were deshaped, Zona pellucida thin and flat membrane tendency to stick, donor having low grade endometritis. ** Left horn not sufficiently flushed. Same is (*) having infection.
40 Out of the 14 recovered viable embryos, two were transferred surgically in recipient (No. 85). Remaining 12 embryos were transferred non surgically as detailed in Table 7. Out of the 7 recipients examined for P.D. after 45 days, three (No. 361, 342 and 369) were pregnant of which one (No. 369) carried a twin. However, on subsequent examination only two pregnancies were confirmed. One turned out to be a case of early abortion. Thus out of the 14 embryos transferred, 28.56% resulted in pregnancies but later only 3 (21.43%) were carried to twins. All the three new borns were females including a pair of twins. The present pregnancy (28.56 %) and calving rates (21.40%) are lower than the observation of Elsden (1980) Newcomb (1982) and Peeples (1984). However, these results are in accordance with those reported by Subramanian et a! (1990) and are suggestive of importance of aseptic precautions in the technology. These trials were preliminary with limited resources and hence need further perfection on more cows.
Table 7. Details of Embryo Transfer in Crossbred Cows.
Sr. Recipient Site of Grade of No.of Method Donor Remark No. cow No. transfer embryo Embryo of tra- No. (ipsilaterai) nsfer
1. 85 R B" Blastocyst 2 S 128 —
2. 94 R "B" Expanded 2 N.S. 128 C.L. Not blastocyst prominent
3. 23 RL B" Blastocyst 2 N.S. 128
4. 361 P Expanded 2 N.S. 128 EED blastocyst
5. 79 R Blastocyst + 2 N.S. 128 Blastocyst
6. 369 AL Blastocyst + 2 N.S. 128 Twin Blastocyst pregnancy
7. 342 1 "C" Hatched 2 N.S. 124 Single Blastocyst 157 pregnancy
N.S. = Non Surgical S. = Surgical A. = Right L. = Left.
41 REFERENCES
Bhattacharya, B.N., Sinha, A.K. and Sinha, S.N. 1987. Workshop on 'Embryo Biotechnology sponsored by ICAR and Dept. of Biotechnology, G.O.I., Delhi. Feb. 27 1987. Cahil, L.P., Lowson, R.A.S. and Parr, R.A., 1976. Cited by Totey, S.M et a! 1987. Christer, J.K., Rove, R.E., Comp., M.R. and Ginther O.S. 1980 Theriogenology 3 (6) : 397-406. Chupin, D.,Combornous U. and Procureur, R. 1985. Theriogenology 23 (1) : 184. Elsden, R.P., Hasler, J.F. and Seidel, G.E., Jr. 1976. Theriogenology 6:523-526. Elsden, R.P., Nelson, L.D. and Seidel, G.E. 1978. Theriogenology : 9 :(1) :17-26. Elsden,R.P., 1980. Bovine Embryo Transfer Proc. Soc. Theriogenology 14 :101-133. Laster, L.B. 1973. J. Reprod. Fertil. 33 : 275-282. Lerner, S.P., W.V. Thayne, R.D. Baker T. Henschen and S. Meredith. 1986. Moore, N.N. 1975. Aust. J. Agril. Res. 26:295-304. Monniaux, D., Chupin, D. and Saumande, J. 1983. Theriogenology. 19(1) : 55-81. Mutter, L.R., Graden, A.P. and Olds, D. 1964. A.l. Digest 12:3. NewComb R.C. 1982. CRC. Pross. Bearaton, Florida, USA. Pawshe, C.H., M.S. Kadu, fasihuddin and S.M. Totey 1972. Ind. J. Anim. Repro. 13 (1) 18-20. Peeples J.G. !984. Proc. NAAB and lETS Ann. Conf. Artif. Ins, and Emb. Tra in beef cattle pp 36-37. Scully, G.M. Boland M.P. and Gordon, I. 1982. Ani. Breed Abstr. 50 (1) 83. Subramanian, A.K.,P. Devarajan and Mc Mohanan 1990. IJAR 11 (2) : 114-11 b. Totey, S.M. Gurpreet Sing., Talwar, G.P. and Singh, G. 1987). Workshop on Embryo Biotechnology G.O.I.,Feb. 1987- Totey, S.M., Gurpreet, Singh, Singh, G. and Talwar G.P. 1988. Paper presented at Nat. Symp. on Anim. Reprod., Trichur, 1988. Yadav, M.C., Walton, J.S. and Leslie, K.E. 1986. Theriogenology. 26 (4) : 509-521.
42 EFFECT OF DIFFERENT COOLING RATES IN PROGRAMMABLE FREEZER ON THE QUALITY OF FROZEN BUFFALO SEMEN
M.R. BHOSREKAR, S.P. MOKASHI, J.R. PUROHIT, S.B. GOKHALE AND B.R. MANGURKAR. BAIF Development Research Foundation, Urulikanchan 412 202
ABSTRACT
Ten murrah buffalo bulls in the age group of 5 to 7 years and weighing 500 to 700 kg were used in a twice a week semen collection programme. After processing of diluted semen the minitube straws were filled and sealed and arranged horizontally on freezing racks. Half of these straws were frozen by conventional freezing method over liquid nitrogen in static vapour for 10 minutes in MVE 320 container and the other half were frozen by using definite cooling rates in programmable freezer (IMV 5300). The quality of frozen semen was assessed by studying post-thaw motility, live count, acrosome maintenance, percent sperm filtered through glasswool-sephadex G- 15 column, and sperm enzyme release in extracellular medium. Definite cooling rates in programmable freezer produced better quality frozen semen as compared to conventional method of freezing. Amongst the three different cooling rates, a rate of -17.32°/minute between -i-4 to -40°C gave significantly better quality of frozen semen.
INTRODUCTION
Deep freezingover liquid nitrogen in static vapour in a wide mouth containerhas been the accepted method worldwide for cryo preservation of bovine semen. Buffalo spermatozoa are more sensitive to cold shock as well as osmotic shock than bull spermatozoa (Tuli et al 1982, Crabo et al 1970, 71, Bhosrekar et al, 1990) and therefore deep freezing technique needs to be modified for buffalo semen. This paper deals with the effectiveness of different cooling rates in programmable freezer on the post thaw quality of frozen buffalo semen.
MATERIALS AND METHODS
Ten murrah buffalo bulls in the age group of 5 years to 7 years having body weight of 500 to 700 kg were selected for this experiment. The semen was collected twice a week from each bull by artificial vagina technique. Four ejaculates under each programme of cooling and a total of twelve ejaculates were considered from each bull for analysing each parameter. The evaluation, dilution, glycerolisation and equilibration
43 was carried out (Bhosrekar et al 1991). After filling and sealing of diluted semen in German minitubes the straws were arranged horizontally on freezing racks and kept for equilibration at 4°C for 3 hours. Half of the total semen straws from each bull were frozen by conventional method in MVE 320 wide mouth container over liquid nitrogen in static vapour for ten minutes as per Bhosrekar et al (1991) and the other half were frozen in programmable freezer (IMV 5300) by stacking the horizontal racks of semen straws. After initial screening of 9 different cooling rates, three cooling rates were finalised based on better post-thaw motility as compared to the conventional method. These were as follows.
Sr.No. Programme Total time taken Total time take n number to reach temp. to reach temp. from + 4°C to -40°C from + 4°C to 140°C
1. 1 107.6 seconds 418.6 seconds
2. 2 116.4 seconds 544.2 seconds
3. 3 152.4 seconds 670.2 seconds
The temperature of the freezing chamber was brought to 4°C before the racks were transferred. The racks were placed horizontally in the freezing chamber one above the other. One of the straws was cut and mounted on the thermocouple probe for recording the actual cooling rate of the semen where the semen was in contact with the sensor. On reaching the temperature to -140°C the racks were quickly removed and were placed in MVE 320 wide mouth container over liquid nitrogen. The straws were then removed from racks with the help of pre-cooled gripping tongs and put in the plastic goblets which were then immersed in liquid nitrogen.
The quality of the fresh semen and diluted semen at prefreezing stage (PF) and after freezing and storage for 24 hours in liquid nitrogen was assessed by quality tests viz, post-thaw motility (PTM),live count, acrosomal maintenance (Bhosrekar et al, 1991), Glass wool-sephadex filtration (Heuer et al, 1983), hyaluronidase enzyme release (HAT) (Linker, 1966), aspartate aminotransferase (AAT) leakage, lacticdehydrogenase (LDH) (Oser, 1965), alkaline phosphatase (ALP) (Kind and King 1954) and acid phosphatase (ACP) (King and Jagatheesan, 1959).
Statistical analysis ot the data were carried out using standard statistical methods described by Snedecor and Cocharan (1976). The percentage data was transformed by Arcsin transformation before it was subjected to analysis.
44 RESULTS AND DISCUSSION
The mean values with standard error for different parameters as affected by the programme of cooling rate is given in Table 1, 2 & 3 while the analysis of variance for each programme is given separately in Table 4 & 5. The mean values in respect of post thaw characters like forward motility, live count and acrosomal maintenance in all experiments were found higher than those of respective controls. The mean percentage of sperms filtered through sephadex G-15 column for first experiment were lower than control; the average values for second and third experiment were however higher than the respective controls (Table 1). Table 1. Effect of Different Cooling Rates On Seminal Parameters Means with Standard Errors.
Experiment No.1 Experiment No.2 Experiment No.3
Parameters Fresh Pre Post freezing Fresh Pre Post freezing Fresh Pre Post freezing freezing freezing freezing Control Prog.1 Contol Prog.2 Control Prog.3
Percent forward 84.3 75.3 62.3 64.4 83.6 5.1 63.1 64.8 84.2 75.8 62.9 67.2 motility ±0.24 ±0.20 ±0.46 ±0.40 ±0.49 ±0.15 ±0.36 ±0.44 ±0.28 ±0.23 ±0.40 ±0.49
Percent Live 93.38 84.34 69.17 72.41 93.31 84.60 69.41 71.89 93.02 84.11 69.36 74.61 count ±0.31 ±0.46 ±0.68 ±0.58 ±0.32 ±0.35 ±0.62 ±0.66 ±0.38 ±0.39 ±0.57 ±0.61
Percent acroso- 95.45 92.78 89.29 89.95 95.41 92.96 89.48 90.02 95.29 92.69 89.34 90.50 mal maintenance ±0.13 ±0.16 ±0.26 ±0.21 ±0.14 ±0.14 ±0.19 ±0.21 ±0.14 ±0.15 ±0.18 ±0.18
Percent sperm 47.84 45.11 49.03 48.56 47.13 52.18 filtered through ±1.00 ±1.06 ±0.73 ±0.67 ±0.65 ±0.56 sephadex G-15
The mean values for HAT, AAT LDH, ACP and ALP releases have been presented in Table 2. The post freezing mean values in respect of HAT, AAT, LDH were found lower in the experimental group compared to their respective controls. The mean value for ACP and ALP were however higher in experimental group than controls.
45 Table 2. Effect of Different Cooling Rates on Release of Sperm Enzymes Means With Standard Errors
Experiment No.1 Experiment No.2 Experiment No.3
Parameters Fresh Pre Post freezing Fresh Pre Post freezing Fresh Pre Post freezing freezing freezing freezing Control Prog.1 Contol Prog.2 Control Prog.3
HAT fresh 10.61 9.28 9.48 (Units/mi.) ± 0.67 ± 0.63 ±0.55
HAT pellet 3.79 5.64 9.07 8.81 5.32 7.29 8.58 9.68 4.35 6.13 10.36 9.52 (Units/mi.) ± 0.30 ±0.41 ±0.64 ±0.68 ±0.31 ±0.63 ±0.59 ±0.79 ±0.30 ±0.42 ±0.79 ±1.03
HAT Plasma 7.56 26.43 52.08 47.75 5.79 24.40 47.13 43.85 6.86 24.02 45.70 41.63 (Units/mi.) ±0.66 ±1.65 ±3.10 ±2.57 ±0.46 ±1.73 ±2.65 ±2.88 ±0.37 ±1.45 ±3.21 ±3.15
AAT 201.44 861.86 1678.43 1638.27 208.05 973.24 1739.10 1699.69 232.68 923.12 1566.55 1393.03 (SF Units/mI.) ±19.99 ±78.51 ±114.53 ±106.72 ±20.25 ±70.13 ±116.95 ±105.10 ±22.31 ±78.92 ± 93.84 ± 88.33
LDH 5159.6036117.40 35742.20 35624.50 5252.80 37299.3036814.20 36709.70 5056.40 33515.90 32829.50 32899.80 (Units/lOOmI.) ±131.55 ±1126.48 ±1098.90 ±1076.83 ±104.09 ±1332.19±1284.97 ±1345.93 ±1 22.53 ±1288.63 ±1277.27 ±1271.13
ACP 58.63 408.61 636.68 608.28 51.65 351.36 502.66 541.41 64.36 340.89 416.38 422.89 (KA unit/ ± 4.34 ± 24.05 ± 32.26 ± 34.33 ± 4.52 ± 16.40 ± 24.62 ± 27,07 ± 6.12 ± 34.02 ± 22.95 ± 26.73 lOOmI.)
ALP 82.29 572.01 800.94 825.83 78.93 511.79 687.99 836.15 90.00 511.59 712.77 697.73 (KA units ± 6.28 ± 36.71 ± 40.62 ± 41.11 ± 8.68 ± 30.18 ± 38.32 ± 35.64 ± 7.18 ± 32.10 ± 40.55 ± 38.77 /lOOmI)
In general it appears that as compared to conventional freezing method all the programmes of cooling rate showed significantly better quality of frozen buffalo semen in terms of percent drop in PTM, live count, acrosome maintenance, HAT release, AAT release and ALP release in extracellular medium (Tables 3 to 5).
46 Table 3. Effect of Different Cooling Rates on Seminal Parameters Means With Standard Errors
Parameters Control Frog. Control Prog. Control Prog. No.1 No.2 No.3
% drop in 13.0 10.9 12.10 10.30 12.90 8.70 PTM ± 0.39 ± 0.36 ± 0.37 ± 0.41 ± 0.36 ± 0.45 % drop in 15.17 11.94 15.19 12.71 14.74 9.50 post thaw ± 0.64 ± 0.56 ± 0.59 ± 0.73 ± 0.54 ± 0.56 live count % post thaw 3.48 2.83 3.48 2.94 3.55 2.19 increase in ± 0.16 ± 0.15 ± 0.16 ± 0.17 ± 0.12 ± 0.12 abnor.acroso. % post thaw 47.84 45.11 49.03 48.56 47.13 52.18 sperm filt. ± 1.00 ± 1.06 ± 0.73 ± 0.67 ± 0.65 ± 0.56 thro.galss wool sephadex G-15. Post thaw 3.44 3.18 1.29 2.30 4.23 3.39 reie.of HAT ± 0.45 ± 0.49 ± 0.45 ± 0.58 ± 0.59 ± 0.67 in sp.pellet. (Unit/mi) Post thaw 25.68 21.32 22.73 19.45 21.69 17.62 rele.of HAT ±1.78 ± 1.29 ± 1.36 ± 1.36 ± 1.69 ± 1.57 in s.plasma (Unit/mI) Post thaw 816.57 776.41 765.86 726.46 643.43 469.91 rele.of AAT ± 44.10 ± 43.53 ± 43.81 ± 37.68 ± 29.09 ± 36.55 in s.plasma (SF units/mi.) Post thaw 375.20 492.90 485.20 589.60 686.40 616.10 rele.of LDH ± 192.89 ± 96.44 ± 131.95 ± 98.07 ±106.57 ± 71.44 acti.in s.piasma (units/100 ml.) Post thaw 228.06 199.66 151.30 190.05 75.48 81.10 rele.of ACP ± 13.28 ±12.91 ± 14.50 ± 16.47 ± 17.11 ± 17.71 in s.plasma (KA units /100 ml.) Post thaw 228.93 253.82 176.21 124.37 201.18 186.14 rele.of ALP ± 20.78 ± 22.22 ± 17.73 ± 16.06 ± 15.83 ± 17.22 in s.piasma (KA units /lOOml.)
The analysis of variance (Table 4) indicated that all the three programmes had significant effect on drop in post-thaw motility, live count and increase in abnormal acrosomes. The bulls as well as collections had significant effect on these characters in programme 3. Amongst the different programmes of cooling rates, programme 3 showed significantly better percent sperm filtered through sephadex column, and less release of AAT in extra celluler medium. Programme 3 also showed better PTM, live count, acrosome maintenance and HAT release than other programmes marginally (Tables 1, 2 & 3) though not significant.
47 Table 4. Analysis of Variance Eftect of Different Cooling Rates on Seminal Parameters
(a) Control v/s programme 1
Source df Mean sum of squares of variance % % % Post thaw Post thaw Post thaw drop in livecount Ab.acro. motility drop increase
* NS NS Bulls 9 30.8 36.9 4.0 NS NS NS S.Collections 7 23.5 59.6 4.5
ProgNo. 1 178.5 417.6 17.4
Error 142 12.4 34.9 2.3
(b) Control v/s programme 2.
** NS NS Bull 9 38.8 76.1 3.8 NS NS ** S.Collections 8 23.1 65.6 9.9
Prog.No. 1 136.9 277.5 13.1
Error 161 13.5 41.2 2.3
(c) Control v/s programme 3
Bull 9 54.8 61.4 3.7
S.Collections 8 28.5 77.5 2.7
Prog.No. 1 777.9 1239.3 66.7
Error 161 13.8 26.1 1.3 * ** = P < 0.05 = P < 0.01 NS = Not significant.
48 Table 5. Analysis of Variance Effect of Different Cooling Rates on Seminal Parameters
(a) Control v/s programme 1.
df Mean sum of squares
Source % Post Post Post Post Post Post of Sperm thaw thaw thaw. thaw thaw thaw variance flit. rele. rele. rele. LDH rele. rele. thro. of of of acti- of of seph- HAT HAT MT vity ACP ALP adex in sp. in s. in s. in s. in s. in s. G-15 pellet plasma plasma plasma plasma. plasma.
NS NS * NS ** ** Bulls 9 293.0 29.1 395.6 238883.4 2649936 54340.4 123973.5 NS NS NS ** NS NS * S.Collect- 3 162.7 27.5 380.4 2041918.2 36307898 27757.8 90721.1 ions. NS NS NS NS NS NS NS Prog.No. 1 149.6 1.4 379.5 32248.3 277183 16134.0 12388.5
Error 66 78.7 22.0 213.0 103100.3 2221708 11607.3 33616.9
(b) Control v/s programme 2.
NS NS NS NS ** ** Bulls 9 92.1 35.9 269.4 210009.5 1598702 77477.7 150029.6 NS * ** * * ** ** S.Collect- 3 16.9 80.8 709.5 414659.0 4229990 64137.3 60142.5 ions NS NS NS NS NS NS * Prog.No. 1 4.4 24.6 215.3 31050.4 218300 30025.1 53540.0
Error 66 44.3 23.1 150.0 149802.9 1186817 14966.2 10617.7
(c) Control v/s programme 3.
** NS NS * * ** Bulls 9 132.0 69.6 418.1 190381.8 1797691 30974.2 64168.0 NS NS NS NS ** NS S.Collect- 3 21.3 268.2 213.6 28801.0 902194 441323.5 41616.5 ions NS NS * NS NS NS Prog.No. 1 509.1 14.1 331.1 602178.6 98982 823.6 4521.5
Error 66 24.6 24.1 247.5 101690.8 686540 11550.0 21688.2 * P<0.05 ** P<0.01 NS Not significant
49 In the present experiment the thermocouple probe was used for recording the temperature withinthe straws (see fig.1,2,3). According to Parkinson and Whitfield (1987) there are three major components in an ideal freezing curve. The rate of decline of temperature from equilibration temperature (4°C), the degree of supercooling which occurs below the diluent freezing point (-7°C) and the efficiency with which the latent heat of fusion is absorbed by the nitrogen vapour input. According to their findings both magnitude of supercooling temperature differential at the time for which sperms are held at or close to the diluent freezing point are the major factors determining survival rate. Whittingham (1977) also reported a similar finding for embryo freezing. Parkinson and Whitfield(1987) have carried out seriesof 5 X 5 latin square experiments using ejaculates from four bulls. The semen was cooled from 5°C to series of intermediate temperatures at various rates before being cooledto 30°C in a programmable freezer. They reported that slow cooling rate is required between +5°C to -15°C and rapid cooling is required between -15°C to -25°C. According to them initial cooling rate showed a quadratic relationship with survival with an optimal rate of -12.2°C/minute (when the data was combined). According to them the cooling rates below -30°C were not critical and loss of sperm below -30°C was found to be small. They also recorded that if the pulsed cooling when extended from below -30°C rapidly to -45°C, gave a small but significant improvement in sperm survival.
No such work on the use of programmable freezer for optimisation of freezing conditions for buffalo sperm were found in the literature. However Bhandari et at (1982) using 0.5 ml French straws, froze buffalo semen in 10, 7, 5 and 3 minutes timing by lowering the freezing racks at different levels above liquid nitrogen without knowing the rate at which temperature decreased inside the straws. On the basis of post thaw motility they reported fastest timing of freezing to be the best. Earlier Landa and Almquist (1979) and Allen and Almquist (1981) used biological freezer to bulk freeze the semen from bulls. They found that the physical packing and the pressure due to the expansion of freezing caused displacement of plugs within the straws, cracking of straws and damage to the integrity of the spermatozoa. The ranges of post thaw survival as determined by direct normal observations were about 2% less in forced vapour unit. Jansen (1989) using different cooling rates ranging from 15.5°C to -129.0°C/mm reported that the rate of cooling of -129.2°C/mm gave the spermatozoa with least normal apical ridges and cooling rate of -26.4°C/mm, the highest. Survival of the sperm also showed a similar trend in their experiment.
All the cooling rates selected gave significantly better quality of frozen semen in the present experiment as compared to conventional freezing over liquid nitrogen in static vapour and amongst the different cooling rates in programmable freezer a rate of -17.32°C/minute between + 4°C to -40°C gave the best results as judged by the laboratory tests. This rate of cooling was slightly faster than the recommended rate by Parkinson Whitfield (1987) and slower than the rate reported by Jansen (1989).
50 Cell death occurs in rapid freezing due to the formation of intracellular and possibly extra-cellular ice. In contrast, slow cooling causes cell death due to the diffusion of intracellular water into the diluent which then becomes increasinglyhypertonic as the diluent water freezes causing intracellular osmotic damage as the cell dehydrates. The cooling rates have to be balanced in such a way that the damage to the sperm cell is minimised. It should not be too fast to cause cell death due to cold shock or too slow to cause death due to osmotic shock. It has also been shown (Farrant 1980) that ultra rapid freezing gives excellent morphogical preservation but with poor retention of function on thawing since a large number of minute ice crystals are formed.
ACKNOWLEDGEMENTS
The authors are thankful to the International Development Research Centre (IDRC) Ottawa, Canada for providing financial support for the project. They also wish to thank Dr. Manibhai Desai, founder President, BAIF for his keen interest and encouragement in publishing the results.
REFERENCES
Alfred Linker, 1966. Methods in Enzymology, Academic press, New York. Vol Ill, PP 650-654.
Allen, C.H. and Almquist, J.O.,1981. Effect of bulk freezingstraws of bovine spermatozoa in programmed freezer on post thaw survival. J.Anim. Sd. 53(6): 1432-1439.
Bhandari, N. Chauhan, R.A.S., Mcthew, A.,1982. Note on the effect of rate of freezing on the freezability of buffalo spermatozoa. Indian J.Anim. Sci. 52(9) 812-813.
Bhosrekar, M.R., Mokashi, S., Purohit, J.R., Gokhale, S.B. and Mangurkar, B.R. 1991. Studies on the effect of deep freezing and seasons on the leakage of aspartate amino transaminase into extracellular medium and sperm morphology of Murrah buffalo bulls. Anim. Repro. Sci. 26: 219-226.
Crabo, B.G., Bower, R.E and Graham, E.F. 1970. The effect of glycerol on bull and boar seprmatozoa in a vitro measured as loss of intracellular glutamic oxaloacetic transaminase (GOT). Proceedings Xl Nord. Vet. Congr. (Bergen) 22-25 July. 76:242 (abstr.)
Crabo, B.G., Bower, R.E., Brown, K.l., Graham, E.F. and Pace, M.M.,1971. Extracellular glutamic oxaloacetic transaminase as a measure on membrane injury in spermatozoa during treatment (Anim. Breed. Astr. 50 No - 41) In : A. lngleman, Sundberg and N.
51 Lunell (Eds). Current problems in fertility Plenum, New York.
Farrant, J.,1980. General observations on cell preservation. Ashwood-Smith M.J and Farrant J. (Eds) In low temperature preservation in medicine and biology. Pitman Medical Kent, U.K. PP 1-17.
Heuer, C., Nazir Tahir, M., Crabo, B.G. Bader H, Shah, M. and Saji M.,1983. A simple method for the assay of water buffalo semen by filtration through sephadex. Pakistan Veterinary Journal 3, 157-161.
Jansen, H.B., 1989. The influence of cooling rates practised in Netherlands on survival of bovine spermatozoa packaged in 0.25 ml French straws In 11th Int. Congress on Ani. Reprod. & A.l.,University College, Dublin, Ireland.
