Swine Genetics for the Next 25 Years
Rothwell, Lincoln LN7 6BJ, United Kingdom
For the last twenty-five years conventional selective breeding, increasingly assisted by computers, has been highly successful in improving lean yield and feed efficiency. More recently this has been extended via BLUP to include traits of lower heritability such as litter size. It has the advantage of being proven, safe, acceptable to the public, and highly cost effective. This extension of traditional methods offers further steady improvement for many years to come.
Yet the revolution in biotechnology from cloning to GM foods now appears in the national press almost daily. To the US pig industry it represents both a threat and an opportunity. The threat is one of public disapproval, competition from alternative food sources, and escalating investment in new technology. The opportunity is for cheaper pork that is healthier, safer, and produced with better animal welfare and greater regard for the environment.
This milestone Year 2000 has already seen the first cloned pigs and the complete DNA sequence of the human genome. As the options for biological change widen beyond comprehension, the expectations of society towards animal production are completely impossible to predict. What then is the role for these new technologies in the next 25 years, and what is the right mix of investment in present versus future technology? Indeed what is the role of genetics in the food chain of tomorrow?
Statistics and information technology
For over ten years cheap desktop computer power has allowed the application of BLUP (best linear unbiased prediction) in pig selection. By using family records this gives more accurate prediction of genetic merit for traits of low heritability, doubling the rate of improvement for example in litter size. However the greatest contribution of BLUP has been that, together with AI acting as the genetic link, it allows direct comparisons of genetic merit among animals measured in different environments. This ability to compare across herds has opened the way for larger more geographically diverse nucleus populations, greater selection differentials and faster improvement.
Shipping breeding stock from a nucleus in one country for production in another is costly in terms of transport and health security, and runs the risk that market requirements may be very different. The solution is to establish separate nucleus populations in key countries selected for local objectives. Where necessary BLUP calculations can easily now be conducted on data transmitted over the Internet using centralised statistical expertise. New genotypes can be introduced via frozen semen or embryo transfer.
This decentralisation also brings the challenge to lower overhead costs by reducing the size of each nucleus population. Conversely, the more accurate the selection the faster the rate of inbreeding. Procedures are therefore now being develop to combine a BLUP prediction of merit with a measure of inbreeding to give a single selection criterion which balances the two (Grundy et al, 2000). Nevertheless BLUP does not overcome the problem that traits such as meat quality or disease resistance are difficult to measure in the live animal.
Substituting a better breed or line will always be a faster method of genetic change than selection. After 30 years of intense selection, some populations of the traditional breeds such as Large White and Landrace are becoming very homozygous. Incorporating a third breed such as the Duroc to restore heterosis and hardiness in a cross greatly adds to the overhead nucleus cost. The dilemma is that unless populations of sufficient size can be maintained, the minority breeds will quickly fall behind.
The solution has been to combine the attributes of different breeds in new composite lines. For example Cotswold has introduced 25% of its White Duroc line into each of Large White and Landrace type dam lines to give new composite lines. When crossed together these give a parent gilt containing 25% Duroc from two rather than three nucleus lines. The two larger nucleus populations result in faster selection. They are also cheaper to maintain with better physical condition than pure breeds due to residual heterosis from the Duroc. Multipliers too benefit from heterosis in what would otherwise have been a purebred GP.
The Chinese Meishan offers eight extra pigs per sow per year, accompanied by very poor growth and carcase characteristics. Since 1987 Cotswold has been developing a 50% Meishan composite dam line by selection for lean growth. Trials at Cotswold’s UK Research & Development Centre in alliance with Imperial College at Wye (London University) show an advantage for the resulting 25% Meishan parent females over non-Meishan parents of 3.8 piglets weaned per sow per year. Backfat of the resulting 12.5% Meishan progeny was increased by 0.7 mm on ad lib feeding to 95 kg live weight, feed efficiency was 1% worse and there was no difference in growth rate. Work continues to improve uniformity and lean distribution.
Completion of the human genome map will greatly accelerate understanding of the mechanism of inheritance at the level of DNA. In the pig the DNA is distributed over 19 pairs of chromosomes and organised into some 100 000 functional genes. The DNA code is made up of sequences of the four bases (A, C, G, T) and a typical gene would be some 5-20 thousand bases in length. Once the sequence for a gene is known, its presence can be detected using a DNA test as in the case of the halothane gene.
