FAO ANIMAL PRODUCTION AND HEALTH PAPER 90

Application of biotechnology to nutrition of animals in developing countries

by R.A. Leng Professor of Nutritional Biochemistry Director of the Institute of Biotechnology University of New England Armidale, NSW Australia

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M-23 ISBN 92-5-103035-9

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FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS Rome, FAO 1991

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Contents
1 Introduction 1.1 Preamble 1.2 Definition of Biotechnology or High Technology 1.3 The Promise of the New Biotechnology 1.4 The Target Animals 2 Background 2.1 Conditions of Ruminants in Third World Countries 2.2 Productivity of Livestock in Developing Countries 2.3 The Implication of Low Productivity 2.4 Feed Resources Available to Ruminants 2.5 Chemical Composition of Low Quality Forages 3 Basic Ruminant Nutrition 3.1 The Rumen and its Micro-organisms 3.2 Fermentative Efficiency in the Rumen 3.3 Meeting the Requirements for Efficient Microbial Growth in the Rumen 3.4 Consequences of the Ruminant Mode of Digestion 3.5 Quantitative Aspects of Fermentative Digestion in the Rumen 3.5.1 A Model of Fermentation in the Rumen 3.6 Protein Utilisation by Ruminants 3.6.1 Ensuring a Balanced Nutrition For Ruminants on Forage Based Diets 3.7 Optimising Microbial Growth in the Rumen 3.7.1 Mineral Requirements of Rumen Microbes 3.7.2 Requirements for Ammonia 3.7.3 Timing of Urea Supplements and the Ratio of Sugars and Starches to Fibre in a Diet 3.7.4 Requirements for Amino Acids/ Peptides by Rumen Organisms 3.7.5 Amino Acid Requirements of Microbes Digesting Fibre 3.7.6 The Roles of Small Amounts of Fresh Forage in Straw Based Diets 3.7.7 Elimination of Rumen Protozoa and Preservation of the Fauna-Free State 3.8 Factors Influencing Efficiency of Feed Utilisation 3.9 Climate, Supplementation and Intake of Low Quality Forages 3.10 Feeding Standards and Feed Evaluation 3.10.1 Implications of low Productivity of Ruminants in the Tropics 3.11 Some Basic Explanations for the Inefficiency of Ruminants on Forage Diets 3.11.1 Inefficiency of Acetate Utilisation 3.11.2 Requirements for Glucose by Ruminants. 3.11.3 Balancing Nutrition for Reproduction/ Pregnancy and Lactation 3.12 Implication of Parasite/ Disease and Nutrition 3.13 Implications of an Increased Nutrient Requirements for Work 3.14 Conclusions 3.14.1 Implications for Areas of Research 4 Research Areas 4.1 Research Targets 4.2 Priority Ratings 5 Present Knowledge and Priority Research Areas 5.1 Adjusting P/E Ratio in the Nutrients Absorbed by Ruminants with Protein Supplements 5.1.1 General 5.1.2 Bypass Protein Supplements 5.2 Adjusting P/E Ratio by Manipulation of the Rumen 5.2.1 Supplementation of the Rumen Microbial Ecosystem 5.2.2 Chemical Manipulation of Rumen Fermentative Efficiency 5.2.3 Other Manipulations of Rumen Ecosystems

5.2.4 Alteration of Protein and Amino Acid Composition of Rumen Microbes Recombinant DNA Technology 5.3 Adjusting P/E Ratios from the Rumen by Manipulating the Feed Base 5.3.1 Feed Technology and Development of Supplements 5.3.2 Supplementation with Naturally Protected Protein: A Case Study in Strategic Supplements 5.3.3 Providing Bypass Protein in Areas Without Protein Resources 5.4 Maximising Digestibility of Fibrous Feeds 5.4.1 Supplementation 5.4.2 Improving the Enzymatic Ability of Rumen Microbes to Degrade Fibre 5.4.3 Potential Targets for Improving Fibre Digestion 5.4.4 State of the Art in Bioengineering of Rumen Organisms 5.4.5 Selection of Anaerobic Fungi for Better Fibre Degradation in the Rumen 5.4.6 Selection of Bacteria for Fibrolytic Activity 5.4.7 Solubilisation of Lignin 5.4.8 Detoxification of Anti-microbial, Toxic and Anti-quality Factors in Plants 5.5 Developing Detoxification Mechanisms in Rumen Organisms 5.6 Treatment to Increase Digestibility 5.6.1 Improving the Digestibility of Crop Residues by Chemical Treatments 5.6.2 Microbial Treatment of Straw and Other Crop Residues to Improve Digestibility 5.6.3 Potential for Increasing the Digestibility of Poor Quality Roughage by Manipulating the Digestive Physiology of the Animal 5.7 Altering the Partitioning of Nutrients Within the Animal 5.7.1 Anabolic Steroids 5.7.2 Growth Hormone 5.7.3 -Agonists 5.7.4 Injected Growth Promotants versus Supplementation to Balance Nutrition 5.7.5 Conclusions 5.8 Cross Breeding to Improve Partitioning of Nutrients into Milk 5.9 Transgenesis and Embryo Manipulation 6 Biotechnology and Monogastric Nutrition 6.1 Introduction 6.2 Potential Areas for Biotechnology in Pig Nutrition 6.2.1 Overcoming Feed Intake Problems on High Fibre Protein Supplements 6.2.2 Possibility of Modifying the Digestive Function Through Development of Transgenic Animals 6.2.3 Porcine Growth Hormone (PSt.) 6.2.4 Transgenic PigsPorcine Growth Hormone 7 Biotechnology and Environment 7.1 Introduction 7.2 Integrated Farming 8 Some Practical Problems for Biotechnology Research in Developing Countries 8.1 Present Research Priorities and Change 8.2 Real World Problems of Capitalising on Modern Biotechnology in Developing Countries 8.2.1 Secrecy in Industrialised Countries 8.2.2 The Need for Expertise in Developing Countries to Capitalise on Various Developments 9 Conclusions and Recommendations 9.1 Priorities 9.2 Overall Conclusions 9.2.1 The Vision of the New Biotechnology 9.2.2 The Future 9.2.3 The Problems 9.2.4 Recommendations A Methods Involved in Modifying Rumen Bacteria B Some Comments on Use of Protein Meals for Use as Supplement for Ruminants Fed Forages C References

Chapter 1 Introduction
1.1 Preamble
Livestock production is a function of:

feed availability and quality the incidence of diseases including intestinal parasites, blood and other parasites of living tissues (e.g. protozoa), viral and bacterial invasive agents the climatic and environmental conditions that prevail in the particular area the genotype which fits it to a particular production system.

In the context of this report, which examines the prospects and perspectives for the use of high technology or biotechnology in nutrition to increase animal production, the target animals must be the domestic ruminants that are managed by small farmers in third world countries. The developing countries, in general, have severe constraints on food availability for humans and are often critically balanced in producing sufficient starch-based carbohydrates and high quality proteins to provide for their human population. Thus, the grains produced are generally required for the resident population, which is often increasing at a rate several times that of the populations of the industrialised countries. Short term food surpluses due to good seasonal growing conditions and/or application of plant/soil research results, will inevitably be converted to deficits by the growing population's demand for food. The only exception to this will be if birth rate is reduced and death rate accelerated in these countries. The impact of the presently untreatable viral disease HIV (or AIDS) will be significant in this area but no predictions are yet available on its effects on future population densities in developing countries. The likely small surpluses of grains over and above that needed by humans indicates that the production of monogastric animals which compete with humans for the basic feed resources (grain) must have good political and or trade advantages before it can be encouraged. However, production of monogastric animals on sugar based products and various by-products may be a future direction for considerable research effort. There is, of course, a middle class in all developing countries which demands meat from poultry and pigs and can afford to pay the relatively high costs of production. This group of people is expanding in the countries that are rapidly industrialising, and this together with the influx of tourists in such countries will increase demand for these products. There is a high correlation between meat consumed and cash income or standard of living (see Brumby, 1989 and Figure 1.1) The husbandry of monogastric animals is already at a very high level and, in general, research on grain-fed domestic animals is probably best left to the industrialised countries where surplus grains are generally available and their production is subsidised (e.g. approximately 40% of the value of grain production throughout the EEC and USA is met by subsidies). In most developing countries pig and poultry production is either based on a scavenger system, (which is restricted to village farmers), or a high cost technology system as used, and transferred from, temperate countries (which is used by the large farmer). Figure 1.1: Food consumption/percentage of diet for meat, wheat and rice, and coarse grains as per capita income increases (Marks & Yetley, 1987-see Brumby 1989)

Monogastric nutrition cannot be omitted from consideration, however, as the appropriate integration of monogastric animals with ruminants in cropping areas is potentially the most efficient systems that might be (or are being) developed to increase animal production overall. Within these systems the production of pig and chicken meats from non-conventional feed sources (e.g. sugar cane, molasses, household swill, tubers, roots and by-products) integrated with such systems as protein production from aquatic plants should be the major consideration in the future. The ruminant animal, because it has pre-gastric fermentative digestion of feed, generally does not compete with humans for vital quality-feed resources and must therefore have the focus of the discussions. Ruminant animals (biomass) in developing countries far outnumber all other domestic animal not only as a source of high quality human food (meat and milk, with blood in some areas), but of fuel (from dung) and draught power. The continuing use of ruminants as domestic animals resides in their ability to:

convert fibrous carbohydrates through fermentative digestion, into nutrients that can be used for growth, and milk synthesis efficiently utilise low protein feeds and non-protein nitrogen in the rumen and synthesise proteins with a high biological value for human consumption convert the carbohydrates of fibrous feeds into nutrients that can be used to carry out work-functions (e.g. ploughing) more efficiently use dietary protein for tissue synthesis than monogastric animals provided it is in a form protected from microbial attack in the rumen but digestible by gastric and intestinal enzymes.

Any biotechnology developments will undoubtedly have to target the improvement of efficiency of these attributes under practical conditions pertaining particularly to the small farmer. There are five major ways by which any technology may significantly improve livestock production:

by altering the feed base to provide a better quantity and balance of nutrients to the animal by altering the digestibility of the feed base by treatment so that more nutrients are extracted. by manipulating the fermentative, gastric and post-gastric digestive processes to extract more and a better balance of nutrients for the animal from the basal feed. by manipulating the efficiency of partitioning of absorbed nutrients into productive processes including those that are involved in life-time productivity by removing or ameliorating constraints that are part of the environment (largely disease, but also the effects of temperature and humidity stress)

Strategies for the future use of animals for work and food production cannot ignore the growing environmental crisis and the need to reduce gaseous emissions from farming systems. Some comments will be made on this in later sections of this report.

1.2 Definition of Biotechnology or High Technology

Biotechnology is a much used in word in research submissions since it conjures up images of research at the cutting edge of science. It also has an overtone of application, potentially patentable products or processes and therefore returns on investment. The promises have been much overrated in recent times and in general the promises for application from, for instance, recombinant DNA technology have failed to materialise in terms of animal production. In general, the results of recombinant DNA technology have been disappointing. For example transgenic animals have been produced but failure to control expression of the introduced genes has resulted in adverse effects often of a nature that horrifies animal welfare groups. This has been given bad press but it has highlighted the need to study the basic aspects of control of gene expression and cell physiology. This does not mean however, that the future for and importance of such work is debatable. In fact, the contrary is true, but it has indicated a major requirement for basic research to understand gene expression and that more time and funds will be needed before many new concepts can be applied. Biotechnology in the context of this report cannot be restricted to recombinant DNA technology. Biotechnology is in reality, a science of great antiquity having its origins in processes such as brewing and wine making. The broad definition used here is the application of biological organisms, systems or processes to production of animal products These include meat, milk, hides, wool or hair and draught power. In particular, biotechnology stresses the integration of microbiology, biochemistry and chemical and process engineering. It is always multi-disciplinary, and its strongest aspect is that it is directed to application. It usually requires disciplinary scientists working together and shepherded by person(s) with major integrative abilities. Young scientists (or disciplinarians) caught up in biotechnology and working in isolation often lose the direction for application or seek out compromise objectives to justify their approach which may or may not be rational. On the tour of institutions prior to the writing of this report, it was an observation that a number of young, newly trained (overseas) scientists in the less well developed countries, were still working in the area of their overseas training without true recognition of the potential

application of their work. They were, thus, in some ways competing with their previous supervisors and are therefore unlikely to produce novel innovations. For the purposes of this report biotechnology research is regarded as a multi-level activity. In animal nutrition this includes research that aims to improve the efficiency of production through manipulation of:

the feed base the animal's digestive system the animal's metabolism

It must also consider the potential for augmenting the feed base with critically deficient nutrients that may be produced locally, particularly from non-conventional sources (e.g. production of protein meals from aquatic plants (algae) grown on biodigestor effluent). It must also consider the possibility of decreasing protein fermentability of plants consumed by ruminants by genetic engineering of plants and other techniques applied to the plant, the microbial digestion system and the animal. The narrower definition will be referred to here as the new biotechnology. This is defined as the application of recombinant DNA technology to the improvement of animal production through improving nutrition.

1.3 The Promise of the New Biotechnology

The beginning of recombinant DNA technology arose from the work of Crick and Watson (Watson, 1968) and their colleagues at Cambridge. Research in this area is often seen as being superior to other forms of biological research. However, like all research it is often the acquisition of technology and instrumentation which triggers development or progress in this field. Example of developments which allowed many groups to begin or progress in their work include the development of techniques to split the ovum, to introduce foreign DNA into chromosomal DNA or to force a plasmid into a cell where it will replicate along with other DNA. This is not meant to be a criticism of modern biotechnology research but it is stated to point out that the new biotechnology is supported by a large group of normal scientists led largely by a few highly capable and creative persons in the same way as every other field of research endeavour. The concept of the new biotechnology as a science for the gifted and the imaginative promises of biotechnology put forward in most reviews, have undermined many other fields of research which have then suffered from financial stringencies. These fields often regain prominence when, for example, innovative gene transfer methodology has failed to produce the expected applied technology (e.g. higher productivity in domestic animals expressing genes for growth hormone). In this case there has been little recognition of nutritional principles; an animal capable of growing say to twice the size and at twice the rate because of the acquisition of a gene for growth hormone may need a totally new nutritional approach. For example, in order to provide for growth and normal bone growth in such animals it will be perhaps necessary to provide more calcium and phosphorus in forms more easily absorbed and assimilated; protein to energy ratios in the nutrients absorbed may have to be increased substantially as may be the availability of vitamins and other minerals. The need is for a suitable balance of research which must be maintained. The new biotechnology needs to advance along with the fundamental and applied research that is needed to ensure that the application of molecular genetic research reaches the end user, in this case the farmer. It is a major theme of this report that it is imperative that research on nutrition must not compete for limited funds with the new biotechnology. It is also a major conclusion that concepts of nutrition transferred from temperate countries have been misleading. Recently developed understanding of nutrition, which has massive effects on productivity, is the area with most promise and with potential immediate application. The great need is for research to find ways of applying these concepts in production systems under differing environmental conditions within countries. With a likely doubling of food requirements of developing countries by the year 2000 the need for research which will result in strategies that can be quickly applied is paramount.

1.4 The Target Animals

This dissertation must deal largely with the economically (in its broadest sense) important ruminants in developing countri es. These are largely cattle, buffaloes, sheep and goats. Other animals that pre-ferment their feed, include camels, alpaca, llama, yak, thimin and certain monkeys as well as certain deer species, and post-gastric fermenters such as the horse, donkey, guinea pig and rabbit must be kept in mind as they are often of major importance in some areas. However, the latter group have not been targeted because of the predominance of ruminant herbivores. Research on the use of non-conventional feeds is given a brief mention but the major research requirements here are to develop the systems which depend on locally available resources and particular within an integrated farming systems concept.

Chapter 2 Background
2.1 Conditions of Ruminants in Third World Countries
The reasons for keeping ruminants in developing countries are not always easily understood by the outsider. They are kept for a number of reasons, which change in priority depending on their location and owners including the following:

for food (e.g. meat, milk and blood or any of these combinations) and also as a reliable food store for years of drought as status symbols of wealth as a means of accumulation of wealth to be cashed for a number of purposes (e.g. life threatening events; to meet marriage costs; to provide for pay-back etc.) as an edge against inflation for fuel (dung for sale or to provide for household cooking) for fertiliser production (e.g. dung) drought power for religious purposes and/or entertainment (e.g. the fighting rams of Indonesia) work purposes (ploughing, puddling of soil for rice etc.) transport hides for leather as investment by city business men to create a stake in agriculture which is often motivated by the possibility of tax relief to make use of poor lands which would not otherwise be used for agricultural purposes.

The desire of the farmer to increase productivity of his animals, is highly dependent on the purpose for keeping the livestock. It is sometimes an advantage to maintain a low level of productivity. For example, if animal numbers are more important than production per animal, it is advantageous to have stunted, thin animals which require low management inputs. In this presentation the target animals are the animals in the herds of small farmers where meat, milk, work, or any combination of these (meat/work, meat/milk, milk/work) are the major objectives as these are the major owners of livestock in developing countries and the target for many of the aid-sponsored projects.

2.2 Productivity of Livestock in Developing Countries

Few developing countries have surplus tracts of land that can be regarded as fertile and therefore produce high quality fodder. In general, human population pressures and the primary need to supply human food ensures that livestock are restricted to:

poor quality (often overgrazed) pasture lands, either infertile or with terrain that makes them impossible to utilise for crops or where erosion has made them unusable.

Table 2.1: Average meat production (kg) per animal of total population of cattle/buffaloes in Europe (representative of the concentrate/forage feeding systems) and in Asia/Pacific (1986)

Cattle Buffalo Europe Asia/Pacific 68 5 50 7

standing crop residues or weeds following cropping when environmental conditions are poor for plant growth and a further crop in that year cannot be taken. crop residues (cut and stored) agro-industrial by-products extensive pastures of poor quality and otherwise sparse (e.g. The Llanos of Colombia and Venezuela).

The net result is in general an extremely low rate of productivity with animals often at 5 years of age at maturity and productivity at 0.10.25 of that of ruminants in temperate countries grazing fertilised pastures or fed high quality feeds based on grain and immature pasture plants. A comparison of the levels of production of cattle between developed and developing countries is shown in Tables 2.1, 2.2 and 2.3. Table 2.2: Average carcass weight (kg) per animal slaughtered (Jasiorowski, 1988) (1986 statistics)

Cattle Buffalo Europe Asia/Pacific 185 120 206 161

Table 2.3: The change in the average milk yield per cow in industrialised and third world countries

Country/Region North America EEC Countries Asia Africa

Average yield/cow Percentage increase (%) (kg/year) 1976 3,250 2,900 620 322 1986 5,200 4,100 700 354 (1976 to 1986) 60 35 13 7

2.3 The Implication of Low Productivity

Biotechnology research in industrialised countries is not generally aimed at low yielding animals. In addition to poor nutrition, adverse climate and disease, other stresses are also generally high, particularly in those countries situated in the tropics. These additional constraints are likely to affect and often remove any advantage of biotechnology transferred from developed countries. The considerable differences in feed resources available in developing countries compared to developed countries (where most biotechnology research is presently centered) and the low cost of production systems employed in the developing world as compared with the industrialised world (where subsidies on agricultural production are often in excess of 40% of the value of the product) indicates that direct technology transfer is unlikely to be successful.