Kind, R.P.N. and King, E.J., 1954. Determination of alkaline phosphatase by measuring liberated inorganic phosphate. J.Clin. Path. 7;323.
King, E.J. and Jagatheesan. 1959. Cited by Herold Warley, In Practical Clinical Biochemistry, 4th edn, William Heinemann Medical Books Ltd, London PP 462-466.
Landa, C.A. and Almquist, J.O. 1979. Effect of freezing large numbers of straws of bovine spermatozoa in an automatic freezer on post thaw motility and acrosomal retention. J.Anim. Sci. 49 : 1190-1192
Oser, B.L. 1965. Hawk's physiological chemistry 14th edn. Mc Graw Hill New York, PP 1124-1126 and 1127-1128.
Parkinson, T.J. and Whitfield, C.H. 1987. Optimisation of freezing conditions for bovine spermatozoa. Theriogenology 27(5): 781-797.
Snedecor, G.W. and Cochram, W.G. 1976. Statistical methods. The Iowa State University Press, Ames IA.
Tuli, R.K. Singh M. and Matharoo J.S. 1982. Effect of different extenders on Glutmaic oxaloacetic transaminase (GOT) and glutamicpyruvic transaminase (GPT) release from frozen buffalo semen. Theriogenology 18:55-59.
Whittingham, D.G. 1977. The freezing of mammalian embryos. In Elliot, K and Whelan, J. (Eds),Ciba Foundation Symposium, 52, Elsevier, Amsterdam 1977. 00 97-124.
52 RECENT ADVANCES IN LIVESTOCK PRODUCTION TECHNOLOGY: GUJARAT'S EXPERIENCE
BK. BHAVSAR Director, of Animal Husbandry, Gujarat State and
H.H. Chhaya Joint Director of Animal Husbandty, Gujarat State.
INTRODUCTION
Livestock production in semi-arid and arid areas is gaining vital importance. This is because of the fact that due to uncertain and erratic rainfaH, agriculture production has become uncertain. This has inspired the farmers to take up mixed farming. This is more true for the small and marginal farmers having scarce resources in terms of land and irrigation facilities. Milk production enhancement through routine programmes has a long gestation period. In Gujarat, besides Animal health, feeds and fodder, cattle development with supply of improved bulls, cross breeding, A.I. etc. have been taken up, which have proved to be useful in general. However, programmes like Embryo transfer, synchronization of heat in cows and buffaloes, and enrichment of low grade roughages through urea treatment have been taken up recently. Utilization of low grade roughages productively is very important because wheat straw and paddy straw are rarely, fed to the livestock. In nutritious fodder scarce areas, enrichment of such straws has great potential in raising the plane of nutrition.
The experiences of Gujarat in the above areas have been given below.
EMBRYO TRANSFER TECHNOLOGY
Embryo transfer technology is a well recognised scientific technique which is extremely useful for livestock improvement. The technique nowadays is used commercially in most of the developed countries. The embryo transfer and embryo cryo-preservation techniques have been standardised and practiced by many agencies in India. Department of Animal Husbandry - Gujarat State has also taken up the embryo-transfer programme since the year 1990-91 at Cattle Breeding Farm - Mandvi, District Surat, on an experimental basis. Under the programme, initially 10 cows were selected as recipients and donors. After giving standardised treatment, flushing programme was carried out. But no embryo was collected initially. Flushing programme was carried out on 1.1.93. 3 good quality viable embryos and one poor quality embryo were collected. This work was carried out under field conditions.
53 The main constraints in implementation of embryo transfer technology are as under
1. The latest scientific instruments/equipments essential for embryo transfer programmes are not available in the country. So to procure such instruments! equipments and to establish scientifically sophisticated laboratory, additional funds are required.
2. Embryo transfer technology requires skilled manpower. To implement ETT programme successfully, special training should be provided to those persons who are actively engaged in embryo transfer work.
HEAT SYNCHRONISATION
The department of Animal Husbandry, Gujarat State has taken up the heat synchronisation programme on an experimental basis in some villages covered under I.C.D.P. areas. During the year 1990-91, the programme was carried out for the first time in which 370 animals (cows/cross bred cows/buffaloes) were treated. After the treatment, 277 animals had shown signs of oestrus and the same were inseminated. The percentage of animals which came in oestrus was 74.19.
The second treatment was given to 69 animals which did not respond in the first treatment and out of these, 56 animals had shown signs of oestrus. In two attempts, 333 animals out of 370 animals came in oestrus after the treatment, resulting in 90% achievement.
All 333 animals which came in oestrus were inseminated. After three months, all the animals were examined for pregnancy diagnosisand 50% animals were found pregnant. Normally under field conditions, conception rate in the animals with Artificial Insemination is 35 to 40%. Thus the heat synchronisation technique is also helpful in increasing the conception rate in animals.
The main constraints of this technique are
1) Non availability of required hormones. 2) Hormonal treatment is expensive.
54 USE OF MINI-STRAW IN Al. WORKS
The department of Animal Husbandry, Gujarat State has carried out an experiment regarding performance of Artificial insemination by using frozen semen doses in ministraw having a capacity of 0.25 ml. These semen doses are being prepared at the frozen semen production centre-Haripura, District, Surat.
During the year 1991-92, 307 cows were inseminated with this type of mini-straw frozen semen doses. On pregnancy diagnosis of these 307 animals, 136 animals were found positive, resulting in 44.29% achievement.
Another experiment was carried out on 69 buffaloes. All the animals were inseminated with similar type of semen and as a result 41 buffaloes were found positive on pregnancy diagnosis resulting in 46% achievement.
The average conception rate with Artificial insemination using frozen semen dose in medium straw is approximately 30 to 35%.
From this it can be concluded that the conception rate with A.l. using frozen semen dose in mini straw (capacity approximately 0.25 ml.) stands higher as compared to medium straw (capacity 0.5 ml.).
There are also other advantages of this method as given below.
1) It maximises the output per bull. 2) Storage is easy and economical. 3) Transport and use are simple. 4) It minimises the cost per Artificial insemination.
ENRICHMENT OF LOW GRADE ROUGHAGES WITH UREA TREATMENT
In the low and erratic rainfall areas, availability of nutritious fodder is a serious problem. However Agricultural by-products like wheat straw, paddy straw are available which are not normallyfed to the animals. Enrichment of such roughages can play a very important role. The State Government has taken up a scheme wherein subsidy Is available to the farmer for enrichment of low grade roughages by urea treatment. This scheme is becoming popular and during 1992-93, around seven Iakh kg lodder was enriched.
55 ANIMAL NUTRITION CONSTRAINTS AND PERSPECTIVES IN DAIRY ANIMAL NUTRITJON IN INDIA
V.D. MUDGAL Project Directorate On Cattle G-123, Meerut 250 005, Uttar Pradesh.
ABSTRACT
The inadequate feed resources problem is prevalent in many developing countries of Asia and Pacific. Due to increasing human population pressure, it will become increasingly difficult to divert arable land for the production of feed and fodder. The approaches to increase milk productivity have been listed.
Under the new technologies the role of "protein by-pass" and "urea molasses blocks" have been highlighted. The results have revealed that the manipulation of rumen function through the removal of the ciliate population appears to have considerable potential for improving ruminant productivity. In these countries, the feed milling industry has to come forward to play its role with special reference to compound feed production based on agro-industrial by products. Perhaps, there is a dire need for making sincere efforts for the education of farmers on the part of private feed industries, cooperatives and Government.
INTRODUCTION
In developing countries, cattle and buffaloes are not just sources of income for majority, they are in fact the major investment of their livelihood. Dairying is an important source of income and means of employment for a large number of people in rural areas. But by and large the animal population (FAO 1990) is low in productivity and also suffers from low genetic potential and meagre feed resources (Table 1).
57 This inadequate feed resource problem is prevalent in many of the South Asian countries where ruminant animals are getting increasingly dependent on low quality roughages which have high cell wall constituents ranging between 65-80% of organic matter with a lignin content of 10-15% of cell wall constituents. The dry matter intake on such low quality roughages is hardly about 50-70 g/kg W 0.75 which is insufficient even for maintenance. This intake on poor quality roughages like straws and stovers is about 60-80% lower than that found on high quality roughage feeding. This is particularly because of their complex physical and chemical structures which are the main barriers in their efficient utilization. For the efficient utilization of these low quality roughages, it is necessary to overcome these physical and chemical barriers.
In developing countries where the buffalo plays an important role, it has been argued that the primary limitation to ruminant productivity is the imbalanced nature of nutrients in available forages and not their low digestibility (Preston and Leng, 1985) and that there are two major strategies for improving milk production i.e. supplementation of balanced nutrients and manipulations to increase digestibility. The principles underlined in the efficient use of mature fibrous resources by ruminants are now well established. The focus on : (i) Creating efficient rumen eco-system and (ii) Balancing the products of fermentative digestion with dietary "escape or bypass" nutrients (largely protein) to optimise productivity (Mudgal, 1989).
CROP RESIDUES - STAPLE DIET FOR RUMINANTS
Due to increasing population pressure, it will become increasingly difficult to divert arable land for green fodder production. Even the present shortage of green fodder may be expected to increase rapidly. Thus, cattle and buffaloes will have to be raised primarily on crop residues such as straws. But to sustain higher production the concentrate feed (based on mainly agro industrial by-products) will inevitably be the essential and perhaps sole source of nutrients even if they can be used only as a marginal supplement to maximise crop residue utilization. An example can be taken from India where milk production which was almost stagnant between 1947 to 1970 (an annual growth rate of just 1%) started responding to the market stimulus and inputs from 1970 onwards, with a growth rate of 6.6% per annum - doubling the milk production by 1985. The per capita availability of milk which declined from 132 g per day in 1951 to 107 in 1970, increased to 158 g/day. Today India has emerged as the third largest producer of milk in the world. Its production of some 50 million tons of milk annually is next only to 106 million tons in the USSR and 66 million tons in the USA. Further 95% of milk in India is produced out of crop residues like paddy straw, wheat straw, maize & millet stovers combined with concentrates prepared - solely out of agricultural by products like oil cakes, molasses, rice polish etc. (Aneja, 1990).
58 CONSTITUENTS OF MILK
The major carbohydrate present in milk is lactose. Because of its spatial configuration, lactose has unique nutritional properties. It is synthesised in the mammary gland from glucose by the intervention of two enzymes, an epimerase and glycosyl transferase. The proteins of milk are mainly caseins, lactalbumins and lactoglobulins and are synthesised in the udder from the amino acids transported from the blood. The fats of milk are partly made in the mammary gland by synthesis de novo and are partly dietary in origin. In essence, efforts in increasing lactation aim at increasing the availabilityof various precursors of milk components, efficiency in the transport of these nutrients into the mammary gland and the facility with which milk is released from the udder (Srinivasan, 1991).
APPROACHES TO INCREASE MILK PRODUCTIVITY
There are several ways in which the routes of biosynthesis of fats, lactose and proteins can be manipulated. Important among them are i. Suitable processing of raw materials. ii. Adjusting the energy/protein ratio. iii. Providing optimum levels of proteinous/non-proteinous nitrogen. iv. Use of the right combination of raw materials. v. Use of the right balance of various fatty acids in the feed and vi. Use of methanogenic inhibitors.
Although all these factors are important in varying degrees, for increasing the productivity of the cattle, the role of the quality and quantity of proteins in increasing milk yield has been discussed.
Protein requirements of ruminants are traditionally expressed in terms of either crude protein or digestible crude proteins. It is normally very difficult to fix a particular level of protein in the feed as being optimum for milk synthesis for the following reasons
1. The right balance of amino acids in the mammary gland for the synthesis of casein and other milk proteins is essential. Because of the complexity of the rumen bio-chemical mechanisms and the extensive degradation of the dietary proteins, the amino acid profile available for the productive metabolism of the animal is completely different from that of the diet. Consequently, it will be difficult to establish the level of crude protein in the feed for optimum lactation.
59 2. There is no clear-cut evidence on the nature of the limiting amino acids. Lysine, methionine, histidine, leucine, arginine - have all been reported to be essential.
For these reasons, greater importance has been given lately to the digestibility of proteins in the rumen, post-ruminal availability of proteins and amino acids.
Several attempts have been made in the past to understand the proteolysis of dietary proteins in the rumen. The proteolytic activity has been shown to be associated with a large number of rumen microbes although most of the studies on the mechanism of proteolysis have been made with a strain of Bacteroides ruminicola.
Solubility of proteins in different solvents was equated with degradability in the rumen. However, this has been challenged. It is now universaHy believed that modification of molecular/tertiary structure of proteins renders them resistant to degradation. The studies on the digestibility of dietary proteinsfrom the different raw materials in the rumen and the post ruminal availability of the various important amino acids with and without chemical modification of proteins of raw materials have been conducted. The advantageous effects of such an approach in terms of enhanced lactation in cows and buffaloes were also investigated in detail.
The following table gives the ruminal and post ruminal degradability of a raw material before and after the modification of proteins.
Table 1.
Before After Processing Processing
Ruminal degradation 78.8 8.6
Post ruminal degradation 10.8 77.0
Protein production can thus offer an opportunity of increased milk production. This approach was exploited by some feed manufacturers and feeds with protected proteins were introduced into the market by them more than eight years ago (Srinivasan,1991). Keeping in view the above discussion, the National Dairy Development Board in India has initiated a massive project on production of concentrate mixure with the following specifications (Table 2).
60 Table 2. Requirements for Bypass Protein Feed.
Sr.No. Characteristics Requirement
i) Moisture, percent by mass, Max 10 ii) Crude protein (N x 6.25) percent by mass, Mm 30 iii) Crude fat, percent by mass, Mm. 3.5 iv) Crude fibre, percent by mass, Max 8 v) Acid insoluble ash, percent by mass, Max 2.5 vi) Rumen undegradable protein (UDP) Percent by mass, Mm 20 vii) Rumen degradable protein (RDP), Percent by mass, Max 9
Note : The values specified for characteristics (ii) to (vii) are on moisture free basis.
Thus the importance of "bypass protein" in the feeding of cattle and buffalo for growth and milk production is evident. Perhaps the technology along with urea molasses block might revolutionise the feeding pattern in developing countries.
A PROJECT IN INDONESIA FUNDED BY THE UNITED NATIONS DEVELOPMENT PROGRAMME (UNDP)
A project in Indonesia funded by the United Nations Development Programme (UNDP) used radio isotopes and concentrated work on native grasses and rice straws to feed their buffaloes. There are many problems with feeding these materials. Firstly, they are extremely indigestible since they contain large amounts of lignocellulose. Secondly, they are low in protein and other materials which are needed to provide a healthy balanced diet and hence animals do not perform well.
The approach was to try and supplement the grass or rice straw. The points kept in mind were that the efficiency of the fermentaflon process which takes place in the fore stomach or rumen of the buffaloes and which is responsible for digesting the feed had to be optimized by providing more of the right kind of nutrients needed by the rumen bacteriaand protozoa. In this way the grass or straw could be more quickly and easily digested. And secondly, the supplements had to be such that a high proportion of their nutrients (e.g. Protein bypass) could escape break down in the rumen and be digested in the animals intestine from where they could be absorbed and used to produce proteins for growth (bypass nutrients).
61 The scientists at BATAN, Indonesia used 14C, 32P and 15N labelled material to measure the efficiency of the rumen fermentation process in animals, fed some of the most commonly available by-products in the country, and determined how much protein should avoid rumen break down and reach the intestine. After further work, these supplements were made into a multinutrient block which could be licked by the animal and thereafter they were distributed to the villagers to be tested for their effect on intake and productivity and their acceptance by the farmers. With such a supplement the animals were able to gain weight at the rate of 3-4 kg/week when given access to a blockconsisting of molasses, urea and local source of bypass protein (e.g. cassava waste, rice bran or soyabean curd waste). In other words, when buffaloes did not receive supplements, 55 tons of grass were needed to produce one ton of body weight gain whereas only 10 tons of grass were needed for 1 ton of body weight gain when 150 kg supplements were also fed. In effect, the efficiency of feed conversion into meat increased enormously. In dairy cattle, a daily increase on milk yield at the rate of 2 litre per animal has been reported (Dargie, 1989).
NEW TECHNOLOGY BEING APPLIED
The scientists have been evolving better feed techniques for high yielding buffaloes and cattle. They have popularised the bypass protein feed that takes the feed directly to the second chamber of the digestive system where it is actually utilized by the animal. The protein, fat and even starch which otherwise depresses the rumen pH causing health hazards is being protected from degradation in the rumen and is carried for hydrolysis in the intestine where it yields 90% glucose. Therefore, it is advantageous to protect the starch and bypass the same to the intestiie for better utilization (Mudgal, 1991).
UREA MOLASSES BLOCKS
The National Dairy Development Board has developed Urea Molasses blocks which contain molasses 45%, urea 15%, mineral mixture 15%, salt 8%, calcite powder 4%, bentolite 3% and cotton seed meal 10% with the following specifications (Table 3).
62 Table 3. Requirements for Urea Molasses Blocks.
Sr.No. Characteristics Requirement
i) Moisture, percent by mass, Max 3.5
ii) Crude protein (N x 6.25) percent by mass, Mm 58.00
iii) Crude fibre, percent by mass, Max 2.0
iv) Total ash, percent by mass, Mm 34.0
v) Acid insoluble ash, percent by mass, Max 3.0
vi) Calcium, percent by mass, Max 4.0
vii) Phosphorus, percent by mass, Mm 1.5
viH) Sulphur, percent by mass, Mm 1.0
ix) Urea, percent by mass, Max 15.0
Note : The values specified for characteristics (ii) to (viii) are on moisture free basis.
The studies carried out at National Dairy Research Institute Karnal indicated that supplementation of UMB to straw based diet led to 25-30% higher straw intake (Gupta & Singh, 1990). Similarly paddy straw consumption in buffaloes increased from 1.5 kg to 1.9 kg/100 kg body weight due to supplementation of UMB lick. In ten villages the average milk sold in the collection centres increased by 0.4-1.1 LID when the villagers commenced feeding UMBs to their buffaloes (Table 6). Other trials showed that concentrate supplementation could be reduced without loss of milk production when a UMB was given.
GROWTH STUDIES
Jersey bulls (350 kg live weight) fed rice straw plus a concentrate (low in true protein i.e. about 15%) trebled their rate of weight gain when fed a molasses/urea block in conjunction (Table 5).
63 In many parts of rural areas, the farmers are not well aware of the need for a balanced feed and do not appreciate the cost - nutrient relationship. The process of enlightening the farmers through extension and education programmes is virtually non existent in the industry. Enlightened rural marketing efforts are required in the cooperative and private sectors of the industry to bring about a greater understanding on the part of farmers. Therefore, there is a strong need for programmes for educating farmers. Perhaps, there is a need to make sincere efforts for farmers education on the part of private industries, cooperatives and Government.
Table 4. FAO Statistics on Livestock Population (1990) (.000).
Country Cattle Buffalo Country Cattle Buffalo
Bangladesh 23,359 1,800 Bhutan 422 4 Cambodia 2,100 750 China 76,965 21,426 India 1,97,300 75,000 Indonesia 10,300 3,400 Iran 8,000 230 Japan 4,760 — Korea' DPR 1,300 — Korea REF 2,051 Laos 842 1,072 Malaysia 652 185 Mongolia 2,580 — Burma 9,150 2,020 Nepal 6,281 3,013 Pakistan 17,573 14,686 Philippines 1,629 2,765 Sri Lanka 1,839 983 Thailand 5,669 5,350 Vietnam 3,199 2,871 Australi 22,602 — Fiji 160 — New Zealand 8,073 — Papua New 103 — Guinea Samao, W. 30 — Tonga 10 — Vanuatu 129 Asia-Pacific Total 4,07,090 135,554 World 12,79,257 140,759
Source: Selected Indicators of Food and Agriculture Development in Asia-Pacific Region, 1980-90, FAQ Regional Qffice for Asia and the Pacific, Bangkok, Thailand.
Table 5. The Effects of Supplying Molasses/Urea Blocks to Cattle Fed Rice Straw PIus 1 kg Amul-dan.
Straw Block Live Wt. Feed cost/kg intake intake chan ge gain (kg/d) (g/d) (g/d) (Rupee/kg)
No block 6.4 0 220 9.3
With block 6.8 530 700 3.7
64 More recently it has been demonstrated that feeding a meal high in bypass protein (low in grain) as compared with normal cattle feed concentrate (Amul-dan) increased milk production and live weight gain without substantially influencing the basal feed intake).
Cattle (Jersey, Jersey X Kankrej) in pens (in groups of 75 animals) were fed 40 kg of green forage per head per day. The forage consisted of 60% legume (mostly lucerne! cowpea) and 40% non-legume (maize, sorghum/oats). A major point here is that the animals in group 1 disposed off nutrients equivalent to 20-25 MJ of energy presumably through futile cycles of metabolism. The extra metabolic heat production would have increased the body temperature by 16.5°C if the animal could have allowed this to happen. The feeding trial was conducted during the cool season. In other words if the environmental temperature was critical for cattle in group 2 then the animals in group 1 would have needed to reduce their feed intake by 20 MJ metabolisable energy.
DEFAU NATION
Manipulation of rumen function through the removal of the ciliate population appears to have considerable potential for improving ruminant productivity. A variety of methods which have been employed to obtain ruminants free of ciliate protozoa can be classified into three categories (Mudgal, 1990).
i) Isolation of new born animals. ii) Chemical treatment - Alkanate 3SL 3. iii) Dietary manipulation - Cotton seed hulls, certain tree leaves found in Dehradun are-(Entrobolium timbova) an exotic plant from Brazil.
A number of experiments have now confirmed that when ciliates are removed from the rumen there is an increase in the flow of non ammonianitrogen (NAN) and bacterial N at the abomasum of sheep. It is important to note that the increase in post ruminal flow of NAN was due to both an increase in microbial protein and dietary protein suggesting that more dietary protein was escaping degradation in the ciliate free rumen. The data produced by Bird (1989) show the outstanding results of increased growth rate and wool growth of the ciliate free weaner lambs and hoggets. Chaudhury & Mudgal (1989,91) also studied the effect of defaunation on growth of buffalo calves under the Indo-Australian collaborative project at Hissar and observed better feed utilization and growth rate in buffalo calves defaunated by chemicals, feeds or tree leaves.
A recent communication (Leng et al, 1991) has confirmed that there will be more microbial and dietary protein available to the ruminant when Protozoa disappearfrom the rumen. It follows therefore, that in the absence of Protozoa in the rumen, there
65 is an increase in the total protein available related to the VFA produced and absorbed from the rumen. The improved P/E ratio will benefit the animal where it is fed diets low in proteins. The work carried out at CIRB, Hissar where the buffaloes were defaunated by drenching the animal with surface active agent increased liveweight gain and improved feed conversion efficiency of heifers on all diets with the response being greatest in animals receiving the most groundnut cake (GNC) supplement (Table 7). Supplementing the basal diet, the 750 g per day of GNC increased the growth rate of the faunated animals from 220 g/day to 400 g/day (82%) and the growth rate of the defaunated animals from 263 g/day to 477 g/day (82%). The combination of defaunation and supplementation with 750 g/day GNC increased the growth rate of heifers from 220 g/day to 477 g/day (117%).
FEED MILLING
n approach which has been initiated in India refers to the coordinated research project and the utilization of agricultural byproducts and industrial waste material for evolving economic rations for livestock funded by Indian Council of Agricultural Research (ICAR) since 1967. Many feed material including non conventional feed, are also being used in formulating rations for livestock and poultry. The feed compounding industry is presently using such material to produce livestock feed which has significantly increased over more than a two and a half decades (Table 11), (Mudgal, 1991).
The compound feed production Afro-Asian countries is given in Table 12.
CONSEQUENCES OF LOW VOLUME OF BALANCED FEED PRODUCTION
Compound cattle feeds today account for only some 10 percent of the total concentrate fed to different classes of livestock in India. Of the total installed annual cattle feed milling capacity of 3.00 MMT, the private and cooperative sector share 1.5 MMT each. In addition feed plants in the small scale sector and poultry feeds constitutean additional 3 to 4 million tons. Installed capacity utilization, however, is only 60 per cent. Therefore, the total quantity of balanced compounded feed produced can sustain only some 10 per cent of the total milk produced in the country. Though cattle feed production in the country is increasing, efforts have to be made to increase the installed capacity to atleast 30 million MT by the end of 1995, to achieve the VIII plan target milk production of 63 million MT (Patel, 1991). We are therefore, faced with a major problem of being able to provide adequate feed.
Perhaps, the low capacity utilization primarily is a reflection of the lack of rural marketing efforts both by the cooperative and private sector.
66 Table 6. The Observations on Response of Feeding Block Licks in Villages (Kunju, 1986)
Milk (kg/d) Milk fat (g/d) Village Pre lick with lick Pre lick with lick
AIwa 4.8 5.9 Punadhara 4.0 4.8 270 340
Fulgenamuwada 2.4 3.5 160 280 Hirapura 4.2 5.2 350 480 Bamroli (N) 3.6 4.2 270 380 Dehgam 4.3 4.7 310 350
Source Kaira District Co-operative Milk Producers' Union Ltd., Anand, India.