Research teams from the USA and Sweden as well as the EU-funded Pig Genome Mapping Project (PiGMaP) are collaborating to map the genome of the pig (Visscher and Haley, 1998). By locating genes on the chromosomes, the objective is to understand how genes are organised and interact with each other, and how they affect all aspects of performance. To date some 2000 DNA sequences showing genetic variation have been placed on the pig maps. These maps are freely accessible on the Internet, along with similar maps for cattle, sheep and chickens.
Most quantitative traits such as growth rate are controlled by many hundreds of genes, each with a small effect. A gene with a large effect such as the halothane gene is very much the exception. Nevertheless much research is now under way to identify possible genes with useful effects on performance. The function of most of the genes so far detected is unknown. They may however be situated on the chromosome close to a gene that does affect performance, for example growth rate, but for which no DNA test exists. Due to genetic linkage the gene, which can be detected
Fig. 1 Hot spots on pig chromosomes affecting reproduction (Courtesy of P R Bampton).
In this case the DNA tested gene is known as a marker, because it marks a section of chromosome affecting performance. The gene whose presence it detects is known as a quantitative trait loci (QTL), with linkage between the marker and the QTL. Possible markers have been reported for all the important traits, and many have been mapped. Some of the “hot spots” from worldwide pig research are shown in Figure 1. Hot spots for litter size exist on chromosomes 1, 8 and 16; lean growth on 4 and 7; and meat quality on 1, 6 and 15.
Marker assisted selection
In the process of marker assisted selection, DNA testing for the marker can be used to increase the frequency of the QTL and lead to an improvement in a production trait. The main benefit would be in traits such as meat quality or disease resistance which are difficult or expensive to measure in the live pig, or in reproduction which occurs late in life in one sex only. There are however a number of problems:
§ DNA testing is still relatively expensive in relation to the small benefits of most markers on performance.
§ There is no further benefit after the marker has been made homozygous.
§ Marker effects are often inconsistent between lines and even families.
§ Due to the high number of candidates and traits, there is a statistically high chance of false positive markers. Already the number of markers reported would explain more than 100% of the genetic variation for some traits.
§ Selecting on markers causes a loss of selection on other traits.
§ Markers may have unknown harmful as well as beneficial effects. There may therefore be good reasons why selection has not fixed apparently favourable QTLs at 100% in these populations.
Current thinking is that the interaction of genes with each other is probably more important that originally recognised, so the implications of changing the frequency of any gene with a large effect may be difficult to predict from one population or even family to the next. A further difficulty is that the information from hundreds of markers of small effect may be difficult to collate. At this stage the use of markers is therefore risky, whereas BLUP selection is already proven and cost-effective.
Marker assisted introgression
As an alternative to selection within a line, a marker can be used to introduce QTL from one line into another by marker assisted introgression. Suppose for example that a single gene for prolificacy is to be introduced from Meishan into Landrace. An F1 cross of the two is then backcrossed to Landrace over several generations, gradually increasing the proportion of Landrace while selecting for the desirable gene. In the absence of a DNA test, this is the method by which Cotswold introduced the dominant white coat colour gene from the Large White into its White Duroc line.
By the same principle other markers can be used to reduce the proportion of background genotype from the undesirable line, for example fatness from Meishan, hastening a commercially viable result. A further application might be the use of markers to retain maximum heterozygosity in any closed population. The risk of introgression is that it takes several generations. During this time the linkage with the marker could break down, selection on other traits may be lost, and the intermediate Meishan crosses could be over-fat and costly. The benefit of the QTL in improved performance would therefore need to be large.
New types of markers
The main disadvantage of existing markers is their high cost and low accuracy. The majority are random segments of DNA of the form CACACA (microsatellites) that show genetic variation in the number of repeats. The inaccuracy stems from the weakness of the linkage in predicting the presence of the QTL. Either closer markers are needed or ideally a method of detecting the QTL directly. Several new options are now appearing:
§ AFLPs Amplified fragment polymorphisms can be generated by enzymes which cut the chromosomes only at specific sequences. The presence of different genes results in DNA fragments of different length which can be correlated with performance traits. Patented by KeyGene NV in the Netherlands and applied in plants, this has the advantage of producing a set of markers specific to one line. It also overcomes any patents on published markers.