2.4 Feed Resources Available to Ruminants

The feeds that are available to ruminants in developing countries are fibrous and relatively high in ligno-cellulose. They are usually of low digestibility and they are often deficient in critical nutrients, including protein, non-protein nitrogen and minerals. As a generalisation, the forages consumed by ruminants in developing countries are almost always below 55% (usually 4045%) digestibility and are often less than 8% crude protein and this protein level is more often around 3 5%, e.g. cereal straws. The only exception to this is in the early growth phase of pasture and when stocking rates are extremely low and the animals are able to select for leaf material. The low levels of production per head and per acre of most grazing systems are indicated by the summary of data given by Walker (1987) (see Figure 2.1). Without supplements, these low levels of production lead to a highly inefficient use of the available feed, with a possibility that up to 30% of the feed consumed is dissipated as heat. This heat has at times great effects on feed intake particularly in the tropics (see Chapter 3).

It is necessary, therefore in discussing the nutrition of ruminants, to appreciate the digestion of forage based diets and the constraints to the utilisation of nutrients that arise largely from a fermentative digestion system, since this knowledge must considerably influence research priorities.

2.5 Chemical Composition of Low Quality Forages

In the literature concerned with the nutritional value of forages, considerable emphasis is placed on the crude chemical composition of the forage. Although analyses that indicate cell solubles and cell wall materials are highly useful for studying the fermentative characteristics of a forage, they bear little relationship to its feeding value to the animal (see later). Considerable efforts is often put into feed analysis which is often totally unwarranted particularly in the developing countries. In this report the overriding effects of a balanced nutrient approach to feeding are emphasised. It is suggested that in most production systems for ruminants based on a poor quality forage, it is the balance of those nutrients providing the major building blocks for tissue synthesis and milk production, that should be the primary concern. With most forages and contrary to the assertations in the past that energy deficiency is the primary constraint to ruminant production on low quality feeds, the efficiency of feed utilisation is the major determinant of the production levels achieved. Therefore in reviewing the need for biotechnology research, this is the area for most consideration. Recent evaluation of data from studies in tropical countries has indicated that medium to high levels of production at very high feed conversion efficiencies can be achieved by ruminants on poor quality forages adequately supplemented with critical nutrients (see Preston & Leng (1987) for review). Of more importance is that the efficiency of utilisation of the metabolizable energy of a straw based diet appropriately supplemented can be higher than that of grain based diets (Leng, 1989b). This suggests that the efficiency of utilisation of metabolisable energy of a forage can be markedly improved simply by supplementation (by up to 10 fold). It also discounts theories that the digestible nutrients from such feeds are less efficiently utilised than from grain based diets. Figure 2.1: Cattle growth on pasture is a function of pasture type, fertiliser applications and legume content. Productivity per unit area is maximised for the different pastures at different stocking rates: 89 kg/ha for native pastures, 223 kg/ha for tropical grass with legume, 682 kg/ha for tropical grass with fertiliser and on temperate pasture (clover) 105 kg/ha. (Source: Walker 1987)

Chapter 3 Basic Ruminant Nutrition

3.1 The Rumen and its Micro-organisms
As the utilisation of forages by ruminants depends on microbial fermentative digestion, the principles of digestion in the rumen are discussed as a framework to view the requirements for biotechnology innovations in nutrition. The rumen is the dominant feature of the digestive tract of cattle. This maintains a medium that supports a dense and varied population of microorganisms. These organisms ferment feed materials to produce mainly shortchain organic acids or volatile fatty acids (VFAs), methane and carbon dioxide and the process provides substrate (the feed) and ATP (energy) for the growth of micro-organisms. The microbial mix in the rumen is complex and highly dependent on diet. The main agents that break down fibre, sugars, starches and proteins in the rumen are all anaerobic and include bacteria, protozoa and fungi. The bacteria are the principal organisms that ferment plant cell-wall carbohydrates (Hungate, 1966) but the anaerobic phycomycetous fungi may at times be extremely important (see Bauchop, 1981). Protozoa are now recognised as having an overall negative effect in the rumen, particularly where ruminants are fed forage diets low in trueprotein (Bird et al. 1990). Protozoa ingest and digest bacteria and reduce the bacterial biomass in the rumen (Coleman, 1975) and consequently the protein supply to the animal. Thus, they decrease the protein to energy ratio in the nutrients absorbed (see later) and increase the requirement of animals for true protein. The net result of the presence of protozoa is an increased requirement for dietary bypass protein and on low protein diets a decreased efficiency of utilisation of feed for growth and milk production (see later) (Bird et al. 1990). The presence of protozoa in the rumen may also reduce the rate at which bacteria colonise and degrade the ingested feed particles. In studies with sheep fed straw based diets, it has been found that the apparent digestibility of dry matter was increased by 18% after protozoa had been removed from the rumen (i.e. defaunated) (Bird & Leng, 1984; Soetanto, 1986). This research indicates that large increases in productivity may be achieved with ruminants fed fibrous diets, particularly those low in true protein by controlling or removing protozoa from the rumen. Other workers have not seen the differences in digestibility and in some instances removal of protozoa from the rumen has led to decreased digestibility of mixed, starch containing diets (Jouany & Ushida, 1990).

3.2 Fermentative Efficiency in the Rumen

A deficiency of a nutrient needed by rumen micro-organisms reduces microbial growth efficiency which reduces microbial biomass and eventually reduces digestibility and feed intake, particularly of fibrous feeds. The first priority in feeding ruminants is to ensure no deficiencies in the diet of nutrients for microbial growth in the rumen. Of major importance is that the efficiency of microbial growth (that is, the amount of microbial biomass available for digestion in the intestines per unit of digestible carbohydrate entering the rumen) also determines the proportion of digested feed that is converted to methane and VFA. Methane production accompanies the formation of acetate or butyrate, whereas methane and VFA production are inversely related to microbial cell production.

3.3 Meeting the Requirements for Efficient Microbial Growth in the Rumen
On most diets based on crop residues and low-digestibility forages, the primary limitation to the growth of rumen micro-organisms is probably the concentration of ammonia in rumen fluid The second consideration is deficiencies of minerals, particularly sulphur, phosphorus, magnesium and certain trace minerals. Ammonia in the rumen must be above a critical level for a considerable period of the day to ensure a high rate of microbial growth and digestion and therefore feed intake. The level of ammonia that supports the optimal population of micro-organisms in the rumen the highest protein to energy ration in the nutrients absorbed, and therefore maximum digestion, will vary among diets. In general on forage based diets the ammonia level should be above 200 mg nitrogen/litre (see Leng, 1991). It must be stressed, however, that any nutrient, (including many minerals required in the growth of micro-organisms), that is deficient in a diet will result in low microbial cell yield relative to VFA and lead to a low protein (from microbes) to energy (from VFA) in the nutrients absorbed (this is discussed under quantitative aspects of fermentation digestion below). The ratio of protein digested and absorbed from the intestines to the VFA produced in and absorbed from the rumen is termed the P/E ratio.

3.4 Consequences of the Ruminant Mode of Digestion

One of the consequences of the ruminant mode of digestion is that fermentation results in up to 20% of the digestible energy intake being lost as heat and methane. A second major disadvantage is that proteins that are fermented in the rumen are not then sources of amino acids for the animal because they are hydrolysed and their constituent amino acids deaminated by microbes. In general, where ruminants are fed forage based diets typical of that available in tropical developing countries, small amounts of extra nutrients are needed to balance nutrient availability to requirements. Proteins which are directly available to the animals and are protected from degradation increase the efficiency of anabolism of the absorbed nutrients in growth, pregnancy, lactation or work. (see Leng, 1991).

3.5 Quantitative Aspects of Fermentative Digestion in the Rumen

The end products of rumen fermentative digestion are governed by the feed, the rate of consumption of feed, the balance of nutrients in the feed for microbial growth and the balance of micro-organisms that develop in the rumen (bacteria, protozoa and fungi). In general, a proportion of the digestible feed dry matter is converted to VFA, methane and carbon dioxide and the balance is assimilated into microbial cells. The pathways of these reactions are well known and a schematic outline is shown in Figure 3.1. Microbial cells, that are synthesised from the feed resource use the ATP that is generated in the formation of VFA from the feed to provide the energy for synthesis. The microbes are lost from the rumen pool either by passage out of the rumen to be partially digested in the intestine or by death and breakdown within the rumen (with formation of VFA, CO2 and methane). Lysis and degradation in the rumen is inefficient as it makes the protein of microbes unavailable as such to the animal. Because microbial cells are more reduced than the substrate fermented, the quantity of microbial cells leaving the rumen per unit of carbohydrate consumed is related to methane production. The efficiency of microbial growth is then a primary determinate of the quantity of methane produced. Figure 3.1: Energetics of rumen fermentation (Leng, 1982)

3.5.1 A Model of Fermentation in the Rumen

For the purposes of the present discussion and to demonstrate the underlying principles of the concepts developed, a model for a 200 kg steer will be used to illustrate the quantitative availability of nutrients from rumen fermentation. The steer consumes 4 kg which represents 25 Mole anhydroglucose or organic matter which is completely fermented in the rumen. It is assumed:

that the fermentation of 1 mole of carbohydrate gives rise to either 2 mole acetate, 2 mole of propionate or 1 mole of butyrate, according to the following stoichiometry:

Hexose 2Pyruvate + 2H2O 2Pyruvate +

Pyruvate + 2ATP + H2 2HAc + 2CO2 + 2H2 + 2ATP

2HPro + 2H2O + 2ATP

8H2O 2Pyruvate + 4H2O CO2 + H2 H Bu + 2H2 + 2CO2 + 2ATP CH4 + 2H2O + 2ATP

In the stoichiometry, H2 indicates reduced co-enzymes, HAc is acetic acid, HPro is propionic acid and HBu is butyric
acid that the animal's rumen is functioning at normal level of fermentative efficiency in which one-third of the organic matter fermented is converted to microbial cells and the rest to VFA, CO2 and CH4. that the moles ATP generated per mole of end-product are for acetate 2, butyrate 3, propionate 3, and methane 1 (Isaacson et al. 1975).

On chemical principles, the equation of substrate use and end products from fermentation of 4 kg of carbohydrate is:

Carbohydrate to VFA 16.7 CHO 21 HAc + 6HPro + 3HBu + 7.5CH4 + 78ATP Carbohydrate to microbial cell precursors 8.3CHO 1.4 polysaccharide + 13.8 pyruvate + 2CH4 + 17ATP Overall: 25CHO 21HAc + 6HPro + 3HBu+9.5CH4 + 1300 g dry cells.

In the example, one-third of the carbohydrate provides the substrate for microbial cell synthesis 1300 g dry microbial cells are produced at a YATP of about 14.5 (YATP is a measure of the efficiency of utilisation of ATP generated in fermentation of carbohydrates to VFA; it is defined as the g dry cells produced per mole ATP available.) The upper level of efficiency (or the theoretical highest level of cell production) has a YATP of 26. On the other hand the lowest efficiency of a microbial growth in the rumen that is deficient in, say, ammonia, is possibly below a YATP of 4. The relationship between the efficiency of cell synthesis and fermentative end products produced are shown in Figure 3.2. These values were arrived at by similar calculations as that given above. Figure 3.2: Relationship between the production of microbial cells and volatile fatty acids and methane in fermentative digestion in ruminants The relative efficiency of the system (indicated as YATP) is governed largely by the availability of essential nutrients for microorganisms (after Leng, 1982). The ranges of YATP are shown for: A. B. C. D. a relatively inefficient rumen (i.e. ammonia deficient) a normal rumen with no deficient nutrient for microbial growth a rumen free of protozoa with no deficient nutrient for microbial growth the theoretical optimum microbial growth efficiency.

Table 3.1: The effect of different efficiencies of microbial growth on the ratio of protein to VFA energy (P/E ratio) available from the rumen of a steer consuming 4 kg of organic matter which is totally fermentable.

YATP 8 Microbial protein* synthesised (g/d) VFA produced (MJ/d) Methane produced (MJ/d) Heat (MJ/d) P/E ratio (g protein/MJ) 14 19 25

500 800 1010

1212

41

34

30

26

9.4

8.5

8.0

7.6

6.4 12

5.1 25

4.3 34

3.1 47

* Microbial protein may be only 7585% digestible and this will change the P/E ratio markedly in the animal.
Based on this model, but assuming a varying efficiency, the microbial cells produced relative to VFA and methane production change are shown in Table 3.1. The main point to emphasise is that, depending on the efficiency of utilization of ATP, the amount of carbohydrate converted to microbial cells can be highly variable. It is the efficiency of microbial growth that largely controls the amount of methane produced by an animal (see Figure 3.2). Ensuring a high ratio of microbial cells (protein) produced relative to VFA (energy) or a high P/E ratio is critical for efficient feed utilisation (see Section 3.10) and mechanisms for manipulating this ratio are discussed in the next section.

3.6 Protein Utilisation by Ruminants

Protein that is fermented in the rumen is largely wasted as a source of amino acids to the animal because:

dietary protein is degraded and essential amino acids are deaminated to form ammonia and VFA fermentation of 1 g of protein generates only half the ATP that would be produced from 1 g of carbohydrate and therefore anaerobic microbial growth on protein is approximately half that on carbohydrate.

In combination these effects result in only 30 to 60 g of microbial protein becoming available to the animal for digestion for every kilogram of dietary protein that is fermented in the rumen. The fermentation of protein is, however, associated with relatively small amounts of methane production. On the other hand methane is not generated when protein bypasses the rumen. Protein that is insoluble, or has a high component of disulphide bonds or is associated with tannins tends to bypass rumen fermentation but is digested in the intestines and in this way it augments the microbial protein and alters the ratio of protein to energy (P/E) in the nutrients absorbed. The better the balance of nutrients for microbial growth the higher the ratio of P/E in the nutrients produced in fermentation. The higher the content of bypass protein in the diet the higher the P/E ratio in the absorbed nutrients.

3.6.1 Ensuring a Balanced Nutrition For Ruminants on Forage Based Diets

From the above discussion the first priority for improving the utilisation of a low digestibility forage by ruminants is to optimise the availability of nutrients from fermentative digestion by:

ensuring that there are no deficiencies of microbial nutrients in the rumen and therefore the microbes in the rumen grow efficiently and, through fermentative activity, extract the maximum possible amounts of nutrients from the forage (i.e. the ratio of microbial cells to VFA produced is high as are production rates of these end products) ensuring that the microbial cells (which provide most of the protein to the animal) synthesised in the rumen are not lysed and fermented in the rumen but are available for digestion and absorption as amino acids from the intestines.

The second objective of a feeding strategy should be to optimise the efficiency of partitioning of absorbed nutrients into product by:

supplementation with critical nutrients that escape or bypass rumen fermentation to augment and balance the nutrients absorbed to those required for maintenance of homeostasis, maintenance of body temperature, exercise (or work), and the particular physiological or productive function.

As the nutrients needed for different functions differ in priority, supplementation strategies will need to vary according to climate, environment, management and production targets in any one location.

3.7 Optimising Microbial Growth in the Rumen

3.7.1 Mineral Requirements of Rumen Microbes
The rumen microbes have specific requirements for both macro and micro minerals to meet the needs of structural components of cells and for components of enzymes and co-factors. Little is known about the requirements of the microbial milieu for trace elements and as a rule of thumb it is accepted that if the animal is not deficient then it is unlikely that the rumen microbes will be deficient. As Suttle (1987) has so aptly put the situation, it will rarely be possible to approach a suspected mineral deficiency situa tion with a table of minor nutrient requirements or biochemical criteria in the hand, and define a scale of the animal health (microbial health) problems. In practice, either no mineral supplements are used or a shot gun mixture is given as a salt licks (McDowell et al. 1984) or as molasses (which is concentrated plant juice rich in minerals) suitably fortified with minerals (Kunju, 1986). As with any deficiency of a nutrient the likely scenario of a mineral deficiency for rumen organisms is first a reduced growth efficiency of microbes (lowered ratio of cells to VFA produced) with or without a decrease in digestibility. As the deficiencies become more extreme the digestibility of forage must decrease along with the decrease in microbial pool size and it is only then that feed intake will decrease. Feed intake however, will be decreased as P/E ratio decreases if the animal is heat stressed (see later). Correction of deficiency will obviously reverse these effects.

3.7.2 Requirements for Ammonia

Ensuring adequate ammonia N in the rumen to supply the majority of nitrogen for microbial growth is the first priority in optimising fermentative digestion of forage. Satter & Slyter (1974) suggested that 5080 mg NH3-N/1 of rumen fluid was the optimum for maximising

microbial growth yield and this has been widely accepted. However, recent studies from two laboratories in Australia have clearly indicated that the minimum level of ruminal fluid ammonia for optimum voluntary intake of low N, low digestibility forage by cattle is about 200 mg N/1, even though the digestibility of the forage (in nylon bags) was optimised below 100 mg NH3-N/1 (Krebs & Leng, 1984; Boniface et al. 1986; Perdok et al. 1988). All these studies were carried out in hot environments and the effects on feed intake are possibly explained by an improving P/E ratio in the nutrients absorbed, which reduces the metabolic heat load. The effects of increasing ruminal fluid ammonia by infusion of urea into the rumen of steers on the intake of rice straw and its digestibility in nylon bags in the rumen are shown in Figure 3.3. Figure 3.3: The effects of the level of ammonia in the rumen on the intake and in sacco digestibility of straw by cattle. The ammonia levels were adjusted by infusing urea in the rumen (Perdok et al., 1988)

3.7.3 Timing of Urea Supplements and the Ratio of Sugars and Starches to Fibre in a Diet
Supplements must provide adequate levels of ammonia in the rumen for continuous growth of both fibrolytic and saccharolytic organisms. The only satisfactory approach to meeting these changing requirements for ammonia is to provide ammonia continuously. One way of doing this is to provide salt/urea or molasses/urea licks and allow the animal to take these as needed. There are indications that cattle and buffaloes given continuous access to multi-nutrient blocks based on molasses/urea are able to control fairly closely their intake of urea. Once buffaloes were accustomed to molasses/urea blocks they adjusted their intake according to the N content of the basal diet (NDDB unpublished data). Lambs given wheat straw and molasses/urea blocks also had similar abilities and consistently maintained their rumen ammonia levels above 200 mg N/1 (Sudana & Leng, 1986).

3.7.4 Requirements for Amino Acids/ Peptides by Rumen Organisms

There has been considerable controversy concerning the requirements for peptides/amino acids by rumen microbes for efficient growth. A number of studies, however, have reported results of in vivo studies which appear to have indicated no apparent requirement for amino acids for efficient growth of rumen organisms (see Leng & Nolan, 1984). The results of studies by Maeng et al. (1989) may explain some of the contradictory results. The studies of these researchers indicated that rumen microbes growing on different carbohydrate substrates have requirements for different N-substrates; celluloytic organisms may not require amino acids to the same extent as organisms growing on starches or sugar as the major substrate. For microbes utilising sugars growing on starches there was an apparently high requirement for preformed amino acids/peptides but this was not so for cellulolytic organisms. Maeng et al. (1989) also showed an increase in efficiency of microbial growth on fibrous carbohydrates with decreasing dilution rate of rumen contents. If true this may be advantageous to ruminants given low quality forages that must be retained in the rumen for a considerable period to allow digestibility to be optimised. At the same time the improved ratio of cells: VFA yielded (i.e. P/E ratio) along with the increased availability of the total nutrients are both advantageous. Such a mechanism would advantage an animal with (1) a comparatively slow turnover rate of rumen contents (i.e. buffalo vs. cow or goat vs. sheep; see Devendra, 1989) or (2) fauna-free vs. faunated animal (see Bird & Leng, 1985) or animals at high environmental conditions versus cold stressed animals (see Young, 1983).