Table 7. The Effects of Replacing Balanced Concentrates With a High Bypass Protein Pellet on Live-Weight Change and Milk Yield of lersey x Kankrej Cows (M.G.P. Kurup, G. Kunju-Pers. Commn).
Crude protein Intake of Milk Live-weight in supplement supplement yield change (%) (kg/d) (KgId) (g/d)
Group I 20 4.7 8.0 -210
Group Il 30 2.6 8.8 +202
Source : National Dairy Development Board, Anand, India.
67 Table 8. Growth rate (g/day) and feed conversion (FCE) of faunated (+F) and defaunated (-F) buffalo given straw based diets, (CJ.R.B., Hissar).
Total DM intake Growth rate FCE kg/day) ( g/day) g DMI/g gain
Diet +F -F -F +F -F a Basal 3.13 2.57 220 263 18.2 11.1 b Basal + GNC (250 g/day) 3.16 3.08 277 370 12.9 9.8 Basal + GNC (500 g/day) 3.50 3.24 360 434 10.1 8.2 Basal + GNC (750 g/day) 3.48 3.14 400 477 9.8 7.3
Basal ration : Urea ensued wheat straw, green feed 3 kg, minerals. a GNC : Groundnut cake b
Table 9. Composition and Cost/kilogram of Urea-Molasses Mineral Block (Heat Treatment).
Feedstuff Parts by weight
Molasses 48
Rice Bran, Dl 31
Urea, (46% N) 10
Bone meal, steamed 03
Calcium oxide 03
Dolomite 03
Common salt 01
Vitamin-mineral mix 01
Total 100
68 Table 10. Composition of Urea-Molasses-MineralBlock (Cold Process).
Feedstuff Parts by weight Molasses 48 CementlCaO 08
Urea 12 Rice bran 26 Salt 03 Mineral mixture 03
Total 100
In cold process, the calcium oxide is used and promising results have been achieved. There is also a lot of scope for energy consumption.
Table 11. Production of Compound Livestock Feeds by CLFMA Members (All figures in 000 tonnes).
Year Cattle and Poultry Total Other feeds feeds 1964 25.0 14.4 39.4 1965 53.3 25.4 78.7 1966 69.4 39.2 108.6 1967 91.5 42.6 134.1 1968 94.5 47.1 141.6 1969 131.3 57.7 189.0 1970 125.4 84.3 109.7 1971 144.8 114.7 259.5 1972 188.5 183.0 371.5 1973 238.3 163.8 402.1 1974 275.4 164.6 440.4 1975 275.3 143.9 419.2 . 1976 313.8 160.5 474.3 1977 401.8 204.2 806.0 1978 428.9 225.7 654.6 1979 517.6 270.4 788.0 1980 549.0 350.2 900.1 1981 590.9 359.3 950.2 1982 618.9 325.3 944.2 1983 663.9 348.9 1012.8 1984 746.4 406.8 1153.2 1985 867.3 502.8 1370.1 1986 924.8 567.4 1492.2 1987 1208.9 630.3 1839.2 1988 1116.1 709.2 1825.3 1989 1151.3 772.5 1922.8 1990 1324.5 833.7 2161.2
69 Table 12. Compound Feed Production.
1974-76 1977 1978 1979 1980 1981 Growth
Average rates 1974-76 to 1981
World 289855 316871 342033 360495 371442 376913 4.5 DEVELOPING COUNTRIES 20385 27772 32867 37011 40683 43236 13.4 AFRICA 595 744 947 1165 1510 1834 20.8 Morocco 80 84 197 165 210 274 27.5 Nigeria 135 210 260 300 400 460 22.5 Tunisia 130 200 250 350 450 600 29.0 LATIN AMER. 13260 16270 18833 20370 21567 22600 9.3 Argentina 1100 1300 1500 1800 1850 1900 9.5 Brazil 6900 8600 10300 11100 11400 11700 9.2 Colombia 290 450 600 700 1100 1200 26.2 Mexico 3167 3780 3930 4400 4600 5000 7.9 Peru 320 340 403 320 467 500 7.7 Venezuela 1233 1500 1700 1700 1700 1800 6.5 NEAR EAST 784 1532 2287 3031 3861 4212 32.5 Egypt 150 490 800 1100 1640 1700 49.5 Iran 100 160 190 200 200 230 14.1 Libya 2 4 6 10 12 22 49.5 Saudi Arabia 15 90 180 250 300 400 73.0 Turkey 484 712 937 1271 1449 1500 20.5 FAR EAST 5676 9026 10500 12045 12725 13140 15.0 India 700 900 1000 1100 1150 1150 8.6 Korea,Rep.of 1070 1890 2700 3300 3500 3600 22.5 Malaysia 319 386 444 465 475 490 7.4 Philippines 747 830 961 1100 1150 1200 8.2 Sri Lanka — 50 90 100 100 100 — Thailand 700 900 1005 1150 1100 1100 7.8 ASIAN CPEs 70 200 300 400 1020 1450 66.0 China 70 200 300 400 980 1400 66.0
Source : FAQ estimates based on statistics provided by governments, the European Federation of Compound Feed Manufacturers, and National Feed and Feed Ingredient Association.
70 REFERENCES
Aneja, R.P. 1990. Dairy Development in a changing world : Lessons to be learned and principles in Dairy Development - The experience in India. Paper presented at 23rd International Dairy Congress, 1990 Montreal, Canada, Oct. 7-12. 1990. Indian Dairyman, XLII : 454-459.
Bird, SH. 1989. Role of protozoa in the Rumen. In Proc. Indo Australian Seminar on improving straw utilization on buffaloes for growth and milk production. 1987 p. 36-45.
Chaudhary, U.B., Gupta, R., Sikka, P., Sengar, S.S. & Mudgal, V.D. 1988. Ciliate population in growing buffalo heifers fed on urea treated wheat straw based diets. Proc. II World Buffalo Congress, Vol. 1 P. 203.
Chaudhary, U.B., Bird, S.H. & Mudgal, V.D. 1991. Effect of defaunation and protein supplementation on buffalo (Bubalus bubalis) Heifers. III World Congress, Bulgaria Vol. 1: 168.
Dargie, J.D.1989. Helping small farmers to improve their livestock. Joint FAO/IAEA Division of Nuclear techniques in Food and Agriculture, IAEA, Vienna, Year book P.B. 44-46.
F.A.O. 1990. Selected indicators of Food and Agriculture Development in Asia-Pacific Region, 1989-90, FAO Regional Office for Asia and the Pacific. Bangkok, Thailand. Asian Livestock Vol. XVI Sept. 1991, P. 107.
Gupta, B.N. and Singh, G.P. 1990. Urea Molasses Mineral Block Lick for Ruminant Feeding. Proc. 15th Dairy Husbandry Officer's Workshop, NDRI, Karnal (India), Dec. 27-28 p. 46-49.
Kunju, P.J.G. 1986. Urea Molasses Block lick : a feed supplement for ruminants. In Rice straw and Related feeds in Ruminant rations (M.N.M.Ibrahim and J.B. Schiere). Proc. of Workshop, Kandy, Sri Lanka. pp 261-74.
Kurup, M.G.P. and Kunju, P.J.G. 1991. National Dairy Development Board, Anand, Personal Communication.
71 Leng, R.A., Bird, S.H., Kleve, A., Choa Boon Singh, Ball, F.M., Asefa, G., Brumby, P., Mudgal, V.D., Chaudhary, U.B., Haryano and Hendratno, N. 1991. The potential for tree forage supplements to manipulate rumen protozoa to enhance protein to energy ratios in ruminants fed on poor quality forages. FAO Experts Consultation on Legume Trees and other fodder trees as Protein Source for Livestock, MARDI, Kuala, Lumpur, Malyasia, 14-18 Oct., 1991.
Mudgal, V.D. 1989. Current trends of Research in Buffalo Nutrition. Animal Productivity, P. 135-146. Oxford and IBH Publishing Co. Pvt. Ltd., New Delhi.
Mudgal, V.D. 1990. Management of Natural Resources: Cattle and Buffalo. Proc. First International Symposium on Natural Resources Management for Sustainable Agriculture. Feb. 6-10. New Delhi, P. 348-394.
Mudgal, V.D. 1991. Nutritional programmes related to Dairy Buffalo Production in Developing Countries. Third World Buffalo Congress, Bulgaria. Vol. IV : 962-976. Agricultural Academy Sofia.
Patel, Amrita. 1991. Keynote address at National Symposium on Technological Innovations in Animal Feed Industry, arranged by CLFMA at NDDB Anand, India, Dec.4.
Preston, T.R. and Leng, R.A. 1985. Matching Livestock Production to available feed resources. ILCA, Addis Ababa, Ethiopia.
Ranjhan, S.K., Faylon, P.S., Momongan, V.G. and Cruz, L.C. 1987. Husbandry of Swamp buffaloes in the Philippines. Field Document 10, F.A.O. & U.N.D.P., Philippines Council of Agriculture, Forestry and Natural Research and Development, Los Banoes, Philippines, 192 pp.
Srinivasan, K. 1991. Some recent trends in Protein Requirements in Cattle. Proc. National Symposium on Technological innovations in Animal Feed Industry. CLFMA, Bombay, India.
72 ROLE OF FODDER CROPS, BUSHES AND TREES FOR LIVESTOCK FEEDING
L. L. RELWANI BA/F Development Research Foundaton, Pune.
ABSTRACT
Top cross bred cows and high yielding buffaloes, stable and remunerative milk prices, greater demand for milk and milk products by urban middle class have in recent times helped to divert small pieces of land to cultiyated fodders. Intensive legume based fodder crop rotations and in mixtures with high yielding grasses like Hybrid Napier and Guinea grass on fertile irrigated areas have shown yield potentials of 150 to 200 t I ha / yr of green fodder. In arid and semi arid areas, management of grass lands by cleaning up of such pernicious weeds like Parthenium and Alternanthera sessilis (Resham Kanta) broadcasting seeds of Stylosanthes hamata, Centrosema pubescens, Macroptilium atropurpureum etc. and filling up the gaps with superior strains of grasses, application of small doses of fertilisers and micronutrients, controlled grazing and interplanting with fodder trees is of paramount importance. The most important fodder trees and bushes with multipurpose uses are Acacia albida, A. nilotica, A. saligna, A. senegal, A. seyal, A. tortiis, Ailanthus exce/sa, Albizia lebbek, Albizia amara, Albizia procera, Artocarpus heterophyllus, Azadirachta indica, Da/bergia sissoo, Dendrocalamus strictus, Gliricidia sepium, Grew/a optiva, Hardwickia binata, Leucaena leucocepha/a, Prosopis cineraria, Sesbania grandiflora, Sesbania sesban and Zizyphus jujuba. The management of such trees for lopping, pruning, branch cutting, coppicing and pollarding, leaf and pod conservation should be recognised as a high priority area of research so that their contribution as supplementary top feeds is well understood by the low resource farmers feeding their animals on tree leaves, fibrous crop residues, stubbles, edible weeds, roadside and community or forest grazing lands. Animal production potential of fodder trees in different agroclimatic zones based on fodder quality including antiquality factors and in different farming systems also needs to be critically evaluated.
INTRODUCTION
The milk and meat production for the escalating population is chiefly dependant upon the steady supply of green, succulent and nutritious cereal and leguminous cultivated fodders all the year round. The other alternatives are, improved management practices for our natural grasslands, optimal silvipastural combinations and top feeds, to sustain high yields. Th package of practices for obtaining high yields and nutritional quality is detailed below.
73 INTENSIVE FODDER PRODUCTION
Under irrigated and high soil fertility conditions, the following intensive fodder crop rotations can be adopted.
1. Maize+Cowpea - Berseem+Oats - Jowar+Guar/Cowpea 2. Maize+Cowpea - Jowar+Guar/Cowpea - Oats - Bajra+Cowpea 3. Maize+Cowpea - Lucerne (Annual) 4. Multicut sorghum - Berseem/Lucerne (Annual) 5. Hybrid Napier interplanted with cowpea in kharif and Berseem in the winter season.
6. Hybrid Napier interplanted with Cowpea/Guar in Kharif season and Lucerne in the winter season. Such type of rotations yield about 1500 to 2500 quintals green or 300 to 500 quintals dry matter and 30 to 50 quintals of crude protein per hectare per year. By feeding the cows at the rates of 20kg, 30kg and 40kg greens per cow/day, the annual requirements will be 75, 110 and 150 quintals per cow respectively. With an average yield of 1500 q/ha/year, it will be possible to feed 20, 14 and 10 cows/ha/year respectively. Thus with a well balanced green fodder supply of cereal and legume mixture in the proportion of 2:1 and additional 2 to 3kg concentrate mixture and 2 to 4 kg of straws, an average yield of 10 litres of milk per day during the lactation period of 305 days can be ensured. But this type of intensive farming requires inputs like assuredyear round irrigation water, large doses of manures and fertilisers, seeds of improved varieties and mechanised cultivation to carry out timely field operations. Since grain and cash crops have a priority over fodder crops, particularly in small and medium sized holding, only a small proportion of the total cultivated area near cities and large townships can be brought under such intensive type of farming.
LIMITED IRRIGATION FACILITIES
In case of lands which are dependent on monsoon rains or those which have seasonal irrigation facilities,, larger areas will have to be allocated for raising fodder crops so that surplus can be conserved in the form of hay and silage for the lean periods. This type of farming is prevalent in Western countries which are generally coveredwith snow in the winter season. Instead of large silos, smaller ones measuring 2mx2mx2m or 3mx3mx2m can be made under Indian conditions to fill them up quickly. Later on they can be opened and silage regularly fed to animals. Berseem, Lucerne and oats can be chatted, sundried for two to three days and stored as hay in barns or tilled in gunny bags with a moisture content around 13 to 14 percent. Since the demand for milk is
74 steadily increasing, particularly from urban and semiurban areas and the support prices are fairly remunerative, conservation of fodders becomes necessary to balance regular supply all through the year.
MANURING AND IMPROVED VARIETIES
The manuring practice should consist of a single application of farmyard manure for the entire rotation at the rate of 15 to 20 tonnes per hectare particularly for the cereal crops in the kharif season for proper decomposition. Since farmyard manure requires minimum 2 to 3 weeks to decompose, it will not be possible to leave frequent gaps of time between harvesting of one crop and sowing of another in the relay cropping system. The cereal-legume mixtures like maize/jowar+cowpea/guar, or oats may in addition be given 60 to 80 kg nitrogen + 20 to 30 kg P205 + 20kg K20/ha whereas lucerne and berseem crops may be dressed with about lOkgN+60kgP205+4OkgK2O/ha. Where the animals are regularly fed with mineral mixtures, there would be no need for application of micronutrients as the manure from such animals will contain adequate quantities to meet the requirements of the crops. The list of improved varieties of fodder crops, region wise currently found to be the best is given in Appendix I.
MANAGEMENT OF GRASSLANDS
Apart from cultivated fodders, the management of grasslands is of vital importance as such lands still constitute a major source of forage for livestock. The community controlled grasslands are shrinking rapidly and are further being infested with such pernicious weeds like Parthenium hysterophorus, and Alternanthera sessilis. The most nutritious grasses like Cenchrus ciliaris, Cenchrus setigerus, Lasiurus sindicus, Dichanthium annulatum, Sehima nervosum, Panicum antidotale, Chrysopogon fulvus, Bothrochloa and Cynodon species from our grasslands are therefore, steadily disappearing due to suppression by these aggressive weeds and excessive pressure from large numbers of goats, sheep and cattle.
Unless these weeds are systematically removed and the pasture lands reseeded with appropriate species mentioned above along with pasture legumes like Stylosanthes hamata, Stylosanthes humilis, Stylosanthes scabra, Atropurpureum macroptilium, Centrosema pubescens etc, the productivity levels will continue to fall and finally degenerate into barren lands. Controlled grazing, manuring, application of small quantities of micronutrients should be rigorously practiced to improve these lands which constitute the largest source of fodder, particularly for the poor farmers.
75 SILVIPASTURE
Trees and bushes spring up naturally in grasslands and keep multiplying through fallen seeds or the droppings of birds. If left to grow and not trampled by animals or cut by humans, they serve multiple uses like fodder, fuel, timber, fibre, gums, resins and even human food. The trees are Acacia ni/of/ca, Acacia catechu, Acacia farnesiana, Acacia leucophloea, Ailanthus exce/sa, Albizia lebbeck, Azadirachta indica, Bauhinia variegata, Butea monosperma, Ficus species, Hardwickia binata, Leucaena leucocephala, Pithecellobium dulce, Pongamia pinnata, Prosopis cineraria, Sa/vadora oleoides, Sesbania sesban, Sesbania grandiflora and Zizyphus mauritiana. Unfortunately their numbers keep decreasing due to excessive lopping in and out of season, cutting whole trees for fuel or timber and trampling by cattle and other animals. Some of the trees particularly nitrogen fixers are quite rich in protein and calcium while most exhibit deficiencies in phosphorus. It is necessary to plant trees in our grasslands to supplement the fodder resources as well as to help prevent soil erosion and conserve water. Apart from the above list of trees, others like Acacia modesta, Acacia senegal, Acacia toflilis, Gliricidia sep/urn, Holoptelea integrifolia, Me/ia azedarach, Moringa o/eifera, Parkinsonia aculeata, Tamarindus indica, Samanea saman, Term/na/ia alata, Term/na/ia arjuna, and Terminal/a chebula (may also be introduced for multipurpose uses) /n the wet tropics, Aegle marme/os, Albizia amara, A.chinensis, A.odoratissima, A.procera, Artocarpus hirsutus, Artocarpus heterophyllus, Syzygium cumin/i, Bauhinia purpurea, Bauhinia roxburghiana and Grewia op/va are ideal. Depending upon soil and climatic conditions, chemical composition and nutritive value of the leaves, age and optimal time of lopping, the quantity of leaf yield per tree, freedom from toxic chemicals, coppicing and pollarding capacity, complementary and competitive effects on the grass-legume understorey, palatability of leaves as well as the socio-economic aspects of the other parts of the trees, about 6 to 8 tree species may be combined according to their growth and canopy and systematically spaced to achieve the desired results.
LOPPING MANAGEMENT OF TREES
Normally trees take about 5-9 years to develop conopies large enough to yield good quantities of leaf fodder. Under optimal moisture and high soil fertility conditions, Leucaena leucocephala, Sesbania sesban, Sesbania grandiflora, Ailanthus excelsa, Moringa oleifera, G/iricidia sep/urn, Dendroca/amus Strictus, Acac/a tort//is, Acacia farnesiana and Zizyphus maur/tiana may start yielding leaf fodder after three to four years. It is therefore better to have a combination of slow and fast growing species. Lopping should be restricted to lower two thirds the size of the canopy so that enough leafy portion is retained at the top to carry on photosynthetic activity. Lopping should preferably be done in summer and winter seasons when the trees have practically
76 stopped growing. Besides, toxicities like tannins, mimosine and hydrocyanic acids are more concentrated in younger than older leaves. Depending upon the recovery period after defoliation, lopping may be done every six months to two years. Forage yields can be as little as only 2 kg/tree to 50kg/tree. With proper manuring, irrigation and weeding, the yields of leaves and small branches can be increased substantially.
TOX1CITES
Depressed performance of animals is an indication of an allelochemical effect and calls for investigation about the presence of toxic or antinutrient factors in the top feeds. Toxicities may principally be caused by any of the following factors.
1. Aversion : This results in reduced intake and low digestibility or induced deficiencies. Gliricidia sepium which is high in digestible protein has low palatability although obviously no toxic factor is involved. The animal rejects it because of a "mousy smell". This can be overcome by wilting the fodder for 12 to 24 hours and initially mixing it with salt or molasses for quite some time to improve its taste. After wilting, the volatiles disappear. The nature of these volatiles is not known.
2. Tannins : Tannins are generally tolerated upto about 5 percent on dry matter basis. But some of the woody species show 10 to 20 %. In extreme cases even upto 50%. They are toxic, irritant and corrosive. They depress N-digestibility by precipitation of proteins resulting in low DCP. Aegle marme/os, Morus a/ba and Grewia opt/va have low tannin content and high digestibilty, while Eugenia jambolana with tannin content of 7.5% shows low DCP.
Hydrolysable tannins have antimicrobial activity and affect rumen fermentation. They cause decrease in voluntary intake, unpalatability and reduction in influx of the nutrients through gut wall. Sulphur and Calcium hydroxide additions reduce their toxic effect(s). Condensed tannins are not absorbed and protect soluble proteins from fermentation in the rumen and also prevent bloat.
3. Lignin : Higher than 1 to 15% adversely affects digestibility through physical encrustation and chemical bonding with structural carbohydrates and proteins.
4. Mimosine : Although mimosine is easily detoxified by the rumen bacteria of the cattle in South and South East Asia, there have been reports of toxicity
77 symptoms from Australia and some other countries. This feed should be limited to 33%of the total ration. Inoculation of rumen microorganisms to alleviate toxicity is quite effective. Ferric salts also reduce uptake of mimosine and DHP from Leucaena leucocephala.
5. Cynogenic : When cynogenic glucocide comes in contact with hydrolases, free glucocide hydrogen cyanide is formed and disappears by diffusion from leaf. it is for this reason that cassava leaves are wilted before feeding as a normal practice.
6. Fluoroacetic: Inhibits the citrate cycle, Its tolerance can be increased adaptations. acid 7. Terpenoids : They are hepatoxic and also cause photo sensitization. and steroids
It may be observed that although fodderof trees serve as an excellent source of protein, energy, vitamins and minerals their antinutrient factors may be closely observed particularly with young and succulent foliage. They should be mixed with grasses, pasture legumes, straws, cultivated fodders, concentrates and mineral mixtures particularly while feeding high yielding animals. They should contribute not more than about 40% of the total diet.
REFERENCES
Blair, G. 1989. Diversity and potential value of shrub and tree fodders. International workshop on the utilization of shrubs and tree fodders by farm animals, Denpasar, Bali 24-29 July 1989. Brewbaker, J.L. 1985. Leguminuous trees and shrubs for South East Asia and the South Pacific. Forage in South East Asian and South Pacific Agriculture. ACIAR proceedings No.12 Proceedings of an International workshop held at Cisarua, Indonesia, 19-23 August 1985. Eds.Blair,G.J, Irovy, D.A, Evans, T.R. Hocking, D. 1993. Trees for drylands. Mohan Primalani, Oxford and IBH publishing Co. New Delhi. Lowry, J.B. 1989. Toxic factors, problems and methods of alleviating these in animals. International workshop on the utilization of shrubs and tree fodders by farm animals. Denpasar, Bali, 24-29 July 1989. National Wastelands Development Board 1986. Report of the Committee on fodderand grasses, Ministry of Environment and Forests, Government of India. Roy, D.R. and Rai, P. 1992. Top feed production and management under silvipastural system for rearing goat. Research in goats. Indian experience published by CIRG,
78 Makhdoon Mathura. pp. 74-81.
Saha, R.C and Gupta, B.N 1987. Tree leaves as feed for Dairy cattle in India. Indian Dairyman 39 (10), 489-492. Sharma, K. Ogra, J.L, Bhattacharyya, N.K. 1992. Development of Agrosilvipasture for goats. Research in goats, Indian Experience. Published by CIRG, ICAR, Makhdoon, Mathura. pp, 66-73. Thakur, S. S and Kundu, S. S. 1987. Tree leaves as livestock fodder, Indian Dairyman. 39 (8), pp 369-371. Table 1. Profits Per Hectare Per Year With Progressive Reduction In Yields Of Green Fodder From 110 To 55 Tonnes/Ha.