§ SNPs Single nucleotide polymorphisms are changes in a single specific coding unit of the genetic code. They are easy to detect and usually occur within the functional gene. Unlike microsatellites SNP tests can be automated on DNA microarray chips.
§ ESTs Expressed sequence tags allow genes to be detected when they are ‘switched on’. This would allow selection for animals expressing rapid early growth, earlier puberty, or perhaps for immune response. ESTs will provide the key to how genes are organised and controlled.
As the number of mapped genes increases, AFLPs are likely to provide alternative markers for QTLs or hot spots that are already known. Microarray technology already allows 30 000 SNP DNA tests to be conducted on a single chip the size of a microscope slide, making this the most likely method for the future. This technology is therefore likely to be both powerful and cheap.
Rather than searching at random for markers, the candidate gene approach uses knowledge of physiology to identify likely QTLs with a major effect. Equally QTLs from human, mouse or other species maps would be candidates for investigation in the pig. Patents have been filed on some markers, but can often be overcome using others which are near to the QTL.
The halothane RYR1 appears to be the functional gene or QTL responsible for all the effects on lean growth and stress susceptibility. In Germany Cotswold has developed a very lean Pietrain-type composite sire line which is approaching homozygosity for the absence of the halothane gene. The oestrogen receptor ESR which affects litter size in some populations but not others appears to be a marker rather than the QTL responsible. H-FABP was discovered to affect intramuscular fat in Durocs and is currently being trialed under licence in other populations (Gerbens et al, 1998). Candidate genes to control boar taint arising from skatole and androstenone are being investigated by several groups (Davis and Squires, 1999).
As an example Cotswold is co-sponsoring a study at Glasgow University in which a knowledge of myosin heavy chain muscle protein polymorphisms is being used to deduce likely sequences of DNA that could act as markers within the genes affecting quality (Beuzen et al, 2000). Certainly markers represent an important opportunity to accelerate genetic improvement, and Cotswold is very actively continuing its programme of in-house evaluation with exploratory selection on a combination of markers and BLUP.
In violation of the simple laws of Mendel, the expression of some genes can be switched on or off in the progeny depending on whether the gene was transmitted through the mother or father. This process of imprinting occurs by methylation of C (cytosine) units in promoter regions of genes carried by either sperm or eggs, shutting off their function. It may have evolved as a means of resolving conflicting requirements of mother and offspring. At least 34 genes showing imprinting are already known in the mouse (Ruvinsky, 1999).
In humans and mice the Igf2 gene (insulin-like growth factor 2) affecting growth appears to be maternally imprinted, and is thus expressed only when inherited from the father. By contrast, the corresponding receptor gene Igf2r is paternally imprinted and expressed only when transmitted by the mother. In pigs the Igf2 gene on chromosome 15 appears maternally imprinted and expressed only via the sire (Nezer et al, 1999). A marker for Igf2 in Pietrain crosses has been patented and is used by one breeding company. There have also been some reports of maternally imprinted genes for fat in the pig. This would allow higher levels of fat in the dam, allowing a long reproductive life, with no adverse effect on the carcase fat of the commercial progeny.
Gene transfer in animals between individuals and species has been possible for some years. The method of micro-injection of DNA into the fertilised egg had a low success rate and could not control where and how many copies of the DNA sequence were incorporated. Dolly type cloning makes gene transfer much easier and cheaper, allowing DNA to be incorporated into cloned cells before transfer into the embryo. The first cloned pigs were announced by PPL Therapeutics in the USA this year.
First attempts to add extra copies of pig or human growth hormone genes brought adverse publicity due to undesirable effects on fertility and physical soundness. Methods are now being developed to control the number of copies, site of insertion, and the degree of expression. The technology of gene transfer is being driven by the use of the pig as a donor of hearts and other organs for humans (xenotransplantation). Human genes are added and pig genes ‘knocked out’ to avoid rejection of the heart as ‘foreign’.
