Review on the Current Status of
the Extent and Use of Cloning in
Animal Production in Australia
and New Zealand.
Reviewer: Professor R. F. Seamark
for SA Consulting Pty Ltd
March 2003
CONTENTS
CONTENTS
...................................................................................................................................II
ACKNOWLEDGEMENTS ....................................................................................................................... III
EXECUTIVE SUMMARY ...........................................................................................................................1
INTRODUCTION
....................................................................................................................................2
SECTION 1
LIVESTOCK CLONING: BACKGROUND INFORMATION ...............................................................3
A)
LIVESTOCK CLONING TECHNOLOGY ........................................................................................................3
B)
LIVESTOCK CLONING: STATUS AND FUTURE TRENDS ...........................................................................10
SECTION 2
LIVESTOCK CLONING IN AUSTRALIA AND NEW ZEALAND .....................................................17
A)
SPECIES AND NUMBERS OF ANIMALS CLONED ......................................................................................17
B)
CRITERIA USED IN SELECTING ANIMALS FOR CLONING..........................................................................18
C)
LIVESTOCK CLONING PROCEDURES IN USE IN AUSTRALIA AND NEW ZEALAND. ...................................20
D)
FOOD PRODUCTS FROM CLONED LIVESTOCK, ITS USE AND EVALUATION ..............................................21
E)
DIFFERENCES BETWEEN CLONES AND THE DONOR ANIMAL ...................................................................22
SUMMARY COMMENTS ..............................................................................................................................23
ADDITIONAL COMMENTS .......................................................................ERROR! BOOKMARK NOT DEFINED.
GLOSSARY
..................................................................................................................................24
APPENDIX 1
Health Status Of Cloned Livestock 1991 ..............................................................27
APPENDIX 2
Report on safety of food products from cloned cattle by the Japan
Research Institute for Animal Science in Biochemistry and Toxicology ...........28
LIST TABLES
..................................................................................................................................35
LIST OF FIGURES ..................................................................................................................................35
REFERENCES
..................................................................................................................................36
II
Acknowledgements
I wish to gratefully acknowledge the willing collaboration of members of the cloning teams
in Australia and New Zealand and their help in compiling this report.
Disclaimer
The information presented in this report has been compiled from the published reports cited
in the review and from interviews or correspondence with the individual researchers named
in the Acknowledgements. Every effort has been made to ensure the accuracy of the data
reported. Views expressed in any commentary on the data are those of the reviewer unless
otherwise stated.
III
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
Executive Summary
Cloning, is the propagation of genetically exact duplicates (clones) of an organism by
means other than sexual reproduction. Cloning of mammals is currently achieved by
nuclear transfer (NT), that is replacing the genetic material of an unfertilized ovum
(oocyte) with that of an embryonic or somatic cell taken from the animal being cloned,
and then transferring the reconstituted embryo to a surrogate mother for rearing.
Globally, all the major livestock species have now been cloned but animal-cloning
technology is still best viewed as being in its infancy. Only 0.1-5 % of cloning attempts
yield viable offspring due to complications caused by incomplete reprogramming of the
nucleus following transfer, imprinting failure and aberrant expression of early
development genes. Furthermore there are concerns about the health status of
surviving animals. Given the public interest in human cloning, this has served to excite
debate and initiate a flurry of regulatory activity worldwide relating to animal welfare and
food safety issues. Researchers are optimistic these concerns can be addressed and the
prospects for medicine, biotechnology and agriculture, offered by this revolutionary
technology, will be realized.
There are six groups with active livestock cloning programs in Australia and New
Zealand (AgResearch, Hamilton, NZ; Dairy CRC, Vic.; CSIRO, NSW; SARDI, SA;
BresaGen, SA, and Clone International, Vic.), and these have so far produced a total of 32
cattle, 11 sheep and 2 pig clones by NT from a variety of cells of embryonic, fetal and
somatic (adult) origin. The animals presently targeted for cloning are mostly of scientific
value and many are genetically modified. Outcome assessments concerning animal health
and welfare or data on comparative food values of clones versus conventionally bred
livestock are likely to remain, at least for a 1-2year time period, extremely limited.
Given the expected genetic similarity of a clone to the parent animal, there is no known
scientific reason to expect that the production traits of livestock clones (and any
offspring), will differ in any major way from the parent and, allowing for any
differences in husbandry, to expect that any food or other products derived from the
clone would differ in nutritional value and other food qualities; although this
supposition has yet to be fully validated experimentally.
The close links in the public mind between potential foodstuffs arising from cloning
technology and genetically modified animals is likely to generate concern that may
demand a conservative regulatory approach.
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Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
Introduction
This review complements and extends a recent report to the National Research Council of
the National Academies (NRC, 2002), defining ‘Science-based concerns associated with
the products of Biotechnology’ (2002), and a pending WHO report on ‘Modern food
biotechnology, human health and development; An evidence based study’ (WHO, 2003;
[email protected]), to address specific issues concerned with application of cloning
technology in Australia and New Zealand and the use of clones or their progeny in food
production. Particular attention is focused on cloning through nuclear transfer, a
technology that allows cloning of adult animals. The review seeks to establish whether
there is science based evidence that food products derived from cloned animals threaten
to defile consumer expectation that 1) all food and food products, for human or animal
consumption, should be free of harmful agents, chemical or biological, or 2) differ in any
regard in nutrition value or other food qualities from product derived from conventionally
bred animals. Given the heightened public interest in cloning technology generally,
evidence has also been sought concerning any aspect of cloning technology as applied to
commercial animal production for food consumption, which might be viewed as socially,
politically and/or ethically contentious.
The Review is presented in two Sections;
Section 1 provides background information on cloning technology, its development and
present and prospective global use in human and animal food production.
Specifically the first section provides:
a) an introduction to, and technical description of cloning technology, together with,
b) an overview of its current global status, the challenges and future trends
Section 2 presents a detailed review of the status and proposed development of livestock
cloning in Australia and New Zealand with particular reference to use of cloned animals
or their offspring in food production.
Specific data is presented for Australia and New Zealand on the:
a) livestock species being cloned,
b) criteria used in their selection;
c) cloning procedures in use,
d) nature of the products derived from cloned animals together with any data
concerning associated food quality and/or compositional data, and
e) differences between cloned and the donor animals
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Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
Section 1
Livestock Cloning: background information
a) Livestock cloning technology
Introduction
Cloning was first introduced into animal production practices in the early 1970s as an
adjunct to embryo transfer programs. Referred to as ‘embryo-splitting’, the technology
was simple, viz. an embryo was cleaved in two and the halves co-transferred to the
surrogate mother to produce genetically identical twins. This elementary procedure
gained wide acceptance in commercial embryo transfer programs both as a means of
increasing the yield of embryos and improving the chance of a successful pregnancy,
gained from having more than one embryo, without introducing the risk of freemartins or
other complications arising from the co-transfer of genetically dissimilar embryos.
Attempts to increase the number of clones by cleaving the embryo into smaller pieces had
only limited success due to limited viability of the fragments. Multiple cloning of
livestock embryos, awaited the later development of nuclear transfer (NT, Willasden,
1986); a revolutionary technical advance that, within a decade, would make possible the
first cloning of an adult animal (‘Dolly’, Wilmut et al 1997); thus offering real prospects
for a future of unlimited asexual replication of superior gene-stock.
Nuclear transfer (NT): NT as its name implies, allows transfer of the nucleus of one cell
(donor) to another (recipient), usually a mature mammalian egg (oocyte), whose
chromosomes have been ablated (cytoplast). As so dramatically demonstrated by
Campbell et al., 1997, even when the donor cell is from a highly differentiated somatic
tissue (mammary gland), the unique environment provided by the cytoplast can restore
the capacity of the donor cell genome to fully direct the development of the reconstituted
embryo and create a clone of the donor animal. The success of NT is thus directly related
to the capacity of the cytoplast to reprogram a donor cell nucleus to correctly express the
genes required for early development. Experiments with the cytoplast of other cells are
ongoing, but thus far, the cytoplast of a mature oocyte remains the only one identified
with this special capacity (Fulka et al., 2001).
Embryo-Cloning: The first application of NT to cloning livestock embryos was made by
Willadsen (1986) who successfully cloned early stage pre-implantation sheep embryos,
using undifferentiated cells from the blastocyst (blastomeres) as donors. Initially cloning
was assumed to be restricted to such undifferentiated cells. This limited the number of
potential clones from an embryo to about 60, the number of cells generally found in
embryo at the morula (mulberry)/ blastocyst stages, i.e. the stages before cellular
differentiation, leading to the formation of trophectoderm and an inner cell mass (ICM), is
initiated. However, as was subsequently demonstrated with both cattle and sheep, it is
possible to increase the potential yield of clones from an embryo by serial cloning, that is
by using a cloned-embryo as a source of donor cells to generate more clones which can
themselves be cloned (see for example Peura and Trounson, 1998). Embryo cloning
technology was enthusiastically adopted by livestock breeders, particularly by the stud
cattle industry, but struggled commercially due to low success rates and calving problems
due to oversized calves at birth (Cloned Calf Syndrome); albeit there are over 10,000
embryo-cloned calves registered with the Holstein Breeders Association in the USA
(Norman et al., 2002)
3
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
Coincident with the pioneering studies by Willasden and his colleagues an alternate, even
more exciting route towards multiple embryo cloning was also being developed in the
UK, based on Embryo Stem cell (ES cell) culture. This approach became feasible
following recognition that each ICM cell still retained totipotency, that is its full capacity
to direct the development of an embryo, and that is was possible to establish conditions
for in vitro culture of ICM cells, which allowed their numbers to be increased without loss
of totipotency, (Robertson, 1987). Furthermore it proved possible to genetically
manipulate the genome of ES cells in culture using the full range of recombinant DNA
techniques available: genetic modifications already achieved via ES cells include the
addition, deletion and/or replacement of genes (Bronson and Smithies, 1994): inclusion of
a cell culture step in the cloning of somatic cells allows application of a similar range of
genomic modifications including, as shown recently, the addition of whole chromosomes
(see Robl et al., 2003). The development of ES technology excited huge scientific interest
as it provided a direct link between molecular genetics and reproductive technologies; and
a nexus that continues to power a series of discoveries in the biosciences and quantum
increases in knowledge, particularly in developmental genetics.1
Adult cell cloning: Until the production of ‘Dolly’ in 1996 the use of adult cells in
livestock cloning was not thought possible, as it was generally assumed that adult somatic
cells would lose totipotency as they underwent differentiation. The production of Dolly
from adult somatic tissue clearly challenged this assumption. As reviewed below, six
years after ‘Dolly’ somatic cell nuclear transfer still remains one of the most exciting of
the many recent developments in biotechnology. Adult animals of all the major livestock
species have now been cloned (Cattle, Cibelli et al., 1998, Pigs, Betthauser et al., 2000,
Onishi et al., 2000), and there is already enough developments to confirm, that with its
capacity to provide a direct link between reproduction and molecular engineering, adult
cloning can be used to capture a wealth of new biotechnological opportunities in both
medicine and agriculture, opportunities first recognized with the development of ES cells
and of a scope which is now generally appreciated as ‘limited only by the imagination’
(e.g. Niemann & Kues, 2003).
