www.publish.csiro.au/journals/ajea Australian Journal of Experimental Agriculture, 2004, 44, 1105–1111

Commercial aspects of cloning and genetic modification in cattle

I. M. LewisB,C, A. J. FrenchA,C, R. T. TecirliogluA,C, G. VajtaF, A. E. McClintockA,B,
K. R. NicholasD, K. A. ZuelkeE, M. K. HollandA,C and A. O. TrounsonA,C
Research Centre for Innovative Dairy Products (Dairy CRC),
Level 1, 84 William Street, Melbourne, Vic. 3000, Australia.
BGenetics Australia Cooperative Ltd, Woolpack Road, Bacchus Marsh, Vic. 3340, Australia.
CCentre for Early Human Development, Monash Institute of Reproduction and Development,
Monash University, 27–31 Wright Street, Clayton, Vic. 3168, Australia.
DDepartment of Zoology, University of Melbourne, Parkville, Vic. 3010, Australia.
EUnited States Department of Agriculture, ARS Germplasm Laboratory, Belstville, MD 20705, USA.
FSection of Reproductive Biology, Department of Animal Breeding and Genetics,
Danish Institute of Agricultural Sciences, Tjele, Denmark.
GCorresponding author. Email: ilewis@genaust.com.au

Abstract. A range of potential commercial applications of cloning and genetic modification in cattle has been
suggested over the last decade. It includes the rapid multiplication of elite genotypes, production of valuable human
proteins, altered production characteristics, increased disease resistance and milk with improved nutritional value
and processing capabilities. However, an economic return from the sale of product is far from reality in any of these
areas. One impediment to achieving economic sustainability is the extremely low efficiency in producing healthy
offspring from transferred cloned embryos. Other significant impediments are societal concerns surrounding such
technologies, animal welfare issues and regulatory requirements. This review will focus on current biological
limitations and technical capabilities in commercial settings, the changes required to allow the production and sale
of products at economically sustainable levels, cryopreservation and the progress towards automation of cloning

Introduction The birth of normal offspring that remain fit and healthy
Amphibians such as frogs were cloned in the 1950s in the long term is the only valid measure of technical
(Briggs and King 1952; Gurdon et al. 1958). The first success from cloning. In addition, commercial success will
successful cloning of livestock, using embryonic cells, was require the production of animals that have added value as a
reported in sheep in the 1980s (Willadsen 1986). In the result of cloning. Reported rates of healthy calves born from
1990s, successful somatic cell cloning resulted in the birth of cloned embryos are extremely low in cattle in studies with
Dolly (Wilmut et al. 1997), probably the most famous animal meaningful numbers, with 95–99% of attempts failing to
ever born. produce healthy offspring (Willadsen et al. 1991; Stice and
Reports on the possible future applications of cloning and Keefer 1993; Wilson et al. 1995; Garry et al. 1996; Hill et al.
genetic modification in livestock are numerous and include 1999, 2000; Kubota et al. 2000; Pace et al. 2002; Wells et al.
those by Willadsen et al. (1991), Anderson and Seidel
2003). Significantly higher success rates are possible under
(1998), Stice et al. (1998), Ziomek (1998), Brink et al.
specific conditions with certain cell lines and quite low
(2000), Niemann and Kues (2000), Lewis et al. (2001a),
numbers (Kato et al. 1998; Wells et al. 1999).
Seidel (2001), Dinnyes et al. (2002), Renard et al. (2002),
Faber et al. (2003), and Galli et al. (2003). For wide-spread agricultural applications, for example
In the late 1980s, cloning research in livestock was driven those that lead to genetic gains equalling or exceeding those
commercially by the lure of large genetic gains from the from artificial insemination (AI) in the dairy industry,
production of numerous low cost, cloned elite livestock quantum leaps forward in cloning technologies are required.
(Willadsen et al. 1991; Wilson et al. 1995). More recently, For such applications, there are 2 major challenges: first, to
the demonstrated ability to link somatic cell cloning with produce technical outcomes similar to the current AI
genetic modification revealed significant potential in both technology (healthy, genetically superior females) at a
the biomedical and agricultural fields (Schnieke et al. 1997; similar price; second, to produce industry outcomes of
Wilmut et al. 1997; Cibelli et al. 1998; Zuelke 1998; Pace significantly increased genetic gains over and above those
et al. 2002; Brophy et al. 2003). achievable by AI.