3.7.5 Amino Acid Requirements of Microbes Digesting Fibre

The organisms in the rumen that are largely responsible for the fermentation of cellulose (Ruminococcus albus, Ruminococcus flavefaciensand Fibrobacter succinogenes (previously called Bacteroides succinogenes) appear to have minimal requirements for amino acids and grow on ammonia (see Leng, 1991 for discussion). Conversely, organisms important in starch hydrolysis (Butyrivibrio fibrisolvens, Bacteroides ruminicola, Selenomonas ruminantium, Streptococcus bovis and Ruminobacter (Bacteroides) amylophilus (Hobson et al. 1988) readily incorporate amino acid N and in many cases peptides (see Leng, 1991). Supplementation of sheep fed a poor quality forage with branched chain VFA has been reported to increase the apparent flow of microbial-N to the duodenum. The apparent stimulation of microbial growth with branched chain VFA has also been shown to increase feed intake on occasions (Hemsley & Moir, 1963). This together with the suggested requirements for peptides/amino acids by rumen organisms (which on the basis of the results of Maeng et al. (1989) must now be questioned) has tempted many scientists to explain the increased feed intake of ruminants, on poor quality forages that are supplemented with bypass protein, to the slow release of amino acids, peptides and branched chain fatty acids to the rumen milieu from the protected protein (see Hunter, 1988; Silva & rskov, 1988a), even though in most studies there was no evidence of increased digestibility with such supplements in predominately fibre based diets. The above discussion indicates that the cellulolytic organisms in the rumen even of cattle on straw based diets, are rarely if ever deficient in amino acids, peptides or branched chain VFA in the rumen (see also Maeng et al. 1989). This is not to deny that these organisms may still need these nutrients in catalytic amounts but that they are rarely if ever at such low concentrations in rumen fluid to bring about a deficiency.

3.7.6 The Roles of Small Amounts of Fresh Forage in Straw Based Diets
Farmers in developing countries have generally recognised the benefits to cattle of adding a small amount of fresh green herbage to straw based diets. These practices, which have evolved through trial and error, may have a number of beneficial effects which include the supply of vitamin A, essential minerals, ammonia, peptides/amino acids in an otherwise unsupplemented diet. Recently it has been shown that where the supplemental forage in a straw based diet given to sheep is of high digestibility a boost to digestibility of the basal diet occurs even at relatively small levels of supplementation (Juul-Nielsen, 1981; Silva & rskov, 1988a; Ndlovu & Buchanan-Smith, 1985). The rate of digestibility of straw depends on the rate and extent of colonisation of fibre and the biomass of adherent organisms (Cheng et al. 1989) and the high digestibility forage supplement may act to seed microbes onto the less digestible straw. On the other hand, other influences cannot be ruled out. For example, in the studies of Silva & rskov (1988a) in the absence of an effect of supplemental forage on digestibility, the rumen ammonia levels were often not significantly below 200 mg NH3-N/1. Where increases in digestibility of the basal forage occurred to supplemental forage the ammonia levels in the rumen were significantly below 200 mg N/1 and the supplement apparently improved the concentration to above the critical level (see Leng 1991).

3.7.7 Elimination of Rumen Protozoa and Preservation of the Fauna-Free State

The physiological research which has shown that there is an increased availability of microbial protein for digestion in fauna-free as compared to faunated ruminant (see Jouany & Ushida, 1990) has supported the feeding trials with large numbers of animals which has demonstrated significantly high feed conversion efficiencies and wool growth rate in sheep without rumen protozoa as compared to control animals. Fauna free cattle on the same intake of a low protein, molasses/urea based diet grew at a 43% greater rate than faunated cattle on the same feed intake. The improved production was therefore an effect of a higher efficiency of feed utilisation (Bird & Leng, 1978). The discussion to follow, on the implications of environmental temperature/humidity for the nutrition of ruminants, will indicate why a major change in P/E ratio in the nutrients absorbed (i.e. the major difference between faunated and fauna-free ruminants) will be more effective in improving ruminant production in the tropics as compared to temperate/cold countries. In the tropical areas the available forages used to feed to ruminants are generally lower in digestibility, lower in true protein and animals are rarely cold, but heat stress at times may be severe. It should also be noted that animals brought into animals houses from fairly could environments may at times suffer severe heat stress through a combination of a well insulated fleece or coat and an imbalanced diet.

3.8 Factors Influencing Efficiency of Feed Utilisation

The efficiency with which absorbed nutrients are converted to ruminant products (liveweight, milk etc.) is dependent on precisely meeting the animal's requirements above maintenance for individual nutrients required for the particular function (see Preston & Leng, 1987). These, at times, are influenced by body condition as affected by previous health and nutritional history (see Leng, 1989b), the demands for body temperature control (Blaxter, 1962) and the requirements for substrate oxidation in exercise (or work). Graham et al. (1959) (see also Blaxter, 1962) showed that the quantitative oxidation of individual nutrients (largely fat) depended on the degree of heat/cold stress of the animal. A cetogenic substrate was largely used to keep an animal warm when it was required to increase its metabolic rate in cold stress. In this report it is assumed that a cold stressed animal will oxidise acetogenic substrate for heat production until surplus acetogenic substrate is totally utilised, after which fat mobilisation provides an extra and often the major source of metabolic fuel. The apparently preferential oxidation of circulating acetate leaves a higher ratio of amino acids (and glucose) in the nutrients available for production than would be available to an animal in its zone of thermoneutrality. Conversely, an animal that is not cold has more acetogenic substrates available for anabolic purposes. The environment can, thus, alter the partitioning of nutrients into productive functions and therefore affect the efficiencies of feed utilisation. The design of supplements, to balance diets for ruminants, needs to account for the varying demands for nutrients brought about by the thermal environment of the animal. It is recognised that cold stress in animals often increases voluntary feed intake and rumen turnover rate. And in this way on some diets it increases microbial cells moving to the lower tract and this has been suggested to increase the P/E ratio in the nutrients available for maintenance or production (see Kennedy et al. 1986). As an example of how environmental factors can change the nutrient balance available to ruminants for anabolism and maintenance, a model used previously to predict the relative availability of specific nutrients to a standard steer (see Leng, 1982) has b een modified to use with sheep and includes the effects of cold stress. The model is based on the sheep (closely shorn) used in the studies of Graham et al. (1959) which were fed on a daily basis 600, 1200, or 1800 g of a dried grass pellets and subjected to short periods at environmental temperatures ranging from 8 to 40C at a relative humidity of 50%. The data in Table 3.2 indicate that the need to maintain body temperature may require a considerable proportion of the available acetogenic nutrients to be oxidised. In the absence of a cold stress considerably more of digestible nutrients (and in particular more acetogenic substrate) is available for maintenance and synthesis. If in thermoneutral conditions the acetogenic substrate cannot be utilised for synthesis of tissue component because of a low availability of essential amino acids and/or glucose (i.e. imbalance in P/E or G/E) (see Preston & Leng, 1987) then acetate must be dissipated as heat. If the animal is able to oxidise the excess substrate but cannot dissipate the heat-generated because environmental temperature and humidity are high then it could allow its body temperature to increase to some extent but eventually it must reduce its feed intake. If the animal's body temperature rises, metabolic rate increases through the oxidation of protein (Blaxter, 1962) which may have implications for protein requirements of ruminants in the tropics and differential responses to supplementation in the tropics as compared to temperate areas. it means that in hot . The requirements of are high. Table 3.2: A theoretical assessment of the effects of environmental temperature on the balance of nutrients available for anabolism (The example used is from Graham et al. (1959), in which closely shorn sheep were subjected to temperatures from 8 to 40C)

Ration (g dried

grass/d) 600 Minimal heat production (MHP) Temperature at MHP (C) Metabolizable energy intake (MJ) at MHP Heat production required to combat 5C below critical temperature (MJ)* Nutrients available (MJ)** from: Acetic acid Butyric acid Propionic acid (G) Total Volatile Fatty Acids (E) Microbial protein available (g/d) P:E ratio (G/MJ)++ Available P:E ratio (g/MJ) G:E ratio (MJ/MJ)II Available G:E ratio 1.90 0.27 0.54 2.71 72 26:1 118:1 0.25 7.71 3.80 0.54 1.08 5.42 148 27:1 45:1 0.24 0.48 4.75 0.74 1.49 6.98 198 28:1 40:1 0.27 0.43 5.8 40 5.1 1200 1800 8.3 33 9.8 10.5 24 13.7

2.2

2.2

2.2

* The heat production for each degree lowering of environmental temperature below the critical temperature was assumed to increase by 0.44 MJ/24 h (Graham et al. 1959). ** The available nutrients are calculated assuming that all the digestible dry matter is digested in the rumen, that the rumen microbes have a YATP 14 and that microbial cell synthesis and VFA production are stoichiometrically related as described by Leng (1982). No allowance was made for a possible increase in dilution rate with increasing feed intake. ++ Calulated microbial protein available (g) for digestion relative to VFA (MJ). II Propionate (MJ): acetate plus butyrate (MJ) available; the glucogenic energy ratio The available P:E and G:E ratios are defined as the nutrient ratios after the acetogenic nutrients have been used for body temperature control at 5C below the critical temperature. They are calculated assuming that the energy for heat production arises from the oxidation of acetate and/or butyrate. Graham et al. (1959) showed fat was the major source of heat and that metabolism of glucogenic or aminogenic substrate is unaffected by cold stress whereas fat (acetogenic substrate) oxidation accounted for the heat produced.
The temperature/humidity at which ruminants are cold stressed depends greatly on the level of feed intake, the insulation provided by the hair or wool coat and the environmental conditions prevailing, e.g. wind, rain and availability of shelter. Thus, the environmental temperatures at which minimum extra heat production to combat the cold stress occurs will probably move through a range of from around 10C to 40C.

3.9 Climate, Supplementation and Intake of Low Quality Forages

There has been vigorous debate on whether supplementation of sheep and cattle on low quality forage based diets with urea and/or bypass protein increases intake of the basal feed resource (see Leng, 1989b). The differences in results may be hypothesised to be a result of an

interaction between climate and the balance of nutrients available from a diet. When research results (Australian) on the effects of supplementation to balance nutrition of cattle on low quality forages are grouped according to climatic zones a pattern emerges (Figure 3.4). It appears to be in the tropics and subtropics where poor quality forage intake by cattle is low without supplementation and where significant responses in feed intake occurs when a non-protein nitrogen deficiency is corrected and extra protein that escapes rumen fermentation is provided in the diet. It is strongly stressed that supplementation with urea and proteinmeals increases voluntary intake of poor quality forages by cattle under tropical conditions to approximately the same level of intake as unsupplemented cattle under temperate conditions (Leng, 1989b). In this situation the supplement is only correcting a depressed intake back to normal intake. Figure 3.4: Intake of low digestibility forages by cattle either unsupplemented or supplemented with bypass protein or bypass protein and urea (Lindsay & Loxton, 1981; Lindsay et al., 1982a,b; Lee et al., 1984; Hennessy, 1984; Perdok, 1987; Kellaway & Leibholz, 1981)

The conclusion that can be drawn from this is that supplements which improve the P/E ratio in nutrients absorbed by cattle fed low quality forage reduces metabolic heat production. Where metabolic heat production in unsupplemented cattle fed low quality forages would increase body temperature then the animal reduces its feed intake. This reduction in voluntary feed intake is ameliorated by the supplement which allows the acetogenic substrate which would otherwise have to be oxidised to be partitioned into synthetic reactions with a resultant decrease in heat production. The concept of small increases in P/E ratio being able to reduce metabolic heat and at times therefore allow an animal to consume more food might explain the effects of increasing levels of urea in a diet on forage intake (when digestibility is no longer increased) and also the occasional effects on feed intake of branched chain VFA supplements. The concept is that it is a supplement that improves microbial growth efficiency which has an effect on feed intake and this is only seen in the hot conditions when feed intake is depressed. The interaction of nutrition and climate may explain why there is a stubborn disbelief by some researchers from developed countries (largely in the temperate areas) of research carried out in developing countries in the tropics. Many of the results of supplementation indicate that a protein that escapes rumen fermentation stimulates both the level and efficiency of production of milk (or live-weight gain) in ruminants fed on crop residues (see Figure 3.5).

The discussion above indicates that ruminants in hot countries have an advantage of not having to oxidise much acetogenic substrate (or body fat) to keep warm. By balancing the diet with supplements, this acetogenic substrate may be captured in products or oxidised to provide ATP for assimilation of the additional nutrients into products. In cold/cool countries supplementation with protein is less necessary, as the utilisation of surplus acetate for heat, decreases the need to balance nutrients. As long as feed intake is high (i.e. the diet is highly digestible and perhaps cold stress stimulates intake) production remains relatively high as the nutrients for heat production are extracted and the balance used in synthetic reactions. Nevertheless, increases in the efficiency of utilisation are obtained when low protein diets are supplemented with a bypass protein even in temperate countries (see rskov, 1970; Silva et al. 1989; Leng et al. 1977) and at times feed intake is also stimulated but it is unknown whether the animals in such studies were actually subjected to hot conditions. Figure 3.5: Schematic relationship between diet quality (metabolisable energy MJ/kg dry matter) and food conversion efficiency (g liveweight gain/MJ ME) (- - -) (from Webster, 1989). The relationships found in practice with cattle fed on straw or ammoniated straw with increasing level of supplementation. Australia (, o, ) (Perdok et al., 1988), Thailand () (Wanapat et al., 1986) and Bangladesh () (Saadullah, 1984). Recent relationships developed for cattle fed silages supplemented with fish proteins (Olafsson & Gudmundsson, 1990) (o) and tropical pastures supplemented with cottonseed meal (Godoy & Chicco, 1990) (*) are also shown. This illustrates the marked differences that result when supplements high in protein are given to cattle on diets of low ME/kg DM

It can be concluded that ruminants in the tropics that are adequately supplemented with small quantities of essential nutrients may produce at the same rate on a lower digestibility feed as an animal on higher digestibility feed in a cold environment. To emphasise the differences in potential thermal stress of animals under different conditions the average temperature humidity index (THI) (which is an index of potential heat stress conditions for ruminants (see Johnson, 1987) on a monthly basis for Cambridge (England), Chittagong (Bangladesh), Bangkok (Thailand) and Armidale (Australia) are shown in Figure 3.6. The critical THI (72) for high milk producing cows as determined by Johnson (1987) is included in the figure. However, it must be emphasised that in addition to temperature/humidities, the critical THI will depend on the insulation provided by the animal's coat and its behaviour in seeking shelter, as well as the incidence of wind and rain in addition to level and quality of feed intake. Many studies have shown that at the same forage intake by ruminants with an already efficient digestion that a supplement of protein that reaches the small intestine increases the efficiency of feed or metabolisable energy utilisation for growth. This is positive proof that wasteful oxidation of nutrients can occurs (See Figure 3.5). It seems reasonable that, because Blaxter (1962) and his colleagues showed that acetogenic substrate are largely burned off that the inefficiency of ruminants on forage based diets is a result of acetate being oxidised wastefully. This points to a major difference in considering the nutrition of ruminants in the tropics as compared with temperate countries. do e nutrition guideline at the end, at the Nicourguious conditious.

3.10 Feeding Standards and Feed Evaluation

Most forages consumed by livestock in developing countries have a low digestibility which rarely exceeds 55% and is mostly in the range of 4045%. The calculated metabolisable energy in the dry matter (ME/DM), thus, ranges from 7.5 MJ down to 4.8 MJ. Feeding standards indicate that feeds with a metabolisable energy content, of 7.5 MJ will support growth rates of cattle of approximately 2 g/MJ of ME intake. On a forage at the lowest level of ME, cattle would be in negative energy balance (see ARC, 1980) (also for reference see Webster, 1989). Figure 3.6: Temperature humidity index (THI) of climates in temperate countries as indicated by Cambridge (U.K.) and Armidale (Australia) as compared to tropical countries as indicated by Bangkok (Thailand) and Mymensingh (Bangladesh). The THI is calculated on the mean of the maximum-minimum temperatures and humidities THI(C) = Temp. (dry bulb) + 0.36 Temp. (dew point) + 41.2C

Contrast this with results of supplementary feeding trials based on balancing the nutrition of animals with urea/minerals and bypass protein, where cattle growth rates equivalent to 18 g/MJ of ME intake have been achieved in cattle fed straw (see Figure 3.6). Obviously the presently accepted feeding standards (see ARC, 1980) have been very misleading and can not be used as a means of predicting animal performance. Of vital importance however, is that the application of the concept of balanced nutrition can improve animal growth by 23 fold and the efficiency of animal growth by as much as six fold over previous estimates (a range of 210 fold). In addition it also shows that although growth rates of cattle are below those on grain based diets cattle on forage based diets can highly efficiently convert feed to product.

3.10.1 Implications of low Productivity of Ruminants in the Tropics

Low productivity of ruminant livestock has been accepted in developing countries as an inevitable result of the poor feed base and a low feed conversion efficiency. The concept being that there is a large heat production (energy requirement) associated with the ingestion, movement of digesta along the tract in animals on forages as compared to concentrates (see rskov & Macleod, 1990). This conclusion is contrary to the conclusions of Leng (1990) and the concept of balanced nutrition presented here.

3.11 Some Basic Explanations for the Inefficiency of Ruminants on Forage Diets

3.11.1 Inefficiency of Acetate Utilisation

The original calorimetric studies of Graham and his colleagues (see Blaxter, 1962) indicated that infused acetate or butyrate were utilised by sheep with low efficiencies, i.e. there was a high heat increment when acetate was given compared with propionate. Considerable effort has since been expended on testing the hypothesis (or disproving it) that acetogenic substrate is used wastefully. Blaxter and his colleagues (Graham et al. 1959) used diets based on dried grass which was chopped and cubed. It had a metabolisable energy content (M/D) of about 8.5 MJ/kg dry matter. As most of the protein in the diet could have been highly soluble, the P/E ratio in absorbed nutrients would have been relatively low. The controversy concerning the efficiency with which acetogenic substrate is utilised may be rationalised at least to some extent by considering the balances of nutrients available to the ruminants in the various experiments and the ability or otherwise of the animals to synthesise fat, dissipate heat or to oxidise substrate to keep warm. For example the presence of small amounts of fish meal, that has a considerable amount of protein that escapes the rumen, in a concentrate diet (see rskov & Allen, 1966) provides an explanation for the differences between these authors' results and those summarised by Blaxter (1962) where sheep were fed dried grass which may have contained a highly soluble source of protein.

3.11.2 Requirements for Glucose by Ruminants

The need to manipulate or supplement diets for ruminants in order to ensure an adequate supply of glucose and of glucogenic compounds was discussed fully by Preston & Leng (1987), who made a comprehensive literature survey. In outline, the rationale that is used to justify the concept of glucose being a limiting nutrient is as follows: Little glucose is absorbed by ruminants but they synthesise considerable glucose from precursors such as propionate and certain amino acids, largely in the liver. Glucose is certainly required, by ruminants, as a major substrate for cell synthesis, as an important oxidative energy supply in the brain and red cells and as an important nutrient for the growing foetus and for milk lactose and fat synthesis in lactating animals. Glucose needs to be oxidised in the adipose tissue, and to a lesser extent the mammary gland, to supply the reduced cofactors for fat synthesis from acetate (in this case of NADPH, generated in the pentose phosphate pathway). It is possible therefore that fat synthesis in an animal may be limited by glucose availability for oxidation. If there is a block in acetic acid utilisation, it cannot be allowed to accumulate in the blood as this would lead to acidosis, the animal therefore, needs to oxidise the excess acetate in a futile cycle in which acetyl CoA is produced with a requirement for ATP and then hydrolysed. Alternatively an animal could increase its muscular activity by standing instead of sitting or walking long distances which would ensure increased acetate oxidation. The animal that is most at risk to a deficiency of glucose is the animal with a big demand for glucose (late pregnant or early lactating) fed forage based diets (high acetogenic fermentation in the rumen) with little bypass nutrients in the diet (receives only amino acids from microbial sources) and is tethered (no requirements to oxidise acetogenic substrate to walk) living in a tropical country (no requirement to oxidise acetate to keep warm). As discussed above, reduction in the need for acetate oxidation and a high glucose demand can establish a situation where a deficiency of glucose could lead to an imbalanced nutrition with a need to burn off excess acetate with a high thermogenic effect. Where such a thermogenic effect cannot be tolerated (high environmental temperatures, high humidity and perhaps heavy insulative coat) the animal needs to respond by reducing its feed intake. Conversely the thermogenic effect may be prevented by balancing the diet with nutrients that supply glucogenic substrates for absorption from the intestine (see Leng, 1991).