However, from an agricultural perspective, whilst there may be ‘proof of concept’ of the
feasibility of cloning adult livestock animals, the technology is still very much in the
development phase with commercialization prospects in a 1-3 year time frame, limited to
programs focused on producing small numbers of high value animals for breeding
purposes and transgenic animals producing high value biomedical products or
preservation of rare breeds (e.g. Enderby Island cattle, Wells et al., 1998, Mouflon sheep,
Loi et al., 2001). Broad-scale use of cloning procedures for generating livestock for use in
feedlots or dairies or other food production enterprises awaits significant improvement in
cloning technology.2
1
Cloning from ES cells is so far limited to laboratory studies using mice and true ES-cells, i.e. those able to
generate both somatic and germline tissues (thus confirming their totipotent as apposed to pluripotent
status) have so far only been generated from two inbred strains of mice. ES-like cells have been generated
from livestock (see for example Cibelli et al.,) but most somatic clone donor cells are still derived through
primary cell cultures that exhibit a high degree of variability in the cell population and have a limited
lifespan
2
As demonstrated in the seminal experiments in amphibia by Gurdon (see Gurdon 1999 for review), adult
cell cloning by nuclear transfer is not restricted to non mammalian species but the reviewer was unable to
find reference to its use in chickens, fish or other non mammalian livestock species.
4
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
Technical Procedures: an overview
Support for the view that livestock cloning is in a developmental phase can be readily
gained from a recent meeting of the International Embryo Transfer Society (IETS)
recently held in Auckland (January 2003), where scientists from 20 countries presented
64 papers reporting improvements in livestock cloning procedures. Thus this review can
do no more than present a snap shot of what is an emerging technology, as revealed by
review of the recent literature and interviews with leading Australian and New Zealand
practitioners.
Animal cloning is a complex multi-step process with success susceptible to minor
variations. Procedures vary, dependant on the species and operatives, but all need to
address 5 tasks: two relating to the selection, isolation and preparation of the 1) Donor,
and 2) Recipient cells, and the remainder the execution of 3) Nuclear transfer, 4) Embryo
culture, and 5) Embryo transfer process. The interrelation of the tasks is presented
diagrammatically in Figure 1.
Figure 1 Animal Cloning: Diagrammatic Representation
Donor Cell
Harvest
*
Culture &
Synchronise
Mature
Oocyte with
Zona
Enucleation
G0/G1
Donor Cell
Recipient
cytoplast
Fusion &
Activation
Reconstituted Embryo
Embryo
Culture
Blastocyst
Transfer
Clone
5
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
1) Donor cells
Adult animals of all the major livestock species have now been cloned using NT from
a range of donor cells, including fibroblasts (derived from a range of embryonic, fetal
and adult tissues), mural granulose cells, cumulus cells, and second generation clonal
cells. The cells are generally isolated from donor tissues, using conventional
procedures employing mechanical and or enzymatic separation, and established in
primary cultures to allow ex vivo control of the cell cycle and, in some studies, genetic
manipulation. Results have been variable with initial overall efficiencies remaining at
generally less than 5% of reconstructed embryos developing into live offspring (Han et
al., 2003 and Table 1).
Table 1: Current efficiency of Somatic Nuclear Cloning (Niemann &Lucas-Hahn, 2003)3
SPECIES
% VIABLE
OFFSPRING
ESTIMATED NO. OF CLONED
ANIMALS
Cattle
12-15
<1000
Sheep
8
<100
<1
<150
3
150
Mouse
<2
>250
Rabbit
<2
6
Pig
Goat
Reviewer Comment: Donor cell-related factors known to determine the success of the
cloning process include donor species, cell type and cell characteristics, such as cell
population doubling times, the stage of cell cycle, and degree of synchronization with
the recipient cell (see Brem, 2002, for recent review). Wilmut and his colleagues
(1997) identified the critical factor, determining their success in producing Dolly, the
worlds first somatic cell clone, as their use of donor cell harvested at a non-dividing
resting (G0), phase of the cell cycle. This specific requirement is now questioned and
more emphasis placed on the need to coordinate donor cell type and cell cycle stage to
maximize overall efficiency (Wells et al., 2003). However most cloning programs are
still based on the use of resting stage donor cells (G0/G1) and still use the
synchronization methods introduced by Wilmut et al. (1997); namely high confluence
culture (G1) or serum starvation, viz. reducing the serum content of the cell culture
medium from the usual 10% to less than 1% (although this is known to cause
irreversible damage to DNA (Kues et al., 2002)). Other ways of controlling the cell
cycle are known and their application to NT is being explored; for example, papers
presented at Auckland include use of reversible metabolic blocking agents such as the
cyclin dependant kinase inhibitors staurosporine (early G1 arrest), aphidicolin (late
G1 arrest) and roscovitine (G1/S-phase boundary, (e.g.Wells et al., 2003).
3
Overall success rates reaching 18% offspring from cloned embryos have been reported, but more
commonly figures of 2-5% or less are found in the literature. If the calculation is based on reconstructed
embryos the average figure is between 0.1-5% successfully cloned offspring. Post-natal development can be
severely compromised and up to 50% of the living offspring can die soon after birth.
6
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
2) Recipient Cell.
Mature oocytes are recovered from hormone treated adults and juvenile donors using
minimally invasive non-surgical (trans-cervical) and surgical (laparoscopy, minilaparotomy) procedures. Alternatively immature follicular oocytes are recovered from
ovaries excised post mortem from slaughtered livestock using simple aspiration
techniques and matured in the laboratory
Reviewer comment: Both procedures are already in widespread use to provide oocytes
for IVF (in-vitro fertilization) programs. Abattoirs provide a valuable source of low
cost oocytes and there are well developed protocols for maturing them in vitro; the
stage of maturity and age of the oocyte is controlled by reference to the time of
initiation of culture in IVM procedures; studies of other, i.e. chemical, ways of varying
oocyte growth rates are ongoing (Anderiesz et al., 2000). The stage of maturity of
oocytes recovered from hormone treated animals is determined by adjusting the timing
of collection relative to the timing of ovarian stimulation/ovulation stimulation
protocols used to increase oocyte yield. Individual oocytes can vary in their ability to
reprogram a transferred nucleus. For example, a recent study, of surgically derived
oocytes, indicates differences in competence of individual oocytes dependant on the
physiological status of the donor animal; thus implicating a multitude of factors that
are known to influence this, such as genetics, age, seasonality and nutritional status,
hormonal treatment regimes etc. (Peura et al 2003).). However, it is unlikely that
variation between oocytes is a major factor determining the presently poor outcome of
NT programs, for example in a study using IVM oocytes as recipient cells, Piedrahita
et al., (2002) found that cytoplasts as dissimilar as those obtained from small (1-3mm)
follicles and those obtained from large (6-12mm) follicles had equal developmental
competence.
Enucleation. In his pioneering study of NT, Willadsen (1986) removed the haploid
chromosomes present in the oocyte, by simply splitting the oocyte and using the
enucleate half to create the recipient cytoplast required for the donor nucleus. This
approach was subsequently refined to use micromanipulation procedures to visually
locate and aspirate the metaphase plate (the body formed by the chromosomes when
the oocyte is in a mature state) from the intact oocyte, still invested in its surrounding
zona pellucida; the enucleation process is facilitated by including a cytoskeletondepolymerising agent, typically cytochalasin B, in the media.
Reviewer comment: This direct, but technically demanding, approach is still the one
most commonly used. However with oocytes from many of the livestock species, direct
visualization of the metaphase plate is difficult even under polarized light, due to the
dense cytoplasm, necessitating the use of potentially damaging chromatin dyestuffs
(most commonly Hoechst 3342) and UV light. Some researchers resort to removing the
metaphase plate without visualization by aspirating a portion of the oocyte adjacent to
the polar body; a procedure which in experienced hands is greater than 80% effective.
Less technically demanding techniques are being actively sought. For example, there
were reports at the Auckland meeting of the use of the antimitotic drug, demecolcine,
to chemically induce enucleation and the use of X–irradiation to inactivate the oocyte
genome in situ (functional enucleation). However one of the more promising low-tech
methods resorts to the Willadsen approach of simply cleaving the oocyte in two and
combining the enucleate halves of two cleaved oocytes to reconstitute a recipient
cytoplast (Vajta et al., 2001).
7
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
2) Nuclear Transfer and oocyte activation procedures.
Nuclear transfer is generally carried out in two steps:
Step 1) Nuclear transfer, is generally achieved by simply fusing the intact donor cell
with the oocyte (electro-fusion) using an electrical pulse or, less commonly by directly
injecting the donor cell (or the donor cell nucleus) into the cytoplast (intracytoplasmic
nuclear injection); fusagenic agents such as viruses (viral mediated) are now rarely
used.