© CSIRO 2004 10.1071/EA03239 0816-1089/04/111105

because of the significant beneficial effects of 26 million beef cattle (Henderson 1998). Then purification of the product they secrete. The cattle are heterosis in F1 crosses. More importantly. Cloned embryos would be of known to disease or that produce milk with added value. survive neonatally and there is a minimum number of Genetically modified cattle that have increased resistance abnormal calves born. Even though clonal lines of animals will show terminal sires (whose offspring are used for meat) in natural considerable phenotypic variation for such traits. One product would be clonal lines of genetically in the dairy industry today (McClintock 1998). produced for less than $50 (Lewis et al. This would lead to significant conformation and production traits are about 25% heritable genetically based production gains. Lewis et al. are suggested as the percentage of healthy calves born per embryo implanted potential commercial uses of the technologies. Artificial breeding businesses are to be built on beef cloning. less than 5% of such Potential agricultural applications of cloning embryos currently result in healthy calves born. a number of exceptional herd annually as natural service sires at average prices . This will depend on the value of the final frequently a significant proportion of abnormal calves born product. especially in economically important reared under extensive conditions with extremely low traits such as fertility and longevity. some other advantage such as efficacy or safety. would be multiplied and the embryos sold in the same way as Alternatively. Cloned beef bulls will need to embryos from a clonal line whose production characteristics compete at realistic prices if sustainable commercial were known in a range of environments. between 1 and 10% of in traditional mating schemes [such as AI and multiple cloned embryo transfers result in live births when significant ovulation and embryo transfer (MOET)] to generate large numbers of cloned embryos are transferred. The northern beef herd (north of One important product would include cloned crossbred the tropic of Capricorn) contains over half of Australia’s embryos. can be nutraceuticals. are purchased currently for about testing will require fewer records for the same level of $2000–5000. be it valuable pharmaceuticals. still must be the ‘best’ clonal line (using the same selection parameters as commercially sustainable. following transfer into recipient cows. disease-resistant and of neonatal deaths (for review. For For such dairy applications to be realised. are exceptions. Each clonal line human proteins will have less stringent economic efficiency (50–100 clones from each ‘exceptional’ crossbred) would be requirements because of the potential value of the end tested in a variety of environments (that is on a number of product. 2002). the cost of producing the required different farms) in the same way as bulls are progeny tested transgenic animals and the subsequent cost of extraction and today with up to 100 daughters each on up to 50 farms. in beef production systems. However. The approaches 50% and the percentage of abnormal calves born production of transgenic founder animals that are then used is reduced to near zero. cloned embryos example. The production of transgenic cows secreting valuable crossbred animals would be cloned. Beef bulls for use as or less. the total production costs those used for daughters of bulls being progeny tested today) must be lower than those in current production systems. clone mating herds. cloned embryos in Australia has a unique opportunity for the use of cloning a straw in place of semen. although this can numbers of offspring with the desired genetic make-up. cloning technology will have selection and production of superior clonal lines of dairy significant implications for genetic gains in the beef females in much the same way that bulls are progeny tested industry. In a scenario that stocking rates. may vary between 0 and 100% (high rates have been achieved allow relatively low efficiencies to be commercially with small numbers of transfers). such as in sex. Most elite bulls for natural mating. such high prices. 1998). there is acceptable. genetically identical elite animals Beef bulls: natural service sires Quantitative geneticists have calculated that large genetic If and when the considerable biological and technical gains could be made if unlimited cloning allowed for the hurdles can be overcome. although widely Under this scenario. Currently. so $40–50 per embryo is commercially viable as long as the nutraceutical and milk processing areas. the post-translation folding of pharmaceutical would need to compete with frozen semen from elite dairy proteins produced in in vivo mammalian systems may result bulls that currently delivers about 50% pregnancy and in increased efficacy over those produced by current calving rates per insemination of one dose of semen costing methods such as transgenic microbial cultures or in vitro cell about AU$20 per dose. dairy farmers would purchase cloned publicised. precluding any form of large-scale artificial allowed for unlimited cloning (with all the biological and breeding.1106 Australian Journal of Experimental Agriculture I. Dairy females: large numbers of low cost. companies would then sell cryopreserved. Much higher prices are occasionally paid for accuracy as progeny testing of bulls (McClintock 1998). Over 97% of calves born to AI culture systems. However. animals or cow’s milk that delivers significantly improved Results from our laboratories demonstrate that cloned processing capabilities or increased health in the form of blastocysts. appearing morphologically normal. That is. the product must be more effective or have semen from the top proven bulls is sold today. M. However. stud animals. Over 100000 bulls are bought into this northern societal constraints overcome). see Renard et al.