Genes for transfer
What are the opportunities for gene transfer in pigs? Take as an example the myostatin gene, a naturally occurring mutation which is the cause of double-muscling in Belgian Blue Cattle. When this gene is ‘knocked out’ of laboratory mice, lean growth rate is doubled and ham weight tripled (McPherron et al, 1997). In a pure Meishan line with eight extra pigs per sow per year, a similar knockout might restore the very fat carcase to normal with dramatic consequences for productivity. Other opportunities might include:
§ Lean growth (eg leptin, Igf)
§ Boar taint (eg androstenone, skatole)
§ Meat quality (muscle proteins)
§ Disease resistance (major histocompatibility complex)
§ Gender determination (SYR on Y-chromosome)
§ Pollution control (eg phytase)
Genes for androstenone and skatole might be knocked out to control boar taint. The SRY region has a major role in determining maleness. Transfer of this onto one of the non-sex chromosomes might allow sires which produce only male or female offspring.
Guelph University has produced pigs transgenic for phytase, which emit less phosphate pollution. In rats attempts have been made to introduce cellulase genes to improve digestion of plant material. This raises the issue of which genotype should be chosen for manipulation: the animal, the fodder plant, or the gut flora. The animal itself should probably be the last choice. An even better long-term solution will be to control the expression of the existing genes and avoid gene transfer altogether. This is a prime area where plant and animal geneticists could work together.
Gene therapy involves a permanent change to the germ line by manipulation in the embryo. Gene therapy attempts to change the individual phenotype by adding genes to the tissues of the live animal for example to replace an enzyme that is missing due to a naturally occurring mutation. These genes are not passed to the next generation. Genes can be introduced by a number of methods from injection to being fired through the skin absorbed onto gold particles.
Researches at the Baylor College of Medicine Houston have recently used this approach to introduce a modified GHRH (growth hormone releasing hormone) gene into the young pig (Draghia-Akli et al, 1999). The DNA sequence in the GHRH gene was altered to greatly extend its life by preventing normal breakdown by protease enzymes. The modified gene was introduced into three-week old pigs by a single injection. An electric current was then passed (electroporation) to integrate the DNA into the cells.
After 65 days the treated animals showed a 37% increase in growth rate with no penalty in body composition. In future the cost of such a treatment might well justify its use in commercial production. While it would not be classified as GM, it would still be open to concerns of ethics and welfare for animals growing ‘unnaturally’ fast.
This major source of loss in pig production has attracted strangely little genetic research. The existence of genetic variation in immune responsiveness within and between breeds has only recently been demonstrated. At Guelph, selection on a BLUP index for high or low immune responsiveness was successful in creating a genetic difference (Mallard et al, 1992). At Iowa State, Durocs showed greater resistance to PRRS virus than other breeds (Halbur et al, 1998).
However, a line with higher immune responsiveness would be expected to show a correlated reduction in lean growth every time the immune system was triggered (Baker and Johnson, 1999). This is a natural defence in response to infection, and is mediated by the cytokines such as interferon and interleukin. One challenge for molecular genetics would be to break this association so that high immune responders could continue to grow normally.
Improvements can be expected in the reproductive technologies such as frozen semen, frozen embryos, and non-surgical embryo transfer (ET). As pigs already have large litters the main benefit of ET will be in establishing and updating nucleus populations, obtaining 100% of the desired genotype with minimum health risk. Cloning the slaughter generation would give 100% uniformity from top pigs in the nucleus, but cloning would need to be repeated each year to keep pace with genetic improvement. Genetic variation would of course need to be maintained in the nucleus to allow continued improvement by selection, which may well cause the nucleus to lag behind the cloned commercial population in genetic merit.
In vitro fertilisation (IVF) together with ET will be enabling technologies for gene transfer. In vitro meiosis to produce sperm and eggs would be the final step that would allow successive generations to be produced entirely in vitro. IVF would be used to produce cloned embryos from which cells would be sampled to conduct marker assisted selection. In vitro meiosis would then give the next generation of sperm and eggs directly from cells of the embryo allowing IVF to be repeated (Haley and Visscher, 1998). Genetic improvement could thus proceed at 5-10 times the pace without the need for any live pigs.