Step 2) Oocyte activation is usually achieved by subjecting the oocyte to a physical or
chemical shock to excite the chemical changes within the oocyte needed to initiate
chromatin remodeling and the sequence of gene activities necessary to ensure the
development of the reconstituted embryo.
Researchers vary the time when activation is induced relative to the time of nuclear
transfer dependant on their view of the period of time (O-3hrs) required for tissue
specific organization to be erased and the essential remodeling of the donor cell
chromatin to take place in a species; reducing the calcium (Ca) ion content of the
medium at the time of electrofusion prevents premature oocyte activation.
Reviewer comment: The cellular and physical factors influencing the success of fusion
process have been studied exhaustively and methods optimized for each species.
Typically, electro fusion is carried out by placing an individual donor cell underneath
the zona pellucida and adjacent to the cytoplast plasma membrane (it should be noted
that the oocyte is 60-80 times larger than the somatic cell), and fusing the couplet
electrically; a DC pulse, typically one pulse of about 1-3. KV/cm for 20-80u secs, is
usually sufficient to achieve fusion rates >80%; robotic cell fusion devices have been
developed (Hematech Ltd). Alternately, in the so called zona-free fusion methods, an
individual donor cell and a zona-free cytoplast (or two or more cleaved cytoplast
halves) are aligned electrically in an AC pulse field and/or aggregated by exposure to
phytohemagglutin (or lectin) prior to electrical fusion; the zona pellucida surrounding
the oocyte is removed mechanically or through the use of enzymes, most commonly,
0.5% pronase. Recent variations on the process include fusing the cells prior to
oocyte-enucleation (‘Reverse-Order’ zona-free method, S.Walker pers com).
Electrofusion can also be used to activate the oocyte (see below), dependant on the
calcium (Ca) ion content of the fusion medium
Nuclear Injection has so far mainly been confined to mice. The procedures used to
directly inject the donor cell nucleus into the oocyte-cytoplast are similar to those used
in Intra-Cytoplasmic Sperm Injection (ICSI) procedures in human IVF programs, and
commonly use similar injection aids such as pizo-driven micropipettes The inclusion of
the further step(s) required to isolate the donor cell nucleus prior to transfer
introduces an additional degree of technical difficulty but has the benefit that it
extends the range of possibilities for nuclear programming prior to transfer (Heymann
et al 2002) It also avoids any adverse effects from co-transfer of mitochondria and
other non-nuclear cytoplasmic constituents of the donor cells (Cummings, 2001, St
John, 2002).4
4
Mitochondria possess their own DNA and are inherited independently of the nucleus (cytoplasmic
inheritance). This has lead to some researchers questioning the use of the term clone in the context of
somatic cell NT via cell fusion, due to the ‘genetic dissimilarity’ resulting from the likely cytoplasmic
inheritance of the mitochondria from the cytoplast and not the donor cell.
8
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
In normal fertilization and IVF, oocyte activation is induced by the fertilizing sperm
exciting orchestrated changes in Ca ion compartmentalization and flux within the
oocyte. In cloning procedures, functionally similar changes are induced by subjecting
the oocyte to an electrical pulse, or exposing the oocyte to chemicals, such as ethanol,
Ca ionophores or sperm extracts. Most cloning procedures also include steps to
control any cyclical changes in the cytoplast to allow closer coordination with the
cycle of the donor cell. This is achieved in various ways, but principally through using
cytokinesis inhibitors, such as cdc2 kinase, or protein synthesis inhibitors, such as 6DMAP.
3) Embryo culture
The reconstituted embryo is maintained in embryo culture for up to 5 days to assess
normalcy of development and allow use, where possible, of non-surgical embryo
transfer (ET) procedures; in these procedures the embryo is transferred directly to the
uterus, via the cervix, thus requiring later stage (morula/blastocyst) embryos. A variety
of media developed for ET are being currently used, dependent on the species and
background of experience. Most are simple, physiological salt solutions supplemented
with vitamins and amino acids and serum or albumin; the once favoured complex coculture systems are now rarely used.
Reviewer comment: The choice of the conditions used to support the development of
the embryo ex vivo has been shown to be a critical determinant of outcome success in
all assisted reproductive technologies with the effects possibly amplified in cloned
embryos (Hill et al 2000). Recent innovations include staged introduction of
dinitrophenol to act as an uncoupler of oxidative phosphorylation (Thompson et al.,
2000), and inclusion of embryotrophic factors, such as LIF and GMCSF (several
reports). Variation is also found in the physical conditions under which the embryos
are maintained, with recent papers citing use of microdrops, ‘well of the wells’ (WOW;
small wells formed within the conventional four-well culture vessel) and
microcapillaries, to name a few of the methods extant. The period of embryo culture
also varies, with a longer period (3-5days) providing opportunity for preliminary
assessment of the viability of the embryo with a shorter period (hrs –2 days) having the
benefit of potentially avoiding some of the known adverse effects of ex vivo culture
(Young, et al., 1998).
4) Embryo transfer
Embryo transfer (ET) procedures suited to the transfer of cloned embryos are already
in wide-spread use throughout the livestock breeding industry and transfer of the
cloned embryos, to hormonally or naturally synchronized surrogate mothers (embryo
recipient) for rearing, is carried out using the same practiced surgical or non surgical
procedures, dependant on the species. Alternately, cloned embryos can be
cryopreserved for long term storage and/or international transport prior to transfer.
In response to birthing issues associated with clones (e.g. Cloned Calf or Large
Offspring Syndrome, see below), many researchers actively manage the parturition
process, by use of timed caesarian section or treating the surrogate-mother with
parturition inducing agents, such as glucocorticoids (long acting and short acting)
and/or prostaglandins, appropriate to the species; most of the methods have been
9
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
adapted from Assisted Reproduction Technologies (ART) programs where they are
used to manage birthing dates.
Reviewer comment: Ways of offsetting the 80-89% loss of clones post transfer and
improving embryo development rates is a subject of active research. In highly fecund
species such as the pigs, benefit is claimed from admixing non cloned “normal’
embryos with the clones or supporting the pregnancy through endocrine
supplementation (Walker et al., 2002). Other researchers are exploring the use of
chimeric embryos created by admixing tetraploid cells with the cloned embryos cell,
with the aim of gaining benefit from the contribution of the extra cells towards the
support of the pregnancy without the risk of the polyploid cells contributing to the
clone germ-line. This approach could be useful when attempting to establish cross
species pregnancies when attempting to clone rare breeds or when cloning (some)
genetically modified animals.
b) Livestock Cloning: Status and Future trends
Status
As previously noted all the major livestock species have now been cloned but the
procedures are presently too inefficient to excite commercial interest in broad-scale
adoption of cloning technology for food production purposes (see Table 2). There are also
well-founded suspicions about the health status of surviving clones, although this is
challenged (Cibelli et al., 2002), leading to concern about the implications for quality and
standards in food and other animal products derived from clones. These deficiencies have
maintained the immediate focus of research and commercial interest on the application of
cloning technologies to the production of animals producing high value biotechnological
and medical products rather than commercial production livestock.
Table 2 Current status of somatic cell cloning technology by NT
Species
Cell-Type
% Success
Pre-transfer
Reference
Post-Transfer
Term
Weaning
Cattle
Fetal fibroblasts
50
37
20
Wells et al., 2003
Sheep
Granulosa
36
18
2
Peura et al., 2003
Pigs
Fetal Fibroblasts
58
5.5
5.5
Walker et al., 2003
Mouse
Sertoli
6.5
6.1
Ogura et al., 2002
Cumulus
1.3
1.1
Fetal fibroblasts
1.3
1.3
Adult fibroblasts
2.6
2.6
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Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
There have already been some achievements. For example, cloning technologies are being
used to help generate and asexually propagate cattle and goats that produce high value
biopharmaceutical products in their milk or serum, and pigs that can serve as donors for a
variety of xenogenic organs, cells and tissues; and this is just the beginning. Agricultural
applications are also pending; and already cloning procedures have been used in the
generation of livestock with natural or GM enhanced growth rates and food-conversion
efficiencies, or superior and or altered qualities in milk, food or fibre production (see
Niemann & Kues, 2003, for recent review).
Future Trends
The future pattern of cloning technology use will, primarily, be decided by two things,
these are: the extent to which the present technical and other limitations can be overcome
and the consumer acceptance of the technology.
Addressing the technical limitations
Livestock cloning can be simply viewed as an extension of a range of reproductive
technologies, already in widespread use in the livestock industry, that are based on
embryo transfer. However the extension is not a trivial one, as cloning requires replacing
the genome of an embryo with another one derived from a cell of the embryo or adult
animal to be cloned (genome donor). To do this necessitates identifying conditions which
re-instate totipotency to the donor genome and provide an environment which allows
embryonic development. Extensive investigation during the last two decades has seen
outstanding progress in determining these conditions but much has still to be done as
judged by the large percentage of cloned embryos that begin to develop then fail, at an
early pre-blastocyst stage, due to developmental arrest and the low percentage of live
births obtained from those clone embryos that are transferred due to gestational and
neonatal failure.
Reviewer comment: An analysis of literature gives repeated support to the view, first
voiced in seminal cloning experiments with amphibians (reviewed by Gurdon, 1999), that
the primary determinant of the success of adult cloning is the extent of differentiation of
the donor cell. Thus cloned embryos derived from ES cells, that require little or no
reprogramming of early developmental genes, develop substantially better post transfer
than NT clones derived from somatic cells (Rideout et al., 2001); recently confirmed in a
study that directly compared the post transfer survival rates of ES and somatic cell mouse
clones, viz. 30-50% and 1-3%, for ES and somatic cells respectively (Jaenisch et al.,
2002).5
In principle the poor post-transfer survival of clones could be due to genetic or epigenetic
abnormalities resulting from the NT process, but the generally agreed explanation, is that
it mostly reflects the inability of the cytoplast to reprogram the epigenetic profile of the
somatic donor cell to that of a fertilized zygote (Rideout et al., 2001).