increased . large offspring syndrome (LOS). Capecchi 2000. placental abnormalities. the cost per healthy bull calf at weaning is evolving to cope with these new production methods. normal animals Biological and technical limitations are minimum requirements in the dairy industry. Therefore. the most important of which is nuclear reprogramming. Public about $8500. 2000). producing pharmaceuticals in their milk has been reported With the current success rates achieved in our (Brink et al. calves implantation rates following embryo transfer compared with with an additional α S1 casein locus have been generated (non-cloned) in vitro-produced embryos and higher foetal with the aim of increasing casein production in milk (Zuelke loss to term following implantation. Complete Cloned. Currently. significantly increased therapeutic proteins in their milk (Ziomek 1998). These include: decreased capabilities (Brophy et al. even at current efficiency levels. Already. 2000) and large commercial investments have laboratories. a technique used to produce numbers of the top sires (up to 100 of each) can be cloned transgenic animals expressing significant levels of and used as natural service sires. 2003). allows a relatively to transgenic animal production is a significant improvement accurate assessment of their economic merit. the price Therefore. Regulatory requirements are still cloned embryos. combined with Small numbers of animals producing high value product information on the dollar returns from the sale of progeny Although still relatively inefficient. significant increases in the efficiencies of technologies in animals for producing human medicines are producing healthy cloned offspring are required before more positive than for the use of such technologies in food economic application of the technology will be possible in production and other agricultural purposes (Cormick 2003). Recently. the birth of healthy offspring depends on the abnormal calves born. 1998. As described above. Daniels et al. French et al. altered milk composition and milk transfer is likely to be faulty epigenetic reprogramming of processing efficiency. cloned cattle capable of producers. the lack of progress in in our laboratories under optimum conditions (using certain getting pharmaceuticals produced by these technologies to selected cell lines) of $8500. If significant on pronuclear microinjection.Cloning and genetic modification in cattle Australian Journal of Experimental Agriculture 1107 between $1500 and $5000. Genetic information through the Potential biomedical applications beef animal evaluation system Breedplan. using certain selected cell lines that give been made in this technology in the hope of significant healthy offspring at rates of about 5% per recipient receiving commercial returns. Other suggested future possibilities include the appropriate demethylation/remethylation patterns increased disease resistance and the production of transgenic required to support successful embryogenesis (Campbell bulls that produce female or male only offspring (Faber et al. attitudes towards the use of cloning and transgenic Therefore. the beef cattle industry. Pregnancy rates approaching 50% following embryo transfer Limitations to realising the potential and the birth of a high percentage of healthy. 1999. the cloning approach from these natural service sires for meat. returns from increased genetic gains are possible to the production of transgenic. ongoing pregnancy rates restrict commercial opportunities. meiotically heritable changes in gene function that cannot be only small numbers of founder animals will be required. The present low levels of success from cloning severely following cloned embryo transfer. This explained by changes in DNA sequence (Cezar 2003). for review see Cezar (modified) bulls will allow the rapid dissemination of the 2003). The primary cause of abnormalities from nuclear production traits. 2003). approach will allow the technology to be used productively Methylation of DNA is the major epigenetic system even though the biological and technical impediments to regulating development and is usually associated with gene cloning prevent the production of large numbers of cloned silencing. transgenic animals in agriculture: small numbers reprogramming is required to enable the transferred genome of founder animals to provide the correct spatial and temporal patterns of gene The technology is moving towards the production of expression required for normal development (Campbell genetically altered cloned animals that have improved 1999). and the demise of several companies involved in these areas indicate Quantum leaps forward are required for large-scale dairy that considerable hurdles remain. 2002. Following nuclear are too low and there is an unacceptably high percentage of transfer. Production of genetically improved the donor nucleus (Jaenisch et al. 2003). Reprogramming of the donor nucleus must induce animals directly. it is possible that currently paid for natural service sires is $1500–5000 an economically viable industry could eventuate from this compared with the current cost of production of cloned bulls use of the technology. Epigenetics is the study of mitotically and/or improved genetics through existing AI systems. hydrallantois. However. applications for both animal welfare and economic reasons.and κ-casein for improved milk processing procedures in animals. despite early promise (Ziomek 1998). market. In our laboratories. genetically modified cows have been A wide range of abnormalities has been reported produced with significantly altered ratios of the milk following the transfer of embryos derived from cloning proteins β. successful completion of a chain of events.

