S.K. Ho,
Centre for Food and Animal Research, Agriculture and Agri-Food Canada, Ottawa, Ontario,
Canada, K1A 0C6
E.E. Lister,
Animal Science Consultant, Ottawa, Ontario, Canada K1Y 1B1
Presented at the Symposium on Animal Industry and Animal Product Processing, the First China International Annual Meeting on Agricultural Science and Technology, Beijing International Convention Centre, Beijing, People's Republic of China - March 20, 1996. Compiled by Department of Animal Husbandry and Health, Ministry of Agriculture, People's Republic of China and Chinese Association of Agricultural Science Societies In "Research Progress in Animal Industry and Animal Products Processing", China Agricultural Scientech Press, Beijing 1996 (ISBN 7-80119-127-7), pp 172-178.
Biotechnology has been with us for a long time. It refers to the use of biological processes and living cells (or parts of cells) in the manufacture of products, or as part of industrial processes. Wine-, bread- and cheese-making are examples of industries based on traditional biotechnology. In recent years, biotechnology has become synonymous with genetic engineering, the science of altering the genetic make-up of cells to produce specific commercial biological products or desired biological activities. This technology is known more formally as recombinant DNA (or rDNA) technology. It is based on knowledge from the rapidly growing field of molecular biology, the study of living cells and their components at the most basic molecular level.
Biotechnology related to animal productivity is sometimes viewed from a very restricted perspective, limited to embryo transfer, growth hormone or vaccine. However, there are other applications of biotechnology that are important for the future of animal production. This paper discusses some of these newer technologies that are being developed and used in Canada to improve animal productivity. It is a simplified representation of these developments. It is noteworthy that the practical implementation of these technologies is not trivial and has taken a lot of research effort. On broader scientific and technological options, the reader is referred to a recent report by the Organisation for Economic Co-operation and Development (OECD
Although PSS has been known as a genetic condition for some time, it was not until the DNA site of the defect was identified (Fujii et al., 1991; Otsu et al., 1991) and a DNA test developed by Canadian researchers, that a reliable and practical method became available for detecting the so-called halothane sensitivity gene that identifies animals susceptible to PSS. The test is used to select against breeding animals carrying this recessive gene.
Through an agreement between the Canadian Pork Council and University of Toronto's Innovations Foundation, swine breeders and hog producers across Canada are now able to become licensed users of this Canadian-developed DNA test to determine the presence of the halothane gene and to use the results to control the incidence of PSS. The Canadian Pork Council, which represents the interests of commercial hog producers, has taken this licensing agreement to make the technology available as widely as possible throughout the Canadian industry. This benefits all segments of the swine and pork industries, with on-farm productivity improvements by both breeders and commercial producers, and with enhanced pork quality for packers and processors.
The most recent work that has been conducted with poultry in Canada has centred on the endogenous viral genes. The genomes of most chickens carry genetic elements that are closely related to the exogenous, field strains of avian leukosis viruses (ALV). These endogenous viral elements behave as other chicken genes and are passed on from parents to progeny. Endogenous viral loci have been implicated in a reduction of performance in layer stocks, and there is clear evidence that certain endogenous viral loci - those that retain the capacity to produce infectious viral particles - can compromise the host's immune response to exogenous ALV resulting in a disease syndrome that is particularly undesirable in commercial flocks (Gavora et al., 1995).
In Canada we have developed polymerase chain reaction (PCR) tests for endogenous viral genes to replace cumbersome and expensive Southern blot techniques. These PCR-based technologies are invaluable for tracking endogenous viral loci in populations of birds, and identifying the association of specific loci with productivity and disease traits (Benkel et al., 1995).
Descriptions of some aspects of recent Canadian research aiming at improvement of natural resistance to disease in animal are contained in a review by Gavora (1996) as given at this Symposium, and by Falconer et al. (1995). Included in the reviews are attempts to genetically engineer new resistance mechanisms such as the production by the host animal of viral envelope proteins that block entry of virus into host cells. Production of such protein by the host could block the receptor sites and prevent attachment by the virus to the cell wall. An example of potential application is the genetic engineering of resistance to rotavirus that causes calf diarrhoea.