During nuclear cloning, the reprogramming of the somatic nucleus must occur within
minutes or, at most, hours between the time that nuclear transfer is completed and the
onset of cleavage of the activated egg begins. In contrast, epigenetic reprogramming is
5
However, it should be noted that clones derived from ES cells commonly show poorer survival relative to
somatic cells in the early preimplantation stages (10-20% vs 60-70%); this is likely due to most ES cells
being derived from cultures maintained in a growth phase resulting in the majority (60%) of cells being in
S-phase and thus incompatible with successful NT; it is also known that between 20-80% of cells in some
ES cell lines can be aneuploid (Eggan et al., 2002)
11
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
normally accomplished during spermatogenesis and oogenesis, processes that in livestock
species may take months. It can therefore be readily understood why prenatal mortality of
nuclear clones could be due to dysregulation of gene expression consequent on
inappropriate reprogramming (e.g. see Daniels et al., 2001, Ogura et al., 2002). Gaining
a better understanding of these epigenetic factors and the way they can be controlled is
therefore key to progress in this area. Once understood the molecular factors in the
cytoplast that determine the competence of oocytes to support NT, can be identified and
the knowledge utilized to achieve better control and/or replicate these conditions in vitro
(see Hakelien &Collas, 2002, for recent review).
In the interim, the focus of research is on procedural improvements targeted at
1) achieving a better coordination between the cycle of the donor cell; cytoplastcoordination is known to be crucial to maintaining normal ploidy in the
reconstituted embryo and benefit has now been shown from modifying
procedures to allow for small differences in cell cycle characteristics, such as
those due to GM (Wells et al., 2003)- and
2) determining the timing of activation; as it is known that the use of pre-activated
cytoplast in somatic cell NT, invariable results in poor development compared
with non-activated cytoplast.
Improving the viability of cloned embryos. The adverse effect of donor cell
differentiation on the post transfer survival of clone embryos has been noted above.
Surviving embryos can also be adversely affected, resulting in respiratory distress, birth
defects and high post-natal losses in cattle and sheep; but interestingly not in pigs (Wells
et al., 2002). The consensus view emerging from extensive studies in both mice and
livestock species is that these anomalies are a further manifestation of faulty epigenetic
reprogramming of the donor nucleus. The long-term post-natal survivors are also likely to
have subtle epigenetic defects but below the threshold that threatens viability (Jaenisch
and Wilmut, 2001).
Reviewer comment: The very early stage of placental development are known to be
subject to epigenetic control (see Jaenisch et al., 2002: for links to the extensive and
growing literature), and disturbances in epigenesis caused by one or more of the cloning
procedures are thus directly linked to the high incidence of large placentas found in
cloned animals. Large placentas in turn provide the most likely explanation for the
significant number of livestock clones surviving to term that are overgrown (Bertolini et
al., 2002); this condition, was first noted with cattle and sheep, and referred to variously
as the cloned calf syndrome (CCS, Wells et al., 2003) or more usually the large offspring
syndrome (LOS, Young et al., 1998). An abnormally large offspring at birth is of concern
because of birthing difficulties (dystocia) which often, in the absence of veterinary
intervention, lead to death. CCS also has clinical association with a range of other
anomalies, including the respiratory distress and circulatory problems that are the most
common cause of neonatal death; and, disturbingly, even the premature death of
apparently healthy survivors from immune dysfunction or kidney or brain malformation.6
6
Interestingly CCS and many of the other anomalies identified in cloned embryos are similar to those found
following conventional IVF and other related reproductive technologies (Thompson et al., 1995, Walker et
al., 1996, Young et al., 1998); where the process of embryo culture is known to significantly alter the
expression of imprinted genes through disturbing epigenetic effects on genome imprinting (Reik et al.,
1993).
12
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
Species vary, in the degree they are affected by CCS, with sheep and cows the most and
goats and pigs the least affected. According to current paradigms, this is a reflection of
the extent to which donor cell nuclei need to be reprogrammed, in each species, to direct
normal development (Kang et al., 2001, Jaenisch et al., 2002, Ogura et al., 2002).
However, there is an alternate explanation. This was suggested by the recent finding that
even highly differentiated adult tissues contain a significant (but variable) number of
tissue specific stem cells with a capacity to trans-differentiate, that is give rise to a range
of other tissues (see Blau et al., 2001), thus the few viable clones presently seen in
livestock cloning programs maybe the result of inadvertent selection and use of
(relatively undifferentiated) ‘stem’ cells rather than de-differentiated somatic cells as
donor cells (H. Niemann, pers. com.). Differences in cloning success between species and
tissues could therefore reflect the varying percentage of stem cells in the donor cell
population dependant on the tissue of origin. If validated, it not only suggests explanation
for species/strain etc. differences in yield of viable clones but gives hope that the
prospects for commercial livestock cloning could be enhanced dramatically when
practical ways of isolating stem cells are developed,
Addressing the Animal Welfare and Food Safety Issues In response to the growing
debate on the animal welfare and food safety issues concerning animal cloning Cibelli
and his colleages (Cibelli et al., 2002) undertook an exhaustive review of the published
literature in the area and came to the view that, in contrast to many adverse reports on the
technology, the majority of surviving livestock clones were seemingly healthy. They
noted, however, extreme variability between studies, in the percentages of apparently
healthy offspring produced, with the inference that improvement in procedures would do
much to minimize the risk of abnormalities.
None of the animal health issues so far identified threaten to alter the food or nutritional
qualities of products derived from an individual clone; given that the surviving animal
clones that enter production are likely to be healthy, to the extent that they would be
selected, by livestock producers, to be free of anomalies threatening production and or
evoking welfare concern.
Reviewer comment:, The main body of experimental evidence supporting this contention
is obtained from a substantive number of laboratory studies with cloned mice (Ogura et
al., 2002), as there is only a scatter of reports on studies with livestock. In mice, most (>
90%) cloned fetuses that develop to term have a normal phenotype and the majority
(>90%), attain puberty and have normal reproductive performance; these figures are
similar to non-cloned mice. Albeit, in mice as with livestock species, cloning technology is
still far from being perfected and the life span of cloned mice can be shortened, or of
normal length dependent on the genotype, with many clones showing laboratory evidence
of liver damage and disturbance in immune function, that makes them more susceptible to
disease
Similar detailed investigations of cloned livestock have yet to be carried out. Albeit, the
evidence from studies in cattle and pigs are very encouraging. For example, Infigen Inc,
one of the most active of the USA cloning companies, recently reported (November 2001,
to a scientific meeting sponsored by the National Academy of Sciences in Washington,
DC), the results of a four year study following the health of 120 live calves and more than
50 piglets into adulthood, that claimed to show that adult-livestock clones had normal
13
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
health and genetic characteristics. In support of their claim, Infigen presented data on
detailed analysis of milk harvested from cloned Holstein cows to show it was safe for
human consumption; including detailed biochemical analysis of fat and protein. The study
also included detailed molecular genetic studies including analysis of global methylation,
a widely accepted metric of the genetic functionality of an organism. Analysis of the
progeny of clones, and clones of clones was also reported as supporting the company’s
claims that the clones were healthy and ‘normal’. To this reviewers knowledge a formal
publication of these results has not been published; and Infigen Inc is no longer
functioning as a company and all clones have been destroyed. However there are formal
publications from other groups, which make similar claims, albeit on fewer animals.
For example, Lanza and his colleagues (2001) reported a study of the health status of
24/30 Holstein cattle clones that developed to term and were vigorous at birth with
normal birth weights and remained alive and healthy 1-4 years later. Supportive evidence
was provided from the physical examination of all animals including objective
(temperature, pulse, and respiratory rate) and subjective (general appearance, eyes,
lymph nodes, and cardiac and pulmonary auscultation) findings; normal results of
abdominal palpitation per rectum (reproductive and gastrointestinal organs and kidneys)
and body condition scores that were appropriate for the animals’ nutritional program.
Social interaction and behavior of the cloned animals were also normal and the clone
herd developed a social dominance hierarchy and a full spectrum of behavioral traits.
Reproductively, the cloned animals exhibited puberty at the expected age (10-12 months)
and body weight (318-365 kg). Conception rates were excellent and two of the cloned
animals had calves at the time of the report that were normal in all respects.
Routine clinical laboratory analysis on blood and urine were also consistently within
normal range and the normality of the clones immune system was confirmed by a series of
functional tests and the animals similar response to periodic infections in the same
manner as naturally conceived, control animals. No milk production measurements are
reported in the Lanza study, but in another recent study of the milk production
characteristics of 2 cloned Holstein cows vs 8 naturally conceived, control cows (Aoki et
al, 2003), no differences were found in lactation or milk composition (milk fat, protein,
lactose, solids-non-fat and total solids).
Health issues of potential concern were identified in the Lanza study, for example several
of the cloned calves experienced pulmonary hypertension and respiratory distress at birth
and fever after vaccinations at 4 months, and similar observations are to be found
embedded in other published reports, including those of Australian and New Zealand
groups (see part 2 of this report).7
Since completing the Draft version of this report Reuters (Reuters Tokyo 14/04/2003),
reported the Japanese government as saying meat and milk products made from cloned
cattle are safe for human consumption. A copy of the report of the 3 year study on which
this claim is based has been obtained (courtesy Dr T Nagai, see Appendix 2)
Offspring: Concerning the offspring of clones, there are well founded scientific reasons,
supported by a mounting body of experimental evidence, to confidently expect that the
7
These issues are likely a reflection of the early stage of development of the cloning technology and or a
manifestation of the fact that all animals produced by ET are at risk of LOS and the health sequela
associated with an altered expression of imprinted genes due to embryo culture
14
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
health profile of any offspring, produced by natural mating, would be entirely normal; as
any imprinting anomalies of the cloned parent would be removed during the genetic
remodeling processes that occur during gametogenesis (see Prather et al., 2003).