1995. later developed musculo-tendonous problems leading to difficulties standing and walking. bacterial infection. 1995. techniques for automation and livestock and other animals (Cibelli et al. including those produced by when transferring in vivo-produced embryos. 2000). Wagtendonk-de Leeuw van et al. M. although at depend both on the development of safe and efficient lower frequencies. Sixteen healthy offspring are sensitive to perturbations (Cibelli et al. In addition.1108 Australian Journal of Experimental Agriculture I. Sinclair et al. Lewis development of commercial applications of cloning and GM et al. However. a very different attitude exists for the production of et al. can lead to nuclear transfer. solving the biological limitations that result in poor cloning Given that some widely used pharmaceuticals such as efficiencies and the birth of an unacceptably high number of insulin are produced almost entirely in transgenic microbial abnormal calves remains the major priority. 1995. the tools and genetic modification of both plants and animals (Cormick techniques used in assisted reproduction including in vitro 2003). 1999. concerning these animal biotechnologies. genetically modified animals from one transfected 2 healthy offspring were produced that survived to maturity cell line. 1992. 1996. Such advances. peri-natal morbidity and mortality. et al.Young et al. Recent results from offspring. 2000. Cibelli et al. Hasler et al. it appears that society accepts the transgenic of post-natal vigour and lymphoid hypoplasia (Willadsen production of potentially life-saving human medicines. 2000. Peterson With all embryo transfer involving livestock. 2002). Hill et al. 1995. Cryopreservation of cloned embryos 1995. percent (25/160) of recipients receiving fresh somatic cell 2002). lack systems. cryopreservation of transferable stage embryos is critical for 1999. 2002). compared with in vivo-produced 1996. technologies in cattle for large-scale agricultural use will Similar abnormalities have been reported. 2001). The 1999. Renard et al. outcomes (pregnancy rates and the birth of healthy offspring) The epigenetic mechanisms required for the birth of were achieved with fresh and vitrified embryos. Behboodi et al. Whether or bovine embryos. 1997. Even those seemingly normal cloned offspring born cloned embryos became pregnant and one healthy calf was may have latent abnormalities resulting from faulty born that survived to maturity and produced a healthy reprogramming (Rideout et al. currently employed in cloning. Nineteen percent (15/78) of recipients receiving our laboratories support this observation. Similar such as IVF. Society miniaturisation are transforming a number of areas of generally has a negative attitude to cloning in animals and to biology (Beebe et al. 1998. successful cryopreservation of cloned embryos. and McMillan 1998a. large-scale applications of the technology. Garry However. Farin and Farin 1995. seemingly normal for the first weeks and months of and were fertile. 2000. have proven difficult to cryopreserve by increased birth weights (Wilson et al. 1998b. In both North recently been described (Beebe et al. However. 1991. Wilson et al. which occurs In vitro-produced embryos. results from our laboratories not the epigenetic perturbations that are likely to be the cause using the open-pulled-straw vitrification techniques of Vajta of significant problems in cloning are responsible for et al. including America and Australia. 1999. Jacobsen et al. 2000). Large-scale agricultural applications for cloning will Limits imposed by societal perceptions eventuate only if the current biological limitations are solved There are real and significant animal welfare issues and cloned embryos can be produced that have markedly relating to the birth of an unacceptably high percentage of increased developmental potential. food for human consumption (Cormick 2003). Garry et al. Campbell et al. In Europe. Wells et al. necessitating euthanasia Progress towards automation of cloning (unpublished observations). 1996. and South America the degree of public angst is considerably combined with other developments described below that are less. A further technical abnormal offspring from cloning. from in vitro fertilisation (IVF)-derived technologies and on a significant change in the way society embryos. Lewis et al. . suggest that significant are presently taking submissions ahead of legislation automation of the cloning technologies is possible. (1997. 1995. periods (up to a few hours) of in vitro culture. 1998) indicate that these methods allow the abnormalities seen in other in vitro embryo manipulations. remains to be demonstrated. 1998. Pace et al. placental abnormalities (Walker et al. the spectre of requirement is for efficient and cost-effective methods of human cloning has had a significant negative impact on the cloned embryo production. Even short cost-effective. traditional techniques. However. the relevant authorities in several western designed to simplify the technically cumbersome techniques countries outside Europe. 2002). Wells et al. Six out of seven vitrified somatic cell cloned embryos became pregnant and cloned. However. 2002). life. Wagtendonk-de Leeuw van et al. Wilmut et al. way society views the application of this technology to Over the last decade or so. including the large offspring syndrome and in general views them. public fear has led to strict years. Kubota et al. The development of microfluidic devices to simplify legislation surrounding both animal and plant cloning and the steps involved in the in vitro production of embryos has genetic modification (GM) in many countries. with the degree of concern depending partly on embryo production and cloning have changed little over the geographical location. 2000.