3.11.3 Balancing Nutrition for Reproduction/ Pregnancy and Lactation

Considerable research has indicated that the level of protein nutrition considerably influences reproduction of both male and female animals and subsequent pregnancy and lactation. Leng et al. (1987) indicated that on forage based diets each physiological function is affected adversely by a low P/E ratio in the nutrients absorbed by ruminants; this could be overcome, to some extent, by feeding protein meals which bypass the rumen. In summary, feeding a supplement which improves the P/E ratio in the nutrients absorbed by ruminants on a low true protein, forage diet has the following potential effects, particularly in a hot climate, on reproductive efficiency of female ruminants:

stimulates liveweight gain of dam or reduces liveweight loss (see Lindsay et al. 1982a) improves ovulation rates (Waghorn et al. 1990) improves placental size (Hinch, 1989) improves birth weight (Stephenson et al. 1981) and so increases survival (Lynch et al. 1990) and because of the increased birth weight possibly lowers the incidence of retained placenta increases milk yield and efficiencies of milk production (Saadullah, 1984).

Prevention of protein deficiency in early life also prevents stunting of final body size (see Preston & Leng, 1987). Differences in size of animals of the same breed, in the same country, is almost always a result of differences in nutrition and not inherent differences. This has recently been emphasised with N'Dama breed which has always been considered to be a small breed weighing up to 250 kg liveweight. With

good nutrition and adequate management the bulls have now been shown to grow to 500 kg liveweight (Murray, 1989). Work from Nigeria and Australia has also shown that young and old bulls are also very susceptible to low P/E ratios in the nutrients absorbed (Rekwot et al. 1988). Young bulls that grow on diets that would have given a high P/E ratio in the nutrients absorbed, compared with animals fed a diet giving a low P/E ratio, had better testicular development and produced larger ejaculates with double the sperm content (Rekwot et al. 1988). Older bulls that go through a period of protein undernutrition have decreased testicular size and probably are less fertile (Lindsay et al. 1982b).

3.12 Implication of Parasite/ Disease and Nutrition

Undoubtedly any parasitic or disease condition that drains protein from the animal will increase the animals requirements for protein relative to energy (Leng, 1982). Similarly, infective agents that utilise glucose may also increase the demand for this critical nutrient. For example, trypanosomes and epyrythrozoan parasites which invade red cells, increase protein requirements by increasing red cell turnover rate and also increase the animals requirements for glucose as this is the major substrate used in the parasite's metabolism. It is suggested that improving the protein nutrition of ruminants through providing bypass protein directly to the animal (i.e. avoiding rumen fermentation) may considerably ameliorate the detrimental effects of intestinal and blood parasites (Leng, 1982) and may assist in development of early immunity. (J. Steele personal communication)

3.13 Implications of an Increased Nutrient Requirements for Work

Light work requires acetogenic substrate probably acetate for muscle contraction but heavy work is probably dependent on long chain fatty acids mobilised from adipose tissue. All working muscles use some glucose and there is always a concomitant increase in glucose utilisation when free fatty acid metabolism is increased by the work load (see Pethick et al. 1983). Imbalanced diets fed to draught animals which result in a low P/E ratio in the nutrients absorbed are not so disadvantageous as they are for a lactating or growing animal. The excess acetogenic substrate is used for work removing the necessity to otherwise enzymatically dispose of the substrate. However, the increment in requirements for glucose oxidation in muscles although small could be a limitation. In addition the need in heavy work to supply long chain fatty acids which can be oxidised to produce ATP at a faster rate than from acetate, indicates that there is an increased need for glucose not only in muscle metabolism but also to aid fat synthesis from acetate in the period of rest. An imbalanced feed (low P/E ratio or low G/E ratio), which is associated with a high metabolic heat production, could also reduce the recovery of body temperature of draught animals when the animal is resting and, thus, reduce feed intake. A decreased fat deposition because of such an imbalanced diet during the non working season may also be a major constraint to draught capacity because of lack of body reserves for mobilisation in the working season. The P/E ratio required to support fat deposition (in periods of low work load) or to reduce heat stress by reducing metabolic heat is likely to be much lower than for example for growth and milk production. A draught animal fed poor quality forage probably requires little more nutrients than can be supplied from an efficiently functioning rumen. This is attained for example by supplying the animal with a molasses/urea multi-nutrient block. Indian farmers soon found the benefits of molasses/urea block for draught animals when these became available (personal observation) and recently the beneficial effects of feeding molasses/urea blocks to draught buffalo have been demonstrated in Vietnam (T.R. Preston personal communication). The results of Preston's studies in Vietnam are given in Table 3.3. Table 3.3: Liveweight change and work capacity of buffaloes given rice straw supplemented with a urea/molasses block (Preston, 1990) There were 22 animals in the trial

Observation

Control (no supplement)

Block

Live weight (kg) Dry Season (no work) Beginning After 1 month change Wet Season (ploughing) 354 346 -814 381 395 + 14

Beginning After 1 month change Work Capacity Area ploughed (m 2/day) Beginning After 1 month Ploughing speed (2 buffaloes) (m/min) Beginning After 1 month Recovery time (min)* Beginning After 1 month

346 334 -11.5

372 370 -2

1919 1508

2243 2141

36 32

44 41

14 16

12 13

* Time needed to recover normal heart rate.

3.14 Conclusions
3.14.1 Implications for Areas of Research
This discussion highlights the manipulable aspects of ruminant nutrition, where substantial improvements in productive efficiency of animals fed largely on fibrous feeds could result. The differences between temperate and tropical conditions also indicate opportunities which are often not apparent in temperate countries and mitigates against direct transfer of results from the former to the latter. In tropical conditions, protein nutrition of ruminants is more crucial than in temperate areas. The general conclusions are that:

the primary constraint to ruminant production from fibrous feeds (in the tropics) is the low efficiency of feed utilisation often coupled with an environmental/physiological effect which results in a reduction in potential feed intake. in practice the most effective mechanisms for improving productivity will be to improve the P/E ratio in the nutrients absorbed because of the large effect on efficiency of feed utilisation. draught animals will also benefit from technologies that increase microbial growth efficiency in the rumen and at times improve digestibility, intake and P/E ratio in the nutrient absorbed. contrary to many statements in authoritative reviews, only when P/E ratios have been optimised does the energy density of the feed become a primary constraint. as the primary constraint is an inefficiency due to a low P/E ratio in the nutrients absorbed it is unlikely that other inputs (e.g. agents to repartition nutrients or to stimulate growth (B-agonists, BSt)) could be successful without first applying strategic supplementation.

Chapter 4 Research Areas

4.1 Research Targets
The discussion in Chapter 3 highlights the areas and suggest priorities for application and continuing research for improving animal production from the available feed resources for livestock in developing countries. The targets are: Maximisation of P/E ratio in Nutrients Absorbed from the Rumen Some methods/techniques to do this are listed below: 1. 2. by combinations of minerals, non-protein nitrogen and protein supplementation to support high efficiencies of rumen fermentation improve microbial growth efficiency by manipulating the microbial populations

3. 4.

to alter the genetic capacity of the microbes to synthesise protein or to produce higher biological value proteins improve dietary protein bypass. Where protein is fed, to decrease protein and/or peptide and/or amino acid degradation in the rumen and allow a greater flow of amino acids, peptides and protein to the intestines from dietary origin development of sources of supplements of protein meals that have natural protection or are manipulated to escape rumen fermentation. This includes the need to:

to obtain more efficient microbes to remove protozoa to adjust the P/E ratio with feed additives

identify protein sources that are protected naturally or in processing identify soluble protein sources and find economic and viable mechanisms for protection of the proteins develop protein crops or by-products that can be grown and processed at the site of ruminant population densities in countries where protein meals are scarce develop rumen manipulation studies to enhance the bypass of soluble proteins.

Maximisation of Digestibility of Fibrous Feeds This is achieved by: 1. 2. 3. 4. 5. 6. providing supplements of essential nutrients required by microbes manipulating microbial populations in the rumen to increase digestibility promote populations of selected, highly fibrolytic fungi in the rumen defaunation treatment of crop residues prior to feeding to enhance their digestibility altering the physiology of the digestive tract (e.g. rumen mobility, turnover etc.) e.g. through the use of immunisation of somatotropin release inhibiting factor (SRIF).

Optimising the Partitioning of Nutrients into Product This includes: 1. 2. by breeding more efficient animals chemical interventionhormonal implants or injections

3. 4.

injections of compounds such as B-agonists, bovine growth hormone development of transgenic animals with particular characteristics e.g. high circulating BSt or enzymes complements previously not present in animals.

4.2 Priority Ratings

Assigning priorities within this list is a most important aspect of this report. Clearly the research requires a differing level of sophistication of technology and different priorities will need to be assigned to different resource bases. Simply stated, the priority ratings for research emphasis are in order of priority: 1. 2. 3. to ensure an adequate P/E ratio in nutrients absorbed to improve the extent of digestion of low digestibility feeds that require microbial fermentation for digestion to improve the partitioning of available nutrients for product synthesis (meat, milk, wool etc.) or drought power generation.

It is clear that an inadequate nutrition will not allow the expression of any manipulations in priority (3). It is also obvious that a super bug cannot be successful in the rumen that is deficient in critical nutrients (for example, ammonia).

Chapter 5 Present Knowledge and Priority Research Areas

5.1 Adjusting P/E Ratio in the Nutrients Absorbed by Ruminants with Protein Supplements
5.1.1 General
The effects of improving the P/E ratio in nutrients available to ruminants on low quality forages have been discussed above. Research into supplementary feeding is well developed in a number of countries along the lines of optimising fermentative digestion (using molasses urea blocks) and optimising the efficiency of use of nutrients from the rumen (by using a bypass protein supplement). It is estimated that about 50 countries are now testing molasses urea blocks as a means of supplementing ruminants on forage based diets with critically deficient nutrients. Some results of studies from Indonesia are given for the effects of introducing molasses urea blocks into ruminant production systems using cut/carry grass (Table 5.1). Many countries that do not have a source of molasses within the country are looking for other means of giving the same mixture of nutrients to their indigenous livestock. It is again stressed that both supplementation of the rumen with deficient nutrients (e.g. phosphorus, sulphur and non-protein nitrogen) and the animal (with a bypass protein) will improve the protein to energy ratios in the nutrients that are available to the animal. This in turn will optimise production from the available resources. Even where the rumen is not optimised a bypass protein can still adjust the P/E ratio significantly. In some respects, where a bypass protein is relatively inexpensive, and/or molasses urea blocks difficult to use or get animals to take, then, a sole supplement of bypass protein may be sufficient to improve efficiency of feed utilisation significantly. In addition the soluble protein that is always present in the practical bypass protein supplements, together with the recycling of urea-N back to the rumen often diminishes the effectiveness of urea/molasses blocks and many applied practitioners have taken advantage of this particularly when dairy cows are fed high levels of bypass protein supplements, the molasses/urea block can become only marginally beneficial. The theoretical calculations of P/E ratios are shown in Table 5.2. Obviously the best option is the use of both supplements.

5.1.2 Bypass Protein Supplements

Providing bypass protein to cattle under small farmer management is often difficult and at times it is too expensive. There is often little information on the locally available protein sources, particularly the level of protection of the protein in the rumen. As a rule-of-thumb, solvent extracted oilseed cakes, fish meal that has been flame dried (but not sun-dried fish meal or fish silage) and protein sources that have been heat treated, have some considerable protection from rumen degradation. The degree of protection is enhanced by pelleting the protein meal in the presence of free xylose, glucose or fructose, (as occurs in molasses) when a mild browning reaction occurs (unpublished observations). The major requirements before wide spread use of these technologies can be promulgated in a country are: Table 5.1: Summarised data on growth and milk production responses to UMB technology in both large ruminants (UMB 400500 g/h/d) and small ruminants (UMB 100150 g/h/d) in West Central Java in 198889. The data are from Hendratno, N and numerous co-operators and were summarised in Entwhistle (1989)

Treatment Species Location Trait measured Control UMB Dairy cattle W.Java (Friesan) C.Java Average Beef cattle (Ongole) W.Java " " " " C.Java 1 Weight gain (kg/d) 2 3 4 5 6 Average Sheep (Garut) Goats (Ettawa) C.Java Milk yield (l/d) 0.795 1.091 +37% W.Java Weight gain (kg/d) Milk yield (l/d) 7.44 5.70 6.57 0.182 0.333 0.277 0.478 0.200 0.446 0.319 0.130 9.92 8.00 8.96 0.400 0.526 0.439 0.465* 0.278 0.689 0.466 0.320 +46% +146% +36% Response to UMB

* Trial 4 was the only beef cattle study in which no growth responses to UMB were noted.
Table 5.2: The effects on P/E ratio in the nutrients absorbed of supplementation with a bypass protein to cattle with a poor or optimised (i.e. supplemented) microbial milieu in the rumen. The values are calculated for a steer digesting 4 kg DM in the rumen (see Leng, 1982)

Rumen environment

Dietary protein bypass (g/d) 0 0

Microbial cells Microbial produced protein (g/d) (g/d)

Total protein (g/d)(P)

VFA produced (MJ/d)(E)

P/E *(g/MJ)

Poor Optimised

830 1680

500 1010

500 1070

41 30

12 33

Poor** Optimised

400 400

830 1680

500 1010

1330 2690

41 30

22 47

* Microbial protein plus dietary protein to VFA energy. * Although the rumen environment is deemed not to change through the addition of protein meal, in fact it will have been improved but may not be optimised to the extent it would by feeding a molasses/urea block. P/E ratio here is underestimated.

determination of the extent of protection of proteins from rumen degradation in the available protein resources identification of the potential protein sources together with development of technologies to inactivate anti-quality compounds and to protect protein (see feed technology later) in some countries there are no recognised protein crops at the centres of ruminant population densities. In these areas there is a need to find new sources of protein. This could include growing trees and forages for leaf, seed or pod production followed by development of appropriate technologies to harvest and protect these proteins when these are not naturally protected the development in ruminants of response relationships to increasing levels of bypass protein supplementation on the available feed resources to allow economic assessment development of the marketing and extension technologies to get the products to the farmers.

5.2 Adjusting P/E Ratio by Manipulation of the Rumen

Microbial growth efficiency can very from as low as 10 g of dry cells/MJ of VFA produced in the rumen to as high as 30 g of dry cells/MJ of VFA (see Table 5.3). The reasons for these variations include differences in population mix of microbes and the deficiency of nutrients specific to micro-organisms that grow in the rumen. Under most management conditions where ruminants are on good quality forage based diets the P/E ratio theoretically is of the order of 17 g cells/MJ VFA; on diets of low quality forage the ratio may be between 4 and 10 g cells/MJ VFA; therefore there is considerable scope for manipulation of this ratio.

5.2.1 Supplementation of the Rumen Microbial Ecosystem

In ruminants correcting a chronic rumen deficiency of nutrients for microbial growth generally increases digestibility, feed intake and the quantities of microbial cells produced relative to VFA. The technologies discussed below all have a prerequisite that the rumen ecosystem has been brought up to a high level of efficiency by supplementation before attempts are made to further manipulate P/E ratio.

5.2.2 Chemical Manipulation of Rumen Fermentative Efficiency

There are a number of chemicals available which, when added to a diet, improve the efficiency of feed utilisation by 520% (see Chalupa, 1980). Among these, the most prominent ones are monensin (or rumensin), lasalocid and avoparcin. However, the improvement that is brought about by balancing the amino acid supply in a forage based diet may increase the efficiency of feed conversion to liveweight of a milch heifer by many times this value (see Leng, 1990). Even though the costs are high and the returns small, monensin is utilised extensively throughout the world in the feedlot industry based on grain. These chemicals have been considered as propionate enhancers (in the rumen) but in all probability they improves P/E ratios by decreasing peptide and amino acid degradation (see Russell et al. 1989) in the rumen and in addition possibly by inhibiting protozoal growth which improve the P/E ratio in the nutrients absorbed. Because these chemicals tend to have side effects in that they reduce intake at least initially, which often removes any overall benefits when added to a diet, they have not been accepted for forage fed ruminants. Methods that decrease peptide and amino acid degradation in the rumen potentially would be a very effective means of stimulating P/E ratios on moderate protein diets but would be highly significant where soluble protein sources are available and would circumvent the necessity to chemically protect proteins. In summary, there is potential for screening chemicals for activity against the micro-organisms that degrade peptides and amino acids as they appear to be different to the ones that degrade proteins to peptides and amino acids (Russell et al. 1989). Manipulation of the Microbial Mix Within the Rumen The rumen microbial populations are composed of protozoa, bacteria and anaerobic fungi as well as viruses and phages. The population density of each group can be highly variable depending on diet but, in general, the end products of fermentative digestion are qualitatively the same. Although there has been major attempts to manipulate protozoal populations (see Leng, 1984) little success resulted until good laboratory systems for defaunation were invented so that large numbers of ruminant animals could be studied (see Bird et al. 1990).

There is little doubt that defaunation results in increased microbial cell outflow to the intestines (defaunation improves the P/E ratio in the nutrients absorbed) (see for discussion papers in Nolan et al. 1989). Jouany & Ushida (1990) have also clearly shown that on diets containing considerable true protein, a greater amount of dietary protein reaches the intestines in addition to the extra microbial protein in animals that are unfaunated compared with those having normal populations of protozoa. Provided that the amount of bypass protein in the diet is below requirements of the faunated animal, an improvement in efficiency of feed utilisation results when protozoa are removed from the rumen. Defaunation has increased ruminant production levels by up to 50% at times (see Bird & Leng, 1978). On high quality concentrate feeds high in bypass protein, the improvement in efficiency that results from defaunation may not be expressed but the level of feed protein can potentially be reduced by 2050% with the same efficiency and production rates. The potential for application of defaunation is highlighted by the improved efficiency of utilisation of the basal feed resource in defaunated or unfaunated animals. Defaunation and maintenance of the unfaunated state has resulted in 3060% increases in growth rate of sheep, cattle and buffalo (Bird et al. 1990) and a preliminary study indicates a promise of up to 2 litres/day more milk in Friesians (Moate, 1989). The problems are:

to identify suitable defaunating or protozoa suppressing treatments which are simple and economic to find mechanisms for administration of anti-protozoal compounds to ruminants under prevailing management systems.