Reviewer comment: This surmise has yet to be validated experimentally. The scatter of
references in the literature to the offspring of both male and female clones all refer to
healthy outcomes (e.g. Lanza et al., 2001), but the numbers are still too few to form
definite conclusions.
There are no published studies directly addressing any food quality issues relating to the
offspring of clones; a reflection of NT still being in a developmental stage, with no
generally agreed common protocols concerning the potential use of cloned livestock in
specific production enterprises
Addressing the Animal Health issues. Theoretical disease risks have been identified in
relation to possible effects of cloning on some intrinsic viruses, such as endogenous
retroviruses (Thebier, 2001) but so far very little targeted experimental work has been
done to scientifically evaluate the risk; Thebier advocates a precautionary approach with
restriction on the movement of NT derived embryos until the risk is better defined.
Concerns have also been raised about the potential impact of loss of genetic-diversity, in
herds and flocks comprising a high percentage of clones, on animal health due to the
expected increased susceptibility to infectious diseases.. .
Reviewer comment: As noted, cloning is an extension of a suite of technologies, in
current use in the livestock breeding industry, based on ET. As with all reproductive
technologies, there are associated animal health risks, but these are being effectively
managed, both locally and internationally, under a framework of procedural guidelines
established by the IETS; the effectiveness of these guidelines has been validated by field
experience with the safe transfer of several million embryos over the past three decades
(Thebiet, 2001). Extension to these guidelines, to include any additional disease risk
introduced by cloning, is under active consideration by the IETS.
The concerns about susceptibility of cloned animals to disease echos those voiced
following the introduction and widespread adoption of cloned plants; and most other
animal reproductive technologies including artificial insemination (AI), ET and embryo
cloning. These concerns are justified, as genetic variance, introduced through sex-based
reproduction, underpins on animals (and plants) natural resistance to disease and
extensive commercial use of clones in livestock production would increase the
susceptibility of herds and flocks to disease risk.8 However the risk is recognized by both
national and international livestock breeding organizations and can be expected to be
well managed, should the need arise.
Gaining Consumer acceptance.
As noted by Faber et al., 2003, consumer acceptance of cloning is dependent on three
factors: economics, societal values and regulatory agencies
8
As noted in the NRC report (p 49), diversity of animal populations, particularly at the major
histocompatibility (MHC) loci, is a major factor preventing spread of disease, particularly viral disease (Xu
et al., 1993; Schook et al., 1996; Kaufman and Lamont ,1996).
15
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
Economics. Economic analysis indicates that bovine cloning in particular holds great
promise for eventual wide-scale application in both the beef and dairy industry once the
problems of pregnancy maintenance and calf viability can be overcome (Faber et al.,
2003). Wide-scale application in the less intensive sheep industry is less certain, but
cloning is already being used to propagate individual animals with superior traits, such as
ultra-fine wool, for use in stud-breeding programs and intensive production enterprises,
and in the generation and propagation of GM stock; applications to the production of
wool with new or enhanced qualities is anticipated. In the pig industry, short-term
applications of cloning technology will probably remain restricted to use in generating
high value animals for medical use, although if the Texas study is validated (Walker et al.,
2002) application to generation and propagation of high value non-GM stock would be
commercially viable.
Reviewer comment: Research in livestock cloning is an expensive venture and, it should
be noted, that Australian and New Zealand groups, active in livestock cloning, have, like
their overseas counterparts, all established industry and commercial links to share the
costs. Various steps in the cloning process are already subject to patent claim, and use of
patents controlled by International commercial interests (viz. use of serum starved donor
cells, Roslin Institute/Geron Corp; non-quiescent cells, Advanced Cell Technology; and
chromatin transfer using nuclear reprogramming prior to nuclear transfer, Hematech;
oocyte activation, Infigen Inc. etc.). Thus the pattern of introduction and initial focus of
cloning may accord more with the commercial view of international investors than that of
the local livestock breeding industry.
Societal values. There is also wide public awareness of the poor health status of some
clones, due to any adverse aspects of the technology being used to influence the Human
cloning debate (Sinclair et al., 2000). Increasingly intense reaction to cloning may also be
expected from the growing body of people who view further intensification of
agricultural, with its increasing tendency to view animals as mere things, as undesirable
(Orlans, 2000).
Reviewer comment: In the past, commercial links with international companies have
often served to inflame debate over concerns about new technologies (e.g. GM debate);
and given the present gap between scientific and public understanding, public concern
about livestock cloning is to be expected. Cloning is already identified in the public mind
with genetic engineering, thus demanding a cautionary regulatory approach.
Regulatory issues. The clone health issues are of concern from an animal welfare and
ethical perspective but there is no scientific evidence to suggest clones present a threat to
human health. Nor would the cloning procedure be expected to alter the nutritional value
or other food qualities or products derived from clones, but, as noted elsewhere, this is yet
to be established experimentally. Offspring of clones, produced by sexual reproduction,
can, according to present scientific understanding, be confidently accepted as normal.
Reviewer comment: As argued elsewhere, food products, from a healthy clone pose no
risk of threat to consumer expectations- a clone is a clone. However cloning, with its
associations to GM and other challenging technologies, does raise public concern-‘an
uneasy state of blended interest, uncertainty and apprehension’- which more accurately
characterizes many of the questions and issues that need to be addressed to sustain public
confidence and trust.
16
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
Section 2
Livestock cloning in Australia and New Zealand
a) Species and Numbers of Animals cloned
Embryo cloning by NT was first introduced into Australia and New Zealand over a
decade ago (Sheep, Cattle, McLaughlin et al., 1990;) but failed to excite strong
commercial support due to concerns about the low success rates and high incidence of
LOS in clones (Lewis et al., 1998); estimates of the total numbers of embryonic clones
produced in Australia and New Zealand during the 1990-2003 period are cattle <500,
sheep<100, goats < 50 and pigs 0 (reviewers compilation)
The first successful clonings of adult animals were carried out in Australia in 2000; cattle,
‘Suzi', (Dairy CRC), sheep, ‘Matilda’ (SARDI); pigs following later in 2002 (BresaGen
Ltd), but, due to the limitation of the technology, the overall numbers of adult clones so
far produced for any species remains small (sheep, <20; cattle <40; pigs < 10, Table 3).
Future predictions: Predictions of the numbers of clones over the next few years
presented in Table 3 assume a continuing focus on procedural development and small
numbers of clone donors; no allowance has been made for commercial expansion into
production livestock which would inevitably follow resolution of limiting technical
issues. However, as argued earlier in this report, given the structure of the livestock
breeding industry and the inevitable lag phase in technology transfer, even if a technology
breakthrough happened in the next few weeks, it could be 5-10 years before significant
numbers of clones (or their offspring) were available to enter the food-product
chain.
Table 3 Current Status of Livestock Cloning in Australia and New Zealand
SPECIES
GROUP
CLONES PRODUCED
CLONES PLANNED
2003
produced surviving
Cattle
Sheep
Pigs
Dairy CRC
20
CSIRO
2
Ag Research NZ
14
Clone International
6
<20
<5
14?
<20
6
4
<10 ?
SARDI
16
3
0
CSIRO
4
0
<5
(2)?
(2)?
<20 ?
BresaGen*
Total all species
approx
80
Reviewer comment: Cloning success, as noted by Cibelli et al., 2002, is highly variable
between groups ascribed to differences in methodology, species/strain of animals, source
of donor nuclei and investigator skills. However, as best can be determined the success
17
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
rates reported by each of the Australian and New Zealand groups indicate they are using
leading edge technologies. For cattle, the spectacular production of 10 healthy calves
from 100 cloned embryos derived from one dairy cow in Hamilton, New Zealand (Wells et
al., 1997) established a new National (and International) benchmark for livestock cloning
procedures which has only recently been consistently bettered; e.g. the AgResearch team
now reports 20% transferred cloned embryos yielding healthy calves at the Auckland
meeting (Wells et al., 2003). However a majority of pregnancies (>60%) from transferred
cattle embryos still fail and a variable number (20-80%) of clones born can still die
shortly after birth from LOS related health issues.
Similarly with sheep, the SARDI group achieve >20% pregnancies to term from
transferred cloned embryos but of these 90% can die prior to weaning (Peura et al 2002)
from a variety of health issues symptomatic of LOS.
Until recently the situation with pigs was also limiting with the BresaGen group reporting
a piglet yield from transferred cloned embryos of <3% (Boquest et al., 2002); a figure
similar to that reported by other international groups. However the situation may now be
changed, as a recent publication on pig cloning, from Texas A&M (Walker et al., 2002)
reports a remarkable 12% of fused doublets transferred yielding healthy piglets.
b) Criteria used in selecting animals for cloning.
Given the deficiencies in present cloning procedures, the animals presently targeted
for cloning are all of high commercial or scientific value with many genetically
modified. Donor selection criteria presently used by Australian and New Zealand teams
is presented in Table 4.
Reviewer comments: Broadly the animals fall under 4 categories
1) Experimental animals used in procedural development studies or to produce
unmodified or modified cell lines and clones to be used in other applied or basic
studies.
2) Unmodified breed-stock or production animals of high genetic merit as regards
production efficiency or product quality or other performance or show measures,
3) Genetically modified animals such as transgenic dairy cattle producing milk, with
enhanced food or production values, or containing high-value biopharmaceutical
products, or pigs modified to be a source of xenogenic organs, tissues or serum;
and
4) Rare or endangered animals.
Compared with the major International groups, there is a greater interest in
agricultural applications, encouraged by strong industry support. Australia has
however played a significant leadership role in medical applications of the
technology, especially in the organ transplantation field (BresaGen).
18
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
Table 4 Criteria used in selecting animals for cloning
CRITERIA
DONORS
OFFSPRING
a) Production Traits
Meat
CSIRO
Milk
Dairy CRC
Clone Int.