Yield of green fodder 110 99 88 77 66 55 (tonnes/ha/year) 1. cost of fodder for 400 1720 1911 2150 2457 2867 3440 days at 30kg/day 2. 2kg/day Bhusa (straws), 900 900 9000 900 900 900 400 days @ Rs.2.25/day 3. Concentrate mixture 3100 3100 3100 3100 3100 3100 4 kg/day for 310 days 2 kg/day for 80 days Cost on 150 150 150 150 150 150 feeds and fodder Interest 5870 6061 6300 6607 7017 7590 @ 12°k for half period 386 399 414 434 461 499 Total cost on feeds amd fodder 6256 6460 6714 7041 7478 8089 4. Additional costs 2681 2681 2681 2681 2681 2681 5. Purchase of a crossbred cow yielding 3100 litres of milk @ Rs.5000 for 400 days. Interest @ 12% on Rs. 5000 658 658 658 658 658 658 Depreciation © 16.67% per year 913 913 913 913 913 913 Total expenditure on one 10508 10712 10966 11293 11730 12341 cow for 400 days. Income for 400 days a. 3100 litres of milk 12400 12400 12400 12400 12400 12400 b. 400 x 10 kg dung/day 400 400 400 400 400 400 Total gross Income 12800 12800 12800 12800 12800 12800 Net Income for 400 days 2292 2088 1834 1507 1070 459 Net Income / day / cow 5.73 2.22 4.59 3.77 2.68 1.15 Number of cows/ hectare 10 9 8 7 6 5 Net profit per hectare/year 20915 17148 13388 9626 5858 2094
79 Table 2. Kharif Crops ( Monsoon Season)
Crops I II Ill IV V
Hilly North West North East Central Southern Cowpea - UPC-5286 UPC-5286 UPC-5286 UPC-5286 (Vigna UPC-5287 UPC-5287 UPC-5287 UPC-5287 sinensis or vigna CS-40 CS-40 CS-4200 IFC-8402
unguiculata) CS-35 IFC-8402 UPC-8705 PC-3 UPC-4200 UPC-4200 IFC-8401 IFC-8503
Guar HFG-119 HFG-119 HFG-119
(Cyamopsis - IGFRI-212-1 HG-202 - psoralioldes) HGS-285 HGS-204 IGFRI-2395-2 IGFRI-1019-1 HGS-284
Val - JLP-6 JLP-4 JLP-4 CAZRI-1258-1 CAZRI-1253-1 JLP-6 JLP-29 APLP-2 (Lab Lab Purpureus) CAZRI-1461 JLP-3 JLP-3 JLP-3 Maize African African Tall African Tall African Tall African Tall Tall Vijaya comp- GBM-84-2(KANKE GBM-84-2 PLP-Local osite Vijaya compo- PLP-Local GBM-84-2 site (Kalyani) Vijaya Comp. Deccan-103
Tesointe Improved Imporved sirsa Improved Improved Sirsa Sirsa Sirsa
(Eucleana - TL-1 TL-16 TL-16 TL-16 mexicana) TL-14 TL-2 TL-1 TL-13 TL-14 Jower - M.P.Chari M.P.Chari MP.Chari M.P.Chari U.P.Chari U.P.Chari U.P.Chari U.P.Chari
(Sorghum biclor) UPFS-21 S-411 UPFS-21 S-171 Early group (70-80 days) S-386 S-41 1 S-386 S-386 S-41 1 S-386 Late Group - HC-136 HC-136 HC-136 HC-136 P0-6 P0-6 PC-6 PC-6 (More than 80 Raj. chari Raj.Chari Raj.Chari Raj.Chari days) S-208 S-208 S-208 S-208 S-194 UPFS-22 S-194,UPFS S-194 UPFS-22 S-194 22,PC-54, PC-63 PC-63
80 Makhdoon Mathura. pp. 74-81.
Saha, R.C and Gupta, B.N 1987. Tree leaves as feed for Dairy cattle inlntha. Indian Dairyman 39 (10), 489-492. Sharma, K. Ogra, J.L., Bhattacharyya, N.K. 1992. Development of Agrosilvipasture for goats. Research in goats, Indian Experience. Published by CIRG, ICAR, Makhdoon, Mathura. pp, 66-73. Thakur, S. S and Kundu, S. S. 1987. Tree leaves as livestock fodder, Indian Dairyman. 39 (8), pp 369-371. Table 1. Profits Per Hectare Per Year With Progressive Reduction In Yields Of Green Fodder From 110 To 55 Tonnes/Ha.
Yield of green fodder - 110 99 88 77 66 55 (tonnes/halyear) 1. Cost of fodder for 400 1720 1911 2150 2457 2867 3440 days at 30kg/day 2. 2kg/day Bhusa (straws), 900 900 9000 900 900 900 400 days @ Rs.2.25/day 3. Concentrate mixture 3100 3100 3100 3100 3100 3100 4 kg/day for 310 days 2 kg/day for 80 days Cost on 150 150 150 150 150 150 feeds and fodder Interest 5870 6061 6300 6607 7017 7590 © 12% for half period 386 399 414 434 461 499 Total cost on feeds amd fodder 6256 6460 6714 7041 7478 8089 4. Additional costs 2681 2681 2681 2681 2681 2681 5. Purchase of a crossbred cow yielding 3100 litres of milk © Rs.5000 for 400 days. Interest @ 12% on IRs. 5000 658 658 658 658 658 658 Depreciation @ 16.67% per year 913 913 913 913 913 913 Total expenditureon one 10508 10712 10966 11293 11730 12341 cow for 400 days. Income for 400 days a. 3100 litres of milk 12400 12400 12400 12400 12400 12400 b. 400 x 10 kg dung/day 400 400 400 400 400 400 Total gross Income 12800 12800 12800 12800 12800 12800 Net Income for 400 days 2292 2088 1834 1507 1070 459 Net Income / day / cow 5.73 2.22 4.59 3.77 2.68 1.15 Number of cows! hectare 10 9 8 7 6 5 Net profit per hectare!year 20915 17148 13388 9626 5858 2094
79 Table 2. Kharif Crops ( Monsoon Season)
Crops II Ill IV V Hilly North West North East Central Southern
Cowpea - UPC-5286 UPC-5286 UPC-5286 UPC-5286 (Vigna UPC-5287 UPC-5287 UPC-5287 UPC-5287 sinensis or vigna CS-40 CS-40 CS-4200 IFC-8402 unguiculata) CS-35 IFC-8402 UPC-8705 PC-3 UPC-4200 UPC-4200 IFC-8401 IFC-8503
Guar HFG-119 HFG-119 HFG-119
(Cyamopsis - IGFRI-212-1 HG-202 - psoralioides) HGS-285 HGS-204 IGFRI-2395-2 IGFRI-1019-1 HGS-284
Val - JLP-6 JLP-4 JLP-4 CAZRI-1258-1 CAZRI-1253-1 JLP-6 JLP-29 APLP-2 (Lab Lab Purpureus) CAZRI-1461 JLP-3 JLP-3 JLP-3 Maize African African Tall African Tall African Tall African Tall Tall Vijaya comp- GBM-84-2(KANKE GBM-84-2 PLP-Local osite Vijaya compo- PLP-Local GBM-84-2 site (Kalyani) Vijaya Comp. Deccan-103
Tesointe Improved Imporved sirsa Improved Improved Sirsa Sirsa Sirsa
(Eucleana - TL-1 TL-16 TL-16 TL-16 mexicana) TL-14 TL-2 TL-1 TL-13 TL-14 Jower - M.P.Chari M.P.Chari M.P.Chari M.P.Chari U.P.Chari U.P.Chari U.P.Chari U.P.Chari
(Sorghum biclor) UPFS-21 S-411 UPFS-21 S-171 Early group (70-80 days) S-386 S-411 S-386 S-386 S-41 1 S-386 Late Group - HC-136 HC-136 HC-136 HC-136 PC-6 PC-6 PC-6 PC-6 (More than 80 Raj. chari Raj.Chari Raj.Chari Raj.Chari days) S-208 S-208 S-208 S-208 S-194 UPFS-22 S-194,UPFS S-194 UPFS-22 S-194 22,PC-54, PC-63 PC-63
80 Table 3. Agro-Climatic Zones Of India
Crops I II Ill IV V
Hilly North West North East Central Southern
Jowar - SSG-59-3 SSG-59-3 SSG-59-3 SSG-55-3 (Multicut) X-988 X-988 X-988 MFSH-5 (Sorghum MFSH-3 MFSH-2 MFSH-7 MFSH-4 biccior) MFSH-7 MFSH-7 MFSH-4 MFSH-7 MFSH-6 MFSH-6 S-199
Bajra PCB-15 L-72 L-72 L-72 L-72 (Pannisetum Composite-i TNSC-i TNSC-i UUJ-i americanum) TNSC-i Composite-9 UUJ-i PCB-15 UUJ-i MBFH-1 TNSC-i
Dma Grass IGFRI - TNDN-i IGFRI-S-56-i TNDN-1 (Pennisetum -S-4-2-i JHP-2 JHP-2 JHP-2 pediceHatum) JPH-4 JHP-4 TNDN-1 JHP-4 GFRI-5 JHP-3 IGFRI-S-56-i 56-1 Guinea Grass PGG-9 PGG-9 PGG-9 PGG-9 PGG-9 (Panicum PGG-i23 PGG-122 PGG-i47 PGG-i51 PGG-123 maximum) PGG-147 PGG-i4 PGG-i29 PGG-147 PGG-26
Hybrid Napier NB-2i IGFRI-7 (Pennisetum IGFRI-7 CN-13 Purpureum) - - - IGFRI-1O CN-8 x IRBN-4 (Pennisetum CN-13 americanum) Anjan Grass (Cenchrus - CAZRI-75 - CAZRI-75 CAZRI-75 ciliaris) CAZRI-1106 CAZRI-123 CAZRI-i23 Cazri -1263 Black Anjan (Cenchrus - CAZRI-76 - CAZRI-76 CAZRI-76 setigerus) CAZRI-4i5
81 Table 4. Agro Climatic Zones Of India Rabi Crops (Winter)
Crops I II III IV V
HILLY North West North East Central Southern
Berseem Mescavi Mescavi Mescavi Mescavi Mescavi (Trifolium Wardan Wardan Wardan Wardan Wardan alexaridrinum) BL-1O BL-63 JB-2 UPB-1iO HFB-481 HFB-476 JHB-i5B-86 JHB-15B-86 JB-2 JB-3
Lucerne - L.L.Comp..-3 L.L.Comp.-3 L.L.Comp.3 L.L.Comp-3 (Medicago RLS-88 IGFRI-112 RLS-88 Anand-4 sativa) Anand-3 IGFRI-2112 Anand-4 IGFRI-1212
Shaftal SH-48 SH-69 SH-69 SH-29 - (Trifolium SH-72 SH-72 SH-48 SH-72 resupinatum) SH-69
Mustard NDFS-1 No.77 NDFS-1 NFDS-1 (Brassica DLC-2 NDFS-1 DLC-2 DLC-2 - compestris) DLM-4 DLC-1 DLC-i
Oats Kent Kent Kent Kent Kent (Avena sativa) OL-9 OL-9 OL-i25 OL-9 OL-265 (Multicut) UPO-220 UPO-94 OL-344 OL-344 OL-219 JHO-829 NDO-9 UPO-219 OL-265 OL-125 JHO-813 OL-265 UPO-225 OL-219 NDO-3 PLP-3 JHO-829 JHO-813 NDO-3 UPO-219 OL-344 P0-287 JHO-841 OS-i48 JHO-861
Oats OS-6 OS-6 OS-6 Kent Kent (Avena sativa) OS-96 OL-9 OS-96 OS-6 OS-96 single cut OL-i29 OS-121 OL-125 OS-96 UPO-2i0 PLP-i OL-125 OL-265 OS-12i OL-125 OL-99 UPO-121 UPO-210 P0-22
82 PRODUCTION AND UTILIZATION OF GREEN FODDER, BUSHES AND TREES FOR LIVESTOCK
V.S. UPADHYAY Indian Grassland and Fodder Research Institute Jhansi, Uttar Pradesh
INTRODUCTION
India has a population of 870 million people but comprises only about two percent of the world geographical area. Similarly India possesses 450 million livestock but the productivity per animal is quite low. Shortage of feeds and fodder in addition to poor genetic potential of animals is one of the major limiting factor. Various estimates have been made about demand and supply of feed resources. As per an estimate made by a committee on fodder and grasses (1987), the dry and green fodder requirement in 1990, was 832 and 992 million tonnes (mt) against which the supply was only about 441 and 250 mt respectively. It clearly indicates the gap between demand and supply of the forage. The alarming gap between demand and availability is further compounded by regional imbalances. Out of 55 regions, only 12 show net surplus led by Punjab and Haryana. At the current rate of fodder production, the deficit of animal feed will be to the order of 15 per cent in case of straws, 64 per cent in case of green fodder and 80 per cent in case of concentrate by 2000 A.D. Therefore, there is an urgent need to increase feed and fodder production in the country.
Fodder Production and Utilization
It is well known that regular supply of green fodder is a pre-requisite for profitable dairy farming because forages are cheapersource of nutrients than concentrate. The common practice of feeding milch cattle and buffalo is to feed them on crop residues with little supplementation of green forages and concentrate. Large quantities of concentrate are required even for low producing animals if crop residues are the only roughages. This results in increased cost of feeding. Feeding of green forages, however, reduces the cost and raises the level of milk production. Cross-bred cattle specially require good quality forages for expression of their genetic potential of milk production.
Presently about 8.1 million hectares (4.4 per cent of the total cropped area) is only under fodder crops in India. Thus there is a need to bring additional area under fodder crops which seems to be a remote possibility. Adoption of advanced technology developed for intensive fodder production, needs to be popularized for increasing fodder production per unit area per unit time. Efforts are also needed to grow short duration fodder crops in between two arable crops. In wheat-jowarfmaize/bajra sequence, a gap period exists between April-June which can be utilized for growing fodder like maize + cowpea, sorghum
83 + cowpèa or balra + cowpea yielding 35-40 t/ha without affecting the kharif grain crop wherever irrigation facility is available.
Under dryland conditions, introduction of short duration fodder crops like cowpea, guar and rice bean can be grown for 50-60 days for fodder followed by oil seed crops. There is also the possibility to grow fodder crops on bunds along with grain crops. Therefore, there is a need for food-fodder production system from the same piece of land in succession and/or association.
Improvement in Grazing Resources
The grasslands provide the main source of roughage to livestock in arid and semi- arid regions. Grasslands also play an important role in hill regions where crop cultivation is not possible. Due to uncontrolled continuous over-grazing for decades, productive grasslands have become wastelands. The productivity of existing grassland is hardly 0.5 to 1.0 t/ha while the potential is ten times more than the present level of production.
The revegetation of grazing land through protection, reseeding, control of bushes and obnoxious weeds, introduction of legumes, strategic use of fertilisers and adoption of appropriate grazing systemis urgently required to mitigate the shortage of forage, sustain the productivity of grasslands and conserve natural resources. Pasture legumes have a special role to play both for increased biomass production and fertility build up of the soil. Atylosia spp, Clitorea and Lab lab purpureusetc. have proved to be excellent pasture legumes for semi-arid conditions.
Silvipasture System
Silvipastoral system has been recognised as one of the ways to develop degraded rangelands and wastelands for forage and fuel production. Browse spp. and trees are likely to play an important role as one of the components of the silvipastoral system. Browsing plants have an advantage over grazing plantsthrough their powerful and often deep rooting systems which may exploit large nutrient resources and make better use of limited water resources. They are the only source of available fodder at the end of the dry season. Pods of browse species are rich sources of protein and are used as supplements. While selecting the browse species for forages, due consideration should be given to their adaptability to local agroclimatic situation, reliability and seasonality of production, palatability, favoured use (e.g for particulartypes of animals) and other critical issues (e.g free from toxic constituents.)
Chemical analysis of browse species preferred by cattle, sheep and goats indicate a wide variation in chemical composition. The crude protein content was found to be
84 adequate but majority of the species had higher lignin and tannin contents which are likely to inhibit the nutirent utilization. However, it was observed that Zizyphusoenoploea, Z nummularia, Flacaurtia indica and Helicteres isora etc. have lower lignin and tannin contents and are therefore preferred by animals. Browse species containing more than 15 per cent lignin and 5 per cent tannin may not be of much value in terms of nutrient availability to the animal. Non-palatable and toxic plants should be avoided.
Trees traditionallyhave served as a major feed resource during lean periods in all parts of India. In the arid zone of the country, graziers depend mostly on the lopped tree leaves for feeding sheep, goat, cattle and camels. In the Himalayan region, farmers lop tree leaves in autumn and store them for feeding in winter. Many studies have indicated that hundreds of varieties of tree fodders are commonly and traditionally fed to livestock in different parts of the country, but not all of these have been studied properly and systematically for their feed value. Chemical analyses of tree leaves indicate, in general that most of them are rich in crude protein and calcium but poor in phosphorus. The dry matter intake varies from 1.0 to 2.5 kg/i 00 kg body weight. Low intake of dry matter thus appears to be a limiting factor for energy supply from tree fodder. Tree fodder like AllantI-,us excelsa, Grewia optiva, Helicteres isora, Leucaeria Iatisiiqua, Sesbania sesban, S. grandiflora, Hardwickia binata and Albizia lebbeck etc. could be used for animal feeding when they are mixed in suitable proportions with dry grasses.
Forage Conservation
Conservation of surplus fodder and grasses available during flush growth period, in form of silage and hay would be an ideal feed during lean period for sustained livestock production. Good quality silage from crops like sorghum, maize, teosinte, oats and natural grasses either alone or in combination with leguminous fodder crops or with 0.5 per cent urea can be made if dry matter of material is between 30-40 per cent
It is therefore, suggested that efforts are further needed to (1) improve the quality of low grade roughages, (2) popularise feeding Urea-Molasses-Mineral Blocks, (3) intensify the fodder production both from irrigated and dry lands, (4) improve grazing resources through pasture and silvipasture and (5) develop a fodder bank to mitigate the shortage of forages and to increase the animal productivity.
85 RECENT ADVANCES IN APPROACH TOWARDS DEVELOPMENT AND RESEARCH IN LIVESTOCK PRODUCTION
D.V. RANGNEKAR BA/F Development Research Foundation Ahmedabad 380 016, Gujarat
ABSTRACT
Besides realisation of the crucial role of livestock production in rainfed areas, which accounts for about 75% of the farmers, it has also been felt that livestock development and research programmes had limited impact on the underprivileged rural families. As a result, a silent but perceptible change in approach to research and development is taking place. A major change, relatively recent for India, is adoption of Farming Systems, Research and Extension (FSRE) approach. The approach necessitates developing a systems thinking, multi-disciplinary approach and encouraging participation of the farmers without considering livestock in isolation.
The paper describes how the BAIF initiated FSR-E type of studies and developed integrated farmer oriented programmes, using livestock programme as an entry point. A few studies undertaken in Gujarat and Rajasthan states are discussed alongwith comparable studies and approach in other livestock programmes. These range from studies of production systems, farmers knowledge and perceptions of livestockand crop production, traditional systems, involvement of women in livestock development and ethnoveterinary aspects to socio-enonomics of various technologies through on-farm research. The other factors that need due consideration are sustainability of the systems, influence on environment, effect of agro-ecology and marketing conditions.
The paper stresses the need for adoption of a participatory approach, with systems perspective by animal scientists and development planners for creating impact on underprivileged families. There is a need to consider farmers and livestock keepers as resource persons, besides the beneficiaries and assess technologies and development programmes on basis of adoption.
INTRODUCTION
Livestock development is emerging as one of the major development efforts in the country. There is growing realisation of the potentialities of livestock production as source of employment and income in rural areas. The technologies, aimed at improving
86 agriculture production (including livestock production) are advancing at a fast pace and the scope seems to be unlimited. However, there is also the growing concern about slow adoption of new technologies by small farmers, which constitute majority of the farming community. Development analysts, including agricultural research scientists, are drawing attention to the fact that significant improvement in agricultural productivity is limited to a few pockets in the country. A variety of workers are critically studying the issues related to technology adoption in developing countries. The studies have resulted in a silent but perceptible change in the approach towards agriculture research and technology generation for developing countries.
Human factor is more critical for the success of livestock development effort, due to close socio-cultural linkage and adherence to the traditional systems. For many traditional livestock keeping communities it is a way of life and not just a commercial proposition. It offers assured subsistence which is preferred over high productivity which is often risky (Rangnekar, 1992). The results of livestock development take a longer time for expression, compared to crops and personal attachment is much more for livestock than crops. The multiple objectives of livestock also include social status, nutritious food, manure and fuel for family through utilisation of crop residues. (McDowell 1985, Dolberg 1982, Devendra 1988, De Boer 1985). Hence the owner (he or she) should be properly oriented and fully convinced before any change in the system can be brought about. The animal connot be bred according to a recommended programme unless the owner accepts it and reports the heat. The cross bred born will not grow and develop properly unless it is fed and well looked after. We can at best vaccinate some animals by force but feeding, milking, and management depends on the owner. The importance of human dimension and human capabilities approach in development is now well recognised (Haq 1988).
This paper is an attemptto draw attention towards relatively recentadvances in approach and outlook towards agriculture research and development. Agriculture scientists in several developing countries and also from International Research Centres have changed their approaches with beneficial results some of which are described and discussed in this paper.
SYSTEMS APPROACU
Adoption of systems approach to research, development and extension has been a major advancement in agriculture related fields. Farming systems research (FSR) approach is strongly recommended for developing countries and is expected to make research and extension more effective. (Simmonds 1985, Raman 1988, Raman and Balguru 1988). It implies stress on interdisciplinary, need based, location specific,
87 problem solving research approach with due weightage to farmers perception and priorities. It necessitates taking a holisticview with full cortsideration to factors influencing production systems like agro-ecology, marketing situation and product demand, available services, resources etc. The FSR approach necessitates a change in the attitude in which the farmer is treated as a resource person and the decision maker. Thus on- farm research with participation of farmers is necessary.
The FSR approach has been adopted in several developing countries in South Asia with good results. The National Workshop on farming systems research at Hyderabad in 1991 stressed the need to change the approach to agriculture research and technology developments and institutionalise the FSR approach to make agricultural research more effective.
Livestock production is part of the entire farm operation and its development or research cannot be dealt in isolation. It is not difficult to realise that various sub-systems influence each other. De Boer (1982) has clearly described various issues influencing livestock production in developing countries which extend from demand and supply to social aspects like traditional resources, systems etc., and Preston & Leng (1987) have recommended the need to link livestock production to feed resource and develop strategic supplementation approach for optimal utilisation of resources. More recently Qureshi (1992), Venkatadri (1993) have described the need for a systems approach for future research and technology development for livestock production in developing countries. They have clearly stressed the need to develop production technologies for each natural resource situation, farming system and give due consideration to sustainability and environmental aspects.
In the livestock development programme undertaken by BAIF, studies, to understand the production systems, feed resource, people's perception, priorities and the constraints to productivity have led to a clear understanding of the situation as it exists in different pockets of Gujarat and Rajasthan.
In the livestock development programmes of BAIF in Gujarat and Rajasthan states, studies were undertaken to understand total farming systems as influenced by agro-ecological and socio-economic factors. Through the cattle development centres, baseline surveys, indepth studies of families, chosen through stratified random sampling method, were undertaken. These observations are supplemented through periodic field recording, covering milk production, growth, health, feeding and such related aspects. The studies have provided very good insight of various relationships between factors like social aspects, agro-ecological conditions, marketing facility, availability of inputs and services etc. These studies have also provided a clear picture of crop/livestock relationship variations between different socio-economic groups. Based on the data collected, attempts have been made at modelling the crop/livestock production systems for critical understanding in interrelationships (Patil et al 1991, Joshi et al 1991).
88 It may however, be mentioned that even the farming systems research approach has become a subject of discussion with regard to definition, methodology, interpretation of results as well as the utility of farming systems studies. (Fresco 1984, Simmonds 1985, Merril Sands 1986). Chand and Gibbon (1990), Gibbon (1992), Bowden (1992) have emphasized the need to develop systems aptitude, focus on farm household and encourage farmers participation and take cognizance of flexibility in FSR approach. The FSR type studies for BAIF have multipurpose utility and is a continuous process, with simultaneous action, rather than a one time research exercise. Thus within the livestock development programmes there have been continuous modifications and improvement to make it more effective and beneficial for the poor families. BAIF has followed a holistic approach which implies learningwhile doing. Starting with cattle cross breeding programme and research on a few production aspects, the programme has become more integrated with inclusion of development of other animals like buffalo, goat and support programmes like fodder development, agroforestry, farmer's training, extension, health control and feed supplementation.
GENDER ISSUE
It is known that much of the livestock management work is done by women and they decide the type and quantity of feed to be offered, milking of animals, care and management of new born, etc. However not much attention is paid to undertake research on gender issue related aspects or plan development and research programmes considering women's perception and priorities.
Adoption of improved methods of management is impossible without convincing the women and training them properly. There is a flood of information and knowledge available with men and women farmers, which has been largely ignored. Women are excellent sources of information about animal behaviour, local feed resources, adapt- ability of the animals, etc. Field studies in BAIF projects have helped in better understanding of the role of men and women in livestockproduction, decision making, perception and variations due to region and socio-economic strata. (Rangnekar 1991a, 1992 A, Singh 1982, Rangnekar & Rangnekar 1992, Vishwanathan 1989). These studies and feedback from the field led to a change in the training and extension programmes in livestock production. These were made practical and the duration and course topics were reduced and timings suitably adjusted. In training programmes, women are involved as resource persons for providing information on various management aspects. Several useful tree species, bushes, grasses specific to certain areas were identified by women. These resources were found to be nutritious on analysis and further studies on these feed material are in progress. (Rangnekar 1991 B.).
89 Learner and Farmer Participatory Approach
To make the development and research efforts effective, it is necessary to take a learner approach to understand the farming systems, perceptions about the systems and more importantly their knowledge about local conditons. Such approaches are being tried in a few projects in India like the Indo-Swiss projects in Orissa and Andhra Pradesh and the integrated watershed development project in Dungarpur, Rajasthan. The small ruminant coordinated research projects taken up in Indonesia, Kenya and Peru are very good examples of inter-disciplinary effort involving animal science, social science, economics and anthropology workers, (Nolan et al 1989). Gilles (1982) has given a vivid description of planning livestock development from indigenous systems studies in African countries. More recently, Kaasschieter et al (1992) reviewed various studies on livestock production and development and suggested approaches for research and development, sustainability and effective livestock development.