Walters E (2002) Microfluidic manual aspiration and automated harvesting.0%) Zona enclosed (traditional) 25 6/25 (24%) 2 (8%) 2 (8%) Simplified cloning procedures Conclusions Traditional cloning techniques are complicated and time Present outcomes from the cloning of livestock are consuming. 2002).9%. over many technology for assisted reproduction Theriogenology 57. Genetics Australia Cooperative Ltd and of each possible step in cloning is desirable to maximise the Victorian Institute of Animal Science. thousands of oocytes.5%) 6 (5. sheep and pigs reported worldwide (for review somatic cells. Seidel GE (1998) Cloning for profit. this represents several million oocytes efficiencies are possible. Even with dramatically improved cloning efficiencies. the general public and regulators. Although manual oocyte aspiration from abattoir. 2003) confirm ways of reducing epigenetic perturbations to levels which that a significant step towards automation of cloning has result in the birth of healthy offspring in numbers that are been made. developed in our laboratories that significantly decreases labour time and costs without affecting overall blastocyst References Anderson GB. 2002) allow cautious up to 100 000 animals (McClintock 1998). the average number of oocytes doi:10. Simplified techniques for cloning embryonic arrival of Dolly demonstrated that somatic cell cloning in cells were developed by Peura et al. Wheeler M. Even the transfer methods. For large-scale agricultural production of cloned embryos (Booth et al. doi:10. of recipients (%) % of recipients used) (as a % of recipients used) Zona free 101 52/101 (51. (1998). 2002).1016/0093-691X(95)00172-5 .2 and 31. acceptable to producers. Anderson GB. However.Cloning and genetic modification in cattle Australian Journal of Experimental Agriculture 1109 Table 1. An automated.1126/SCIENCE. Other reports using similar commercial production of transgenic cattle producing simplified zona-free cloning techniques for the successful certain valuable human proteins. although the number of calves surviving current low efficiencies are likely to be adequate for the long term was lower (Table 1). Coupled with the observation that mammalian development Automated harvesting of oocytes normally is somewhat tolerant to epigenetic abnormalities The optimum size of genetically ‘identical’ clone family (Rideout et al. Murray JD (1995) Birth of large calves that developed day 7 blastocysts rates were 27. development rates. Zeringue H. zona pellucida-free techniques seemingly healthy and fertile cloned livestock. The techniques do not require expensive see Cibelli et al. since they are not embryos that can be produced in a given time (Lewis et al. collected per ovary were 10. Science 280. respectively from in vitro-derived bovine embryos. doi:10. In controlled replicate trials comparing 1400–1401. large numbers of cheap oocytes will be Acknowledgments required for an economically viable industry based on We acknowledge the numerous scientific. and require expensive equipment and highly disappointing. and the Medrano JF. significantly reduce the skill levels generation clones appear to be epigenetic and erased from required and significantly increase the number of cloned one passage through the germline. Vajta et al. produced and the techniques gave equivalent pregnancy rates It is unlikely that all epigenetic faults arising from these and live births when compared with traditional nuclear technologies will be eliminated all of the time. zona-enclosed NT techniques using the same donor cell line Nuclear transfer No. 125–135.280. the task will be to find significant numbers of offspring (Oback et al.1016/S0093-691X(01)00662-8 Behboodi E. BonDurant RH. goats. 2001. 2001). of recipients No. including for embryonic cells have been successfully modified for cattle. zona-free nuclear transfer (NT) techniques and traditional. technical and cloning. Cargill SK. transmitted to offspring produced following sexual 2001b. (Lewis et al. whose dedicated overall efficiencies for economically sustainable large-scale work over the last 12 years has helped significantly to agricultural applications. There are now hundreds of laboratories. mechanical progress the technologies discussed in this review towards method of harvesting oocytes from ovaries has been large-scale application.5368. 2001) and applications of cloning in livestock. these simplified.9%) 2 (2. Abnormalities seen in first micromanipulators. Kreuscher BR. 2003). Healthy offspring have been reproduction (Shimozowa et al. Comparison of pregnancy rates.1 and 7. automation and Development.3 respectively. (unpublished observations).1400 oocyte recovery and blastocyst development rates between Beebe D. In our mammals was possible. administrative staff at the Monash Institute of Reproduction derived ovaries is relatively quick and efficient. pregnant at day 30/ Live births (as a Calves surviving >6 months method (2–3 embryos per recipient) no. 227–232. only 7 years have passed since the skilled labour. live births and calf survival between simplified. Theriogenology 44. At current optimism that the required quantum leaps forward in cloning cloning efficiencies. 1998). new approaches to nuclear lines for the Australian dairy industry is likely to be large — reprogramming (Hakelien et al.

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