CSIRO
Wool
Elite Beef Bull (1)
Elite Beef Cow (1)
Elite Dairy Bulls (2)
Elite Dairy Bulls (2)
Parasitic resistance (2)
Ultra Fine wool (2)
Total
0
0
6
4
0
0
10
Ewes
Elite Cows
Elite Ewes
Ewes (6)
1
14
2
2
Elite Ram
Sow?
Elite Ram
Elite Ewe
1
2
2
3
27
b) Scientific
Cell Lines
Adult
fibroblast SARDI
granulosa AgTech
SARDI
cumulus CSIRO
Fetal
fibroblast SARDI
fibroblast BresaGen
Embryo
Ag Tech
Total
c) Genetically Modified
Meat
BresaGen
Milk
Dairy CRC
Medical
Xenogenic BresaGen
Other
Porcine Somatotrophin gene
Alpha S1 Casein Gene
?
7
Various
?
Total
7
d) Rare & endangered breed
Livestock
Native
Other
AgTech
Enderby Is. Cow(1)
1
Total
1
Grand Total
45
19
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
c) Livestock cloning procedures in use in Australia and New Zealand.
None of the procedures used by Australian and New Zealand teams differ radically from
those generally used as described in Section 1.
Donor cells: Cattle are being cloned from primary cultures of adult and fetal fibroblasts
and mural granulosa cells, sheep from cumulus cells and adult and fetal fibroblasts and
pigs from fetal fibroblasts and in many instances the cells or the animals have been
genetically modified. All groups pay particular attention to synchronization of the donor
and recipient cell cycle stage.
Recipient cells: Recipient cytoblasts are being derived both surgically and from abattoir
sources. Enucleation is generally being carried out using micromanipulation of zona intact
eggs but there is a move to the simpler oocyte bisection procedures
NT: All groups are presently using electrofusion methods.
Activation: The timing of activation, using ionophores, relative to the timing of
electrofusion varies between 0-9 hrs depending on the species and cell cycle stage.
Embryo culture: All groups are very experienced in embryo culture as part of the
extensive experience in ET. The cattle and sheep groups use a simple media based on the
known composition of sheep oviductal fluid (SOF) supplemented with amino acids and
albumin (Gardner et al., 1994) and generally extend the period of culture until the day of
embryo compaction (4-5days); the modified SOF media has been shown to reduce the
incidence and severity of LOS associated with extended periods of embryo culture in
cattle and sheep (Young et al., 1998). The AgResearch team has recently introduced the
novel step of adding dinitrophenol into the culture medium on days 4-5 to reduce
oxidative phosphorylation in an attempt to more closely mimic metabolic changes
occurring in vivo (Thompson et al., 2000)
Embryo transfer: In cattle, all groups use non-surgical transfer procedures and the
pregnancies and birthing process are actively observed and managed both for scientific
record and to reduce complications from cloned calf syndrome. For sheep and pigs,
embryo transfer is carried out using minimally invasive surgical procedures and the
conservative pregnancy management practices, used in routine ET, followed.
Future prospects: All groups anticipate significant technical breakthroughs within a 1-2
year time frame stemming from a better understanding of the genomic repacking and
reprogramming process following NT. There is major interest in the recently developed
Chromatin Transfer procedure developed by Phillipe Collas and his colleagues from
Hematech Ltd (Collas et al., 2003, in press, A. French pers.com)9. Breakthroughs are also
anticipated in the culture of livestock ES cells following the huge medical investment in
stem cell technology.
9
In this procedure, the donor cell is permealised, using a Streptolysin O preparation, and the chromatin
exposed to enzymes to remove all existing RNA; the stripped chromatin is then ‘repackaged and
reprogrammed’ in an extract prepared from mitotic cells, i.e. cells undergoing cell division, prior to NT,
using electrofusion. According to Dr Collas, this radically different approach results in a major reduction in
number of cloned cattle embryos lost post transfer; and, if as successful as claimed, promises to make cattle
cloning attractive to livestock breeders and potentially livestock producers.
20
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
In the interim, continuing improvement is anticipated in further attention to detail
(tweaking) aimed at improving the efficiency of each step; this is of critical importance in
developing complex multiuse procedure such as cloning.
Reviewer comment: Concerning future developments in cloning technology, the four
principle research groups involved in the development of the technology in Australia and
New Zealand are in active contact with all the major overseas groups, allowing quick
uptake of any break-through technologies. Information exchange is also facilitated by
frequent researcher contact and exchange, attendance at International meetings and
ongoing professional contact through joint membership of national and international
professional societies such as The International Embryo Transfer Society, The Society for
the Study of Reproduction (USA) and the publication of a growing number of
International specialist journals featuring cloning and stem cell technology such as
Nature Biotechnology, Cloning and Stem Cells, Theriogenology, Animal Reproduction
Science, Reproduction Fertility and Development, Journal Reproduction and Fertility,
Biology of Reproduction, Xenotransplantation etc.
d) Food products from cloned livestock, its use and evaluation
If products from the donor animal are acceptable to the consumer (see below), then
products from the clone and its ‘identical’ offspring should be equally acceptable. As
reviewed in Section 1 preliminary data on the production traits of cloned livestock is
encouraging but apart from a few studies on milk yield and quality there is a major deficit
of science based data relevant to the assessment and assurances of quality for food
products for clones of any species, although collection of relevant data has begun.
Cattle: There are a few preliminary studies indicating normality of milk yield and quality
but no other data on composition or quality of other food products from cattle.
Reviewers comments: Detailed analysis of the milk from one cloned dairy cow has been
carried out by the Dairy CRC showing it to be normal (table 5) but there are no
corresponding detailed evaluation of meat yield and quality or any other measure of food
composition available for food product derived from any of the clones, or any of their
offspring, produced so far.
Table 5 Suzi Milk Test Results (Updated 6 February 2003)
TEST
Fat
% m/v
Protein
% m/v
Lactose
% m/v
Somatic Cell Count
/ml
Total Solids
% m/m
Total Nitrogen
% m/m
Non Protein Nitrogen
% m/m
Non Casein Nitrogen
% m/m
Casein
%
Whey Protein
%
Density
g/ml
Ash
% m/m @ 550°C
DM Cell Count
/ml
AVERAGE
3.75
3.12
4.54
400,625
12.07
0.50
0.029
0.13
2.34
0.67
1.026
0.79
17,048,375
RANGE
1.17 - 5.00
2.60 - 3.47
3.84 - 5.05
30,000 - 1,690,000
9.30 - 14.70
0.422 - 0.560
0.022 - 0.040
0.105 - 0.150
1.89 - 2.69
0.53 - 0.77
1.021 – 1.030
0.70 - 0.87
9,000 - 130,000,000
Calving Date:
11/07/2002
Period of Sampling:
24/07/2002 to 9/01/2003
N= 8
Independent
Interpretation of Results:
“Overall the milk
composition results from
this cow are within
normal ranges for a
healthy dairy cow in
early lactation”. Martin
Auldist Research
Scientist – Milk
Composition DNRE
Ellinbank
21
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
Sheep: In regard to cloned sheep or their offspring, no data is available on meat
composition or quality
Pigs: No data on quality or composition of food products from cloned pigs is yet
available.
e) Differences between clones and the donor animal
The core benefit of cloning to any agricultural enterprise is its use to make essentially
identical copies of an animal with superior production traits. However, as the review
reveals, cloning technology has yet to be perfected and a significant number of clones still
differ phenotypically from the donor, most notably in size at birth, a reflection of as yet
uncontrolled epigenetic events (see section 1 of this Review).
Cattle: The pattern of growth and development of cloned cattle produced in Australia and
New Zealand and their general health status and reproductive capacity are consistent with
the view that surviving clones are generally healthy; and productivity assessments, such
as quality of semen from bulls, calving rates and milk yield as quality measures for cows
are also consistent with this view. Concerning the offspring of cattle clones, the
experience of Australian and New Zealand researchers is limited, given that the first adult
clones were only produced in 2000. ‘Suzie and ‘Mayzi’, the first cloned dairy cows
produced in Australia, have both given birth to normal healthy calves through artificial
insemination (AI)
Sheep: Matilda, the first adult sheep clone produced in Australia, recently died (Feb 2,
2003) at 3 years of age of uncertain causes but the other two cloned ewes (Annabel and
Frida) and a cloned ram, Macarthur, produced by the SARDI team, remain healthy.
Concerning the offspring of cloned sheep, the data is limited. The two surviving CSIRO
cloned ewes were not mated and were euthanased at two years of age. Of the SARDI
clones, Matilda and Annabel have proven fertile but Frida is yet to conceive. Matilda
produced 3 lambs by IVF at 3 months of age (two males, one female) and a lamb
following natural mating with Macarthur, which died at birth. The IVF lambs are now two
years of age, one of the males is cryptorchid, i.e. the testes have been retained within the
abdominal cavity, and the female is yet to conceive.
Macarthur, the cloned ram, mated with two other females, which were non-clones but
derived by ET, to produce 2 lambs; one described as normal, the other having an unusual
hemi-lateral facial palsy; the significance of these health anomalies cannot be determined.
Pigs: No health problems were noted for the two cloned pigs produced by the Bresagen
group. As noted above, recent reports indicate pig-cloning procedures have recently
improved dramatically (Walker et al., 2002, cited above). Importantly, only one
abnormality was noted, by the A&M team, in the 28 piglets cloned from porcine fetal
fibroblast cells, and this was possibly unrelated to NT; notably there were no signs of
LOS, commonly seen in cattle and sheep cloning programs but seemingly at low
incidence or absent from cloned pigs. If porcine cloning is as effective and the clones as
healthy as claimed, the method, which can be undertaken by a single micromanipulator at
reasonable cost, is a breakthrough and makes broad commercial utilization of porcine
cloning technology a distinct possibility in the near future.