The idea of raising antibodies to remove unwanted X or Y sperm is not new. However a recent breakthrough in Guelph now offers the prospect of a commercial method for doing this within 3-5 years. Based on the knowledge that the DNA on the X chromosome of all mammals is very similar (Ohno’s Law), it assumes that proteins on the surface of the sperm must also be very similar between species. If so, then injecting male porcine material into a male rabbit will not raise antibody to male-specific proteins, but will raise antibodies to non-sex-specific proteins. These antibodies can then be used to remove the non-sex-specific proteins, leaving the male-specific molecules available for retrieval. From these, sex-specific antibodies can be raised by injection into the opposite sex (Blecher et al, 1999). The plan is to prepare monoclonal antibodies that will be added in solution to the semen. Sperm of the unwanted sex can then be made to clump together and filtered off using glass wool.
The main benefit of semen sexing lies in improved feed efficiency and carcase lean content. Compared with a castrate, a gilt has up to 15% better feed efficiency and 3% more lean. An entire boar shows roughly the same advantage again over a gilt. For an industry practising castration, switching to 100% gilts would give an annual advantage of over $60 per sow place. Switching to 100% entire boars could give over $180 per sow place. Single sex production also avoids the need for split-sex feeding, better meeting nutritional requirements and improving uniformity.
Through its owner Ridley Inc, Cotswold has made a strategic investment of $1 million in the Guelph University spin-off company Gensel Biotechnologies Inc set up to commercialise this process. The strategic investor for cattle is Genus, and Monsanto is a collaborator for protein biochemistry. If successful the semen sexing technique will be easy and cheap to apply for on-farm AI collection. It involves no genetic manipulation, and is safe and acceptable to the public. In the short term sex determination probably represents the greatest single potential step forward in pig production and is therefore well worth the risk. The slower method of physically sorting stained sperm by laser (flow cytometry) would not be fast enough to supply the high numbers of sperm per insemination in pigs.
Putting the technologies together
The new technologies could be brought together to revolutionise the pig industry. Suppose for example that a Meishan type containing the myostatin knockout could give single line production with an acceptable carcase and 32 pigs per sow per year. Semen sexing could be used to give 100% entire male offspring, and androstenone/skatole knockouts could remove boar taint. This would immediately improve production efficiency by some 30%.
Another possibility would be to produce surrogate mothers from an F1 cross of say Meishan with Fengjing. These would receive cloned and frozen embryos from a dedicated sire line containing myostatin. Dam lines would be bred only for uterine capacity and the sire lines only for slaughter pig attributes. As well as 32 pigs per sow per year this could give better lean growth with faster genetic improvement of finishing traits.
Of course no-one is advocating these methods, but their adoption by rival industries could pose a threat in terms of lower production cost. On the other hand there could be a huge benefit for the animal and human by manipulating genes for say disease resistance or sustainability.
Genetic modification of plants
Much of the public debate on gene transfer has centred on plants, for which the applications to date have included:
Herbicide tolerance - bacterial genes to provide an alternative metabolic pathway
Resistance to viruses - insertion of plant virus genes
Resistance to insects - genes for toxin from Bacillus thuringiensis (Bt)
Frost resistance - Arctic Flounder ‘antifreeze’ gene
Drought resistance - water retention
Antibiotic resistance - genes used during the cloning process
Terminator genes - introducing sterility to prevent breeding by crossbred offspring
Well known examples would be ‘Roundup Ready’ soya, and ‘Bt’ insect-resistant maize. Future prospects include changes to the amino acid profile of grains to improve nutrient value, for example by increasing lysine content. In addition anti-oxidant levels of say vitamins A, C or E might be increased. To benefit human consumers directly, the ellagic acid content of strawberries might be increased to fight cancer, or the allicin content of garlic might be increased to lower cholesterol. GM plants therefore offer the prospect of better animal nutrition and welfare, but may also be seen as contributing to improved human health as well as relieving starvation in the Third World.