Defaunating Agents Rumen protozoa are susceptible to a wide range of chemicals, particularly those that disrupt cell membranes (i.e. they are surface active). From a very small survey of natural products (less than 100 forages were surveyed), two sources of anti-protozoal agents which can be used in small doses to suppress or eliminate protozoa in the rumen were identified (Assafa, G., Klieve, A. & Leng R.A. unpublished). The rich tropical floras of many of the developing countries may be sources of many other anti-protozoal compounds or forages. A priority area for research is to establish a simple anti-protozoal assay in a number of laboratories in differing climatic zones and to quickly identify potential sources of antiprotozoal drugs. Initially, it would be simple to look at the effects of increasing quantities of a soluble extract of a forage on the viability of protozoa incubated with the materials for, say, up to 2 hours. This would obviously have to be followed by animal experimentation and, finally, growth studies to determine whether the other effects of these anti-protozoal compounds could be detrimental to the animal. Mechanisms of Defaunation The only practical means for manipulating the protozoal population of the rumen is likely to be through strategic supplements containing a defaunating agent, either given daily or on a periodic basis. As discussed earlier defaunation has a marked stimulating effect on P/E ratios and efficiency of feed conversion. Research will be needed to examine the administration of any potential defaunating agent through the basal feed, in a protein supplement or through a multi-nutrient block to the animal. The latter will be potentially the most practical routes, but it limits the dose rate of the drug to quite small quantities as intake of blocks tends to be a small fraction of total intake. A highly active antiprotozoal agent might be delivered by an intraruminal device capable of sustained release of a drug (see Ellis & Costigan, 1989) but again costs are likely to mitigate against this approach.

5.2.3 Other Manipulations of Rumen Ecosystems

By chance, recent research has demonstrated that small amounts of a clay mineral (bentonitea montmorillonite clay), added to a diet of sheep, improved wool growth by up to 20% in faunated sheep but only by 5% in unfaunated sheep (Fenn & Leng, 1989; Fenn & Leng, 1990). The mechanism of action of the bentonite is unknown but it either improves the P/E ratio or specifically the outflow of sulphur amino acids from the rumen as these are the main controlling nutrients for wool growth. The problems are:

to understand the mechanisms of action of bentonite as this will be crucial in identifying the sources of bentonite to use and the systems in which it will improve animal productivity finding a mechanism for administration of the bentonite through supplements.

5.2.4 Alteration of Protein and Amino Acid Composition of Rumen MicrobesRecombinant DNA Technology
Rumen microbial protein appears to have a relatively constant amino acid composition, independent of the mix of microbes in the whole population (Czerkawski, 1986). The primary limiting amino acids for wool growth in sheep are methionine and cysteine and for growth are possibly the same amino acids plus lysine and threonine. Undoubtedly, if rumen microbes could be engineered to produce protoplasmic proteins high in the deficient amino acids then improvements in wool production would result. Methionine is relatively slowly broken down in the rumen and peptides and proteins high in sulphur amino acids appear to be also relatively slowly degraded by the resident microbes (Nugent & Mangan, 1981). Therefore, establishment of rumen organisms in the rumen that produce these amino acids could increase the outflow of the sulphur amino acids and hence the efficiency of wool growth. The problems will be to isolate, identify DNA en-coding for the necessary enzymes; develop vectors and transform rumen microbes. Following this, the transformed rumen microbes must be able to grow extensively in the rumen and be retained, particularly during periods of nutritional stress for the rumen microbes.

5.3 Adjusting P/E Ratios from the Rumen by Manipulating the Feed Base
5.3.1 Feed Technology and Development of Supplements
Supplementary feeding to ensure an efficient digestion in the rumen of cattle on poor quality forages, undoubtedly increases the efficiency of productivity from local feed resources. This has been well demonstrated by research of the National Dairy Development Board (NDDB) of India. The NDDB has developed molasses/urea multi-nutrient blocks that are available at cost to village farmers. Similar developments are now occurring in some 50 developing countries in the tropics. However, few countries have realized the potential to develop and use bypass protein concentrates. A specific case history of the application of these feed technologies in India is discussed below.

5.3.2 Supplementation with Naturally Protected Protein: A Case Study in Strategic Supplements
In two milk sheds (Kedah and Baroda), through their feed mills, a high (30%) protein concentrate that contains a high proportion of bypass protein is manufactured and sold (roughly 100 tonnes/day in Kedah alone). The protein meals selected are all from the oil seed industry and have received a degree of protection from the processing techniques which include solvent extraction and application of heat. In addition, the final protein concentrate is pelleted with about 8% molasses which appears to give additional protection (presumedly due to heat in the press and some browning reaction of sugar with the lysine units that provide from the protein surface). In a commercial operation and using a mill with a capacity for 100 tonnes/day, mixtures of proteins from different sources are necessary and quality control essential. It is well recognised that protein sources vary in the extent to which they escape the rumen and pelleting seems to ensure that protection is improved. Some comparisons of the use of bypass proteins in milk production systems are shown in Table 5.3.

5.3.3 Providing Bypass Protein in Areas Without Protein Resources

In many countries of the developing world, proteins for livestock are scarce. Sometimes highly soluble and easily degradable proteins are available (e.g. lupins and whole cotton seed) but these are only as effective in altering the P/E ratio as supplementation with urea. There is a great need for the development of inexpensive technologies for protection of protein meals. Proteins can be protected from degradation in the rumen by insolubilisation processes which include:

heat (depending on the protein source); temperatures from 120 to 180C will denature the protein and the subsequent insolubility affords considerable protection from microbial attack when it is in the rumen. chemical reactions which include:

reactions with aldehydes, such as formaldehyde and gluteraldehyde or mixing with tannins or selection of plants with tannins.

Table 5.3: Some practical results from commercial milk producing systems where feed resources are based on low quality forages fed to Friesians (13) and cross bred cows (4)

Basal Feed

Supplements

Milk Production

1. Tropical grass/maize silage or Free choice molasses/urea other crops plus 1-2 kg rice blocks + protein pellet (30% straw/day CP) (350 g/kg milk) 2. Rice straw/millet straw (8 kg/d) 3. ad lib. mixture of cottonseed hulls (46%; molasses (17%); cottonseed meal (18%); sesame seed meal (15%); crude lecithin (4%) and 10 kg freshly harvested kikuyu grass (2 kg DM/d)

5000-6500 kg/305 d

Bypass protein pellet 300 g/kg 25 l/day (at 3 months) milk

6200 kg/300 days (2nd calf cows); 5700 kg/300 days (1st calf heifers)

4. Cane tops (50% more than daily intake) + cut/carry grass as Cottonseed meal 250 g/kg milk 2800 kg/lactation/yr (4) available

References: (1) NDDB Anand (2) Personal observationsin village system one animal only (3) C.E. Payon, V. (personal communication) (4) Boodoo et al., (1988)

by low heat application with protein meal and a monosaccharide when a mild browning reaction occurs (see Lewis et al. 1988) protection of proteins by coating with a resistant polymer or a calcium soap of long chain fatty acids.

Most Likely Technologies It is most likely in the future that to process proteins to ensure that they bypass the rumen, feed technology will make use of the browning reaction or the use of calcium soaps of long chain fatty acids (particularly for dairy cows where the long chain fatty acids can be used in milk synthesis and fat is low in the diet) to coat the protein meals. The use of aldehydes to protect proteins has undoubtedly been successful but is often not economic, nor readily applied in most developing countries. It has been phased out in some countries because of the potential carcinogenic effect of a volatile compound produced when formaldehyde and acids react. In recent years the development of mild treatment of protein meals with xylose (a 5 carbon sugar pentose) at low temperatures has become a possibility. Results from Prof.T. Klopfenstein's laboratory (Lewis et al. 1988) at the University of Nebraska, have clearly shown that small quantities of xylose heated with soyabean meal substantially protects the protein from rumen degradation and improves efficiency of feed utilisation of cattle fed forage/concentrate based diets. This technology has been singled out because it is simple, the process has enormous scope for development by imaginative scientists and the sources of xylose are readily available in most developing countries. Processing technologies for generating the xylose, however, must be developed prior to application. Xylose is a major constituent of hemicellulose and a useful source includes sugar cane bagasse and cottonseed hulls. It can be released in a mixture of sugars by simple acid hydrolysis or by application of steam and pressure. It is also present in significant quantities in the black liquor (e.g. sulphite liquor) from the acid bleaching in paper manufacture. Where bleaching depends on alkali then the sugars are probably destroyed and absent from the black liquor. Unfortunately the latter process is the one mostly used in developing countries.

Methodology for protection of proteins must be a priority research area in countries where the proteins in the available meals are highly soluble; for example, where unprocessed oil seed cakes, seeds such as lupins or pressure extracted oil seed cakes, forages or tree forages are available. The application of heat also removes anti-quality factors and, therefore, broadens the potential protein feed base (e.g. canavalia seed may become more usable). Extrusion systems appear to be a potential manufacturing systems for the future. Use of Protein Banks to Provide a Source of Bypass Protein The concept of the protein bank which is used strategically for supplementing grazing cattle when the pastures are dry and have low protein content, is an important development in some countries where extensive rangelands are an important source of animal products. These protein banks provide a ready source of nitrogen and minerals for the rumen organisms and the animal. The strategy involves the development of an area of land, within a range land, with a protein crop which is a legume and to feed or graze this strategically with ruminants in the dry season. Small inputs of legumes into a diet of low quality pasture are undoubtedly beneficial, providing minerals and nitrogen for the rumen organisms and also stimulating digestibility of the lower quality feed that is the bulk of the diet. However, it is unlikely that such supplements alter the P/E ratio other than through its direct effect on the rumen environment and therefore is only equal in value to a molasses/urea block. A much more effective use of such protein banks would be made by harvesting and protecting the protein and possibly developing alternative sources of NPN, such as molasses/urea blocks to feed along with this protein. A legume with tannins may on the other hand provide substantial bypass proteins and a molasses/urea block would then be extremely complementary. Protein banks can be forage legumes, tree leaves, seeds or seed pods of crops, forages or trees (e.g. Enterolobium, Prosopis and Leucaena) or any other source of leaf protein. There is a need to develop appropriate technologies to capitalising on such protein sources in those countries where proteins are expensive or unavailable. It may be necessary to commence with plant and agronomic studies to find suitable plants and to develop the correct method of harvest, followed by techniques to protect the protein. Potential of Trees as a Source of Bypass Protein The potential of tree leaves, tree seeds and pods or seeds has been vastly underestimated. For example mature trees of Prosopis juliflora have been shown to produce 400600 kg pods/tree/year in the rainfall areas of 400 mm annually (range 200600 mm) (F. Riveros pers. comm.) with 1620% crude protein. Many rangelands in the semi-arid areas produce less than 2,500 kg/annum of pasture biomass of which only 200400 kg is harvested by cattle (see Ellis & Swift, 1988). A few trees per acre therefore will have little effects on the available pasture biomass but a huge effect on available biomass and will also supply a soluble N source and provide the option of producing bypass protein meal. Trees cultivated for protein sources have a huge potential in the range lands. The case for growing Prosopis spp. is obvious and has been put (see Riveros, 1988). The step missing is to marry the approach with the recent advances in protein nutrition of ruminants and to develop:

harvesting techniques for pods/seeds. technologies for drying grinding and protecting the protein (if necessary) techniques to concentrate the protein and protect it methods to market the feeds research to demonstrate the response of cattle to using such feeds.

There are a large number of trees that have potentially similar attributes, however, in the semi arid zone the use of Prosopis to provide protein is a major opportunity. Fractionation of the pod to produce a high protein component would be a useful first step prior to developing a protection technology. It should be noted that in many areas Prosopis is regarded as a potentially dangerous weed. In these areas it must not be grazed when pods are falling because of the spread of seeds through the faeces of cattle which may result in its establishment and spread to extensive areas. However, this is rather easily managed in plantation culture. Possibilities of Using Plant Biotechnology Plant biotechnology may be very important to the use of trees as forage components, as there is considerable variability in seed production among trees, cloning and plant tissue culture of seedlings from high yielding varieties could be a considerable advance. However, similar attempts with coconuts and palm oil have resulted in poor or no inflorescence in plants produced by tissue culture and therefore some caution is needed.

Prosopis spp. are only given as an example here and there are many other under-developed potential sources of protein for the purpose discussed. The potential of developing legumes with tannins at appropriate levels is a worthwhile objective as Barry and his colleagues have demonstrated that Lotus sp with 36% tannins apparently have greater bypass protein in the leaf than similar species without tannins (Barry & Blaney 1987). This has potential application within the legume bank concept. Again the problem may be met in gene expression since too much or too little tannins are both disadvantageous. The level of tannin is often directly related to the soil type and rainfall (G.Blair, personal communication).

5.4 Maximising Digestibility of Fibrous Feeds

5.4.1 Supplementation
A deficiency of any nutrient in the rumen must first affect the rumen ecosystem by reducing microbial growth efficiency which, as the deficiency becomes more acute, will eventually reduce microbial pool size and inevitably digestibility and intake of roughage by ruminants, Decreased digestibility will only be seen with time as the deficiency of the nutrient becomes progressively greater. Supplementation of a critically deficient animal with nutrients for the rumen will increase microbial growth efficiency, microbial pool size and digestibility of forage which will almost always increase feed intake. The feed intake effect, however, often depends on the degree to which the limiting nutrient is deficient. The most likely deficient nutrients, are non-protein nitrogen, P, S, Mg, Na and also trace elements such as Co, Cu, and Zn. Supplementation with multi nutrients of the type listed above is generally successful in increasing intake and digestibility but the large increases in productivity can be anticipated from combining such supplements with a source of bypass protein. Sources of Mineral Nutrients and Molasses Urea Blocks One of the best sources of multiple trace minerals in developing countries is a concentrated plant juice, such as molasses or palm oil sludge. Multi-nutrient blocks are no better than the mixtures of materials from which they are made. Blocks are useful because they form a package readily handled, marketed and used by the farmer with little impact on the time he has for other activities. The major problem with these multi-nutrient blocks occurs in the grasslands where there is a need for appropriate means of encouraging stock to consume them in sufficient amounts. In these areas molasses is often unavailable or is too expensive. More research is needed to produce suitable packages (e.g. blocks without molasses) for such localities. The importance of block supplementation relates more to the access it allows to livestock managed by small farmers. It may be possible in the future to apply innovations through inclusions in the blocks of chemicals and other materials that will manipulate the rumen (e.g. antiprotozoal chemicals) and manipulate the animal (e.g. protected chemicals that are absorbed from the intestines) or even control disease and parasitism (anthelminthics). In this respect the production and testing of blocks incorporating anthelminthic is appropriate and being undertaken already by the Commomwealth Scientific and Industrial Organization in co-operation with the Australian Centre for International Agricultural Research (ACIAR).

5.4.2 Improving the Enzymatic Ability of Rumen Microbes to Degrade Fibre

This is an area that has received emphasis and has aroused considerable expectations. The concept is simple, if rumen microbes can be developed with greater abilities to degrade fibre, or its components, the microbes will be able to extract a greater amount of the available nutrients from the feed consumed. This often receives a high priority because of the concept that animals fed low quality pastures are primarily energy deficient. In fact, these animals more often are inefficiently using the available nutrients. It must be stressed that the application of balanced nutrition is the first priority and if other perturbations of the system are to succeed an animal must have an efficient rumen and also metabolism. However, in an animal fed a roughage diet, with an efficient rumen and balanced for protein, it is possible that for every 5 units increase in digestibility of the basal roughage resource, there will be 50% increases in liveweight gain on the same basal feed resource (see Figure 5.1 Perdok & Leng, 1990). Silva & rskov (1988b) demonstrated that the extent of breakdown of a fibrous feed was dependent on the rumen medium. Untreated ground straw placed in nylon bags, in the rumen of sheep fed ammoniated straw, was fermented to a greater extent than the same untreated ground straw placed in the rumen of sheep fed untreated straw. This difference might be due to a higher concentration of enzymes in the rumen of sheep fed ammoniated compared to untreated straw. Figure 5.1: Growth response to different levels of protein meal supplementation of cattle given ammoniated (NH3-treated straw) or untreated rice straw (untreated straw)

The two observations discussed above when combined indicate the great potential for improvements in productivity from a successful establishment of a super bug in the rumen, i.e. a micro-organism engineered to produce more, or a greater variety, of enzymes for fibre digestion.

5.4.3 Potential Targets for Improving Fibre Digestion

There are potentially three ways to enhance the ability of rumen microbes to degrade fibrous feeds:

selection of microbes, particularly with a high fibrolytic enzyme secretion or that secrete fibrolytic enzymes of high specific activity creation of microbes with greater spectrum of enzyme secretions by recombinant DNA means, e.g. create cellulolytic capacities in xylolytic organisms and vice versa create microbes with enhanced fibrolytic activity by recombinant DNA means.

In all cases, the prerequisite will be that the organisms are able to grow in and maintain their space in the rumen. These same prerequisites also apply to the production by genetic engineering techniques of high biological value proteins, other secretions of proteins, amino acids and peptides by rumen organisms (see earlier discussion).

5.4.4 State of the Art in Bioengineering of Rumen Organisms

There are number of well established ways of going about incorporating DNA encoding for specific enzymes into anaerobic bacteria by recombinant DNA technologies. The approach is universally similar, and an outline is given in Appendix Athis suffices to point to the uncertainty of such research and its complications. Progress has been relatively swift, with at least 4 groups having now achieved transformation of rumen microbes with foreign DNA. Virtually all transformations have been achieved with plasmid constructs and through electroporation methods. None of the engineered organisms have been reintroduced into the rumen to test their stability and in only one case was the engineered organism a recent isolate from the rumen. The approach of using laboratory strains of rumen organisms which have definitely changed in culture clearly demonstrates that the research is at the basic level in which the transformation was the major object and not the improved digestibility that might result. A major constraint has been the lack of suitable monitoring systems for identifying the engineered organism within the milieu of the rumen. DNA probes are not proving very appropriate because of the genetic diversity of bacterial strains within a species (Hudman & Gregg, 1989). The above discussion is intended to emphasise that their is a great need for considerable basic research before modified organisms that could survive in the rumen is developed. It should also emphasise the long time lag that is likely from the beginning, where virtually no information was available to application.

5.4.5 Selection of Anaerobic Fungi for Better Fibre Degradation in the Rumen
Filamentous structures of the rumen anaerobic fungi penetrate and substantially weaken the xylem cell walls of forages in the rumen, whereas the bacteria do not adhere to, nor degrade these highly resistant plant tissues to any significant extent. Rumen fungi, but not bacteria, penetrate the cuticle barrier that protects the leaf's surface. Research indicates that these fungi have unique enzymes, or enzymes with higher specific activities, that give them the ability to weaken and degrade the most limiting structural barriers to degradation. On the basis of these observations it appears that research aimed at promoting the biomass of fungi in the rumen or the establishment of highly active rumen fungi (e.g. by selection) could be a major step forward in increasing the digestibility of poor quality forages by ruminants. The identification of strains of anaerobic fungi that most rapidly degrade the cell walls of forage plants is a necessary prerequisite to the successful inoculation of fungi into the rumen and which will nutritionally benefit the host. Culture, selection of strains and the identification of new species of anaerobic fungi, testing of their ability to weaken plant fibre and improve the rate of digestion are worthwhile research areas for development. The developing countries may be well placed to initiate and develop this research as they have access to animals under extreme dietary conditions that have been adapted over long periods of time and therefore could have already selected fungi for high fibrolytic activity. The research must include developments which allow selected strains to multiply in the rumen and to prevent the competition from wild strains. As rumen fungi are spread by resistant spores (Ho et al. 1990) which are passed in faeces and then, when taken in by the animal in feed, multiply by sporulation in the rumen, the support of specific species to ensure the survival is a research area with a high priority. The selection criteria will include the measurement of the rate of solubilisation of fibrous carbohydrates by selected fungi and the weakening of the forage stems following a period of incubation with the forages.