In the BAIF project, studies were initiated on traditional systems of livestockproduction with various social groups for better understanding and also to take benefit of their experience and knowledge. This included studies on tribal families, pastoralists, farmers, etc. (Rangnekar, 1992, Pradhan et al 1991). The studies revealed that many of the traditional systems can be very well compared with approaches recommended as modern and scientific. (Rangnekar 1992). The traditional livestock keepers were found to be very critical in selection of breeding animals. In animal feeding, the material identified as beneficial and traditionally well accepted like cotton seed, cotton seed cakes, copra cake, rice polish, rice bran, tree leaves, pods etc. are now found to be excellent source of protein (slowly degradable protein) or energy. The farmers were found to assess nutritive quality of the feed material, (roughage and concentrate) in their own way but generally the laboratory or research results and farmer ranking more or less coincides.
PARTICIPATION OF FARMERS -
It is necessary that the farmer is involved while developing or testing technology or evaluating livestock development programme. Participatory evaluation is now well ccepted in various fields of development and research, particularly in crop production area. The close linkage between the farmer family and livestock necessitates adoption of such an approach in livestock related programmes.
On - farm trial approach with involvement of the farmer during planning, execution and evaluation stages is necessary for proper and critical assessment of the technology or scientific recommendations under field conditions. Amir and Knipscheer (1989) have clearly described various aspects related to this subject, spelt out the methodology,
90 choice of approach and ways of analysing the data so that the results can be meaningful and helpful in decision making. On farm trials with technologies like Urea treatment and use of steam treated sugarcane bagasse have been very useful in understanding scope and limitation of such technologies. (Rangnekar et al 1986). For a technology like urea treatment, farmers (particularly women) devised ingenious ways of adopting the treatment, with desired results.
The studies on feed resource, animal productivity and farmers preferences carried out at a few selected centres of the project helped in developing farmer friendly recommendations which were acceptable and proved to be beneficial. (Rangnekar et al 1989). In these studies, attempts were made to assess nutritional status of animals by studying feed resources, feeding practices and animal productivity. It was found that protein was the most limiting factor. Discussions revealed that the farmers had identified some tree leaves, pods etc. to be beneficial to the animals. These were found to be rich in protein, on analysis. These materials were then recommended as supplements rather than the more popular protein supplements. It was found that such recommen- dations were more readily accepted and benefits were clearly observed.
Environment and Sustainability Issues of Livestock Development
These issues have attracted attention since the last decade or so and the concern is justified considering rapidly deteriorating environmental conditions/natural resources. Livestock production was adopted as an integral part of farming system to make it sustainable by covering or spreading the risks. Livestock, particularly ruminants, were found to be extremely useful for converting low quality crop residues into high quality human food, energy for draft power and manure. Many traditional livestockmaintaining families firmly believe that as long as they own livestock(which may be low producing), they will not starve.
Studies on range ecology and desertification have shown need for a radical change in thinking that grazing many animals is the main cause of desertification (Behnka & Scoones 1991). Report of studies on goat keeping in India by a commitiee formed by Govt. of India (Hanumanth Rao 1987) has clearly shown that goat per se cannot be blamed for loss of vegetation and the desired approach can make the system environmentally sound.
Farmers and pastoralists are known to adjust their herd/flock size and breeding programme according to feed resources (Rangnekar 1992). Studies in Africa have shown that social control is exercised on grazing, herd size and use of water by pastoralists (Oxby89). Nevertheless there is a need to pay attentionto resource management while developing technologies to enhance livestock production.
91 Another environment related aspect is methane production by ruminants. This gas is associated with global warming and ruminants are the third largest source of methane. The production of methane is more in animals on low quality feed. While it is not practical to think of replacing straws with less fibrous feed, rumen manipulation may be possible to reduce methane production. Field studies in this regard will be initiated through a joint project involving NDRI-NDDB & BAIF.
REFERENCES
Amir Pervaiz and Knipscheer Hendrik C. 1989. Conducting On-farm Animal research Procedures & Economic Analysis.
Badve, V.C., Nisal, P.R., Joshi, A.L. and Rangnekar, D.V. 1991. Variation in quality of Sorghum stover. Proc. of the International Workshop on Feeding Ruminants on Fibrous residues. NDRI, Karnal. Feb. 4-8.
Behnke, R.H. and Ian Scoones. 1991. Rethinking range ecology : implications for rangeland management in Africa. Overview of presentations of the Technologcial Meeting on Savanna Development & Pasture Production, Woburn, UK 1990. Mimeo.
Bowden, R. 1992. Of systemics and farming systems research. A critique in farming systems Research in India. Ed. Raman K.V. and T. Balguru. Pub. Director NARM, Hyderabad, India, pp.115-125.
Chand, S.P. and Gibbon D. 1990. Samuhik Brahman; a rapid and appropriate method of prioritizing and replanning agricultural research in Nepal. Journal for farming systems research and extension 1 (1) : 1-15.
De Boer, J. 1985. Some basic features of Asian livestock production systems and economic factors underlying productivity improvement programmes. Proceedings of Regional workshop on Livestock Production Management-Asian Development Bank, Manila, Jan.
De Boer J.A. 1982. Livestock development:The Asian experience. In Livestock in Asia: Issues and Policies. Ed. Fine J.C. and Latimore R.G. Pub. International Development Research Centre, Ottawa, Canada. pp 13-28.
Devendra, C. 1988. Forage supplements: Nutritional significance and utilisation for draught, meat and milk production in buffaloes. Proceedings of the 2nd World Buffalo Congress, Vo. 2. ICAR, New Delhi, India.
92 Dolberg, F. 1982. Livestock strategies in India. Pub. Institute of Political Science, University of Arhus, Denmark.
Fresco, L. 1984. Comparing anglophone and francophone approaches to farming systems research and extension. 4th Annual farming systems research symposium, Kansas State University, Manhattan, USA.
Gibbon, D. 1992. The future of farming system research in developing countries. Proc. National Workshop on Farming Systems research in India. Strategies for implementation. Ed. Raman K.V. and 1. Balagun. Pub. National Academy of Agricultural Research Management, Rajendra Nagar, Hyderabad pp. 161-178.
Gilles, J.L. 1982. Planning Livestock development, themes from indigenous systems. Agri. Administration, Vol. V. pp 215-225.
Haq, M.U. 1988. People in development, Development 2/3 : pp 41-45.
Joshi, A.L, Walli, T.K. and Tamminga, S. 1991. The Cow Model. Proc. of the International Workshop of Feeding Ruminants on Fibrous residues. NDRI, Karnal, Feb. 4-8.
Kaasschieter, G.A., R. de Jong, Schiere, J.B. and Zwart, D. 1992. Review Papers. The Veterinary Quarterly. Vol. 14. No. 2 April.
McDowell, R.E. 1985. Meeting constraints to livestock production systems in Asia, Proceedings of the Regional Workshop on Livestock production management., Pub. Asian Development Bank, Manila, Jan.
Nolan, M.P. Lipner, M.E. and Corkle, G.M. 1989. Sociology in the SR-CRSP. Research highlights and dilemmas of participation. J. Animal Sci. 67: 3111-3117.
Oxby Clare. 1989. African Livestock Keepers in recurrent crisis. Pub. International Institute of Environment and Development. London.
Patil, B.R., Singh, G.B, Rai, S.N. and Rangnekar, D.V. 1991. Feed availability and requirement for dairy animals in high and low potential areas. Proc. of International Workshop on Feeding Ruminants on Fibrous Crop Residues. NDRI. Karnal Feb. 4-8,
Pradhan, P.K, Jape, A.S. and Rangnekar, D.V. 1991. Initial report on Traditional Livestock Management and Feeding Systems in tribal areas of Gujarat and Rajasthan - a part of systems approach to development and extension. Presented at the International Workshop on Feeding Ruminants on Fibrous Crop Residues. NDRI, Karnal Feb. 4-8.
93 Preston, T.R and Leng, R.A. 1987. Matching ruminant production systems with available resources in the tropics and sub tropics. Pub. Penambul Books, Armidale, Australia.
Qureshi, A.W. 1992. Framework for future research and technology management within given socio-economic environments, Proceedings of the workshop on National Resource Development and Utilisation. International Agriculture Centre, Wageningen, July.
Raman, K.V. and Balguru, T. 1988. NARP - an innovative approach towards FSR in India. Agriculture Administration and extension 30:203-213.
Raman, K.V. 1988. FSR: its implication in future research strategies in India. Farming systems research newsletter 2 (2) 3-5.
Rangnekar, D.V. 1989. Dairy cattle development programme of the Bharatiya Agro Industries Foundation, a non-government organisation. Proceedings of the 6th International Conference of institutes for tropical veterinary medicine, Wageningen, August28-Sept.1.
Rangnekar, D.V. 1991 a. Farmer perceptions of quality and value of feeds, fodder and feeding systems. Proceedings of the International workshop on "Feeding ruminants on fibrous crop residues" of IndoDutch bioconversion project at National Dairy Research Institute, Karnal, Feb. 4-8.
Rangnekar, D.V. 1991 b. Feeding System based on traditional use of trees for feeding livestock. Presented at FAO Expert Consultation on Legume Trees and Other Fodder Trees as Protein Sources for Livestock held at MARDI, Kuala Lumpur, Malaysia, Oct. 14-18.
Rangnekar, D.V., Joshi, A.L., Badve, V.C. and Thole, N.S. 1986. Studies on steam treatment of sugarcane bagasse for feeding of dairy cattle: a review. Proceedings of an International Workshop held in Kandy, Sri Lanka, March 24-28.
Rangnekar, S.D., 1992. Role of women in livestock production and health management. Proceedings of International Workshop on Village Animal Healthcare in Kenya, Mar. 15.
Rangnekar, S.D. & Rangnekar, D.V. 1992. Involvement of women and children in Goat keeping in some villages of Gujarat and Rajasthan. Pre-Conf. proceedings of Vth International conference on Goats held at Delhi, India. Abstts. of Contributory papers vol. 1 pp. 36.
94 Rangnekar, S.D. 1992. Studies on indigenous knowledge, work sharing and perceptions of livestock production by women from pastoralist families. Paper presented at the Workshop on Transhumant pastoralism in Gujarat, Inst. of Rural Management, Anand, Gujarat. July 24-25.
Rangnekar, S.D. 1992. The perception of women farmers regarding animal health and production services as affected by socio-economic conditions. Presented at the Seminar on Livestock Services for Smallholders, Yogyakarta, Indonesia, Nov. 15-21.
Rangnekar, S.D. 1992. Women in Livestock Production in rural India, Proceedings of 6th AAAP Animal Science Congress, Bangkok, Thailand Nov. 23-28.
Hanumantha Rao, C.H. 1987. Report of the task force to evaluate the impact of Sheep & Goat rearing in ecologically fragile zone. Govt. of India, Ministry of Agril., New Delhi.
Merrill Sands, D. 1986. Farming systems research, clarification of terms and concepts. Experimental agriculture, 22:87-104.
Scoones, Ian. 1989. Economic and ecological carrying capacity - implications for livestock development in the dryland communal areas of Zimbabwe.
Simmonds, N.W. 1985. The state of the art of farming systems research. World Bank, Washington D.C., USA.
Singh, R.V. 1982. Fodder trees of Indial Pub. Oxford and IBH Publishing Co. Pvt. Ltd., New Delhi.
Venkatadri, S. 1993. Farming Systems Research. J. Rural Recons., 26(1).
Vishwanathan. 1989. Women in livestock in India - a desk study. FAO Regional Office for Asia and the Pacific, Bangkok, Thailand.
95 ANIMAL BREEDING BREEDING STRATEGIES FOR BUFFALO IMPROVEMENT IN INDIA
S.C. CHOPRA
Central Institute For Research On Buffaloes Hisar, Haryana
INTRODUCTION
India occupies the most important position since it accounts for about 75 million buffaloes in the world, of which 36 percent are reared for dairy purpose (FAO Buffalo Statistics 1961-90). According to these estimates, about 27 million buffaloes produced around 26.80 million tons of milk in the year 1990, thus giving an average of nearly 1000 litres of milk per annum (Table 1). The situation in Pakistan is comparatively better but when compared to North Western plains of India, it is in no way different. This is due to the fact that Pakistan has equally meritorious breeds of buffaloes viz. Nih Ravi and Kundi as compared to Murrah in Haryana and Punjab in India. Buffaloes in India are distributed almost all over the country but in the past the geographical barriers and local preferences led to the development of a number of breeds and types. This species is a traditional rural domestic animal and finds great significance in the social and cultural life of the farmers in the country. Mostly the land cultivators are subsistence farmers and have average holding of 1-2 buffaloes per house-hold for milk and draught. Income from sale of male calves, surplus female stock and manure of animals is also substantial. Farmers hardly give consideration to economics in raising buffaloes as most of the animals are managed under zero to very low input husbandry. Consequently, buffaloes suffer from poor quality feeding, management, health cover and indiscriminate breeding with below average bulls. All these factors are responsible for maturityat a later age, seasonality of breeding, low conception rate, high calf mortality and morbidity, poor milk yield, long calving intervals and dry period. Soni (1991) categorised all the constraints resulting in low productivity into a single major constraint designated as 'neglect' and further contended that if no serious efforts were made in the immediate future, the developing countries would gradually be deprived of good quality buffaloes as well as of their highly nutritive products.
97 Table 1. Population and Production of Buffaloes in top ten leading countries of the World (1990).
Country Number Percent of Milk Percent Annual Percent (million) world popu- (million World pop- yield Buffalo lation tons) ulation. animal for (kg) Dairy
India 75.00 53.24 26.80 61.28 1089 36
China 21.39 15.19 3.85 8.80 504 18
Pakistan 15.00 10.65 4.85 11.09 2047 32
Thailand 4.72 3.35 0.01 0.02 NA NA
Indonesia 3.46 2.46 — — NA NA
Nepal 2.95 2.09 0.71 1.62 812 24
Vietnam 2.87 2.04 0.06 0.14 1000 2
Philippines 2.76 1.96 0.03 0.07 594 1
Egypt 2.55 1.81 1.25 2.88 1160 49
Bangladesh 2.05 1.46 0.05 0.11 411 2.6
Total 132.75 94.25 37.61 86.01 1283 30.18
World 140.86 43.73 1134 27
Source : FAO buffalo statistics 1961-1 990 published in Bulletin on Buffalo Production around the World; Problems and Prospects. International Buffalo Information Centre, at Kasetsart University, Bangkok.
PERFORMANCE LEVELS
The performance of water buffaloes bred and managed at the organised farms has been studied extensively in the Indian sub-continent. It is seen from Table 2, that Murrah breed and types of buffaloes under good breeding and management system has great potential for improving growth, reproduction and milk production both in terms of magnitude and efficiency. By adopting scientific feeding, management and disease control measures, juvenile and adult mortality can be minimised thereby making available young progeny for replacement.
98 Table 2. Average Performance of Buffaloes in Various Countries.
Country Breed No.of Lact. Yield (kg) Av.Lact. Calving records 1 2 3 length interval (Days) (Days)
Bulgaria Murrah 1645 1891 2129 263 436
gradde " Italy 13000 1791 1979 1816 262 405
Iraq 1022 1391 1298 240
Vietnam Murrah x 3000 — — 1400 270 515 Swamp
India(MF) Murrah 210 1559 1753 1856 300
lndia(HAU) Murrah 245 1717 1972 2079 293 473
India Murrah 1453 to 2688 348 470 CIRB&
AICRP Surti 987 to 1600 278 425
Nili-Ravi 1765 to 2057 318 571 grade
Pakistan Nili-Ravi , 2301 1682 1932 1959 282 509
Source : Post Congress proceedings of 3rd World Buffalo Congress held at Varn in 1991 and Annual Reports of Institutes & Bulletin on Buffaloes of Pakistan (1991).
It may be seen that Murrah breed in its native tract has excelled for lactational milk yield while the characteristics determining sexual maturity and reproduction efficiency i.e. age at first calving and calving interval were comparable to the performances in other countries as well as with other breeds or breed crosses. As the fitness traits like growth rate, survival, reproduction etc. are largely governed by environmental influences and are less inheritable, these can be modified or improved by suitable managemental corrections. Milk yield is the trait of direct economic interest and has been reported to be moderately inherited. Continued genetic selection can go a long way in enhancing lactational milk yield and efficiency of milk production.
99 GERMPLASM RESOURCE
In the preindependence period and even tl the year 1970, there was no concerted effort or thinking of the planners and scientific community to conserve and improve the best germplasm of Murrah buffaloes for which India could take pride. Military dairy farms and small institutional dairies were established with the prime function of providing fluid milk to the armed forces and staff, but subsequently buffaloes from the Military farms were gradually replaced by crossbred cows. In fact these establishments were the best congregates of superior germplasm of buffaloes reared underideal management regimens.
Data collected from these herds were used for scientific investigations of many Agricultural Universities, Scientists and post graduate students. After complete elimi- nation of the buffaloes from most of the Military farms, there was a vacuum and it was difficult to lay hand on any large sized organised buffalo herd for research investigation and also which could act as a continuous and dependable source of good bulls.
Livestock developmental agencies of various states including Animal Husbandry Departments and National Dairy Development Board procured bulls from the native tract i.e. in Haryana under IRDP and later on in the operational flood programme. Large scale dairies to meet the requirement of fresh buffalo milk came up around metropolitan cities e.g. Arey Milk Colony in Bombay, resulting in slow depletion of good germplasm with the farmers.
AWARENESS ON BUFFALO RESEARCH
n the year 1966, under the sponsorship of ICAR, a progeny testing programme was started at Hissar by the Haryana State Animal Husbandry for proving sires. Similarly through AICRP on Buffaloes, investigations on large and medium sized buffaloes were started with the establishment of large buffalo herds at NDRI, Karnal and PAU, Ludhiana and for medium size buffaloes at RAU, Vallabhnagar (Udaipur), UAS, Dharwad. This programme was later on modified for Murrah and Surti. Likewise, many Agricultural Universities and Central and State Governments sett up small to medium sized buffalo herds to meet the teaching and research requirements for students and to act as bull mother farms.
But with the lack of modern facilities of proper sire evaluation, germplasm storage, and relatively less interest and success in the use of artificial insemination in buffaloes, the impact of these efforts could not be properly realised. However, the public and private sectors started realising the need for salvaging the efforts made in the direction of genetically improving and preserving this national livestock wealth.
100 INFRASTRUCTURAL DEVELOPMENT
Establishment of a Central Institute for Research on Buffaloes, at Hissar in the year 1985 by taking over the erstwhile Progeny Testing Station and continued support to the AICRP on buffaloes were the important milestones to restore the past glory of the country's possession of best buffaloes of the world. Semen freezing facilities at many centres in the country have been developed. Trained manpower for effective use of frozen semen has been created on a large scale. Importance of proper data recording has been realised and the country is well set to move ahead for launching buffalo improvement programmes on the lines of other developed nations in dairy production.
BREEDING PROGRAMME
Animal Breeders highly trained in modern methods of animal genetic improvement have not been successful in their efforts for obtaining expected genetic progress in milk production in the developing countries. This is mainly due to the fact that the animals are reared in traditional and suboptimum environments. Lack of basic infrastructure for animal production and its recording for large part of the population are major reasons. Enormous geographical variation and non availability of tested genetic material for different regions and varying animal protein needs, act as impediments in adopting a unified breeding plan.
Indian buffalo population therefore, needs to be categorised into three sections.
i) Nucleus herds ii) Multiplier herds iii) Commercial herds
In the true sense, these sections do not conform to the categories of breeding herds of the developed nations, but one can utilize the total livestock resource assuming it under these categories. Nucleus herds will be those which are maintaiied by various institutions and are appropriately managed, fed, bred and their data recorded. Such herds are most suitable for selection programmes. Their resources of animals, skills, infrastructure can be put to use for multilocational progeny testing. Associated progeny testing programme has been started by CIRB as the nodal agency under National Network on buffalo breeding for Murrah and Surti buffaloes. In the network, the existing centres of the AICRP on buffaloes, ICAR institute0 herds and SAU herds have been included. About 20-25 sires will be tested using 800-900 breedable female Murrah buffaloes as test herd. About 100 top buffaloes will be identified for elite herd and will be given nominated matings with the proven bulls from the previous testing. The Sons of the elite mating will be retained for subsequent set of bulls for testing. About
101 20-25 percent of the bulls under test will be proven for large scale multiplication and germplasm conservation. The remaining bulls may be spared for development agencies to upgrade the non-descript buffalo population through A.l. and natural service in remote areas. A similar approach is being followed for Surti breed of buffaloes.
At present no multiplier and commercial herds are in existence except that the native tracts of the recognised breeds can serve regular supply of both male and female germplasm of relatively higher genetic superiority. Till large sized herds in the public and private sectors are established, the proven germplasm can be used in the native tracts to boost the genetic merit of the buffalo population. Participation of ICAR, NDDB Department of Dairying & Animal Husbandry, Govt. of India, State Animal Husbandry Department, BAIF and private breeders is a prerequisite for the success of buffalo breeding efforts.
REQUIREMENTS OF BREEDING BULLS
Bulls required for improving the whole lot of Indian buffaloes are difficult to estimate as no exact figures are available about the female population covered through artificial insemination and natural service. Moreover our country is lacking in developing infrastructure for artificial breeding. The response of breeders to accept modern technology is lukewarm more so in farflung areas, where artificial breeding in buffaloes cannot be provided in the near future. In light of this, based on the calculation arrived at by Glodek et al (1990), the proportion of 20:80 for A.l. and natural service is be assumed for our country (Table 3). For about 27 million buffaloes, the number of breeding bulls required will be 246852 and 1404 for use through natural service and artificial insemination respectively. With the adoption of A.l. in larger proportion of the buffalo population, the total requirement of breeding bulls will be reduced.
Table 3. Number of Breeding Bulls required for Natural Service and Artificial Breeding for Female Population 250000.
Present Buffaloes N.S. Bu Ils A.l.'s Bulls for Total bull N.S. under N.S. needed per A.l. needed/yr. year.
20 50000 667 300000 50 717 30 75000 1000 262500 44 1044 40 100000 1333 225000 38 1371 50 125000 1667 187500 32 1699 60 150000 2000 150000 25 2025 70 175000 2333 112500 19 2352 80 200000 2666 75000 13 2679
102 At present the country is not yet prepared to produce such a large number of superior breeding bulls and therefore, the genetic improvement work will be more meaningful if it is restricted to traditional buffaloes and some selected pockets of different states having resources and infrastructural facilities for buffalo production. Intensive selection programmes can be started at the organised herds, from where the breeding bulls and their semen after testing will be passed on to the breeding pockets. These pockets will serve as multiplier herds for supply of better germplasm to remaining untouched areas. But culling of uneconomic and surplus animals must be ensured as a regular feature to achieve the desired rate of genetic gain i.e. around one percent for milk yield.
REFERENCES
Glodek, P., Alemu, M., Chopra, S.C., Dausal, M., Kasonta, J.S., Buxaders, M., Kaczmarek, A. and Lukaszewiez, M. 1990. Report on case study-Ill Animal Science Papers and Reports. Proc. of the FAQ Conference on ONBS Systems held at Bialobrzegi, Poland, June 11-19. pp. 191-196.
Soni, B.K. 1991. Buffalo Production in South Asia its Problems and Prospects. Post Congress Proc. 3rd World Buffalo Congress Varna, May 13-17. pp. 66-72.
103 FIELD RECORDING AND SIRE EVALUATION UNDER INDIAN CONDITIONS
S.B. GOKHALE AND B.R. MANGURKAR BA/F Development Research Foundation Urulikanchan, Pune
ABSTRACT
The field performance recording as a part of Progeny Testing Scheme of BAIF is discussed. The crossbreds in the initial phase of the programme were recorded fortnightly both in the morning as well as evening and later on at a fortnightly interval adopting alternate AM/PM recording. The lactation yields were calculated from test-day records using Test Interval Method. Regression factors are used for extending lactations in progress.The animals drying within 150 days from calving and those in lactation of less than 90 days from calving were eliminated from analysis.
The data on 4185 lactations from field Holstein crossbreds were used for the present analysis. The average 305 day milk yield of field Holstein crossbreds from first to fourth lactation was observed to be 2677.56 ± 22.12 Kg., 2735.71 ± 22.45 Kg., 2860.29 ± 27.l6Kg,and 2917.82 ± 35.27 Kg. respectively. The locations (Centre), year and month of calving and exotic blood level of animals significantly affected the lactational milk yield.
Under sire evaluation programme, 45 Purebred and 20 Crossbred Holstein bulls were under test. The data on 40 sires (23 purebred and 17 cross-bred) having minimum 4 daughters were used for this purpose. The Estimated sire Merit was calculated using different sire evaluation methods. Namely Simple Daughter Average (SDA), Herd Mate Comparison (HMC), Contemporary Comparison (CC), Least Square (LS) and Best Linear Unbiased Prediction (BLUP).
The product Moment Correlations and Rank Correlations between sire evaluations based on different methods ranged from 0.7101 to 0.9297. Standard Error of sire estimate (used as a measure to compare different methods) for BLUP was least.
Amongst bulls tested, ten purebred and eleven crossbred bulls were found to have reliability of proof of more than 60%. The predicted difference in milk yield of 12 bulls exceeded the average population by more than 5%.
104 INTRODUCTION
The field level progeny testing of bulls gains importance from the point of view of selection of bulls for the traits favoured by the farmers.