22
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
Summary Comments
Until the current technical problems are overcome, the numbers of cloned animals
produced world wide are likely to remain small (in the thousands), relative to the millions
of animals produced using conventional breeding procedures, and commercial interest
restricted to intensive production enterprises centered on high value products most likely
sourced from genetically modified animals. Thus in the immediate future, food production
issues are unlikely to present a major challenge as the clones and their offspring will not
enter the food-chain. Researchers are optimistic that the technical problems can be
overcome (2-5 year time frame?), but it will probably be a gradual process10. Industry
demand exists, as evidenced by the number of requests from producers to clone individual
animals of high genetic merit, but the rate of uptake and penetration of cloning into the
industry will be governed by the economics of the times. The situation is somewhat
analogous to the early days of ET (or embryo cloning), and, if adoption of cloning by the
livestock industry follows a similar pattern to ET, cloning will be initially adopted by the
‘top’ studs for use in breeding high value stud animals for daughter studs and intensive
commercial production enterprises. Thus the initial impact on food production is likely to
be from the offspring of clones rather than the clones themselves as these will remain
relatively few in number and unlikely to be utilized as food product until they had reached
the end of their useful breeding-life (5-10 years); as argued previously there are no
scientifically based reasons to view any sexually produced offspring of clones as other
than normal in every respect. The direct use of clones in food production, in a
numerically significant way, will only occur when current limitations and health issues
related to cloning technology are fully mastered and cloned embryos ready for transfer
can be produced in vitro at an acceptable price (<$50?) to primary producers of food
stock; this is viewed as achievable and is anticipated, but a reasonable consensus view is
that this is unlikely to happen anywhere within the next decade.
10
An indication of this is provided by the targeted milestone for the Dairy CRC in 2003- calving rates, for
cloned embryos that are greater than 25% of IVF embryo (Dairy CRC Annual report 2001-2)
23
Current Status of Cloning in Animal Production in Australia and New Zealand
Glossary
Glossary
Adult clone: Clone derived from adult parent, usually from a somatic cell
AgResearch: the Reproductive Technologies Group at the AgResearch Rurakura
Research Centre, Hamilton, New Zealand
Aneuploid: a cell (or organism) that has an abnormal number of chromosomes
ART: Assisted Reproduction Technologies
Biosecurity: Measures to protect from infection
Bresagen: an SA based biotech company (www.bresagen.com.au)
CCS: cloned calve syndrome also known LOS (see Large Offspring Syndrome)
Cell cycle: a regular sequence of changes occurring within a cell during cell division;
notated as G0 (resting stage), G1 and S
Chimeras: Animals (or embryos) composed of cells of different genetic origin
Cloning: The propagation of genetically exact duplicates of an organism by a means
other than sexual reproduction
Clone International: a Melbourne based biotechnology company (www.)
Cumulus cells: cells surrounding the ovum in the ovarian follicle.
Cytokinesis: changes occurring within the cell outside the nucleus during cell division
Cytoplast: an enucleated cell
Cytoskeleton: structural elements with a cell helping retain its structure and function
Dairy CRC: Cooperative Research Center for Innovative Dairy Products –a collaboration
between the Dairy Research and Development Corporation, Monash Institute of
Reproduction and Development, Genetics Australia Cooperative Limited and the
Victorian Institute of Animal Science. (http://www.dairycrc.com)
Differentiation: the acquisition by the cell of characteristics or functions different from
the original type.
ES: see Embryo Stem cell
Embryo clone: clone derived from an embryo
Embryo Transfer (ET): Embryo transfer
Embryonic Stem (ES) cell. Cell lines derived from early embryos that have the potential
to differentiate into all types of somatic cells as well as form germ lines, and hence
whole animals, through NTT
Embryotrophic: factors able to stimulate embryonic growth and development.
Epigenesis: regulation of gene activity without alteration of genetic structure.
Epigenetic: relating to epigenesis
Fibroblast: a type of relatively undifferentiated cell found in many parts of the body
involved primarily in wound healing. Fibroblasts are relatively easy to grow in
culture, and are often used for this purpose
Gametogenesis: the process of formation and development of gametes (eggs, sperm).
24
Current Status of Cloning in Animal Production in Australia and New Zealand
Glossary
Genetically modified: an organism whose genotype has been modified by application of
molecular biological techniques.
Genomic imprinting: the parent-of- origin control over expression of genes. Imprinting
creates a functional haploid state by silencing an allele of the imprinted gene
depending on its paternal or maternal origin. To date approx. 40 imprinted genes
have been identified primarily in humans and mice. Imprinted genes are controlled
by methylation marks that are established during the later stages of gametogenesis;
importantly, when lost, these marks cannot be restored except by passage through
the germline. The effects of imprinting are widespread and implicated as potential
factor in abnormalities associated with NT, e.g. in normal embryos, regions of
imprinted allele- specific methylation are strictly maintained and are crucial for later
development of the embryo, whereas in cloned embryos created by NTT, there is
evidence of abnormal methylation and associated failure in the reactivation of
critical developmental genes
Genome: a complete set of chromosomes derived from one parent.
Genotype: the genetic identity of an individual
G0: A state characteristic of cells that have exited the cell cycle and entered a resting
phase
G1: The first stage of the cell cycle
Germline cells: cells that contain inherited material that comes from the eggs and sperm,
and that are passed on to the offspring
Granulosa cells: cells lining the ovarian follicle
IETS: International Embryo Transfer society
In vivo: within a living body
In vitro: outside of a living body, lit., in a glass container.
Introcytoplasmic sperm injection (ICSI): fertilisation by direct injection of sperm into
the cytoplasm of an egg.
IVF: see In Vitro Fertilisation
In vitro fertilization: fertilization of the egg in the laboratory
IVM: see In Vitro Maturation
In vitro maturation: maturation (of the oocyte) in the laboratory
LOS: see Large Offspring Syndrome
Large Offspring Syndrome: a syndrome associated with livestock produced by ART
based on embryo transfer characterized by large birth-weight offspring.
Major Histocompatiblity Complex (MHC): a key part of the bodies immune defence
system. Different MHC types recognize different viral or bacterial epitopes encoded
by pathogens for presentation to the immune system. A population of genetically
uniform cloned animals is at risk as pathogens more easily evade host immune
responses (Yuhki and O’Brien, 1990).
Metaphase Plate: structure formed by chromosomes in a mature oocyte.
MHC: see Major Histocompatibility complex
25
Current Status of Cloning in Animal Production in Australia and New Zealand
Glossary
Microinjection: injection through a very fine needle
Nuclear reprogramming: restoration of the correct embryonic pattern of gene
expression in a nucleus derived from a donor cell and introduced into an oocyte
Nuclear transfer (NT): the generation of a new animal nearly identical to another one by
injection of the nucleus from a cell of the donor animal into an enucleated oocyte of
the recipient
Oocyte: an immature ovum
Polar Body: a small portion of the egg extruded during meiosis, and containing
chromatin,.
Placenta: organ linking fetus and mother.
Resting stage: see cell cycle
Risk: the likelihood of a defined hazard being realized.
SARDI: South Australian Research and Development Institute (www.sardi.sa.gov.au)
Somatic cells: cells of body tissues other than the germline.
Telomeres: the simple repeated sequences at the ends of chromosomes that protect them
from loss of coding sequence during replication.
Totipotency: an ability to create all the somatic and germ tissues of a species
Transgenic: genetically modified, usually through the introduction of DNA into the germ
cell by injection into the nucleus of the ovum.
Xenogenic: modified to allow xenotransplantation.
Xenotransplantation: Transplantation of cells, tissues or organs from one species to
another.
Zygote: a fertilized oocyte
26
Appendix 1
Health Status Of Cloned Livestock 1991
Current Status of Cloning in Animal Production in Australia and New Zealand
Appendix 1
Report on health status of cloned livestock as presented on the Infigen Inc web site in 1991.
“Studies presented today at the national academy of sciences' national research council
meeting also determines milk from cloned Holsteins is identical to that from non-cloned
cows found in the general population”
DeForest, Wis. (November 27, 2001) - Nuclear transfer-based cloning at Infigen, Inc., has
been found to produce cows and pigs with normal health and genetic characteristics, based
on a four-year-long study that has followed these animals into adulthood. The study results
were consistent for transgenic cattle (clones genetically altered to produce pharmaceutical
proteins on a commercial scale in their milk) and for non-transgenic cattle. The Infigen study
is the largest of its kind to date, and included nearly 120 live calves, and more than 50
piglets, derived from unique non-embryonic derived (somatic) cell lines. Further, the
reported efficiency of the Infigen nuclear transfer technology for both cattle and pigs marks
it as a commercially viable process, according to the study.
Infigen is a privately-held biotechnology company combining genomics and reproduction
technologies to advance human health. The company's data is being presented today at a
scientific meeting sponsored by The National Academy of Sciences in Washington, DC.
Infigen also presented the first detailed analysis of milk harvested from cloned Holstein
cows. A preliminary biochemical fat, and protein analysis found that the milk is identical to
milk taken from non-cloned cows found in the general population, and is safe for human
consumption, according to the company. Results of the study will be published in a peer
reviewed scientific journal in the first half of 2002.
"Infigen has demonstrated that its proprietary nuclear transfer-based cloning technology is a
commercially viable platform that can produce valuable livestock for a variety of human
therapeutic and agricultural applications," said Michael Bishop, Ph.D., President and Chief
Scientific Officer at Infigen. "General health exams, microarray analyses and other tests, as
well as the longevity and normal levels of care afforded these animals, all demonstrate that
Infigen clones are the equivalent of their non-cloned counterparts in every appreciable
respect." Exact data from the studies are being withheld pending publication in a peer
reviewed scientific journal in the near future.
The study included an analysis of global methylation, a widely accepted metric of the
genetic functionality of an organism. Analyses of the progeny of clones, and clones of
clones, also supported the study's conclusions, Dr. Bishop said.
"Nuclear transfer-based livestock cloning is creating whole new industries for therapeutic
production of pharmaceutical proteins; an important potential limitless source for
replacement cells, tissues and organs for human transplantation; and the preservation of
valuable genetic traits found in certain breeds of animals," said Dr. Bishop. "Infigen is
confident that this extensive report, as well as subsequent scientific publications, will help to
advance the potential use of this new technology."