5.4.6 Selection of Bacteria for Fibrolytic Activity

Selection of bacterial strains for improved cellulolytic activity appears to be less vital as it is the fungi that are relatively more important in the breakdown of the most resistant components of the forages, particularly the low quality forages.

5.4.7 Solubilisation of Lignin

Lignin is found within the cell walls of forage plants where it is closely associated with hemicellulose, forming a matrix surrounding the orderly cellulose microfibrils. Lignin is also found in high concentrations in the middle lamella between fibres where it functions as a binder for contiguous cells. Polymeric lignins are not degraded by any known anaerobic organisms. The aerobic white rot fungi Basidiomycetes degrade lignin more rapidly and extensively than any other studied microbial group. It is generally accepted that the delignification of a ligno-cellulose material will increase the rate of attack upon the cellulose/hemicellulose compounds when this enters the rumen. Under aerobic conditions there are two mechanisms of biological delignification:

mineralisation, with lignin being converted directly to carbon dioxide solubilisation when lignin is released (solubilised) from ligno-cellulose with a variable amount of hemicellulose material attached (see Paterson, 1989).

Solubilisation of lignins of grasses is particularly important for its degradation in soils. Something similar to solubilisation seems to occur in the rumen, as considerable lignin appears to be precipitated in the abomasum and therefore, is soluble in the rumen fluid (this is often referred to as acid-precipitatable polymeric lignin (APPL)). The enzymes for solubilisation of lignin, if present in rumen organisms, may be capable of being boosted by genetic means or by selection. In the event that they are not present in rumen organisms, the potential for inducing lignin-solubilising enzymes becomes a major possibility for improving digestibility of fibre in the anaerobic ecosystem of the rumen.

5.4.8 Detoxification of Anti-microbial, Toxic and Antiquality Factors in Plants

Plants in order to survive insect, fungal and bacterial attack, have developed secondary compounds which detract from these organisms colonising the leaf tissues. Some shrubs and trees respond to leaf damage as occurs by grazing and produce greater quantities of secondary compounds which often make them inedible. Recently it has been shown that the grazing of an Acacia tree caused it to release ethylene which within minutes, caused the adjacent acacias in the group to commence synthesising tannins which then caused the grazing deer to cease consumption of the leaves of these trees. Many secondary plant compounds are toxic to the rumen microbes or directly or indirectly toxic to the animal following microbial metabolism in the rumen. This restricts the feed base of ruminants and, at times, prevents stocking of land. Goats and probably other grazing ruminants are able to detoxify many of these secondary plant compounds in the liver.

5.5 Developing Detoxification Mechanisms in Rumen Organisms

Research into the possibility of detoxification of secondary plant compounds was given impetus by the discovery that organisms in the rumen of goats in Hawaii that was able to degrade the breakdown product of mimosine (i.e. dihydroxypyridine) in Leucaena leucocephala, but ruminants in Australia were unable to do so as they had lost the specific rumen microbe with the necessary enzymes (Jones, 1981). The microbe isolated from the rumen of the Hawaiian goats degraded dihydroxy-pyridine, the toxic breakdown product of mimosine (see Hegarty, 1982). Introduction of the microbe from goats in Hawaii into ruminants in Australia prevented the symptoms of mimosine toxicity in previously susceptible ruminant livestock. This has raised the possibility of genetically engineering into rumen organisms the ability to degrade such toxic compounds. Research at the University of New England is in progress to attempt to introduce genes into bacteria for defluorination of fluoroacetate, a lethal compound present in Acacia gidyea, which often kills cattle in northern Australia when consumed in the dry season (see Barry & Blaney, 1987). The successful transformation of rumen bacteria with a single gene for defluorination of fluoroacetate would indicate the possibility of the utilization of a range of other plants, which may be weeds (e.g. lantana), or potential high biomass producers (some legumes and legume trees) and also high protein crops (e.g. canavalia). The problems are universally the same. The first is identification of bacterial DNA encoding for the suitable enzyme followed by development of methods to introduce this into an appropriate bacteria, stabilisation of the DNA in the cell and the last problem is whether the modified organisms will survive in the rumen and be capable of expressing the gene. The major research area would be in finding, isolating and packaging the DNA for the appropriate enzymes which must be capable of expression of being switched on easily and function in an organisms in the rumen ecosystem. There is a danger in developing the ability of ruminants to degrade toxic compounds, in that often these are the last defense of plants being eaten and in the arid areas could lead to deforestation and soil erosion. Figure 5.2: Sites of action of chemicals that, when added to straw, improve its digestibility

5.6 Treatment to Increase Digestibility

5.6.1 Improving the Digestibility of Crop Residues by Chemical Treatments
There are a number of acid and alkaline hydrolytic processes that will solubilise lignin, cause disruption of fibre by swelling or improve the potential digestibility of fibrous roughages in other ways. Figure 5.2 shows the sites of hydrolysis of a number of chemicals in a fibre which improve digestibility. It seems that there is adequate information available on the technology of treatment and on the benefits of treatment of low quality forages (see Sundst;l & Owen, 1984). There is therefore little stimulus in pursing research of the methods. A new and novel approach will at times, however, require research but at present it can be safely assumed that alkaline hydrolysis of wet straw by ammonia generated by urea (usually 4% of the dry matter) by microbes in the straw, is probably the most feasible treatment for application in developing countries. The use of anhydrous ammonia application is also potentially applicable (see Sundst;l & Owen, 1984). The ammonia in the straw has two effects:

it improves the fermentability of the straw in the rumen by breaking some of the ligno-hemicellulose bonds and it provides non-protein-nitrogen for growth of microbes in the rumen when the forage is consumed.

Although urea ensiling of straw can be economic and effective the technology has not been widely taken up by small farmers in developing countries in general, although there are a few reports of individual farmers regularly using the technology. It is necessary for researchers to understand the reasons for non-acceptance of such technology which is almost always associated with a slow return on funds expended and sociologic constraints (see Sansoucy, 1989).

5.6.2 Microbial Treatment of Straw and Other Crop Residues to Improve Digestibility
In recent years considerable emphasis has been placed on the improvement of fibrous crops by the strategy of growing non-toxic fungi on the straw. In particular, the white rot fungi have been used because of their ability to delignify the plant material. The major problem that has arisen is that the technology, although relatively simple, is more complex than that of urea treatment of roughages and although protein is produced and digestibility of the residue increased, a considerable proportion of the total biomass is lost, particularly when white rot fungi is allowed to proceed to the mushroom formation stage. To some extent the loss of biomass can be prevented by a reduction in the incubation time of the straw and forages, such that biomass losses are reduced to less than 10% (see Rai et al. 1989).

Dr. B.N. Gupta and his colleagues at the National Dairy Research Institute (NDRI) in Karnal, India have developed a two stage process using an altophilic fungus (Coprinus fimetorius). The technology is two-stage and relies on (1) alkali treatment (urea ensiling) and (2) a second incubation period where a fungi is cultivated on the straw. The technology is relatively simple, although the two stages make it more complex than the urea treatment alone for application by the small farmers. Both chemical and microbiological treatments may, however, be very effective where large amounts of feed must be stored and moved to areas where forages are scarce because of drought. However, again caution is needed in that to obtain maximum benefits from such feed, cattle will require supplements of bypass protein (see Leng 1986).

5.6.3 Potential for Increasing the Digestibility of Poor Quality Roughage by Manipulating the Digestive Physiology of the Animal
Goats compared with sheep, and buffalo compared to cattle, tend to retain fibrous materials in the rumen for longer periods and extract a few more units of digestibility at the same time. Within these comparisons, goats and buffaloes recycle a higher amount of urea-N back to the rumen. In the same way, Bos indicus cattle recycle more urea-N back to the rumen that Bos taurus cattle (see Leng, 1990 for review). Mechanisms that slow rumen turnover, increase rumen volume and allow greater recycling of urea-N may therefore improve the utilisation of poor quality forages. Immunisation against somatotropin release inhibiting factor (SRIF) has recently been demonstrated to increase flow rate through the gut, leading to increased digestibility and greater availability of protein to sheep on concentrate/forage diets (Sun et al. 1990). The effects of immunisation against SRIF on cattle on forage/bypass protein diets would be extremely interesting. However, the technology will not be easily applied to village farmers because of the numbers of injections required at the present time to effect immunity.

5.7 Altering the Partitioning of Nutrients Within the Animal

A whole range of injectable compounds will lead to more efficient utilisation of nutrients by livestock. In particular, repartitioning of nutrients into lean meat production or into milk production. These include anabolic steroids, growth hormone, and other hormonal growth factors, beta agonists and techniques such as the suppression of hormones by immunisation. These compounds in general, act through manipulation of hormone status and they lead to improved lean meat in the carcass and a greater efficiency of feed conversion into product. For a recent review of this area, the reader is referred to the publication by Buttery & Dawson (1988).

5.7.1 Anabolic Steroids

Natural sex steroids are used to promote growth and reduce the fat/protein ratio in meat in many countries of the world but their use has been recently banned in Europe. The injected hormone alters the influences of endogenously secreted hormones (oestradiol injections into cattle usually lift growth hormone levels in blood) and growth hormone is more tightly bound to sites in the liver. As is usually the case there is a marked interaction with nutrition and on imbalanced diets in general the effect of exogenous hormones is less obvious than for instance when a diet of silage is balanced by a bypass protein (fish meal) (see Gill et al. 1987). Again this stresses the differences and therefore the need for a new approach to biotechnology in developing countries where ruminant diets are usually low in true protein.

5.7.2 Growth Hormone

Recent developments in fermentation technology together with recombinant DNA technologies have made it possible to produce large quantities of peptide proteins which appear to be the same as bovine or porcine growth hormone (BSt. or PSt.). These growth hormone injected into animals stimulates growth and dramatically decreases fat content of the animals carcass (see Buttery & Dawson, 1988 for review). They also stimulate milk production by some 2025% in dairy animals. In the initial studies with growth hormone injections milk production increases appeared to result from repartitioning nutrients and mobilisation of nutrients from tissue deposits (see McCutcheon & Bauman, 1985) in recent times a long term feed-intake effect has been reported (Chilliard, 1988). In addition the stimulation of milk production was dependent on the level of management and was not apparent in herds with poor management (Bauman, D. 1990, pers. comm.). In Italy and India in well managed buffaloes, milk yield has been increased by exogenous growth hormone by up to 25% (see for example Ferrara et al. 1989) but the cost of the injections may limit this application.

5.7.3 -Agonists
-agonists given by mouth, increase lean meat deposition and reduce fat content of the carcass in pigs and ruminants (Hanrahan, 1987) but with only small increases in liveweight gain. Again it is more than likely that the responses are dependent on the nutrition of the animal.

5.7.4 Injected Growth Promotants versus Supplementation to Balance Nutrition

There are strong similarities between the response observed to growth hormone/ -agonists etc. and the response to correcting the P/E ratio in nutrients absorbed. At least one paper reports an interaction of bypass protein and response to exogenous hormones (see Gill et al. 1987). It is thus possible that the optimisation of P/E ratio in the nutrients absorbed elicits responses in hormonal release that mimic the injection of growth hormone etc. Conversely and if true, this means that effects of exogenous hormones may not be so obvious where the P/E ratio in nutrients absorbed is optimised. In addition the requirements for protein relative to energy in the transgenic animals expressing growth hormone will be much increased as will requirements for minerals and vitamins.

5.7.5 Conclusions
The important conclusion from the paper by Buttery & Dawson (1988) is that: the current technology has given many exciting possibilities but there are, however, still major gaps in the knowledge of the major metabolic control processes associated with growth which will have to be filled before a universally accepted method of growth promotio n is developed This is a reservation which is repeatedly used by biotechnologists and indicates that there will be an ongoing requirement for basic research at the level of metabolic control.

5.8 Cross Breeding to Improve Partitioning of Nutrients into Milk

Many scientists believe that selection among individual breeds for milk production is too slow to produce the highly significant increases in milk production needed to meet increasing population growth and demand in developing countries. Cross breeding of native animals with temperate-country high yielding dairy cows is universally accepted as the way forward. The high yielding dairy cow is considered to have the ability to partition nutrients more efficiently into milk then the indigenous animals. The concept is accurate when management is optimum, nutrition balanced and the feed of high digestibility. To obtain effective milk production from cattle in the tropics it is highly essential to control disease and supplement the poor quality forage to balance nutrition otherwise, a low reproduction rate, coupled with a high death rate, often eliminates the advantages of cross-breeding. Recent experiences in India, however, suggests that this is not necessarily the scenario of crossbreeding. Case Study (India). The milk yield of imported Friesians at The NDDB station in Anand, in India, is an outstanding example of the potential improvements in milk that can result when a package of technologies are applied. These cows are kept under institutional conditions, disease is under control and they are fed fresh forage (up to 40 kg daily), supplemented with maize silage and rice straw in the dry season and given 350 g of a bypass protein concentrate (30% CP) for each litre of milk produced. Average yields of first lactation heifers were above 5,000 litres/305 days with exceptional animals achieving 6,500. The animals are all in their second lactation now, with indications of an average milk yield in excess of 6,000 litres. This level of production has been achieved by using molasses urea blocks and a bypass protein feed supplements. The only grain concentrate, representing 10% of the bypass protein mixture, is used for manufacturing purposes to provide a good pellet composition. This example is given here because it indicates that even in the most difficult climates in Asia, where temperatures often exceed 40C, and reach 50C at times, Friesians can produce to at least of their genetic capacity. It al so indicates a very important concept that must be emphasised in this report; genetic improvement is not a panacea for livestock improvement in developing countries and improvement from breeding programs will only be successful when nutritional and disease management is optimised (see Table 5.3).

5.9 Transgenesis and Embryo Manipulation

Although embryo transfer has little importance in nutritional manipulation, it is a required technology as a forerunner to the production of transgenic animals. The development of techniques to isolate, manipulate and transfer defined pieces of DNA into early embryos removes the potential barrier for the movement of genetic information between totally different living organisms. For example, DNA encoding for enzymes normally produced by bacteria may be inserted into the genome of an animal and it may be expected to express the generation of the enzymes in the animal after birth. The technology consists of the digestion and legation of appropriate sources of DNA with specific enzymes to accurately define and rearrange particular DNA fragments, the molecular cloning of these fragments into bacteria and phages or as plasmids, the nucleotide sequencing of cloned DNA and the technology to transfer the cloned DNA into cultured cells and early embryos. Transgenesis research is largely aimed at developing new genomes (the total complement of genes in the animal) beyond the scope of the random assortment of genes during sexual reproduction, augmented by mutation, which is restricted by the available genetic variation and species boundaries. Furthermore, individuals selected for breeding often transmit some of its undesirable genes to its offspring.

Transgenesis, which brings together the technologies of recombinant DNA, embryo manipulation and embryo transfer offers extra opportunities in the elaboration of new genomes. The boundaries of potentially useful developments of new genomes in animals are only limited by the imagination of the researcher and the definition, isolation and packaging of the DNA with suitable methods for insertion into the early embryo. An example of the highly imaginative approach is the introduction of genes for the synthesis of S-amino acids into sheep, but again although transgenic animals have been produced they have not increased wool growth and expression of the genes is the most important problem facing such research. Most emphasis, to date, has been on developing the technology and introduction of DNA for the expression of growth hormone. This has been successful in a wide variety of animals. In a recent review of these developments, however, Dr. C. Polge (1990, pers. comm.) concluded that the major problem for research into transgenesis is the inability to control the level of expression of a particular gene together with a very low success rate in achieving transgenesis and a low success in expression of the gene. The general conclusions are that a return to the study of the factors that allow or control expression of genes is needed before any potential development can be made. This must be considered in any allocation of funds for this particular development. Archibald (1989) recently concluded an article on transgenesis by the following statement; Transgenesis offers new opportunities to make new genomes. In the short term, transgenic animals are more likely to increase the understanding of the genetic control of performance than they are to make a contribution to agricultural production. This clearly indicates that at the present time research aimed at developing new genomes in bacteria, other organisms or animals should be clearly oriented to advancing fundamental knowledge. This is not to deny that these may have some practical application in the future but the future could be 50100 years hence.

Chapter 6 Biotechnology and Monogastric Nutrition

6.1 Introduction
Most developing countries are experiencing difficulties providing sufficient food (staples) for their resident population. In general in most third world countries food production is increasing at a slower rate than the population growth rate. Developing countries on average are increasing food imports by 10% per year and this is likely to continue into the foreseeable future. Whilst ruminants need not compete with humans for food there are few economic, large scale systems for pig and poultry production established in developing countries which do not rely on grain based concentrate or on crops such as potatoes and other root crops which also take up land that could potentially be used for human food production. The recent developments of sugar cane as a source of feed (juice) for pigs, however, has a major advantage which resides in the high biomass production per hectare and a great potential to form the basis of an integrated farming system (see Preston, 1990). There has been far too little emphasis on developing systems of production of pigs and poultry on agro-industrial by-products. The exception being the molasses and swill-feeding systems for pigs that have developed in Cuba (Figueroa, 1989). By-products high in sugar such as molasses, palm oil sludge and fruit and vegetable pulps lend themselves to feeding to monogastrics. The constraints to production are a low protein content and in some cases an associated high level of fibre which is an intestinally indigestible carbohydrate. Pigs are able to utilise some fibrous feed, possibly up to 30% of their digestible feed intake because of caecal fermentation. It appears desirable that in the future, pig production should utilise at least a proportion of fibrous carbohydrate polymers. Free-range pig production, where breeding stock obtain a fair proportion of their feed from pastures is a good beginning forced on producers in Europe by the high cost of grain feeding in intensive fattening systems. If the microbial ecosystem in the caecum of the pig is well developed, basal diets high in sugar and low in fibre combine well with vegetable proteins such as cottonseed meal which are fibrous.

6.2 Potential Areas for Biotechnology in Pig Nutrition

6.2.1 Overcoming Feed Intake Problems on High Fibre Protein Supplements
The use of oil-seed protein residues for pigs and poultry are generally limited by the fibrous components and in some cases the presence of secondary plant compounds which may be toxic or simply lower production (e.g. gossypol in cottonseed meal). To overcome the constraint created by too much fibre it may be possible to develop pre-treatment to protect the protein and then find mechanisms for decreasing the fibre (e.g. growing fungi, such as the white rot species on the materials to decrease fibre and increase protein) or find methods to hydrolyse the cellulose to simple sugars before feeding. Chemical treatment of the feed to hydrolyze fibre to monocarbohydrates may also be potentially possible (e.g. with pressurised steam and sulphur dioxide). Delignification with say Coprinus fungi and treatment by pressure/steam of fibrous crop residues to produce simple sugars for inclusion in pig rations is a further possibility requiring research. At least one group in the U.K. is attempting to introduce cellulase genes from Clostridial sources into embryos of pigs with the concept of producing pigs with a capacity to digest more cellulose. The enzyme, if expressed will be controlled by a promoter sequence encoding only in the pancreas (this is given in a little more detail below as an example of (a) the possibilities, (b) the problems and (c) the requirements for basic research prior to any developments which might be applied (Hazelwood & Gilbert, 1989)).

6.2.2 Possibility of Modifying the Digestive Function Through Development of Transgenic Animals
By virtue of their hindgut fermentation, pigs have about the same capacity as humans for digesting plant structural polysaccharides (Engelhardtet al. 1985). In theory, substantially more energy would be available to pigs (and other simple animals) if cellulose and hemicellulose could be digested in the small intestine and the sugars absorbed. In addition the complex carbohydrates of grains (e.g. -glucans of barley), fibrous protein sources (e.g. cotton seed meal) could be more efficiently used. In the latter case it would allow a much higher proportion of protein from such sources to be incorporated into the diets replacing more expensive less fibrous protein meals such as fish or meat meal.