A wide range of variability exhibited by the village cattle in India in respect of production and reproduction traits necessitates closer examination for their use in arriving at an appropriate sire selection procedure to be useful at the field level. This becomes more important when the herd size at farmer level is very small.
Although a few attempts have been made to examine the methods of sire evaluation relevantto field level testing of bulls under Indian conditions (Singh et a! 1992, Chauhan et al 1991, Raheja 1991, Chauhan 1991 etc.), much is yet to be done to understand the approach to sire evaluation under field conditions.
The present investigation was undertaken with a view to explore the possibility of arriving at a reliable and practical sire evaluation method which can be used at field level.
MATERIALS AND METHODS
DATA
The data set on 4185 lactation milk records of Holstein crossbreds spread over 10 clusters of villages in Ahmednagar and Pune Districts of Maharashtra were used for the present investigation. Total lactation records were spread over the period 1990 to 1993. The milk yield was recorded at a fortnightly interval using alternate AM/PM recording method. The first record was taken within 60 days of calving. The lactation yield was calculated using Test Interval Method. The animals drying before completing 150 days of lactation were eliminated from the study. The lactations in progress but of less than 90 days lactation period were also eliminated. Regression factors were arrived at for extending the lactation in progress to 305 days lactation milk yield. Since the animals included in the recording programme were at different stages of lactation, separate regression extension factors were developed for each stage of lactation and missing yield was calculated by using these factors.
The lactation records were adjusted for the effects of lactation sequence and recording stage before they were subjected to statistical analysis. The latter lactations were adjusted to the first lactation base.
Estimated sire merit, (ESM) was computed using the following sire evaluation procedures.
105 1) Simple Daughter Average (SDA). The average 305 day milk yield of daughter of each sire was expressed as deviation from Centre Average.
2) Herd Mate Comparison (HMC). The records of each daughter was expressed as a deviation from average of progeny of other sires calving at the same centre in the same year.
3) Contemporary Comparison (CC). The method described by Johansson and Randel (1969) and mentioned in other animal breeding texts has been used. The daughter records of each sire were expressed as weighted average deviations from contemporaries (calving in same centre year season) and were then regressed to account for number of daughters and heritability of 305 day milk yield. The mathematical expression of the contemporary comparison is as follows:
— — ninj CC = ( Di - Dj) x ni+nj ne x ni nj/(ni + nj) ne + 12
Where Di = Mean of the daughters of i th bull in a sub-class Dj = Mean of the daughters of all bulls other than that of i th bull. ni = Number of daughters of i th bull in a subclass. nj = Number of daughters of all contemporary bulls. ne = Effective number of daughters = (ni nj/(ni÷nj)
ne = Reliability ne + 12
3) Least Squares: The least square estimation for unbalanced data has been described by Harvey (1960). The Least Square analysis was carried out by using the following model. u + + S + + + Yijklm=/ CY1 Bk M Elikim Where = Observation on mth daughter of 1th month of calving,kth breed,jth sire 'ijkm th and centre year of calving.
106 /u = Over all mean
CY. = Effect of jtI centre year of calving
= Effect of sire S, jth Bk = Effect of kt1' breed of animal M = Effect of 1th month of calving Ei.k = Random Error
4) Best Linear Unbiased Prediction (BLUP): The model detailed in the Least Square analysis was used to arrive at BLUP estimates for each sire, except that the effect of sires was considered in random and all other effects fixed.
RESULTS AND DISCUSSION
The average lactation yields from first to fourth lactations of the field H.F. crossbreds was observed to be 2677.56 ± 22.12 kg, 2735.71 ± 22.45 kg, 2860.29 ± 27.16 and 2913.82 ± 35.27 kg. respectively. An analysis of variance was calculated to study the effect of nongenetic factors affecting lactation milk yield. The effect of centre, year and month of calving significantly affected lactational milk yield. The exotic blood level of the animal also showed significant effect. The results of the analysis are presented in Table 1. The average milk yield for animals calved in different months of the year is graphically presented in figure 1.
The estimates of sire merits of different sires for lactation milk yield, computed by five methods of sire evaluation, are given in Table 2. The range of sire merit value was found to be smaller for BLUP method compared to other methods of sire evaluation studied. Sires were ranked on the basis of sire merit obtained from all the methods studied. The results revealed that sire 5001 was ranked highest by HMC, CC and BLUP methods while it was ranked second by SDA and LS methods. In general sire rankings estimated by different methods were not the same. The wide range of variation in the progeny number per sire and their distribution among different herds of small size might have led to this variation.
Spearman rank correlation among ranks and simple product moment correlation coefficients among estimates of sire merit calculated from five methods of sire evaluation are shown in Table 3. The estimate of rank and product moment correlations indicate that the evaluation by contemporary comparison and Best Linear Unbiased Prediction were highly correlated. Our estimate of rank correlation between Contemporary Comparison and Best Linear Unbiased Prediction were higher than those reported by
107 Raheja (1991) in Sahiwal breed of cattle. The estimates reported in the present investigation are in a similar range as those reported by Gurunani and Nagarcenkar (1990) in Tharparkar Breed.
The choice among various methods to a great extent would depend upon computational facility and relative accuracy. The criterion used to assess accuracy was standard error of estimate. The standard error of sire merit estimates by SDA, HMC, CC, LS and BLUP were 41.09, 37.56, 34.02, 39.43 and 27.69 respectively. The variance of BLUP estimate was significantly smaller than that of simple Daughter or Least Square procedures. It did not differ from either Contemporary Comparison or Herd Mate Comparison methods. The absence of appropriate weightings for differential number of progeny per sire and non consideration of heritability of character limits the utility of Herd Mate Comparison procedure. These limitations have been corrected ri Contemporary Comparison method. The estimates derived in the present investigation suggest that BLUP or CC methods can be used for evaluation of the sires under field conditions.
The estimated sire merit of bulls by CC method indicated that, amongst 17 crossbred H.F. bulls under evaluation, 11 bulls proof (4690, 4610, 4620, 4020, 4040, 4670, 4680, 4630, 4660,4330 and 4650)exceeded the repeatability index of more than 60%. In case of pure bred H.F. bulls, out of 23 bulls, 10 bulls proof (5100, 5112, 5103, 5111, 5109, 5105, 5104, 5107, 5108 and 5102) exceeded the repeatability of 60%. The repeatability of progeny test of two crossbred H.F. bulls and three purebred bulls ranged from 50 to 60%. There was no crossbred bull having repeatability less than 35% while there were five bulls among purebreds who on account of smaller number of their daughters showed repeatability less than 35%.
The predicted difference in milk yield of 7 bulls - four each of purebred H.F. ( 5001, 5100, 5096 and 5112) and three crossbred H.F. ( 4080, 4150 and 4340 ) exceeded 5% of the average population ( 2719 kg). These bulls were declared as proven bulls.
108 REFERENCES
Chauhan, V.P.S. 1991. Indian J. Dairy SC1. 44 (10) : 602-611.
Gurnani, M. and Nagarcenkar, R. 1992. Proc. 2nd World Congress on Genetics applied to Livestock production. Madrid, Spain 4-8 Oct., 195-99.
Harvey, V.R. 1960. Least Square analysis of data with unequal subclass numbers. USDA, ARS, 11-4.
Johansson, I. and Rendel, J. 1969. Genetics and Animal Breeding. WH. Freeman Co., San Francisco, California, USA.
Raheja, K.L. 1992. Indian J. Dairy Sci., 45(2) : 64-69.
Singh, B.P. Vinod Kumar and Chauhan V.P.S. 1992. Indian J. Anim. Sd. 62 (8): 749-753
Table 1. Least square analysis of variance of milk yield in field H. F. crossbreds.
Source of variation D.F. Mean squares
Centre year subclasses 63 7941256.83 Month of calving 11 1773290.49 Breed of animal 5 2962895.27 Residual 4104 527535.66
109 Table 2. Estimated sire merit and sire rankings of H.F. and Crossbred H.F. Sires by different methods of sire evaluation.
SIRE PRO- EFF NO OF SDA HMA CC LS BLUp GENY PROGENY
4150 7 6.84 755.70 (1) 55.96 (14) 175.20 (3) 632.17 (1) 395.07 (2) 5001 6 5.92 744.70 (2) 732.06 (1) 240.66 (1) 484.85 (2) 484.75 (1) 4080 13 12.81 505.09 (3) 399.65 (2) 199.95 (2) 317.64 (4) 278.08 (3) 5100 29 27.83 375.84 (4) 296.75 (4) 171.65 (4) 135.53 (8) 209.15 (5) 4610 31 29.86 291.82 (5) 164.98 (9) 57.977 (13) 100.24(11) 89.26 (11) 5072 13 12.66 281.63 (6) 260.00 (5) 109.88 (7) 51.70(13) 122.78 (8) 4020 58 54.39 257.69 (7) 29.33 (17) 10.46 (19) 102.21 (10) -32.85 (19) 5111 25 24.25 254.84 (8) 251.21 (6) 70.862 (12) 17.36(18) 73.09 (13) 5096 6 5.87 235.96 (9) 364.09 (3) 96.11 (9) -85.63(26) 169.96 (6) 5071 .4 3.97 233.90 (10) 116.95 (10) 29.60 (16) 334.06 (3) 85.38 (12) 4340 11 10.76 223.32 (11) 209.50 (7) 134.52 (6) 198.00 (6) 215.76 (4) 5112 44 41.97 129.43 (12) 113.07 (11) 165.50 (5) 118.58 (9) 156.39 (7) 4630 22 21.37 91.94 (13) -50.03 (22) -95.21 (36) -20.10(22) -108.00 (28) 5109 30 29.09 88.14 (14) 17.59 (19) 19.01 (17) 36.03(15) -54.77 (22) 4040 22 19.66 87.97 (15) 186.68 (8) -48.18 (25) 278.04 (5) -35.47 (20) 5107 61 58.60 73.05 (16) -95.09 (27) - 58.09 (28) -185.50(30) -129.13 (31) 5103 50 48.13 61.68 (17) 50.18 (15) 73.56 (10) -51.95(23) 24.05 (16) 5085 18 17.48 52.02 21.45 33.73 29.07 (18) (18) - (14) 19.15(17) (15) 5101 18 17.49 17.65 (19) -26.35 (21) 8.98 (21) 42.73(14) -27.30 (18) 5075 4 3.94 16.50 (20) -190.12 (33) 18.35 (18) -328.87(36) -5.65 (17) 5104 54 51.55 15.16 (21) -70.52 (24) -26.01 (23) -60.89 (24) -57.84 (23) 4690 30 28.56 -3.40 (22) 68.03 (13) 106.30 (8) 15.82(19) 114.71 (9) 5108 47 45.30 -16.91 (23) -135.78 (29) -65.51 (31) -134.02(29) -150.32 (32) 4620 43 40.30 -20.53 (24) 72.55 (12) 32.32 (15) 77.15(12) 56.49 (14) 5102 53 50.36 -28.42 (25) -141.86 (31) - 90.49 (34) -189.22(31) -155.93 (34) 4670 26 25.02 -37.80 (26) -85.18 (26) -54.56 (27) -10.43(20) -84.28 (27) 5212 8 7.88 -48.21 (27) -52.54 (23) + 71.84 (11) 150.48 (7) 97.90 (10) 4680 44 42.05 -61.41 (28) -77.24 (25) - 72.04 (32) 28.96 (16) -76.83 (26) 4660 23 22.42 -66.68 (29) -140.66 (30) - 97.12 (37) -64.91(25) -150.90 (33) 5105 57 54.55 -67.05 (30) 29.61 (16) 6.28 (20) -99.93(28) -44.15 (21) 5031 5 4.91 -105.38 (31) -223.50 (34) - 93.72 (35) -364.99(38) -220.89 (38) 5093 4 3.97 -121.07 (32) -146.81 (32) - 36.45 (24) -254.89(35) -121.83 (29) 5314 11 10.76 -129.26 (33) -129.71 (28) -13.69 (22) -16.17(21) -67.47 (24) 4060 14 13.66 136.21 (34) -227.85 (35) -52.45 (26) - 94.88(27) -72.64 (25) 5020 5 4.91 -179.86 (35) -252.18 (36) - 63.79 (30) -423.21(39) -181.32 (35) 4110 12 11.79 -210.70 (36) 14.69 (20) - 72.09 (33) -238.21(32) -126.92 (30) 4650 27 26.14 -277.45 (37) -420.70 (39) -258.82 (40) -244.91 (34) -338.27 (40) 5065 4 3.81 -320.13 (38) -387.07 (38) - 62.40 (29) -701.72(40) -184.72 (36) 4320 9 8.91 -373.48 (39) -376.61 (37) -167.58 (39) -348.85(37) -298.31 (39) 4330 26 25.15 -488.49 (40) -451.41 (40) -130.05 (38) -239.77(33) -205.17 (37)
110 Table 3. Product moment (above diagonal) and rank correlation (below diagonal) among estimated sire merit calculated by different sire evaluation methods.
SDA HMC CC LS BLUR
SDA — 0.8482 0.7984 0.8145 0.8851 HMC 0.8156 — 0.9003 0.7337 0.8642 CC 0.7891 0.8415 — 0.7068 0.9297
LS 0.7101 0.7929 0.7413 — 0.8069 BLUP 0.8026 0.8764 0.9240 0.8114 —
Note
1. The bulls with prefix '4' were crossbred bulls and those with prefix '5' were purebred HF bulls.
2. The figures in parentheses indicate relative ranks.
111 ANIMAL HEALTH STRATEGIES IN ANIMAL HEALTH PROGRAMME
K.P. Mallick, RN. Kaul and D.S. Balain Indian Veterinary Research Institute Izatnagar, Uttar Pradesh.
INTRODUCTION
Livestock and poultry constitute a way of living in India, where there are about 199 million cattle, 74 million buffaloes, 105 million goats, 55 million sheep, about 9 million pigs and 183 million poultry including 9 million ducks. The contribution of livestock to agriculture and gross domestic product (GDP) in India is 16% and 8.1% respectively. Of the total energy required by the agriculture sector in India, 54% is provided by the 85 million working cattle and buffaloes, estimated to yield about half the gross national product (GNP) of the country. (Rama, 1991).
Rearing of livestock as a traditional practice has been a part of rural life in India consisting of 5,75,000 villages where about 75% of the rural population are engaged in raising animals in small numbers. Most of the animal wealth in the country is concentrated in rural areas. In fact, livestock rearing is currently an intergral part of crop farming and rural employment. While most of the livestock are meant to provide animal protein, there are some species reared for fancy, protection and affection. The target for producing 640 lakh tonnes of milk is to be achieved by 2,000 A.D.
Livestock owners sustain serious economic losses due to various animal diseases causing high morbidity and mortality. The utility of animals is adversely affected due to diseases. The diseases induce marked reduction in milk yield in lactating animals, poor growth in young animals, poor quality of hides and wool and decreased quality of faeces.
Considering the manifold utilities of livestock, there is a need to develop sound strategies for animal health in order to protect them against diseases. Past experience has shown that without adequate health care, attempts to improve the productivity of livestock and through it the national income cannot be successful. Further in the absence of effective disease control measures, population of unproductive animals of poor genetic potential may be created, particularly when there is restriction on planned slaughter.
The following aspects have to be considered in developing definite strategies for animal health programme.
113 EPIDEMIOLOGY OF DISEASES
In India, we lack reliable epidemiological data on animal diseases particularly those occurring in rural areas. Hence, there is a need to conduct systematic epidemiological studies so that we can have a correct picture of disease occurrence. This will be of immense help while developing better strategies for disease control.
The diagnostic laboratories particularly those located at district level need to be well equipped and deployed with experts of different fields duly trained in diagnostic techniques. The services of trained persons should be exclusively utilized for laboratory diagnosis.
STRATEGIES FOR ANIMAL HEALTH CARE DELIVERY SYSTEM
The facilitiesfor health care measures are presently being provided through about 19,340 veterinary clinics and hospitals in the country. There is a need to create more units to intensify animal health services. There should be sufficient number of veterinarians at block level for effective implementation of all aspects of animal health and production programme. As per the report of the National Commission on Agriculture, (1976) there should be provision for one veterinarian for 5,000 livestock population by 2000 AD. It may not be difficult to achieve this target in view of the large number of veterinarians produced by 28 veterinary colleges in India.
VETERINARY PUBLIC HEALTH
Almost every animal health activity has some bearing upon human health, so a close liaison has to be maintained between human and animal health. Abundance of vectors because of conducive climate, low socio economic status of overwhelming population and inadequate human and animal health care have given rise to occurrence of zoonoses in the country. Brucellosis alone is estimated to cause an annual loss of approximately 30 million man days in addition to annual economic loss of Rs. 240 million through this disease in cattle and buffaloes (Rama, 1991). Japanese encephalitis has emerged as another zoonosis of great public health significance over the last decade or so. Rabies, tuberculosis, leptospirosis, anthrax, hydatidosis, food-borne diseases, snake bite etc., are a few other zoonoses.
BIOTECHNOLOGY IN ANIMAL HEALTH
Biotechnology has emerged as a frontierarea of science with useful application in animal health and production. The advent of recombinant DNA technology and genetic engineering has shown better prospects for the production of a new generation of vaccines against diseases, health products and newer methods of disease diagnosis.
114 The development of immunological and molecular tools has paved the way for rapid and specific identification of infectious agents. These tools are monoclonal antibodies (Mab), DNA probes and the combination of DNA probes and Polymerase Chain Reaction (DNA + PCR).
STRATEGIES FOR CONTROLLING IMPORTANT EQIZOOTIC DISEASES
The usual methods for the control of epidemics consist of mass medication of clinical cases and carriers, removal of vectors and reservoirs, quarantine, sanitation, immunoprophylxis, genetic measures, environmental measures etc.
There is need for streamlining the reporting system of outbreaks. The outbreak of any disease should be promptly reported so that quick investigation of the disease can be made for confirmation of diagnosis and adopting of control measures.
CONTROL OF EMERGING VIRAL DISEASES
Blue Tongue In Sheep
A suitable vaccine for Blue Tongue is yet to be evolved. The problem of emerging newer strains of B.T. virus is also a constraint in the development of a suitable vaccine. The general control measures can be undertaken. Vector (Culicoides spp.) control is the most important aspect for controlling this disease.
Sheep Pox
The outbreak of sheep-pox is often reported in our country with high mortality rate. A tissue culture sheep-pox vaccine containing Romanian strain of sheep-pox virus is available which confers fairly good immunity for a period of at least one year. Goat Pox
Goat pox in India is an emerging problem. Outbreaks have been reported from some parts of the country. The mortality rate in goat-pox is also reported to be high. An inactivated vaccine is presently used for immunization of goats. But there is a need to develop a suitable vaccine. Buffalo Pox
The outbreaks of buffalo-pox have been occurring off and on in some of the buffalo tracts of India. (Mehrotra et.al. 1981, Malick and Dwivedi, 1982) Sometimes the morbidity rate is very high (75-80%) and the infection spreads in a vast area as it occurred in
115 the state of U.P. in 1978. Both generalised and localised lesions are found in buffalo pox. The localised lesions are not only encountered on the udder and teats, but also in and around ears and eyes. The mortality rate is not reported but several complications develop like mastitis which result in severe damage to the ears and eyes. There is marked reduction in the productivity. The infection of buffalo-pox has been reported to be transmissible to man also with the presence of lesions on the face and hands. The detailed epidemiology of this disease is not yet clearly understood. A vaccine can be developed if felt necessary for mass immunization of buffaloes.
Infectious Bovine Rhinotracheities (IBR)
IBR disease of cattle is caused by virus of herpes group. Though the occurrence of the disease has been reported from different parts of the country, the prevalence is more in southern states.
No single potent vaccine is available for the control of this disease. Efforts are going on to develop safe, potent and cheap vaccine. It is suggested that semen of the bull used in artificial insemination should be free from IBR virus.
BVD
The High Security Animal Disease Laboratory (HISADL) under I.V.R.I. located in Bhopal (M.P.) gives'thrust to research on this disease.
Calf Diarrhoea by Rota virus - The causal agent of neonatal diarrhoea was supposed to be mainly due to E. co/i, but Rota virus infection has now been detected to be an emerging problem. An epidemiological investigation in respect of Rota virus infection is needed to evolve control measures.
Para Influenza Virus
This has been reported from pneumonia cases in calves. Further studies on the occurrence of this infection is needed to know its epidemiology.
Maedi / Visna Viruses In Sheep And Goats
These viruses are responsible for chronic pneumonia in sheep and goats. Visna virus alone causes meningo-encephalitis. There is a need to investigate the occurrence bf diseases due to these viruses.
116 Caprine Arthritis - Encephalitis (CAE)
It is a persistent viral disease of goats characterized by encephalitis in young goats and insidious arthritis in adult goats. The etiology of this disease is a retro-virus. This disease has emerged in India. Many things about this disease are unclear. As the retro- virus is a biological threat to man and animals, there is an urgent need to conduct detailed studies on this disease to evolve control measures.
Emerging Viral Diseases Of Equines
Some viral diseases of equines have emerged in India, viz., Equine Influenza, Equine Infectious Anaemia, Equine Coital Exanthma and Equine Viral Arteritis. There is a need to conduct regular epidemiological surveillance of these diseases.
BACTERIAL DISEASES
Mycoplasmosis In Goats
Mycoplasma infection in goats is one of the important health problems causing pleuro- pneumonia, arthritis, keratoconjunctivitis and mastitis. (Verma 1988; Singh 1988). The incidence of mycoplasmosis has been reported from several states, viz. Andhra Pradesh, Bihar, Haryana, Punjab, U.P. and West Bengal. Pleuropneumonia in goats caused by Mycoplasma mycoides subsp capri is a highly contagious disease causing high mortality particularly in kids. It is also known as Contagious Caprine Pleuro-Pneumonio (CCPP). Mortality rate up to 80% has been reported.
For the control of mycoplasmosis in goats, no effective vaccine is presently available. There is an urgent need to study the epidemiology of various Mycoplasma infections in the states where it is prevalent so that a suitable vaccine can be evolved.
HAEMOPROTOZOAN DISEASES
Theileriosis
Theileriosis, a tick-borne infectious disease is mainly the problem of cross-bred cattle in India. The severity of infection is more in the young calvesthan adults. The indigenous cattle generally do not suffer from clinical theileriosis. They remain in a carrier or subclinical state becoming a constant source of infection to susceptible animals through the movement of ticks. There are two methods for the control of this disease, control of vector ticks and immunization of cattle. The control of ticks can be done by the spray of insecticides which can be successful only in organised cattle herds. The use of insecticidal spray may not be feasible in rural cattle due to certain socio-economic
117 factors. Cell culture schizont vaccine has been developed at some centres under All India Coordinated Research Project on Haemoprotista under I.C.A.R. This vaccine has been reported to be immunogenic after varying number of passages which indicates the possibility of variation in the virulence of Theileria annulata strains used in the preparation of vaccine.
Before development of any suitable vaccine there is a need to conduct thorough epidemiological studies in the country to understand the magnitude of the problem. The strain variation in T. annulata is also to be ascertained preferably using the biotech- nological tools. Considering the strain variation, the vaccine may be developed by a master strain or a combination of strains of the parasite so that the vaccine may be effective against all possible variants of the parasite.
For treatment of clinical cases of theileriosis, a drug known as Butalex has been made recently available in the market by M/S Cadila vety. This drug is said to be specific for theileriosisas and is effective against both the schizontal and piroplasmal stages of the parasite. Since the drug is very costly, it will be desirable to use it after diagnosis of the disease.
Babesiosis
Babesiosis is a tick-borne infectious disease occurring in cattle, buffaloes, horses and dogs. Sheep and goats may also suffer from this disease. The susceptibility in cross- bred cattle is more as compared to indigenous cattle. Mostly the adult animals suffer from the disease. Effective babesicidal drugs are available in the market for treatment of clinical cases. A critical assessment for the need of vaccine is desirable in view of the incidence of this disease and economic losses due to morbidity and mortality. The control of tick vectors may be helpful.
Anaplasmosis
Anaplasmosis is a haemo-parasitic disease caused by Anaplasma organisms which have now been included under rickettsia. Cattle, sheep and goats are susceptible to Anaplasma infection. Among cattle, the crossbreds are more susceptible than indigenous ones. Bovine calves below 6 months of age are resistant. Sheep and goats are generally carriers of infection but they may suffer clinically. No vaccine is available. The transmitting vectors can be controlled by insecticidal spray.
Strategies for Controlling Diseases caused by Helminth Parasites in Livestock
No definite strategies have been developed for the control of helminthiasis in rural areas. The livestock owners sustain economic losses due to helminthic disease. There is a
118 lack of systematic epidemiological data in respect of various helminthic diseases and so the economic losses cannot be estimated.
In a country like India, there are many constraints in developing proper strategies for the control of helminthic infections. There is no system for rotational grazing and the chemical and biological methods for the control of intermediate hosts cannot be applied due to certain soclo-economic reasons. So, deworming with appropriate anthelmintics is the only answer for the control. A deworming schedule should be developed considering the life cycle of helminth parasites and the seasons in which occurrences are high during the year. The systematic epidemiological studies will be valuable while developing the deworming schedule.