Risks of genetic manipulation
So what are the risks of gene transfer in animals? The nightmare scenario would be genetic modification of pathogens (bacteria or viruses) which might then cause an epidemic, perhaps lethal, in man or across a range of species. Production of toxins or allergens would be relatively easy to avoid on any scale by judicious trials in advance of widespread release. Any accident causing sterility would of course be self-eliminating. Risks to animal welfare clearly exist from production stress or physical malfunction. While these risks are very small, those involving pathogens at least have the potential to affect a much larger proportion of the population than say mountaineering or air travel.
The real risk surely lies in the fact that our knowledge of the molecular mechanisms of inheritance, and particularly of the way genes interact with each other, is still rudimentary. Sexual reproduction is set up to produce new genetic variation and is therefore inherently unpredictable. It is therefore difficult to predict the consequences of inserting a gene with 100% certainty. In the event that some undesirable genetic transformation did occur, there is the danger that the knowledge needed to avert a disaster would simply not exist. The prion factor causing BSE and the absence of a solution to cancer would be seen by some as gaps in knowledge. As soon as there is complete understanding of the mechanisms of gene action, the risk will evaporate.
The pig industry will continue to compete on the low cost per kilo of lean meat. With the move to larger more integrated production pyramids serving specific needs of retailers, there will be increased emphasis on the quality and uniformity of the meat. The way meat is produced will come under closer public scrutiny, including ethics, naturalness, traceability, the environment, sustainability and animal welfare.
In the pig industry today arguably some 20-30% of genetic potential is not realised on the farm. There are several reasons for this. The first is poor herd health, with multifactorial diseases such as porcine respiratory syndrome in which PRRS, influenza and pneumonias act in concert. A second reason is incomplete knowledge or application of the nutritional needs of the modern improved genotype. A third reason is poor husbandry and particularly overstocking leading to lower feed intakes. Genetic objectives are therefore twofold: to continue to raise genetic potential, but also to increase the probability that this potential can be realised on the farm.
Future genetic strategy
So what strategy should be adopted for future genetic improvement and research? There are three components:
· Maintain maximum rates of improvement using existing BLUP methods.
· Close the gap between genetic potential and actual performance on farm.
· Secure access to the new technologies and be prepared to quickly deploy them if required to defend the competitive position of the industry.
To close the achievement gap Cotswold has a large investment in better understanding the nutrient requirements of modern genotypes, especially of the young growing pig where energy intake may be limiting lean growth. There is also a need to increase research on the immune system of he pig.
To access the new technologies Cotswold has developed an international science base run by its five full-time PhD geneticists. As well as in-house research this is supported by research fellowships and alliances, collaborative research agreements and research contracts with biotech companies, and shareholdings in semen sexing company Gensel and DNA typing company Rosgen. The aim is to be closely informed on new ideas and technology with the opportunity for early experimentation where appropriate.
Conclusions: the next 25 years
Through gene mapping, gene therapy, gene transfer, control of gene expression, and manipulation of reproduction, the revolution in molecular biology can only be expected to accelerate. Ultimately our knowledge of genetics must reach a point where meat can be produced without a live animal. The extent to which this technology is adopted by the livestock industry depends on the balance among three factors:
1. The cost and capabilities of the technology itself
2. The nature of the competition
3. The wishes of society
Irrespective of livestock, the technology will be driven forward by biomedical applications. Competition will come from other livestock species, novel sources of food, or from livestock industries less scrupulous about the risks of unproven technologies. Society will have to draw the line somewhere, and indeed judge whether there is to be a livestock industry at all.
The course of the next 25 years is impossible to predict. The only sensible strategy therefore is to ensure that the industry keeps all its options open by retaining access to the new technologies, and that it can respond quickly to changes as they arise. The industry must be prepared to join the public debate on biotechnology and to ensure that politicians are correctly informed. Trade barriers may be needed to restrain imports of meat produced by methods outlawed at home.
In the short term the goal for the pig industry remains the production of high quality lean meat at minimum cost, but with increasing regard for public approval, animal welfare, wholesomeness and the environment. Vertical integration of technology if not ownership will continue in ever larger meat production pyramids. The breeding companies will be integrators of a range of technologies, providing a package of genetic services to the food chain.
Meantime, it is important to realise that modern selective breeding programs have been successful, cheap and safe, and that for the present there is little pressure to introduce biotechnology. The industry should use this period wisely to carefully evaluate its position in relation to the new technologies and the public.
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