The ability to digest cellulose and hemicellulose by intestinal enzymes is not present in any animal. It is probable that in early evolution these abilities were not advantageous. However, if encoding a gene for microbial cellulase or hemicellulase could be incorporated into an animal's genome and expressed in the pancreas it may allow these enzymes to be produced and secreted along with the other pancreatic secretions. This is being tested by Hazelwood & Gilbert (1989) by introducing into the early embryo of mice (and they suggest they will do this with pigs) the cel E gene ofClostridium thermocellum which encodes a thermostable endogluconase with xylan-hydrolysing activity. Expressions of this gene in transgenic mice will be regulated by the elastase I promoter/enhancer region (from rats) which is located upstream of the elastase gene and is responsible for restricting the synthesis of the digestive enzyme elastase to target sites of expression of other genes, to the exocrine enhancer cells in the pancreas of transgenic mice. To modify porcine digestive enzyme secretion to include the enzymes, cellulase and hemicellulase, will require tissue specific expression of the cel E gene and secretion of mature active endogluconase enzyme into the intestinal lumen. For this reason gene constructs incorporating the elastase promoter/enhancer and cel E coding sequence have been designed. The concept of transgenic pigs is at the ideas stage, the research will undoubtedly require a return to basic investigations, undoubtedly over and under expression of the genes when transferred will be a major problem. Fibre breakdown in the pig by cellulase is likely to be slow and therefore gut capacity is likely to be a major constraint even when transgenic pigs are produced.

6.2.3 Porcine Growth Hormone (PSt.)

Undoubtedly porcine growth hormone injected in controlled amounts into pigs on grain based concentrates increase their growth rate, efficiency of growth and reduce fat deposition. The place of PSt in pig production, as it pertains to developing countries, is unknown. The small production units, the likely high cost of injections and the scavenger system generally operated at the small farmer level in developing countries suggests that PSt is unlikely to be used. The exception may be in grain-based feeding systems for pigs aimed at the markets provided by the higher income groups and tourists.

6.2.4 Transgenic PigsPorcine Growth Hormone

The technology for production of transgenic pigs has certainly been developed. Problems of over and under expression of the genes are still apparent. Overexpression results in considerable problems of leg weakness and reproductive inefficiencies whereas under expression gives no additional benefits. The nutrition of pigs expressing for growth hormone again has been under-studied with the usually emphasis being placed on the thrust to produce transgenic animals. The need to develop special feeding and management systems for pigs seems to have been ignored. Where these techniques have been applied in developed laboratories the absence of disease is mandatory. It is conceivable that in addition to a special type of nutrition there may be special requirements for disease and parasite control. Disease and parasitism a re more likely to impact heavily on pigs and other animals with a greater potential for growth. The problems to be faced in pigs transgenic for porcine growth hormone are well summed up in a paper by Wilmut et al. (1988): The pig exhibiting human growth hormone did not grow any faster than normal pigs but they had less back fat which might be of potential value to pig farmers. Unfortunately, these transgenic pigs also suffered from a number of abnormalities, including arthritis, lack of coordination of their rear legs, susceptibility to stress, anoestrus in gilts and lack of libido in boars. So it is back to the bench to attempt to redesign the gene to avoid these problems.

Chapter 7 Biotechnology and Environment

7.1 Introduction
Undoubtedly the environmental crisis arising from global warming or the commonly called greenhouse effect m ust develop as a major issue in the near future and it will inevitably influence aid programs. Global warming, attributable to the accumulation of gases in the atmosphere will have enormous repercussions on agriculture. Inevitable changes in sea levels, climate and temperature are forecasted with repercussion for both plant and animal agriculture. The patterns of change are being modelled and predicted but our present level of knowledge suggests that these models will invariably be wide of the mark. Global warming is likely to reduce world food production perhaps largely through the disruption that will occur through changing climate. The conclusions are universally that the world will suffer and therefore attempts must be made to ameliorate the rapidly increasing levels of gases in the atmosphere (see IPCC report 1990). The relative contribution of various gases to global warming is shown in Figure 7.1, the changing CO 2 or CH4 levels in the atmosphere in Figures 7.2 and 7.3. Figure 7.1: Relative contribution (%) of greenhouse gases to atmospheric warming (Source: World Resources Institute)

Figure 7.2: Trends in emissions of CO2 (Source: World Resources Institute)

Figure 7.3: Trends in atmospheric methane accumulation (Khalil & Rasmussen, 1986)

Clearly CO2 production from fossil fuel sources is the major contributor and a reduction in fossil fuel combustion is required, coupled with increased capacity for long term carbon dioxide uptake (e.g. reduced tree felling and increased tree planting). Global methane production must also be curbed. The source of atmosphere methane are shown in Figure 7.4. Ruminants contribute a significant amount (18% of the total) of the world production of methane and are targeted as a source which is one of the few easily manipulated (see IPCC report 1990). As discussed in this report the levels of ruminant production on roughage based diets are in general well below that possible from the available resources and often between 10 and 30% of the genetic potential of the animal species. The reasons for low productivity is complex but it has been argued that the poor feed base which provides an imbalanced nutrition is by far the greatest limitation (see Leng 1990). Figure 7.4: Relative contribution of biological resources to the global production of CH4 in the atmosphere (Bolle et al., 1986)

Figure 7.5: (A) The effects of improving the efficiency of rumen fermentative activity on methane production/kg of digestible energy consumed (B) The production of methane/kg gain in supplemented cattle (feed conversion efficiency (FCR) 9:1) or unsupplemented cattle (FCR=40:1) fed straw based diets (after Saadullah, 1984)

Methane is produced in the pre-gastric fermentative digestion of ruminants at a rate which varies between 8 and 17% of the digestible energy consumed (see Leng, 1982, 1990). A ruminant that grows slowly matures over say 5 years, as against the potential of finishing it at between 12 18 months, may produce up to 4 times the amount of methane per unit of product (see Figure 7.5). The numbers of ruminants in the world and their location is shown in Table 7.1. Potentially any technology which improves the efficiency of conversion of feed into livestock products lowers the number of animals required to produce meat, milk, wool and other products. The reduction in numbers that can potentially be achieved can have a significant effect on methane emissions.

Lowered numbers of animals will also feed back on available land requirements for livestock production allowing greater emphasis on alternative use of pasture lands including tree crop production. Table 7.1: Estimates of methane emissions from animals (adapted from Crutzen et al., 1986)

Animal type and regions Cattle; developed countries Cattle; developing countries * Buffaloes Sheep; developed countries Sheep; developing countries and Australia Goats Camels Pigs; developed countries Pigs; developing countries Horses Mules, Asses Humans Wild ruminants and large herbivores TOTAL

World Population (x 106) 573 653 142 400 738 476 17 329 445 64 54 4670 100500

CH4 Prod. (kg/hd/year) 55 35 50 8 5 5 58 1.5 1.0 18 10 0.05 150

Total CH4 (Tg)** 31.8 22.8 6.2 3.2 3.7 2.4 1.0 0.5 0.4 1.2 0.5 0.3 26

* Includes Brazil and Argentina. * 1 Tg = 1 teragram = 1012 grams = 109 kilograms = 106 metric tons. Total estimate for emissions from domestic animals (cattle, buffalo, sheep, goats, camels, pigs, horses, mules and asses) has an uncertainty factor of 15%
Biotechnology, feeding, management and breeding can be combined to improve animal production and could potentially increase animal production to the extent that in the developing countries (which contain approximately half the livestock in the world) it may be possible to reduce methane generation by up to 60% from the same product (see Leng, 1990). Similar reductions in methane emissions from ruminants in developed countries can also be achieved where the production systems are also often inefficient.

7.2 Integrated Farming

One of the major reasons for the development of livestock production in cropping systems in developing countries is to utilise crop residues, thus, eliminating waste and optimising the use of the total biomass produced within the small farm system for the benefit of the small farmer. The small farmer has often a requirement for draught power with animal production as a secondary consideration. Integrated crop and livestock production systems are highly efficient; potentially crop residues are used as livestock feed; the waste products (e.g. faeces and urine) are fed into biogass digestors and the effluent used to fertilise ponds for aquatic plant/algae production, with fish farming as the terminal activity. These systems are very worthwhile pursuing as a means of providing nutrients/fuel for the family, minimising fossil fuel combustion and methane generation and, thus, reducing environmental pollution (Preston, 1990). The array of integrated systems that could be developed is large. They all have as a central core a basic flow of nutrients through a number of systems. At each of these steps research can be brought to bear to optimise the partitioning of the available biomass into food, fuel and residues (see Figure 7.6). The environmental attributes of such systems are that methane emissions into the atmosphere and fossil fuel use are minimised. In addition the efficient and almost total harnessing of the energy from high producing crops reduces the land areas required per unit of product (see Preston, 1990). The problems are the necessary high level management that must be exerted by the small farmer, which can often be beyond his presently developed skills. Figure 7.6: Flow diagram showing the potential recycling of feed and faeces biomass from crop residues

Figure 7.7: An example of an integrated farming system based on sugar cane and forage trees fractionated to provide feed for pigs and poultry (the juice and tree leaves), sheep (the cane tops and tree leaves), fuel for the family (bagasse and firewood) and litter for sheep and earthworms (bagasse), with recycling of excreta through biodigesters to provide fuel (biogas) and fertiliser (the effluent) for water plants in ponds and for the crops (Preston, 1990)

A complete discussion of these systems is beyond the scope of this document but two examples are: 1. 2. the use of aquatic plants/algaes grown on biodigested effluent for protein production for feeding to pigs and ruminants particularly in the humid tropics and the farming of suitable fish in biogass digesta fluids.

A system incorporating these aspects is currently being investigated and developed by T.R. Preston in Colombia (see Preston, 1990) (and Figure 7.7).

Chapter 8 Some Practical Problems for Biotechnology Research in Developing Countries

8.1 Present Research Priorities and Change
Biotechnology research from its broadest definition (any biological manipulation) to its narrowest definition (recombinant DNA technology) requires the same mechanisms to optimise progress in livestock production. They involve:

the training of scientists in the known principles and practice of animal science development of research and research technology to expand the knowledge base documentation of information.

Major nutritional constraints to livestock production in developing countries may be categorised according to feed availability and quality. In the cropping areas, animals are sustained largely on crop residues and by-products in which deficiencies of nutrients is the major problem. In the rangelands, the feeds available are deficient in nutrients and there is a seasonal shortage of pasture in the dry season when the available feed resources are even more deficient in nutrients than they were during the grazing season. These problems may be overcome to some extent by supplementary feeding strategies which emphasise the balance of nutrition of animals and include methods to increase feed digestibility (see Preston & Leng, 1987). Although a wide range of biotechnologies can be brought to bear on these two strategies, within the nutritional emphasis of this report it is feed technology that is likely to produce the greatest advances for the small farmer in the developing countries. Recombinant DNA research in the developing countries is at a preliminary stage of development and is fraught with difficulties and lack of knowledge of, in particular, control of genetic expression. It is largely exploratory, even in the developed countries. In the advanced research institutes, and at the university level, it is most useful in expanding knowledge. On the contrary the large input by business interests, at the potentially practical, and therefore commercially applicable-level is highly secretive and often advanced. The markets of multi-national companies are likely to be in the more wealthy developed world and are unlikely to have an impact upon the developing countries with its different interests and objectives and the largely unpredictable success rate of an innovation to increase animal production. Biotechnology research in all developing countries lacks the infrastructure of, and skills in, the integrated sciences. There is a shortage of trained personnel and those that are trained are tending to develop along the lines that they began as trainees, PhD or Masters students. What is more, a significant number of these people are skimmed off by institutions in the developed world if they show apti tudes as there is a shortage of trained personnel in these countries. The developing countries must consider the balance of research within a country. The need to partition the available funds and resources into areas that are likely to increase productivity in a reasonably short term and more importantly are likely to be accepted by small farmers. The politics of research funding in every country where the majority of scientists are concerned only with the development of their own science, will ensure the dedication of government funds to recombinant DNA technology and in general, this will have priority over, what appears to be the more mundane animal nutrition research. This seems inevitable even though feeding trials using basal poor quality feeds given to ruminants have indicated that balancing nutrition has the potential to increase animal productivity by 510 fold depending on location. On the other hand recombinant DNA technology has a high potential to fail; it is costly and breakthroughs will be at least as difficult to apply as the present research on supplementary feeding. Developed countries have improved milk production per cow by 60% from 19761986, and this has been achieved by altering the feed base and simultaneously breeding of cows to take advantage of the improved feed base. At the same time, Asia and Africa have improved their yield per cow by 13% and 7% respectively (Brumby, 1989). This indicates the enormous unused potential and the priorities for application. These are the manipulation of feed base with simultaneous genetic improvement. Research and application in India has already demonstrated that a change of feed base with the introduction of cows with a high potential for milk production results in the large increases in productivity needed to lift production towards the demand for high quality protein by humans. The research application by NDDB and by others in a number of countries has indicated that high, realisable yields from Friesians or other breeds fed on local feeds (see Table 7.7) This re-emphasises the less sophisticated biotechnology research concepts that are available to improve animal production from the available resources and which can be applied now. The need is to fit the technology to local conditions.

8.2 Real World Problems of Capitalising on Modern Biotechnology in Developing Countries

Many authors have suggested that the developing countries, in order to participate in the wealth that might be generated by modern biotechnology, (in this case largely dealing with recombinant DNA technology) must develop their research capacity in this area.

A country obtains the benefits of such developments from the new technology packages which when applied, result in improved animal productivity. However, the developing countries will at the present time have to purchase many developed packages which have not considered the particular constraint to production in their country. This may result in no benefit (or even a negative effect) as the purchase price of the technology will often be higher than the value of the increased productivity (e.g. the lift in milk yield of 25% by daily injections of BSt into buffaloes does not pay for the cost of the injections in India). The other way the country will benefit is by its scientists being the inventor, preserving the world patent rights or by preserving at least the production and distribution rights from any invention for the country. The major problems here will be the same as for small-return businesses in the industrial world attempting to develop with limited finance. The potential of large companies being able to move in, take over, buy out or simply compete for promising inventions are obvious with a recent example in the takeover of Genotec. Unless the developing country concerned are prepared to act in the same way as a multi national company, then any potentially highly-profitable invention is likely to be lost to the industrialised giants. This can happen even before the research scientists have identified the importance of their research. The developing countries in general have only embryonic staff, trained in the new biotechnology largely by the scientists from the industrialised countries. The research groups in developing countries are centered around a few high quality scientists who recognise the need to develop new areas (rather than to compete with the scientists with whom they were trained); they lack funding and they will have major problems if they make a major discovery with the scaling up of a development and in defending their resultant patents. It is imperative that along with the development of modern biotechnology there must be development of expertise in patent application/patent protection, deciding on commercial partnerships, scaling up operations and market research. Given the business ethos it would be highly improbable that a breakthrough in biotechnology that would be applicable on a world basis would be fully capitalised by the inventor or his/her country. The greatest problems come after the inventive stage when commercialisation is needed. For example, to produce commercial amounts of a new chemical, scaling up of the laboratory research costs about 10 times the expenditure for the experimental research, the costs of commercialisation can be even 100 times higher than this figure. The lessons of recent years from the take over of new biotechnology discoveries in developed countries suggest that funding of biotechnology research should:

recognise the need for good managers and lawyers have good market research. In this respect specific in-country problem solving may be better than competing in a world market. recognise the need for international co-operation specifically in start up inventions recognise early the need for considerable increases in funding with development and scaling up from the laboratory level.

Modern biotechnology research that is funded by Aid-Agencies is unlikely to be kept secret, with the present requirements and extent of the need to report back to the agency. The need for secrecy stems from the potential of discoveries to be valuable to commercial enterprise. Without support for patent development, scaling up operations etc. then any invention is likely to end up through a variety of ways as the property of a large company.

8.2.1 Secrecy in Industrialised Countries

The communication of information between scientists has long been recognised as a great facilitator of research. However, the high likely value of inventions (e.g. BSt was calculated to have an annual global market of US$500 million) in modern biotechnology has destroyed considerably the ethos of communication and study in science. At the present time even PhD graduates may be sworn to secrecy and their thesis only published when the data is old hat or a patent or process fully protected. This represents a considerable disadvantage for the scientists in developing countries who have restricted access to the literature, no access to industrial personnel and limited funds to travel. Most of the modern biotechnology is now financed by commercial enterprise who have picked off and employed the scientific experts in many fields and have tight security on their developments. For any start up researcher to get ahead and stay ahead of such commercial research it will only be possible where the research is only of local interest or is aimed at sections of the community that do not have the capacity to pay and therefore the inventions/strategies are to be made available at no cost.

8.2.2 The Need for Expertise in Developing Countries to Capitalise on Various Developments
The development of a capacity for biotechnology research and teaching and for scientists with a depth of knowledge in the field allows a country to access product development and also to pick-up technologies applicable to local or small markets and discarded for economic reasons by large companies. It also allows that country to capitalise on inventions following patent expiration. The question now is how to retain such expertise in countries with limited ability to develop a research resource in modern biotechnology. The only option appears to be specialised national institutes funded from within a country.

Chapter 9 Conclusions and Recommendations

9.1 Priorities
The previous discussion suggests that the order of priorities for research in biotechnology in animal nutrition that should be promoted by agencies, such as FAO, are as follows: Feed technologies

Development of appropriate supplements that balance nutrition of ruminants fed poor quality forages, crop residues or agroindustrial byproducts. The research requirements include:

development of appropriate supplements of mineral/non protein-nitrogen/other microbial factors identification of protein resources naturally protected from rumen degradation processing of protein resources to protect them from rumen degradation

Where protein is scarce in a developing country, the development of systems to produce protected protein resources become a priority. These include: proteins from crops, trees or by-products aquatic plants algae and animals.

Manipulation of digestibility

by chemical treatment by microbiological treatment by improving fermentation in the rumen using:

modification of the ecosystem modified organisms.

Manipulation of metabolism within the animal

by chemicals and hormonal implants by production of transgenic animals.

9.2 Overall Conclusions

The views of the author, are summed up by the following:

9.2.1 The Vision of the New Biotechnology

Many review writers start their review as follows: In the near future, improvements of domestic animals for the production of meat and fibre are poised to undergo a revolution by the utilisation of recent breakthroughs and advances in molecular genetics, embryo manipulation and gene transfer systems. The Reality is that: Most research projects have come up against barriers which require a return to basic research. The simplistic approach of expecting transformed organisms and animals to function in a production system have not come to fruition. There is now a recognition among biotechnologists that considerable basic research is needed to complement the technological breakthroughs (of for instance development of the ability to introduce foreign DNA into cells) before an application is forthcoming. The promise of the technology is still there but the targeted time for application of soon after 2000 is now obviously too short.

9.2.2 The Future

Animal production in most developing countries could be increased many fold by finding ways and means of applying already established concepts. These include applying the newer concepts of supplementation of ruminants that have developed in the last 10 years that emphasises the use of local resources balanced with critical nutrients. The need is largely to research mechanisms and/or means for applying these concepts in various localities where animals are managed on poor quality forages. Nutrition appears to represent the most severe constraint to production. It is recognised that disease is still of paramount importance but in reality could be largely controlled by present day technologies. A major point is that without improved nutrition it is unlikely that any modern biotechnology can be successfully applied to ruminants in developing countries.