STRATEGIES FOR CONTROLLING DEFICIENCY DISEASES
The problem of nutritional deficiency diseases may be more in rural livestock where there is no definite system for balanced feeding. Some of the areas may have natural deficiency of minerals in the soil. The impact of the deficiency diseases is shown by poor growth, unthriftiness, weakness, decreased productivity, anoestrus and repeat breeding. These factors may be responsible for economic losses.
DISEASES IN LIVESTOCK DUE TO ENVIRONMENTAL POLLUTION
Environmental pollution has become a problem of serious health concern in industrial and agricultural dominated society. This problem may be said to be man made by disturbing the ecological balance and environmental stability.
Livestock, the victim of environmental pollution, will be a liability rather than an asset to mankind because of widespread diseases resulting in morbidity, infertility, drastic reduction in productivity, toxicity of food, and mortality. The following strategies should be adopted for control.
(i) It should be mandatory on the part of manufactures of herbicides, pesticides, soil and crop spraying agents and drugs to furnish the chemical/toxicological properties of the agents to the professionals before the products are marketed.
(ii) Removal of animals from the source of poisoning. There should be a ban on grazing in industrial areas. The access of livestock to such areas may be prevented by fencing or other means.
(iii) The forest areas should be preserved to maintain the ecological balance for welfare of wild animals.
119 (iv) The livestock owners should be educated about the hazardous effects of toxic agents.
DISEASES OF POULTRY
Amongst the diseases of poultry, there is a need for development of a suitable vaccine against Marek's disease. The emerging viral diseases of poultry, now established in India are: (1) Infectious Bursal Disease (IBD) which is also known as Gumboro Disease. It is caused by a retro virus like HIV causing AIDS. This virus is a potent immunosuppressive. The disease is prevalent in Maharashtra, U.P. and Bihar (ii) Egg Drop Syndrome (EDS)-76 is another emerging problem. The infection of EDS-76 virus in laying birds causes a drop in egg production. There is a need to develop suitable vaccines against these two diseases.
REFERENCES
Khera, S.S. 1979. Bull Off. Inf. Epiz . 91(34):331-347.
Mallick, K.P. and Dwivedi, S.K. 1982. Clinical observations in an outbreak of buffalo pox. Indian Vet. J. 59: 397-398.
Mehrotra, M.L., Verma, K.C. and Somvanshi, R. 1981. Isolation and characterization of buffalo pox from an outbreak among buffaloes in Northern India, Indian Vet Med. J., 5: 155-159.
Rama, U.V.S. 1991. Emerging Zoonotic diseases with particular reference to Rabies in domestic animals. In compendium of ISVM Decennial convention and National Symposium on Recent advances in the control of diseases of corss-bred animals. 19- 21 December, 1991, pp 9-19.
Reports on National Commission on Agriculture, Govt. of India Publication, 1976.
Sen, G.P. 1973. Presidential Address, Section of Medical and Veterinary Sciences, 80th Indian Science Congress, Goa p.4.
Singh, M.P. 1985. In caprine mycoplasmosis current status in India. Lead paper presented by Verma, B.B. (1988) at VIIth ISVM convention and National Symposium on "Recent developments in livestock health care and its impact on rural economy". Hissar, Oct. 27-29.
120 Singh, N., Sharma, V.K., Rajani, H.B. and Sinha, Y.R. (1982). Incidence, economy and test efficacy of subclinical mastitis in dairy animals. Indian Vet. J. 59:693-696.
Verma, B.B. (1988). Caprine mycoplamoses - current status in India. Lead paper presented at VlIth ISVM convention and National Symposium on "Recent Developments in Livestock Health Care and its impact on Rural Economy" held at H.A.U., Hissar. Oct. 27 - 29.
Wells, B.B. (1962). Clinical Pathology, 3rd ed., W.B. Sanders Co. Philedeiphia.
121 APPLICATION OF BIOTECHNOLOGY IN VETERINARY DIAGNOSTICS AND IMMUNOBIOLOGICALS.
M. A. GAJENDRAGAD AND C. NATARAJAN Indian Veterinary Research Institute, Hebbal, Bangalore.
INTRODUCTION
During the past few decades there has been accelerated progress in biological sciences. Such an acceleration is significant especially when discoveries are usually made and applied more slowly in biological sciences than in other scientific disciplines. This has resulted in a certain apprehension of the way in which this field directly affects our environment, even though new discoveries of unexpected progress hold promise. The number and types of discoveries relevant to the veterinary sciences place this field in the forefront of such progress.
For the purpose of this paper the definition of biotechnological procedures will include all those procedures which result in the practical application of our knowledge of biology, microbiology and molecular biology, contributing to improved health and productivity of animals.
It will not be out of place to mention some of the major milestones in the genesis of the biotechnological procedures with respect to diagnostics.
The diagnosis of a disease was earlier based principally on the clinical signs exhibited by the animals. As developments in the scientific field took place, the clinician started utilizing the laboratory findings to correlate his diagnosis. Thus 'Clinical Pathology" was born. Simultaneously microbiologists isolated the etiological agents and identified them with specific diseases. Later on, the knowledge of antibodies further improved the diagnosis. Based on these disciplines viz., clinical pathology, microbiology and immunology, many techniques were developed including hematology, serum analysis for various proteins, carbohydrates and enzymes, different varieties of media for bacterial culture and organ and tissue culture for virus propagation. These techniques were modified and improved upon based on the rapid changes in the knowledge of host- agent interaction. Thus tests such as agar-gel immunodiffusion, serum neutralization (tube test) and complement fixation test (tube test) were used for diagnosis. Today, these tests have undergone modification and simplification which will be explained later on in this paper.
Advancement of immunological knowledge has given a thrust to differential diagnosis of a disease. Thus, the area of diagnosis, in particular immunodiagnosis, is currently
122 becoming popular and is likely to extend well into the future. The main aim of immunodiagnostics is to improve the speed of diagnosis, simplicity, cost effectiveness, the convenience of its use and visual interpretation of the results besides of course, accuracy. The eventual aim will be to produce a hand-held instrument which, after the addition of a small volume of the test samples, for example, milk, plasma, tissue fluids or extracted toxin, will give a direct reading of the amount of analyte present. Although many shortcomings to conduct such a test are visualized, efforts are being made to turn it into a reality.
An attempt has been made in this paper to list the diagnostic techniques developed and currently being used for the diagnosis of various diseases of livestock.
Agar Gel Immuno Diffusion Test (AGID)
AGID is the basic immunological test based on the principle of precipitation of the antigen- antibody complexes, which can be read visually. Although it is the earliest developed technique, it is still being used for the diagnosis of many important animal diseases e.g. Rinderpest, Blue Tongue etc. The advantage of this test is that it is simple, economical and does not need any special instruments. The results can be read within 24-36 h. The main disadvantage of the test is that it needs purified reagents to obtain a specific diagnosis.
Immuno Electrophoresis
This is a modification of AGID. Here two methods are combined, gel electrophoresis followed by immunodiffusion. A suitably buffered agar gel is used as the medium and the two steps of the analysis are performed in the same plate. The method is more advantageous to identify and quantify the antibodies than the antigen detection. However its modification, counter immuno electrophoresis is being used for diagnosisof Rinderpest. Another modification Rocket immuno electrophoresis is applied for measuring antigenic concentration. This test is also a laboratory test and requires an electrophoresis apparatus.
Serum Neutralization Test (SNT)
The knowledge of specific antibodies neutralizing homologous antigens has been exploited in conducting SNT. Depending on the nature of diagnosis needed, either the antigen or the antibody is suitably diluted and mixed with corresponding antigen or antibody. This mixture is allowed to react for a specific time interval and then layered on a cell system, usually a cell line grown as a monolayer. After incubating for a period of 24 to 72 h. the results are read, based on the cytopathic effect (CPE) seen in the cells. As indicated, the test can be used for both antigen and antibody titration, depending
123 on the need. The advantage of this test is that it simulates the in vivo mechanism of antigen-antibody reaction. The test is more specific. There are many inherent difficulties in conducting this test; the important ones being the need for well developed laboratory facilities to grow and maintain primary cells and cell lines and also an efficient and disciplined method of disposal of all the laboratory waste especially tissue culture media, plates and glassware. The results, (i.e. CPE) can be read with the help of a microscope. The test is considered as the ultimate for a specific and confirmatory diagnosis. The test is also a necessity for knowing the strain variation and identifying the extent of immunity breakdown in a herd.
The test was initially carried out in test tubes, which apart from requiring a large volume of test reagents was cumbersome. With the advent of biotechnological tools such as 96 well microplates, micro-pipettes, multichannel pipettes and disposable tips, laminar flow workbenches, carbon dioxide incubators, the test has been scaled down and now it can be conducted using the reagents in as a small quantity as 50 ml. The use of microplates has overcome the difficulty of clean glassware of tissue culture grade and many samples can be tested on a single plate. SNT is basically a laboratory test and use of modern biotechnological tools has made it easier to perform.
Immunofluorescence Test (IFT)
A few dyes when hit with invisible U.V. light, emit specific fluorescence which is visible. Using this basic principle, immunofluorescence test was developed. These dyes combine with antibodies, i.e. proteins, without affecting their activity. When such a conjugate is applied to antibody and allowed to react, depending on the specificity of the antibody, an antigen antibody reaction takes place. Whenjhis antigen-antibody complex is observed under a fluorescent microscope, as in the direct immunofluorescent test a specific fluorescence results. A modification of this, an indirect test, can be used for detection of both antigen and the antibody. Although many dyes are employed for the test, the most commonly used dye is Fluorescein isothiocyanate (FITC) which is a yellow compound and can be readily conjugated with the immunoglobulins. This compound when irradiated with U.V. light emits visible apple green light at about 525 urn. The advantage of this test is that it is simple, sensitive and specific. The major disadvantage is that it requires a fluorescent microscope which many laboratories cannot afford. Secondly it requires fresh antigen and any preservatives or fixatives used will lead to nonspecific results.
Radio Immuno Assay (RIA)
RIA is another test which exploits the formation of antigen-antibody complexes for the specific diagnosis of the diseases. The assay involves precipitation of viral polypeptides by a test serum or blood product and developing system usually a second antibody
124 125 125 conjugated to radio isotope (directed against serum) or labeled protein A which binds to Fc portion of lgG to reveal the presence of antigen-antibody complex. Alternatively 35(S) methionine is being used to label either virus antigen in tissue culture or in cell free system and subsequent analysis for binding studies after selection by immunoprecipitation with a monospecific antiserum. This test is also a lab test and needs a sophisticated lab with facility to handle radio active material. The test cannot be used as a routine test for diagnosis. Because of its specificity the test is employed in certain laboratories for strain differentiation and other studies of academic interest.
Enzyme Linked Immunosorbent Assay (ELISA)
The research on simple and sensitive method for detecting and quantitating antigen and antibody without using particle agglutination, fluorescent or radio labelled reagents has led to the development of a versatile immunodiagnostic technique, EIJSA. The sensitivity and specificity of ELISA compare well with that of RIA and IFT. Basically the principle of ELISA is similar to earlier described immunological techniques. It also makes use of antigen antibody complex. The antibody is conjugated with an enzyme. The formation of the antigen-antibody complex is visualized by the property of formation of a coloured complex by the enzyme when the substrate is added. Many enzymes are being tried in the assay. Horseradish peroxidase is the most common enzyme used in the assay. The other enzymes used in the assay are alkaline phosphatase and penicillinase. There are many modifications of the basic ELISA technique. These techniques can be broadly divided into two categories (1) Non-competitive and (2) competitive. The non-competitive method is widely used for routine diagnostic purposes. ELISA requires immobilisation of either antigen or antibody to a solid support. This permits the separation of immunologically reacted from unreacted material during the test. At present the use of specially designed microtitre plates made of polystyrene or polyvinyl has a major advantage over other solid supports. The advantages of this assay are that it can be performed in the field, reagents are stable, the precautions required in disposal of radio isotopes are not needed and the results can be visually read. Further, the test can be modified to be a field test wherein it can be performed in any laboratory or dispensary. For this purpose the test has been suitably modified. In place of 96 well plates, either strips of 8-12 wells or 1 cm sq. piece of nitro-cellulose paper are being used. Because of its relative easy protocol, ability to detect minute quantity of antigen and specificity, ELISA has become the test of choice for diagnosis of many diseases. In the veterinary medicine ELISA has been developed for almost aH the diseases and many laboratories have stopped performing any other tests for disease diagnosis. Depending upon the need, EL1SA has been suitably modified. The modifications of ELISA are 1) Indirect ELISA, 2) Double antibody sandwich ELISA, 3) Competition liquid phase blocking ELISA and 4) Immunoperoxidase or in situ ELISA.
125 Immuno Electro Transfer Blot Technique
This technique is commonly known as Western blot. The technique involves separation of viral proteins by SDS-PAGE, transferring the resolved antigens to a nitrocellulose sheet and identifying the presence of specific antigens through their reaction with specific antibodies. Western blotting techniques are being extensively used to confirm ELISA positive results in sero-epidemiological investigations, in characterization of monoclonal antibodies and to identify potential translation products from partially purified mRNA.
Hybridoma Technique for Production of Monoclonal Antibodies
Hybridoma technique is the culmination of research in cell fusion experiments wherein the antibody producing cells are immortalized by fusing them with myeloma cells forming "Hybridoma" capable of secreting monospecific antibodies eternally. The immunized mouse spleenocytes are fused with myeloma cells and polyclonal hybridoma thus produce monoclonal antibodies (MAbs) specific to the virus epitopes. The MAbs can be used for a wide variety of research applications. The important areas being antigenic characterization, antigenic variation, immunological function, receptor structure and function. The MAbs are used in immunodiagnostics too. In the ELISA technique, the MAbs replace the polyclonal antibodies thereby making the ELISA much more specific and sensitive.
Nucleic Acid Hybridization Techniques
Nucleic acid hybridization is a fundamental tool in molecular cloning. It is the formation of double stranded nucleic acid macromolecule by base pairing due to sequence specificity of complementary single strands. The nucleic acid is immobilized on nitrocellulose membrane or charged nylon membranes and is probed with labeled sequences by molecular hybridization. This technique is applied for identification of specific clones in cDNA and genomic DNA libraries, the analysis of the organization of specific regions of genomic DNA, analysis of gene transcription and IRNA processing, riuclease foot-printing experiments and RFLP studies etc. The probes generally used are P radio-labelled complementary DNA or RNA strands. Due to these reasons, research is being carried out to replace the radio labelling with nonradio labelling of the probes. The research in this direction yielded such nonradio labeled probes. The chemical Digoxigenin, is being used successfully. Further research is being carried out to make this test a field diagnostic test.
Polymerase Chain Reaction (PCR)
The principle involved in PCR method is in vitro amplification of target DNA using two oligonucleotide primers that hybridize opposite strands and flank the region. This consists
126 of a series of cycles involving template denaturation, primer annealing and the extension of the annealed primers by DNA polymerase, resulting in an exponential accumulation of a specific fragment. Since the primer extension products synthesized in one cycle will serve as a template in the next, the number of target DNA copies approximately doubles at every cycle. Invention of a thermostable DNA polymerase (Taq polymerase) from Thermophilus aquaticus transformed the PCR into a simple and robust reaction with enormous applications in molecular biology. In case of RNA genomes PCR can be employed after producing cDNA by reverse transcription reaction. The application of PCR technique is to enhance gene probe detection of specific gene sequences. The PCR technique is being exploited for diagnosis of a disease that could not be diagnosed by any other test. However the cost of the test makes it prohibitory to be used as a routine diagnostic test. The types of PCR techniques generally used are reverse transcription PCR (RT/PCR) and antigen capture PCR. The latter is highly sensitive and does not require a virus passage.
Nucleotide Sequencing
Many antigens undergo mutation in nature due to various causes. These antigens coming in contact with the susceptible population can cause the disease which could be highly virulent and may not conform to classical clinical signs and lesions. Further it becomes still intriguing that the disease is seen in immunologically protected animals. Under such circumstances it becomes a necessity to understand the exact nature of the antigen. Thus techniques were evolved to know the precise nature of the nucleotides involved in the antigen and to compare it with similar studies conducted on a known stable antigen. Thus nucleotide sequencing of many viruses and their variants have been done and their library is maintained.
OTHERS
Certain techniques that are extensively used in the human medicine are gaining entry into the veterinary field. The knowledge of X-rays brought in a visible change in the field of surgery and internal medicine. Many diseases which were undiagnosed could be diagnosed. However, the harmful effects of the X-rays especially on young and growing cells restricted its use on all the subjects. This in fact, led to the discovery of alternative diagnostic tests to replace the X-rays. Ultrasound method of diagnosis is one such means. This test is extensively used in human beings especially in the fields of gynaecology, cardiology, orthopaedics and oncology. It is slowly gaining entry in the veterinary field. The Nuclear Magnetic Resonance Imaging technique are currently used in modern disease diagnosis. Another biotechnological invention which has made valuable contribution in human medicine is CT-scan. Time is not far when these techniques will be used in veterinary medicine also.
127 IMMUNOBOLOGICALS
The development of vaccines to prevent human and animal diseases is one of the great accomplishments of biomedical science. Although few diseases have been eliminated through the use of vaccines, there have been marked reduction in incidence, morbidity and mortality. In fact, vaccines have been so successful in safeguarding humans from many of the great epidemic diseases that world wide eradication, as in the case of small pox or regional elimination, as in the case of yellow fever and poliomyelitis, have become realities. Vaccines against the important diseases of livestock and poultry have also been used so successfully that they have allowed the development of the modern high-density husbandry systems now common to industrial agriculture. However, many infectious diseases of humans and animals still represent unresolved burdens to mankind. In each case, just as in the past, we continue to turn to vaccination for cost-effective, efficient approaches to disease control. Since the first documented use of a vaccine by Jenner in 1798, vaccines have undergone many changes. Many vaccines have been developed. Yet infectious diseases are still a major problem in the world today and account for untold numbers of animal death worldwide. Thus newer techniques are needed to make vaccines safer, more effective and most importantly, available throughout the world. As in most fields of endeavour, major developments follow certain basic advances in concept or technology. In vaccinology, today's application of recombinant DNA technology represents such an advance.
Expectations for biotechnology have been and continue to be great. Much of the excitement for the application of biotechnology to the development of new and improved vaccines arose from the coalescence of two disparate areas of research. By the 1970's, it had become apparent that isolated structural components, particularly proteins, of infectious pathogens were generally immunogenic. Such noninfectious 'Sub unit vaccines" often evoked protective immune responses with fewer undesirable side effects generally associated with inactivated whole-agent vaccines. However, at that time, their production for practical purposes was prohibitively expensive. During this same period, advances in molecular biologyand recombinant DNAtechnology, particularly with respect to the development of generalized microbial expression systems, provided the potential for inexpensive, high-level production of virtuallyany gene product of interest, prokaryotic or eukaryotic. Thus came the new era of "Recombinant DNA subunit vaccines". Under this the new term "Biosynthetic vaccine" also came into vogue. A biosynthetic vaccine is a formulation containing a noninfectious protective subunit immunogen that is produced in or by a biologic system. The term immunogen is limited to immunogenic polypeptide. The concept of a subunit vaccine, containing only those immunogenic components necessary to elicit a protective response and excluding others unnecessary for immunity, has severa' attractive features. First and foremost is the element of safety. Subunit vaccines lack infectious material. No infection either acute, persistent or latent, can
128 be established in the vaccine/vaccinate, substantially reducing complications arising from vaccination of immuno compromised or pregnant individuals. Adventitious agents can be eliminated. Extraneous antigens are lacking, reducing or eliminating pyrogenic, allergenic, immunosuppressive and other undesirable reactogenicity. Subunit vaccines also exhibit considerable stability potentially allowing for wide distribution and prolonged storage. Another advantage of the subunit vaccine is that it allows a ready identification of vaccines/vaccinates. Simple serologic tests can readily distinguish vaccination from infection.
Subunit vaccines have been tried in the following diseases: Anthrax, ovine foot-rot caused by Bacteroides nodosus, mycobacterial infection, bovine brucellosis, foot-and- mouth disease, Herpesvirus infections (BHV-1, pseudorabies).
Considerable variation in antigenic structure of viruses led to the study of chemical structure of viruses. Molecular biologists showed that a synthetic peptide corresponding to the antigenic segment of the virus can elicit a neutralizing antibody. This knowledge paved the way for the development of chemically synthesized vaccines. Gene coding for the immunogenic protein of several viruses is already known and the amino acid sequencing of these proteins enabled the scientists to develop the vaccines. The chemically synthesized vaccines have been produced against FMD, hepatitis B and malaria. Although the concept of such vaccines is new, it will not be far fetched to have them in the open market.
The third type of vaccines developed through biotechnology are vectored virus vaccines. There are two types of such vaccines. One class comprises replication-defective vector viruses that produce infectious progeny only via complementation by specifically transformed ceUs or by helper-virus superinfection. The second class comprises replication - competent viruses. Herpes virus, adenoviruses and poxviruses are the notable examples of viruses which serve as vectors.
The disadvantage of the chemically synthesized subunit vaccines is that they are poorly immunogenic and many times do not stimulate the cell mediated immunity. Thus the current research in this field is to synthesize a peptide vaccine which can contain both immunodominant B-cell and T-cell epitopes. To achieve this an immunostimulating complex (ISCOMS) i introduced alongwith the peptide. ISCOMS are prepared by mixing peptide antigens with detergent and a glycoside. These three interact to form an antigen. ISCOMS are currently being tried against influenza, measles, hepatitis B and HIV.
The knowledge of anti-idiotype antibody has motivated the scientists to use it as a potential vaccine as it offers the possibility of achieving a state of immunity without having to expose the recipient to a dangerous pathogen. Much work has been conducted
129 on laboratory animals viz, mice and rats, using such vaccines against hepatitis, rabies, polio, Venezuelan equine E. myelitis, Listeria, E.coli, Trypanosoma and Schistosoma.
Many viruses undergo latency after establishing an infection in the host. This is more so in case of DNA viruses wherein the viral DNA gets integrated with the host DNA. Under such circumstances it is the CMI response which plays a key role in host defense. The T-helper cells produce interferons which act by inducing an antiviral state in the cells. This property of interferons especially IFN - alpha has been used for cancer therapy. Recombinant IFN are also available. Use of IFN in veterinary field is limited but likely to gain momentum in the near future.
Advanced knowledge in parasitology and vaccinology has led to development of vaccines for parasitic diseases such as Theileriosis, Babesiosis, lung worm infections, Anaplasmosis,coccidiosis, fascioliasis. Many of these vaccines are attenuated stages in the parasitic life cycle. However, research is being carried out to develop a vaccine using DNA recombinant technology.
Cancer is a disease characterized by uncontrolled growth of abnormal cells at the site of the initial tumor and at other sites in the body. It is now known that many viruses initiate the cell division which eventually form a cancerous mass. Studies are being conducted to prepare vaccines against these viruses which eventually act as vaccines for cancer. Effective vaccines against tumor antigens have not yet been produced and use of immunomodulators may help in cancer therapy.
130 BIOTECHNOLOGICALAPPROACHES IN DIAGNOSIS AND CONTROL OF PATHOGENS TRANSMITTED THROUGH SEMEN
S.N. Singh, P.K. Gupta, S. Kilari, R.S. Kataria, N. Pandita & M.N. Takale BAIF Research Institute of Animal Health, Wagholi, Pune
INTRODUCTION
Livestock population in India plays an important role in our economy. Despite a recent decline in the value of output from agriculture as percentage of GDP from 46.45% to 37.00%, livestock output value of GDP has increased from 8.65% to 9.5%. India has become the second largest milk producer (54.9 million tons) in the world with a cattle and buffalo population of around 206 million & 61 million respectively. By 2000 AD. milk production target of 78.0 million tons has been projected by the Planning Commission. The progress in milk production can be achieved by an intensive cross breeding programme through artificial insemination using frozen or liquid semen of superior bulls. But it has been seen that an improvement in the genetic make-up of animals can only contribute upto 30% of production and the remaining 70% is dependent on micro environment like proper nutrition, management and health care. Though cross breeding programme through artificial insemination (A.l.) has improved productive traits in our livestock, at the same time, disease resistance of our native livestock has gone down. Various methods of semen processing like cryopreservation provides a conducive environment for contaminating micro-organisms. The semen will mainly become contaminated either from the infected animal itself or collection or at the time of processing of the semen. Insemination of cows by contaminated semen leads to infertility problems like repeat breeding, retention of placenta, endometritis, still birth, abortions etc.
MICROORGANISMS IN BOVINE SEMEN
The extensive use of artificial insemination in cattle has facilitated the exchange of desired genetic characteristics both at national and international levels. At the same time possible contamination of semen with semen-borne pathogens is spreading the pathogens across the country. Thus there is a need to screen contaminated semen by regulatory authorities in countries like India where Al is being practiced. The contaminated semen may contain either pathogenic or nan-pathogenic micro-organisms. But ISI arbitrarily permits maximum 500 non-pathogenic bacterial load per insemination dose. However, pathogenic organisms like bacteria, fungi, virus, protozoa, mycoplasma, chiamydia, rickettsia should not be present in semen. Bacterial infections such as
131 Brucellosis, Vibriosis, Trichomoniasis, Leptospirosis, Leishmaniasis, Listeriosis in breed- ing bulls lead to impotentia generandii and their transmission to cows through semen causes serious infertility problems.
