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.
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 400–500 g/h/d) and small ruminants (UMB 100–150 g/h/d) in West Central Java in 1988–89. The data are from Hendratno, N and numerous co-operators and were summarised in Entwhistle (1989)
|Species||Location||Trait measured||Treatment||Response to UMB|
|Dairy cattle||W.Java||Milk yield (l/d)||7.44||9.92|
|Beef cattle||W.Java||1 Weight gain (kg/d)||0.182||0.400|
|Sheep||W.Java||Weight gain (kg/d)||0.130||0.320||+146%|
|Goats||C.Java||Milk yield (l/d)||0.795||1.091||+37%|
* 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)||Microbial cells produced (g/d)||Microbial protein (g/d)||Total protein (g/d)(P)||VFA produced (MJ/d)(E)||P/E * (g/MJ)|
* 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.
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.
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.
There are a number of chemicals available which, when added to a diet, improve the efficiency of feed utilisation by 5–20% (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 20–50% 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 30–60% 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.
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.
By chance, recent research has demonstrated that small amounts of a clay mineral (bentonite—a 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.
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.
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.
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.
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 180°C 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 (1–3) and cross bred cows (4)
|Basal Feed||Supplements||Milk Production|
|1. Tropical grass/maize silage or other crops plus 1-2 kg rice straw/day||Free choice molasses/urea blocks + protein pellet (30% CP) (350 g/kg milk)||5000-6500 kg/305 d|
|2. Rice straw/millet straw (8 kg/d)||Bypass protein pellet 300 g/kg milk||25 l/day (at 3 months)|
|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)||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 available||Cottonseed meal 250 g/kg milk||2800 kg/lactation/yr (4)|
(1) NDDB Anand
(2) Personal observations—in 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 400–600 kg pods/tree/year in the rainfall areas of 400 mm annually (range 200–600 mm) (F. Riveros pers. comm.) with 16–20% crude protein. Many rangelands in the semi-arid areas produce less than 2,500 kg/annum of pasture biomass of which only 200–400 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 3–6% 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).
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).
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 & Ørskov (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.
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).
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 A—this 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.
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.
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.
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.
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.
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
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 Sundstø;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 Sundstø;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).
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).
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.
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).
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.
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 20–25% 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.
β-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.
There are strong similarities between the response observed to growth hormone/ β-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.
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 promotion 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.
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 40°C, and reach 50°C at times, Friesians can produce to at least ⅔ of their genetic capacity. It also 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).
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 50–100 years hence.