9.2.3 The Problems

Undoubtedly the promise of modern biotechnology is still there but the timeframe is so much longer than enthusiastic scientists have suggested in the past. The question How can the developing countries benefit from the advances that will arise as biotechnology research progresses?, is difficult to answer. As this discussion is restricted to nutrition, perhaps the answer is that with the present commercialisation of biotechnology the main beneficiaries are likely to be the stock-holders of multi-national companies unless the application is uncontrolable, e.g. an introduced rumen super bug is readily transmitted from animal to animal and is robust and will survive. There are niches for modern biotechnology in developing countries. There is a need for such countries to have people working in these areas in order to be able to recognise (if the information is not kept secret) particular benefits that might be of local interest. As the concepts of balanced nutrition are extended and applied to more and more livestock in developing countries, the application of modern biotechnology will become more appropriate. How long before this happens? The author's view is that in the next 10 years, application of present knowledge will be slow, but as global warming becomes more evident and recognised by people generally, the stimulus and pressure to improve the efficiency of ruminant production will be stronger. Within 10 years nutrition of ruminants may then be vastly improved along the lines set out in this report and ruminant numbers will be reduced. Further the application of such technologies will improve the feed base for the remaining animals and there will be space for considering a biotechnology approach to further improve the efficiency of production. The need for both applied and basic research is ongoing in these areas and must not be neglected for modern biotechnology which requires enormous funds to support basic research before it can be applied. The modern biotechnology should advance together one step at a time with good nutrition and disease research

9.2.4 Recommendations
1. In as many countries and regions as possible put major emphasis now, on local research to manipulate through feeding technology the microbial ecosystem of the rumen and the animals metabolism to make ruminants as efficient as possible on the feeds that are locally available. When the direct and indirect effects are taken into account this could result in the large improvements in productivity which could meet increasing food demand by humans and allow animal numbers to decrease. The most important milestone will be the production of packaged supplements to complement the available feed resources at the site of dense livestock populations. Modern biotechnology should be developed but only in a few selected research institutes and it should be funded from National resources. The areas of research should be carefully chosen to have a local emphasis that is not being researched elsewhere. These same scientific groups must have a watching brief on modern biotechnology as it develops.

2. 3.

Appendix A Methods Involved in Modifying Rumen Bacteria

Although there are a number of groups around the world that are already researching the possibilities of modifying rumen bacteria most research is in its preliminary phase. The general methods are given in outline below: Isolation of Genes for Fibrolytic Enzymes These usually follow the following course:

the first step follows standard methods for extraction of genomic DNA from fibre digesting bacteria (and attempts are also being made with fungi) the DNA is fragmented by partial digestion with restriction endonucleases and followed by selection of suitably sized fragments using gel or gradient fractionation. (For plasmid libraries a suitable size is 38 kilobases (kb) or 3040 kb for cosmid libraries)

For the next step there are then two alternative approaches:

the DNA fragments are ligated to appropriate vectors such as pUC.18 or pUC.19 plasmids and the recombinant DNA can then be used to transform E. coli. An alternative is to ligate fragments to cosmid vectors, package the DNA into lambda phage particles and transfect into host E. coli recombinant colonies will then be selected for their ability to digest cellulose, hemicellulose or xylan. For cosmids, fragments of inserted DNA require sub-cloning into plasmids for closer analysis and these must be re-screened for enzyme activity in the plasmid subclones the subcloned fragments are trimmed down by using restriction enzymes to delete DNA fragments progressively from the ends of the isolated DNA. In this way the limits of the gene should be defined the trimmed down recombinant genes will then be sequenced by the Sanger dideoxy chain-termination procedure (Sanger et al.,1977) once the genes have been defined, they may be excised for insertion into an anaerobic bacterial plasmid.

Preparation of Anaerobic Bacterial Plasmids Suitable for Gene Insertion A shuttle plasmid has proven to be the most appropriate vector. This is because transformation of rumen anaerobes is likely to be relatively inefficient in the early stages and the ability to grow large quantities of plasmid in E. coli may be essential. The first plasmid has been prepared by combining parts of a Butyrivibrio plasmid with parts of an E. coli plasmid (Gregg, K. and Ware, C., unpublished). The essential features included in this are as follows:

an E. coli origin of replication and antibiotic resistance gene an origin of replication and antibiotic resistance gene active in the anaerobic host a multiple cloning site, cleavable by a series of restriction endonucleases for insertion of genes manageable size of the recombinant plasmid (e.g. 56 kb) to ensure optimum transformation capability, with minimal risk of DNA deletion or rearrangement within the anaerobic host.

Location of Gene Control Factors Located Externally to the Enzyme-Coding Sequences For this purpose it will be necessary to test cloned genes for suppression by simple carbohydrate nutrients. Where this feature is an intrinsic part of the enzyme gene, additional control sequences may be necessary. However, it is possible that separate DNA fragments from the original donor genome may be required to provide a workable control system. Furthermore it is quite likely that gene control may require the combined action of multiple genes and at this stage the use of cosmids, with their capability for larger DNA fragment insertion, may be essential. Integration of Introduced Genes into the Chromosome of the Host Bacterium The likely approach here is as follows:

a short segment of the host bacterial DNA will be introduced into the gene-bearing plasmid the plasmid will be inserted into the host bacterium and the culture treated, with a physical or chemical agent, to induce recombination repair. Examples include the use of UV-light or chemicals such as ethyl methane sulphonate the bacteria must then be grown under curing conditions to encourage loss of free plasmids from the bacteria. Suitable curi ng agents to test might be acridine orange or deoxycholate

to identify the organisms that have integrated the introduced DNA into their chromosomes, the bacteria must then be selected for retention of antibiotic resistance and for their ability to digest fibre, in the absence of plasmids.

Direct Transformation of Rumen Anaerobes Plasmids capable of replication in rumen anaerobes have already been identified (Teather, 1982) and some recombinant plasmids have been constructed for growth in human colonic anaerobes (Smith, 1985). Problems that need to be studied are:

the possibility that host bacteria may contain uncharacterised restriction endonucleases, capable of digesting any DNA introduced the possibility that plasmids already constructed for growth in colonic species (e.g. Bacteroides fragilis) may not possess the features necessary for maintenance in rumen species of Bacteroides.

The predictable results of these problems are that the presence of uncharacterised restriction endonucleases may lead to extremely low transformation efficiency, although, once grown in a particular species, the plasmid will be protected for further transformation in that species. If the features necessary for maintenance of rumen species are absent from available plasmids, then this would lead to complete failure of the plasmid to grow and may necessitate locating naturally occurring plasmids in rumen bacteria, to obtain suitable replication control sequences. The Requirements for Research once Recombinant Rumen Bacteria are Developed There seems to be every optimism, with the rapid development of molecular genetics, that new strains of rumen micro-organisms will be developed with enhanced capabilities to digest (ferment) fibre. The required capabilities are not entirely clear but include microbes with the following characteristics:

enhanced enzyme production multi-enzyme systems allowing new strains to cope with a wider range of nutritive substrates (e.g. a largely pectin fermenter may be given the ability to digest cellulose and hemicellulose) multi-enzyme systems for the uptake of ammonia; this could be extremely important for survivability of bacteria. If a bacterium could switch enzymes to adapt to changing ammonia levels, this would effectively protect its survival in the rumen and may give it a competitive edge, particularly if it could also use a number of carbohydrate substrates possession of enzymes modified by protein engineering, which may allow more efficient function in vivo.

Establishment and growth of such organisms in culture should have few difficulties, since antibiotic resistance will also be cloned into them. However, the maintenance of these organisms in the rumen will be extremely difficult because of the interactive and competitive nature of the complex microbial ecosystem within the rumen. Considerable research is necessary to develop techniques to examine the ecological niche of these organisms, their survivability, the factors involved in maintaining them in the rumen and their growth characteristics. Monitoring the numbers of an individual species will become necessary in order to estimate their representation within the rumen biomass and their growth characteristics. Microbes in the rumen are generally in two major pools which reversibly exchange but which are not necessarily in equilibrium (i.e. the pool free in the liquid medium (the colonizing pool) and the pool of microbes attached to particles (the sequestered pool)). This will make the task of estimating the contribution of individual organisms quite difficult. Isotope technology coupled with genetic engineering must be put into action to solve the problem. In preliminary work in our laboratory, 15N, 35S, 14C and 3H labelling of bacteria is being undertaken both in vivo and in vitro. The specific radioactivity time relationships of labelled microbes in the rumen are being followed in order to quantitate bacterial growth rate. Only mixed populations of rumen microorganisms are being examined at the present time, but the technology must eventually lead to labelling of individual organisms that are then returned to the rumen to label the pool of these organisms. To follow the dilution (mixing and therefore pool size and subsequent growth of the organism) individual species will have to be identified in mixed populations of rumen organisms. This may be achieved by developing DNA hybridization probes for individual rumen species. Once the growth, turnover and survivability of the rumen microbes have been tested under laboratory conditions, then the laboratory technology must be adapted and tested under the conditions pertaining to the livestock producer.

Appendix B Some Comments on Use of Protein Meals for Use as Supplement for Ruminants Fed Forages
Protein Sources for Animal Feeding Whilst crop and agro-industrial by-products (e.g. cottonseed meal) will remain the major supply of proteins for domestic livestock production these are not always available at the sites of livestock production and at times are scarce and expensive. In the future it will be essential to produce protein sources at, or close to ruminant production sites and to ensure protection, either during manufacture or processing or by selecting or creating plants that contain proteins that are associated with secondary plant compounds which protect the protein on chewing and ingestion. If the source of protein had also an associated antiprotozoal component the protein source will be much more active in increasing the P/E ratio in ruminants when supplemented to the basal diet. The research will require a search for potential protein sources that will fit into the prevailing agricultural system. The likely targets are:

certain crops grown specifically for the purpose (e.g. lupins) legume forages (containing tannins or harvested and protected) e.g. Lotus pendiculatus tree leaves readily harvested and protected by processing e.g. Glyricidia spp. seeds and seed pods e.g. Prosopis spp. aquatic plants e.g. Azolla spp. algaes e.g. Chlorella spp.

The options are open but on the savannahs the forage legumes and tree leaves/seeds are likely targets and in the hot dry areas on acid sandy soils, lupins might be considered. In the intensive subtropical areas the production of aquatic plants/algaes in waste waters from many sources is feasible (see Pheng Siew Moi, 1990). The attraction of trees (e.g. Acacia, Prosopis, Leucaena, Erythrina etc.) is that the useful biomass produced with only a few trees/hectares is often the same as the biomass that can be harvested by cattle from grasslands (which seldom reaches 30% of the biomass and is often closer to 10%). The drawbacks are that it is necessary to know how to manage the trees under various conditions and usually harvesting of the leaves is hard work and expensive. However, few real attempts have been made with plantation management and mechanical harvesting. The aquatic plants/algaes can yield up to 30 tons/ha/year of wet materials with 2050% crude protein, they are mostly grown on enriched (by sewerage or other effluent) waste waters (see Pheng Siew Moi, 1990) and are used for cleaning purposes, again the mechanics of harvesting are major constraints. There is no real knowledge of the problem of toxic waste concentration in these sources nor of the extent of protein protection or how to effect this after harvest. The various uses of Azolla have been summarised in a recent FAO publication (Van Hove, 1989). With Azolla as a protein source the costs of harvesting, drying and processing will be the main limitation. The biotechnology of production of local protein resources is an area worthy of much effort. The costs of production as animal feeds could be subsidised by the production of a few fine chemicals (e.g. arachidonic acid) that are produced by algae (see Pheng Siew Moi, 1990).

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FAO TECHNICAL PAPERS FAO ANIMAL PRODUCTION AND HEALTH PAPERS: 1. 2. 3. 4. 5. 6. 7. Animal breeding: selected articles from World Animal Review, 1977 (C* E* F* S*) Eradication of hog cholera and African swine fever, 1976 (E* F* S*) Insecticides and application equipment for tsetse control, 1977 (E* F*) New feed resources, 1977 (E/F/S*) Bibliography of the criollo cattle of the Americas, 1977 (E/S*) Mediterranean cattle and sheep in crossbreeding, 1977 (E* F*) Environmental impact of tsetse chemical control, 1977 (E* F*)

7 Environmental impact of tsetse chemical control, 1980 (E* F*) Rev. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. Declining breeds of Mediterranean sheep, 1978 (E* F*) Slaughterhouse and slaughterslab design and construction, 1978 (E* F* S*) Treating straw for animal feeding, 1978 (C* E* F* S*) Packaging, storage and distribution of processed milk, 1978 (E*) Ruminant nutrition: selected articles from World Animal Review, 1978 (C* E* F* S*) Buffalo reproduction and artificial insemination, 1979 (E**) The African trypanosomiases, 1979 (E* F*) Establishment of dairy training centres, 1979 (E*) Open yard housing for young cattle, 1981 (E* F* S*) Prolific tropical sheep, 1980 (E* F* S*) Feed from animal wastes: state of knowledge, 1980 (E*) East Coast fever and related tick-borne diseases, 1980 (E* S*)

20/1. Trypanotolerant livestock in West and Central Africa Vol. 1 - General study, 1980 (E* F*) 20/2. Trypanotolerant livestock in West and Central Africa Vol. 2 - Country studies, 1980 (E* F*) 20/3. Le btail trypanotolrant en Afrique occidentale et centrale Vol. 3 - Bilan d'une dcennie, 1988 (F*) 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. Guideline for dairy accounting, 1980 (E*) Recursos genticos animales en Amrica Latina, 1981 (S*) Disease control in semen and embryos, 1982 (E* F* S*) Animal genetic resources conservation and management, 1981 (E*) Reproductive efficiency in cattle, 1982 (E* F* S*) Camels and camel milk, 1982 (E*) Deer farming, 1982 (E*) Feed from animal wastes: feeding manual, 1982 (E*) Echinococcosis/hydatidosis surveillance, prevention and control: FAO/UNEP/WHO guidelines, 1982 (E*) Sheep and goat breeds of India, 1982 (E*) Hormones in animal production, 1982 (E*) Crop residues and agro-industrial by-products in animal feeding, 1982 (E/F*) Haemorrhagic septicaemia, 1982 (E* F*)

34. 35. 36. 37. 38. 39. 40. 41. 42. 43.

Breeding plans for ruminant livestock in the tropics, 1982 (E* F* S*) Off-tastes in raw and reconstituted milk, 1983 (E* F* S*) Ticks and tick-borne diseases: selected articles from World Animal Review, 1983 (E* F* S*) African animal trypanosomiasis: selected articles from World Animal Review, 1983 (E* F*) Diagnosis and vaccination for the control of brucellosis in the Near East, 1983 (E* Ar*) Solar energy in small-scale milk collection and processing, 1983 (E* F*) Intensive sheep production in the Near East, 1983 (E* Ar*) Integrating crops and livestock in West Africa, 1983 (E* F*) Animal energy in agriculture in Africa and Asia, 1984 (E/F* S*) Olive by-products for animal feed, 1985 (Ar* E* F* S*)

44/1. Animal genetic resources conservation by management, data banks and training, 1984 (E*) 44/2. Animal genetic resources: cryogenic storage of germplasm and molecular engineering, 1984 (E*) 45. 46. 47. 48. 49. 50. 50/2. 51. 52. 53. 54. 55. 56. 57. 58. Maintenance systems for the dairy plant, 1984 (E*) Livestock breeds of China, 1985 (E*) Rfrigration du lait la ferme et organisation des transports, 1985 (F*) La fromagerie et les varits de fromages du bassin mditerranen, 1985 (F*) Manual for the slaughter of small ruminants in developing countries, 1985 (E*) Better utilization of crop residuces and by-products in animal feeding: research guidelines 1. State of knowledge, 1985 (E*) Better utilization of crop residuces and by-products in animal feeding: research guidelines 2. A practical manual for research workers, 1986 (E*) Dried salted meats: charque and carne-de-sol, 1985 (E*) Small-scale sausage production, 1985 (E*) Slaughterhouse, cleaning and sanitation, 1985 (E*) Small ruminants in the Near East: Vol. I - Selected papers presented at Tunis Expert Consultation, 1986 (E*) Small ruminants in the Near East: Vol. II - Selected papers from World Animal Review, 1986 (E* Ar*) Sheep and goats in Pakistan, 1985 (E*) Awassi sheep, 1985 (E*) Small ruminant production in the developing countries, 1986 (E*)

59/1. Animal genetic resources data banks, 1986 (E*) 1. Computer systems study for regional data banks 59/2. Animal genetic resources data banks, 1986 (E*) 2. Descriptor lists for cattle, buffalo, pigs, sheep and goats 59/3. Animal genetic resources data banks, 1986 (E*) 3. Descriptor lists for poultry 60. 61. 62. 63. 64. 65. 66. Sheep and goats in Turkey, 1986 (E*) The Przewalski horse and restoration to its natural habitat in Mongolia, 1986 (E*) Milk and dairy products: production and processing costs, 1988 (E* F* S*) Proceedings of the FAO expert consultation on the substitution of imported concentrate feeds in animal production systems in developing countries, 1987 (E*) Poultry management and diseases in the Near East, 1987 (Ar*) Animal genetic resources of the USSR, 1989 (E*) Animal genetic resources strategies for improved use and conservation, 1987 (E*)

67/1. Trypanotolerant cattle and livestock development in West and Central Africa Vol. I, 1987 (E*) 67/2. Trypanotolerant cattle and livestock development in West and Central Africa Vol. II, 1987 (E*) 68. 69. 70. 71. 72. 73. 74. Crossbreeding bos indicus and bos taurus for milk production in the tropics, 1987 (E*) Village milk processing, 1988 (E* F*) Sheep and goat meat production in the humid tropics of West Africa, 1988 (E/F*) The development of village-based sheep production in West Africa, 1988 (E* F* S*) Sugarcane as feed, 1988 (E/S*) Standard design for small-scale modular slaughterhouses, 1988 (E*) Small ruminants in the Near East, Volume III: North Africa, 1988 (E*).

75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92.

The eradication of ticks, 1989 (E/F*) Ex situ cryoconservation of genomes and genes of endangered cattle breeds by means of modern biotechnological methods, 1989 (E*) Training manual for embryo transfer in cattle, 1991 (E*) Milking, milk production hygiene and udder health, 1989 (E*) Manual of simple methods of meat preservation, 1989 (E*) Animal genetic resources a global programme for sustainable development, 1990 (E*) Veterinary diagnostic bacteriology a manual of laboratory procedures of selected diseases of livestock, 1990 (E*) Reproduction in camels a review, 1990 (E*) Training manual on artificial insemination in sheep and goats, 1991 (E*) Training manual for embryo transfer in water-buffaloes, 1991 (E*) The technology of traditional milk products in developing countries, 1990 (E*) Feeding dairy cows in the tropics, 1990 (E*) Manual for the production of anthrax and blackleg vaccines, 1991 (E*) Small ruminant production and the small ruminant genetic resource in tropical Africa, 1991 ( E*) Manual for the production of Marek's disease, Gumboro disease and inactivated Newcastle disease vaccines, 1991 (E*) Application of biotechnology to nutrition of animals in developing countries, 1991 (E*) Guidelines for slaughtering, meat cutting and further processing, 1991 (E*) Manual on meat cold store operation and management, 1991 (E*)

Availability: June 1991 Ar Arabic C Chinese E English F French S Spanish * Available ** Out of print *** In preparation The FAO Technical Papers are available through the authorized FAO Sales Agents or directly from Distribution and Sales Section, FAO, Viale delle Terme di Caracalla, 00100 Rome, Italy.