Fungus infections due to Candida sp., Rhizopus sp., Absidia sp., Zygomycetes, Aspergilus sp., Mortierella sp. etc. and presence of these in semen cause mycotic abortions. Similarly Mycoplasma infection also transmits through semen from breeding bulls to inseminated cows.
Among the protozoan infections, Trichomaniosis causes early abortions ( 2-4 months) leading to repeat breeding and is transmitted mainly through natural service but rarely through Al. This can be easily diagnosed either by direct microscopic examination or by isolation in modified culture media.
Apart from the above micro-organisms, viral infections are also causing reproductive losses in breeding bulls and dairy cows. The semen may become contaminated with virus extrinsically, during collection and intrinsically, by systemic or local viral infection through shedding of virus from testes, accessory glands or preputial excretions. The viruses such as Bovine Herpes virus (BHV), Foot and Mouth disease virus (FMDV), Bovine Viral Diarrhoea virus (BVDV), Blue Tongue virus (BTV), Bovine Leukemia virus (BLV) associated with semen, are preserved under freezing and storage and are transmitted through Al.
Among the Bovine Herpes viruses (BHV-1, BHV-2 and BHV-4), BHV-1 is most commonly found in bull semen without any concurrent systemic or local clinical signs. Its spread through semen presents a threat to the cattle industry as a variety of genital disorders including abortions, repeat breeding are associated with it.
FMD virus is present in semen due to viraemia, preputial lesions and contamination during ejaculation. FMDV is preserved by freezing and storage of semen and an inseminated animal can be infected which becomes a carrier.
ANIMAL HEALTH STRATEGY IN PATHOGEN FREE SEMEN
It is clearly understood that due to lack of proper animal health programme at Bull Mother Farms and lack of pathogen screening at the Frozen Semen Bank, the diseases are spreading without notice. Therefore, a defined strategy'to ensure the pathogen free semen, prevention of carrier infection and detection of the latent infection in the breeding bulls (cattle and buffalo) should be organised on top priority to ensure economics in production through increasing conception rate, prevention of abortions, reduction in the inter-calving period and finally a disease free herd for higher milk production for the benefit of the dairy industry.
132 - Detection of micro-organisms in semen, clinical examination of breeding bull and serological testing of breeding bulls.
- All semen coilection centres and frozen semen banks should adopt standard procedures regarding screening for presence of pathogens in semen through different sensitive, cost-effective and rapid diagnostic tests. - From semen collection to freezing in straw, the process should be under 100 class standard of GMP.
- Semen diluters/extenders with proper antibacterial and antifungal agents. - Strategy for proper vaccination regime.
- Improved management practices and feeding standards.
Semen used for insemination should be free from any pathogenic organisms. The presence of micro-organisms should be checked by isolation and identification in selective or differential media. Viruses can be isolated in susceptible cell cultures or chicken embryo. Precautions should be taken while collecting semen as maximum permissible non-pathogenic bacterial load is 500 organisms per insemination dose but preferably it should be at zero level as per the latest GMP regulations.
The breeding bulls used for semen collection should be healthy. However, periodical testing for important bacterial and viral diseases should be undertaken. Serological testing should be done for Brucellosis by plate agglutination, standard tube agglutination and ELISA and tuberculosis paratuberculosis by single intradermal test with PPD and Johnin, respectively. For detection of viral diseases, virus isolation from the animals, serum neutralization test (SNT), agar gel immuno diffusion test (AGID), enzyme linked immunosorbent assay (ELISA), fluorescent antibody test (FAT) and complement fixation test (CFT) should be done to check the clinical infection in breeding bulls.
Use of anti-microbial agent should be done in semen extenders to check the bacterial transmission through semen. These agents to date have not been able to control viruses in semen. However addition of gamma-globulins from hyperimmune serum has been reported to eliminate BHV-1 infectivity in semen without affecting its fertility. Various treatments for semen like trypsin treatment and treatment with specific monoclonal antibody (Mo Ab) have been recommended to check the viral pathogens spread through semen.
133 BAIF'S IBR VACCINE AND RAPID DIAGNOSTICS
BAIF has a unique role in animal health care and inputs through Immunobiologicals, diagnostics with ensured cold chain and delivery system at grassroot level. Apart from proper health care package, BAIF is also engaged in developing diagnostics for important diseases like BHV, FMD and Brucella and new vaccines for economically important emerging diseases. Considering the prevalence of BI-IV-1 in India, BAIF has devebped IBRIVAK, which is under field trial (a cell culture inactivated oil adjuvanted vaccine against BHV-1). It will help in preventing the spread of this economically important reproductive disease through semen. The diagnostics based on indirect ELISA has been standardized for rapid screening of BHV-1 in breeding bulls and in cows at field level. A pre-filter membrane has been used to collect blood from animals and this blood absorbed filter membrane was used to elute antibodies for test system. The concentrated cell culture BHV-1 antigen was immobilized on nitrocellulose membrane as dot and antibodies eluted from pre-filter was tested for dot ELISA to detect antibodies to BHV- 1. This system can be easily performed at field level for screening. A test based on polymerase chain reaction (PCR) is under experimentation which will help in detection of virus (BHV-1) in semen as well as from nasal, lachrymal and preputial swabs.
REFERENCES
Afshar, A. and Eaglesome 1990. Virus associated with Bovine Semen. Vet. Bull. 60 (2), 92-103.
Hafez, E.S.E. Reproduction in Farm animals. Lea and Febiger, Philadelphia.
Mehrotra, M.L., Rajya, B.S. and Kumar, S. 1976. Infectious Bovine Rhinotracheitis in calves. Ind. J. Vet. Path. 1, 70-73.
Roberts, S.J. Veterinary Obstetrics and Genital Diseases (Theriogenology), Ithaca, New York.
Singh, B.K., Sreenivasan, M.A., Tongaonkar, S.S., Kant, R. and Roy Choudhary, P.N. 1986. md. J. Anim. Sci. 56(8), 823-826.
Skirrow, S.Z. and Bon Durant, R.H. (1988). Bovine Trichomoniasis. Vet. Bull. 58(8), 591 -603.
Prof. L.A. Babiuk and Prof. Vikram Misra, Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada (Personal Communication).
134 STRATEGIES IN ANIMAL HEALTH PROGRAMME
DR. V.A. SAPRE Nagpur Veterinary College, Nagpur
INTRODUCTION
Livestock industry has an ultimate aim of contributing to food production and a veterinarian's role in this achievement is undisputed. Importance of food from animal origin has been universally accepted. Not only milk, meat and eggs have acquired a definite place in human nutrition but draft power of bullocks which has a great importance in rural economy, has to be ôonsidered as an item under production.
There was a time, not very long ago, when Veterinarians were busy exclusively in controlling mortality due to deadly and devastating diseases like Rinderpest, Anthrax, H.S., B.Q. in cattle and Ranikhet in poultry. Rearing livestock and poultry were considered the most risky enterprises. The sciences of livestockbreeding, management and nutrition had no relevance in such a situation.
Veterinarians have done a great and commendable job by giving us the most effective and dependable vaccines against these deadly diseases and therefore today we are in a position to plan strategies for livestock production.
Veterinarians thereafterhad time to divert their research programmes towards evolving breeds of livestock and poultry which have immense capacity to produce milk, meat and eggs with economical inputs. The situation seemed most alluring but the problems never ended and they came up in different shapes. The high producing breeds of livestock and poultry are the most delicate biological systems as converters of feeds into milk, meat and eggs and require vigilant supervision, management and feeding. They are also prone to react adversely to petty disturbances in environment, and require a superior health cover from experts. The prestige of the Veterinarian is almost at stake to see that a high pedigreed heifer calf not only survives but maintains optimum health and grows well in time and gives expected high returns for the investment by livestockowner.
Nationwide implementation of the crossbreeding programme has received a good response in the country, though not uniform. This has attracted many resourceful livestock owners who have developed good herds with crosses of Jersey and Holstein breeds. The impact study of crossbreeding indicates that annual milk production in Maharashtra State which was 1.062 million tonnes before implementation of crossbreed-
135 ing programme in 1970-71 has risen to 2.49 million tonnes in 1987-88 recording a 2.5 times increase. The crossbred population has increased from 1467 in 1970-71 to 2,08,879 in 1987-88 recording a 142 times increase.
The progress in districts of Western Maharashtra is noteworthy and we find high yielding cows maintained under ideal conditions. As has been the usual case with any new scheme, the educated and well to do farmers have derived more benefits and have demonstrated that maintaining a few good crossbred cows is more rewarding than having a large number of low and medium producers. Out of the 197 million cattle population in India, 80 to 85 percent are non descript and only 12-13 million are crossbred. Though we account for 25% of the world's cattle population, our milk production is only 5.1 percent with an average milk yield of 157 kg per lactation.
This programme of livestock development by crossbreeding has been rather slow in some parts of the state for example in some districts of Vidarbha which have problems of parasitic and protozoan infections due to peculiar agro-climatic conditions. These are endemic areas and the parasitic infections are prevalent throughout the year and on a large scale resulting in poor growth, delayed maturity, irregular breeding and low production. This is an important constraint in the rapid development of livestock industry in these areas. Recent survey indicated that nearly 70 to 75% cattle in these areas are harbouring one or the other type of parasitic infections. Metabolic profile studies indicate suboptimal levels of calcium, phosphorus and other essential trace elements necessary for vital body functions and optimum production.
Well maintained dairy herds located mostly in suburban areas are also having certain problems of nutritional and hormonal imbalances, metabolic disturbances resulting in suboptimal milk yield, and irregular breeding performance causing worry to the unit owner who has to fight against increasing feed cost, and overheads to maintain the production cost within limits.
Frequent changes in the quality of feed ingredients, lack of quality control measures on feed manufacturers, inadequate storage facilities and possible fungal and residual insecticide toxicities through agricultural by-products in the form of straw, has made feeding problem complex and difficult to handle. This is particularly true with units which are solely dependant on purchased feeds and fodders. Low grade feed toxicities over a prolonged period can impair the river functions slowly, thereby causing complex health hazards resulting in suboptimal production. The type of indigestion has no more remained simple and ingestion of plastic waste packing materials thrown in garbage heaps in towns has reached a level of menace necessitating ruminotomy operation in most of the cows and buffaloes who are let out 'for a walk" during their free time between milking.
136 Infections of udder and uterus - the most vital organs directly related to production are probably the most neglected ones. It is estimated that out of the total annual losses of 2000 millions, half are due to mastitis. The need to keep these vital organs under strict vigilance is probably not adequately emphasized to the livestock owners. Indiscriminate and delayed treatment of udder and uterine infections has created a emerging problem of antibiotic resistant strains of bacteria and can become a big public health hazard because most of the bacteria do not discriminate between men and animals and the antibiotic resistance is transmitted from generation to generation in bacteria.
Hemoprotozoan infection like Theileriasis though not causing havoc as it used to do earlier, still remains a problem which needs strict vigilance.
It will thus be appreciated that an effective and efficient Veterinary health cover is essential if the livestock production unit has to be economically viable.
HEALTH DELIVERY SYSTEMS
There is a wide variation in the degree of development in livestock industry in the State, depending on the agroclimatic zones, type of local breed and road communication facilities. The livestock population could be broadly classified under the following categories.
Category A - Livestock population in rural 75 - 80% interiors comprising mostly of the nondescript breed and some crossbred population.
Category B - Animals in urban and suburban 10 - 15% areas with good transport and communication facilities. Comprise mostly of high yielding buffaloes, crossbred cows and good indigenous animals.
Category C - Animals maintained in commercial 5 - 10% and organised units with selected high yielders either indigenous or crossbred cows.
The veterinary health cover needed for each of the above categories is not the same and therefore a distinct health delivery system will have to be evolved for each.
137 Category A
Requires a rural veterinary aid centre and a mobile clinic to deliver veterinary aid to the farmers at their doorsteps rather than big hospitals.
The veterinary health cover should provide
• Regular preventive vaccinations against all preventive diseases of livestock and poultry.
• Regular deworming of at least productive animals.
• Ectoparasitic control.
• Veterinary First Aid Education.
• Education to prevent udder and uterine infections though adoption of hygienic measures.
• Education to distinguish between an ordinary ailment and an emergency.
Category B
In addition to the above,
• Providing optimum management, hygiene and nutrition.
• Neonatal calf care to ensure optimum growth.
• Maintenance of an efficient reproductive cycle.
• Surveillance and timely dealing with udder and uterine infections.
Category C
In addition to the above,
• Early detection of digestive upsets with special tests like rumen function tests to evaluate rumen flora activity.
• Monitoring regular feed supply of dependable quality, free from any possible toxicities of feed origin. Periodical feed analysis.
138 • Metabolic profile studies carried out periodically.
• Regulating reproductive cycle, investigating causes of repeat breeding, anoestrus, anovulatory oestrus, early embryonic mortality, low grade and chronic infections.
• Monitoring subclinical udder infections with resistant strains.
This category needs a systematic and methodical approach by the Veterinary Physician and a rational therapy guided by timely and relevent investigations like metabolic profile study, rumen function evaluation, hormonal assay, antibiogram of bacterial isolates, etc.
AVAILABILITY OF DISEASE DIAGNOSTIC FACILITY
It is obligatory that minimum diagnostic facilities with trained/experienced graduates be made available at taluka level where routine hemogram, faecal examination etc. is done so as to obtain 'on the spot' diagnosis in common disease conditions.
At a district diagnostic centre at the Veterinary Polyclinic, the facilities to test common blood profile such as glucose, calcium, magnesium, serum electrolytes etc. and facility for bacteriological examination of milk and uterine discharges with antibiotic sensitivity test should be provided.
At regional level it is essential to have a well equipped Disease Investigation Laboratory with facility to detect food toxins, conduct serological tests, histopathology and biological tests etc. The Veterinary Colleges in the Agricultural Universities should also collaborate for the establishment of sophisticated Disease diagnostic facilities at regional headquar- ters, with good mobile unit facilties for spot visits in emergencies.
REFERENCES
Govitrikar et al, 1990. "Impact of crossbreeding programme on rural economy in Maharashtra".
Chaudhary, P.C. 1991. Presidential address at National Symposium et al. I.S.V.M. Decinniel Convention.
139 GROUP DISCUSSIONS AND RECOMMENDATIONS SESSION I
ANIMAL REPRODUCTION
Chairman : Dr. Kiran Singh Co-Chairman : Dr. B.K. Bhavsar Dr. A.A. Nimkar
1. Embryo Transfer and associated technologies can be taken up to produce male calves and heifers from elite donors and the superior germplasm so obtained can be exploited through Al programmes.
2. ONBS along with MOET system may be used to prove the sires at selected farms.
3. Synchronisation of oestrus may be taken up to promote breeding. This is of special importance for remote areas.
4. Simple RIA/EIA technique for milk and plasma progesterone estimations may be used for monitoring the reproduction status in cattle and buffaloes.
5. Ultrasonography is an important aid available for obtaining an insight of the internal organs. It may be used in monitoring ovarian activity in Al and ET.
6. Additional research is needed on pathobiological and bloengineering aspects of embryo transfer.
7. Fertility of much stock in this country is greatly dependent upon nutrition and management. To improvethe reproduction, these aspects should be taken care of.
8. Embryo transfer may be taken up in the field for application whereverpossible.
141 SESSION II
ANIMAL NUTRITION
Chairman : Mr. J.B. Schiere Co-Chairman : Dr. D.V. Rangnekar
1. Fodder production programmes should be an integral part of the Livestock Development Projects.
2. Conservation of forages during surplus production season should be given proper attentionto alleviate the nutrient deficiencies commonly observed in dry season.
3. Top feeds from leguminous trees should be exploited to bridge the gap between availability and requirements of nutrients through promotion of multipurpose tree species acceptable to farmers.
4. Newer technologies involving usage of bypass nutrients through molasses lick blocks need to be widely tested.
5. Systematic field level studies should be undertaken to criticallyassess the role of crossbreds in farming systems in different regions.
SESSION III
ANIMAL BREEDING
Chairman : Dr. V.D. Mudgal Co-Chairman : Dr. I.S. Bakshi
1. Performance evaluation should be done on a regional basis as there are many states with considerable variation in resources and agro-climatic conditions.
2. For cross-breds the exotic inheritance should range between 3/8th and 5/8th. The cross bred development should result from this blood level.
3. Progeny testing should be taken up at farmers herds and associated herds to identify superior bulls. Reliable Progeny Test of bulls can be obtained using farmer level data.
142 4. Reliable procedures of progeny testing should be developed and tested for small herds. It appears that BLUP or contemporary comparison methods are superior in accuracy if 30-50 daughters per bull are recorded.
5. ONBS using MOET should be adopted whereverfeasible and utmost precautions should be taken to safeguard the health of the breeding stock.
6. The view point of farmers must be considered while deciding the breeding policy. It may be location specific.
7. Conservation and improvement of buffalo breeds like Surti and Mehsana need to be considered on top priority. The breeding policy°should not disturb the home tracts of these animals. Similarly herds of these breeds of animals established in other states should be considered as associated herds and extended all possible support.
8. Socio-anthropological, environmental and gender issues should be considered while dealing with livestock development programme.
9. A holistic farm approach should be taken and livestockshould not to be isolated.
10. Greenhouse effect due to production of methane gas by the ruminants is a burning issue. It is strongly recommended to examine the problem in details.
143 SESSION IV
ANIMAL HEALTH
Chairman : Dr. M.C. Prasad Co-Chairman : Dr. Raghunathan
1. Attempts should be made to develop immuno-diagnostic kits for diagnosis of important diseases of livestock and poultry.
2. Pathobiology of semen and embryo should be rigorously studied before their extensive use.
3. Investigations of diseases pertaining to reproductive systems (male and female) should be given top priority and steps should be taken in totality to contain them.
4. The State Animal Husbandry Departments should attempt to select proper personnel for expertservices at district level for disease investigation and clinical diagnosis of conditions including repeat breeding in high yielding livestock.
5. A endemic map should be prepared for the last three years for each district and the vaccination programme should be followed accordingly.
6. ELISA test should be standardised and the standard protocolshould be followed in all the laboratories.
144 LIST OF PARTICIPANTS
Mr. V.C. Badve Dr. H.H. Chhaya BAIF, Urulikanchan Joint Director, Dept of A.H., Pune 412 202 Paldi, Ahmedabad 6 Maharashtra Gujarat
Dr. LS. Bakshi Dr. S.C. Chopra Director Director Dept. of Animal Husbandry Central Inst. for Res. on Buffaloes Talab Tilloo Haryana Jammu Tawi Jammu & Kashmir . Dr. Manibhai Desai Former President, BAIF Dr. R.N. Bhatnagar Pune 411 016 Jt. Director (Project) Maharashtra Dept. of Animal Husbandry Jaipur Dr. B.B. Raiasthan Deshpande Asstt Dean Faculty of Vety. Science Dr. B.K. Bhavsar MAU, Parbhani. Director Maharashtra Dept. of Animal Husbandry Ahmedabad 6 Paldi, Dr. G.R. Guarat Deshpande BAIF Laboratories, Waghoi Pune 412 207 Dr. M.R. Bhosrekar Maharashtra BAIF, Urulikanchan Pune 412 202 Maharashtra Dr. M.R. Gajendragad Sr. Scientist IVRI, Hebbal Dr. T.S. Bukshetwar Bangalore 560 024 BIRD - K, Karnataka Parvana Bank Colony Sangmeshwarnagar Gulbarga, Karnataka 585 103 Dr. P.N. Girdhar Dy. Director Haryana Vety. VaccineInst. Hissar Haryana
145 Dr. S.B. Gokhale Dr. S.A. Joshi BAIF, Urulikanchan Asstt. Director (AH) Pune 412 202 Directorate of Animal Husbandry Maharashtra Pune 411 001 Maharashtra
Dr. G.R. Hegde BIRD - K Dr. M.S. Kadu Sanmati Layout, Prof. & Head K. C. Park Post Office Dept. of Animal Reproduction Dharwad 580003 PKV Akole Maharashtra
Dr. S.K. Jam 61, Sarva Ritu Vilas Dr. S.V. Kakade Udaipur, Dy. Director Rajasthan DIS Pune 411 007 Maharashtra
Mr. V. K. Jere 22, 408/410, Mukundnagar Dr. A.G. Koppikar Pune 411 037 Raman & Weli Pvt. Ltd. 10, Abhilasha Society, Chinchwad, Pune 411 033 Dr. A.G. Joshi BAIF Laboratories, Wagholi Pune Mr. P. Kothadiya Maharashtra BAIF, Pune 411 016 Maharashtra
Dr. A.L. Joshi BAIF, Urulikanchan Dr. R.K. Mahuli Pune 412 202 BAIF, Urulikanchan Maharashtra Pune 412 202 Maharashtra
Dr. D.N. Joshi BAIF Laboratories, Wagholi Dr. B.R. Mangurkar Pune 412 207 BAIF, Urulikanchan Maharashtra Pune 412 202 Maharashtra
146 Dr. RS. Manik Dr. P. Padmakar Scientist Dy. Director NDRI, Karnal 132 001 Key Village Scheme Haryana Directorate of Animal Husbandry Jaipur Rajasthan Dr. A.C. Mathur C-35 Hauz Khas New Delhi 110 016 Dr. A.B. Pande BAIF, Urulikanchan Pune 412 202 Dr. V.M. Mehta Maharashtra Research Scientist & Head Reproductive Biology Res. Unit GAU, Anand 388 110 Dr. A.M. Pande Gujarat Prof & Head Dept. of Animal Management PKV Akole Dr. S.K. Mitra Maharashtra Dy. Director AH & Vety. Services Frozen Semen Bank Dr. S.G. PatH Cuttack 753 010 BAIF, Sangamner Orissa Maharashtra
Dr. V.D. Mudgal Dr. V.K. PatH Director Dean Project Directorate on Cattle Faculty of Veterinary Science G-123 Shastrinagar MPKV, Shirval Meerut 250 005 Dist. Pune Uttar Pradesh Maharashtra
Mrs. M. Newale Dr. Y.P. Phadnis BAIF, Pune 411 004 BAIF, Urulikanchan Maharashtra Pune 412 202 Maharashtra
Dr. A.A. Nimkar Dist. Al Centre Mr.A.S. Pore Ahmednagar BAIF Laboratories Maharashtra Wagholi 412 207 Maharashtra
147 Dr. B.S. Prakash Dr. D.V. Rangnekar Sr. Scientist BAIF, P.B.No. 3080, Asarwa Road Embryo Biotech. Centre Ahmedabad 380 016 NDRI, Kamal 132 001 Gujarat Haryana
Mr. Ramesh Rawal Dr. M.C. Prasad BAIF, 109, MahavirBhavan Assistant Director General, C-2, Karampura Complex ICAR, Krishi Bhavan Najatgarh Road New Defhi 110 001 New Delhi 110 015
Dr. J.R. Purohit Prof. L.L. Reiwani BAIF, Urulikanchan BAIF, Pune 411 016 Pune 412 202 Maharashtra Maharashtra
Dr. J.P. Sabe Dr. K. Raghunathan BAIF, (UP) Dirctor, Dept. of A.H. A 552, Guru Teg Bahadur Nagar Pondicherry Kareli, Allahabad211 016 U.P. Dr. M. Rajamani Director, AH. & Vety. Services Dr. K.L. Sahni Directorate of AH & Vety. Services Prof. & Head Cuttack Dept. of Animal Reproduction Orissa 753 001 JVRI, Izatnagar Uttar Pradesh
Mr. A. Rajan BAIF, Pune 411 016 Dr. V.A. Sapre Maharashtra Prof. & Head Dept. of Vety. Medicine Nagpur Vety. College Dr. M. Rajasekhar Nagpur Project Coordinator Maharashtra AICRP on Disease Surveillance Bangalore Karnataka
148 Mr. J.B. Schiere Dr. V.P. Singh DTAP, Agri. University Director (MF) Wageningen Kirkee The Netherlands
Dr. K.S.S. Somayajulu Mr. B.R. Seth TSO, NABARD IBP Co. Ltd., Sriniketan, 7th Floor A-4 MIDC, Nasik Worli, Bombay 400 018 Maharashtra Maharashtra
Dr. V. Sethuraman, Dr. G.M. Srivastava Dy. Advisor (AH & DD), Dept. of A.H. Planning Commission Lucknow Govt. of India Uttar Pradesh New Delhi
Dr. M.V. Tandale Dr. V.J. Sidhaye Dy. Director (AH) BAIF, Pune 411 016 Directorate of A.H. Maharashtra Pune 411 001 Maharashtra
Dr. C.M. Singh President Mr. N.S. Thole Indian Vety. Council BAIF, Urulikanchan New Delhi Pune 412 202 Maharashtra
Dr. KiranSingh Assi. Director General Dr. V.S. Upadhyay ICAR, Krishi Bhavan Head New Delhi 110 001 Div. of Plant Animal Relationship IGFRI, Jhansi Uttar Pradesh Dr. S.N. Singh BAIF Laboratories, Wagholi Pune 412 207 Mr. M.K. Vohra Maharashtra Chairman & M.D. DAAPS Breeding Products Pvt. Ltd. New Delhi
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