In Canada, one avenue of current research is oriented towards the use of transgenic techniques to add value to animal products. The dairy cow has an enormous capacity to produce milk proteins. Molecular techniques are being developed in Canada to harness and direct this capability to synthesize non-milk, pharmaceutical proteins, and use the dairy cow as a "bioreactor".
The use of dairy cows as bioreactors is seen as a potential means of providing valuable pharmaceuticals at lower cost and in greater quantities than are available at present. Cows with cells modified to express exogenous genes could produce milk containing high levels of valuable protein pharmaceuticals. Transformations of bovine cells so that only the milk secreting cells contain and express the gene of interest, rather than insertion of the gene into germline cells, would be most effective both in terms of cost and flexibility.
Falconer et al. (1995) described their program of research in this area which is being carried out in collaboration with the Canadian dairy industry. The uniqueness of the research approach is its orientation to modification of only the milk secreting epithelium of the udder rather than the entire animal. These Canadian researchers concentrate on two main areas: (a) making and testing plasmids that contain the foreign DNA of interest, and (b) examining ways and means to introduce these constructs into the mammary cells of the udder. Instead of concentrating on a specific pharmaceutical protein, they are at present using marker proteins to show that the technology results in expression of exogenous proteins. The intent is have a technology available so that commercial firms can use it to produce specific pharmaceuticals.
A high proportion of protein available to the ruminant is microbial in origin. Protein present in the feed, like other components, is largely digested by the rumen microbial population, which uses it as a source of metabolic precursors for producing its own proteins and energy. It is this microbial protein that is in turn digested by the ruminant. Rumen bacterial protein however, does not contain sufficient of those amino acids that are essential to milk production. This can lead to a deficit in several amino acids and the cow must either limit the amount of protein in milk or break down body tissue to cover the deficit. High producing dairy cattle in peak lactation are therefore often fed high quality protein supplements. Given the degradation of a large proportion of this protein in the rumen, this practice is expensive and inefficient. Genetic engineering offers an option of engineering rumen bacteria to produce a protein better suited to the needs of the lactating dairy cow. In Canada, researchers have designed a gene to express a new protein rich in the deficient amino acids and have inserted it into rumen bacteria. Thus, in addition to the normal array of proteins the bacteria usually produce, they will make a new protein with no function other than to supply essential amino acids to the cow (Beauregard, Teather and Hefford, 1995).
In addition to serving as the protein source of essential amino acids, rumen bacteria through their fermentation of feed, supply the dairy cow with carbon sources in the form of volatile fatty acids (VFAs) for both gluconeogenesis and lipid biosynthesis. The end products of fermentation differ for different rumen microorganisms, but two of the major VFAs produced in the rumen are propionate and acetate. Propionate is a major metabolic precursor for the gluconeogenic pathway in cows. The glucose produced, in addition to producing energy, also provides the carbon skeletons used in many major synthetic pathways. Acetate is the starting point for fat synthesis. By altering the ratio of propionate to acetate produced in the rumen fermentation process, the relative availability of substrate for milk fat synthesis can be changed.
The ratio of propionate to acetate production can be changed by altering the relative abundance of rumen bacteria producing each of these VFAs (Leng, 1982) which in turn can be effected by various additives such as ionophore antibiotics (Russell and Stoble, 1989). However, such a treatment affects a broad spectrum of bacteria and like any antibiotic agent there is a risk of bacterial resistance.
Current Canadian research is devoted to evaluating the use of natural rumen bacterial bacteriocins to modify rumen fermentation in a much more selective manner (Hefford, Teather and Forster, 1995). Bacteriocins are small protein toxins produced by the bacterium itself to eliminate other bacteria, generally closely related competitors, and thus protect its own ecological niche. A given bacteriocin can have either a wide or narrow range of organisms on which it exerts its effect. Thus, population alteration by bacteriocins affords the possibility of a more controlled and selective effect than the use of broad-range ionophore antibiotics. Specific rumen bacteria are being engineered to either produce an effective bacteriocin or develop immunity to endogenous rumen bacteriocins to obtain a specific and controlled alteration in the levels of specific bacteria in the rumen. Such an alteration could result in improved production efficiency, reduced methane production, a change in milk composition in dairy cows, or all three.
This is another project that researchers have undertaken in a close working relationship with the Canadian dairy industry. One of the challenges is to put these approaches together into a coherent technology that can be commercially applied
Advanced reproductive technologies include cloning of animals by enucleation technology. This technology has been applied to cattle embryos, but commercial use is not yet common. The process is complex. Microsurgery of embryos to produce twins or triplets is a simpler cloning technique but the numbers of clones are limited. In international trade, the destruction of the zona pellucida that is inevitable in this type of surgery (embryo splitting) may have implications for animal health regulations, since the processes applied to render the embryo free from specific pathogens requires an intact zona (Singh, 1993).
Newer reproductive techniques include the use of embryonic stem cells. Embryonic stem cells are cell lines developed from the pluripotent cells of pre-implantation embryos, which retain the ability to differentiate into all tissues of the developing fetus (Carnegie, 1995). They offer potential for modifying the genome of poultry and other species and for the use of such cryopreserved cells as a means of transferring and preserving genetic resources (Etches and Gibbons, 1994; Carnegie, 1995). Embryonic stem cells are expected to be cryopreserved and stored for long periods in liquid nitrogen. Ultimately, they could be thawed and used to produce clones. Embryonic stem cells are seen as a means of conserving genetic resources for long periods for those species for which we lack techniques for cryopreservation of semen and embryos, or as an alternative to such techniques.
The driving forces behind this technology have been a variety of factors, including the rising costs of existing instrument-based analytical procedures, increasing sample loads, improved antibody production methods, and the successful commercialization of test kits. This change in analytical approach is rational and inevitable as efficiency and cost-effectiveness are an integral component of control and regulation. Although for the time being, immunochemical assays tend to be used to complement existing detection methods, this emerging technology is the way of the future. The recent development of engineering antibodies through rDNA technology will revolutionize this field even further (Choudary et al., 1995).
The pursuit of easier, faster, more accurate, and less expensive immunoassays however depends on the availability and quality of the antibody to be incorporated into the assay. Researchers in several Canadian government laboratories (Agriculture and Agri-Food Canada, and Health Canada) are producing and characterizing antibodies to detect contaminants considered food safety priorities. These activities are in various stages of development or commercialization, and include deoxynivalenol and other mycotoxins (Savard, Personal Communications), fumonisin B1 and metabolites (Prelusky, Personal Communications), pesticides (Yeung, Personal Communications), and pathogenic microorganisms (Blais, Brooks, Garcia, Johnson, Nelson et al. Personal Communications).
Conventional anaerobic digestion of animal manure in farm scale digesters was tried at several locations across Canada between 1975-1985. It was not successful for several reasons as reviewed by Van Die (1987). Anaerobic digestion of municipal waste water and animal manure at psychrophilic temperature has been reported in previous studies (O'Rourke, 1968; Stevens and Schulte, 1977; Chandler, Hermes and Smith, 1983; Cullimore, Maule and Mansui, 1985; Lo and Liao, 1986). Most of these studies were aimed at bio-gas production while little consideration was given to odour reduction, waste stabilization or increases in fertilizer value or plant nutrient availability. There was a wide variation in the reported experimental results. In most of these studies the slurry solids content was very low (less than 2%) compared to the typical solids content of manure slurry at Canadian farms. It is unlikely that farmers would dilute manure slurry for anaerobic digestion because it would require larger storage facilities and substantially increase the volume of liquid manure to spread on the land.
The biotechnological process - psychrophilic anaerobic digestion in sequencing batch reactor that is being developed in Canada is unique (Massé et al., 1993; Massé, 1995). It has a low capital and operational cost because it (a) makes use of existing manure storage and handling equipment, and (b) operates at relatively low temperatures ranging from 10 to 20 oC. which are typical of farm manure in Canada. Therefore, it does not require energy input to heat manure prior to feeding the digester. The flow regime of a sequencing batch reactor is highly suitable for low temperature anaerobic digestion because it provides optimal conditions to retain the slow growing anaerobic bacteria in the bioreactor. More importantly, this biotechnological process is easy to operate, requires minimum skill and does not interfere with regular farm operations.
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