27
Current Status of Cloning in Animal Production in Australia and New Zealand
Appendix 2
Appendix 2
Report on safety of food products from cloned
cattle by the Japan Research Institute for Animal Science in
Biochemistry and Toxicology
August 13th 2002
Outline of Investigation on the Attributes of Cloned Bovine Products
Commissioned by the Livestock Technology Association from FY1999, we conducted an
investigation on the attributes of cloned bovine products. The following are the results of
this investigation.
1.
OBJECTIVES
To conduct an investigation on the blood attributes of cloned cattle (BNT 11cloned cattle or
SCNT cloned cattle), and analyze the components of cloned bovine products (milk and
beef), as well as to conduct a study on animal feeding of feed additives from cloned cattle,
and obtain data comparing cloned bovine products and existing foods (products from
ordinary cattle produced by artificial insemination, etc).
2.
OUTLINE OF INVESTIGATION
(1) Blood test
(Material and method)
Blood was sampled from ordinary cattle and cloned cattle at 3, 6, and 9 months of
pregnancy and 3 and 6 weeks after birth in the case of dairy cattle (Holstein), and 3 to
4 times during a period from 21 to 28 weeks after birth in the case of beef cattle
(kuroge-wagyu). The sampled blood was subject to hematological testing (12 items
including red blood cell count, white blood cell count, and hemoglobin) and
biochemical examination of blood (25 items including total protein, and total
cholesterol) and compared.
(Results)
None of the animals showed abnormalities in performance status. There were also no
biologically significant differences* in any of the test values between ordinary cattle
and cloned cattle, for both the dairy and beef types.
* A biologically significant difference means a difference that could possibly have an effect on factors
such as health and survival evident between the study groups. There are no problems if a biologically
significant difference and statistically significant difference are in accord, but even if there is a statistically
significant difference between the study groups in general, and that the difference is within the range of
normal values it is unlikely that health would be affected, so one could not say that a biologically
significant difference exists. In the investigative report, biologically significant difference was studied in
addition to statistically significant difference. The same applies hereafter.
(2) Analytical study of milk and meat components
(Material and method)
The general components (water content, protein, lipids, and sugars), amino acids (18
types), and fatty acid (21 types) content (content per 100 g) in milk and slices of meat
(9 sites) sampled from ordinary cattle and cloned cattle were measured and compared.
(Results)
11
BNT, Blastomere nuclear transfer cloned cattle; SCNT, somatic cell nuclear transfer cattle.
28
Current Status of Cloning in Animal Production in Australia and New Zealand
Appendix 2
Although there were slight variations seen among individual cattle, no biologically
significant differences were evident in the general components, amino acids, and fatty
acid content between ordinary cattle and cloned cattle for both milk and different sites
of meat (Table 1).
(3) Milk and meat digestion study
(Material and method)
A study of digestion of pieces of meat sampled from ordinary cattle and cloned cattle
by artificial gastric juice and intestinal juice, and a study of digestion of milk and meat
that had been frozen, dried, and powdered (freeze-dried food) and added to feed, using
rats were conducted, and the digestion rates that were regarded as parameters of
protein were compared.
(Results)
There were no biologically significant differences in the rates of digestion of the feed
additives due to artificial gastric juice and intestinal juice using rats between ordinary
cattle and cloned cattle (Tables 2 and 3).
(4) Allergen testing of milk and meat by mouse abdominal wall method
(Material and method)
We sensitized mice with an intraperitoneal injection of extract from freeze-dried milk
and meat slices sampled from ordinary cattle and cloned cattle. Fourteen days later we
retracted the abdomen and induced an allergic reaction by re-injection in the
abdominal wall. Allergen activity was compared based on the extent of vascular
permeability (diameter of dye leakage) seen due to the sensitization treatment.
(Results)
For both milk and meat slices there were no statistically significant differences in
allergen activity between ordinary cattle and cloned cattle (Table 4).
(5) Feeding test by the supply of a combination feed of milk and meat using rats
(Material and method)
Freeze-dried milk and meat of ordinary cattle and cloned cattle were each combined
with basic feed at concentrations of 2.5%, 5%, and 10% in the case of freeze-dried
milk, and 1%, 2.5%, and 5% in the case of freeze-dried meat, and fed to rats (20 per
group (10 males and 10 females)) for 14 weeks.
The general sign, body weight, food consumption, urinalysis (8 items), sensory and
reflex function, spontaneous movement frequency, general function, reproductive
cycle, hematology at autopsy (11 items), blood chemistry (23 items), autopsy and
organ weights (brain, pituitary gland, cerebral gland, thyroid gland, heart, lungs, liver,
pancreas, adrenal bodies, and reproductive organs) of rats given the feed were
compared among a basic feed group, ordinary cattle group, and cloned cattle group.
(Results)
There were no biologically significant differences in each of the items observed and
tested over time in rats at any concentration of feed additive for milk and meat
between ordinary cattle and cloned cattle (Table 5).
(6) Mutagenicity by milk and meat supply using mice (micronucleus test)
(Material and method)
29
Current Status of Cloning in Animal Production in Australia and New Zealand
Appendix 2
Feed produced in the feed test by the supply of a combination feed of milk and meat in
(5) was given to mice for 14 days whereupon the incidence of bone marrow
micronucleus-possessing erythrocytes appearing was tested (micronucleus) and
mutagenicity (clastogenicity) was studied.
(Results)
Clastogenicity was negative and mutagenicity was not evident for milk and meat feed
additives from ordinary cattle and cloned cattle (Table 6).
3.
SUMMARY
The results (see above and following Tables) revealed no biologically significant differences
in component analysis testing and feed additive animal testing between products of BNT
cloned cattle and SCNT cloned cattle (milk and meat), and the products of ordinary cattle.
For further information contact Yoshitake, Livestock Breeding Technology Center.
The results of this study were submitted as reference material for the “investigative research
on the safety of animal foods that use cloning technology” currently being conducted at
Japan’s Ministry of Health and Welfare
30
Appendix 2
Current Status of Cloning in Animal Production in Australia and New Zealand
Table 1. General components
(1) Milk
Classification
Cattle No.
Protein
(g/100 g)
Fats
(g/100 g)
Sugars
(g/100 g)
Ash
content
(g/100 g)
Ordinary cattle
Min. value
Max value
3.0
3.4
2.2
3.3
4.6
4.6
0.7
0.7
88.1
89.7
100
110
8
10
Mean value
3.3
2.7
4.6
0.7
88.9
105
9
No.1
No.2
2.9
2.9
2.3
3.6
3.0
3.5
0.8
0.7
91.1
89.3
95
105
9
9
No 1
No.2
No.3
3.1
3.3
3.3
4.3
2.6
3.1
4.6
4.4
4.5
0.7
0.7
0.7
87.4
89.1
88.5
120
115
115
9
11
10
BNT
cattle
cloned
SCNT
cattle
cloned
Water
content
(g/100 g)
Calcium
(mg/100 g)
Cholesterol
(mg/100 g)
Note: The analytical values for each animal are the mean of the analytical values for milk sampled at two points – 3 weeks
and 6 weeks after delivery.
(2) Meat
Cattle No.
Protein
(g/100 g)
Fats
(g/100 g)
Sugars
(g/100 g)
Ash content
(g/100 g)
Water
content
(g/100 g)
Min. value
Max value
17.8
19.6
13.8
22.9
0.4
0.8
0.9
1.0
58.0
64.8
50
68
Mean value
18.4
19.3
0.6
0.9
60.8
59
BNT cloned cattle
17.4
21.2
0.4
0.9
60.2
56
SCNT cloned cattle
16.8
23.8
0.5
0.9
57.9
68
Classification
Ordinary
cattle
Cholesterol
(mg/100 g)
Note: The analytical value for each animal is the mean value of the analytical values of 9 sites: shoulder, chuck loin, rib
loin, loin end, brisket, round, silver side, rump, and tender loin.
31
Appendix 2
Current Status of Cloning in Animal Production in Australia and New Zealand
Table 2.
Digestive
juice
Artificial
gastric juice
Artificial
intestinal
juice
Rates of meat digestion by artificial digestive juices
Rate of digestion after the start of incubation
Sample
Course
Start
0.75 hr
1.5 hr
3 hr
6 hr
12 hr
Ordinary beef
0
68
79
-
95
90
Somatic cloned beef
0
59
78
-
91
90
Ordinary beef
0
-
20
40
66
67
Somatic cloned beef
0
-
28
38
67
63
Note: The digestion rate shows the protein rate of digestion.
Table 3.
Digestion rates of milk and meat in rats
Number of animals
Digestion rate
(mean±standard deviation)
Ordinary cattle
5
83.0±2.6
BNT cloned cattle
5
82.7±2.0
SCNT cloned cattle
5
81.3±3.4
Ordinary cattle
5
83.8±6.6
BNT cloned cattle
5
82.3±4.7
SCNT cloned cattle
5
84.9±3.6
Sample
Milk
Meat
Test group
Note: Milk and meat were each freeze-dried and combined in feed. The digestion rate shows the protein digestion rate.
Table 4. Allergen study of milk and meat by mouse abdominal
wall method
Sample
Milk
Meat
Test group
Number of
animals
Diameter of dye leakage (mm)
(mean±SD)
Ordinary cattle
Control group
Test group
7
10
7.0±3.7
18.0±2.9
BNT cloned cattle
Control group
Test group
7
10
4.7±3.2
18.0±3.9
SCNT cloned cattle
Control group
Test group
7
10
4.9±4.6
17.9±4.2
Ordinary cattle
Control group
Test group
7
10
5.3±5.0
13.0±5.9
BNT cloned cattle
Control group
Test group
7
10
7.0±4.9
12.5±3.5
SCNT cloned cattle
Control group
Test group
7
10
5.7±4.2
13.1±5.0
Note: Milk and meat were each freeze-dried and the extracts were used as samples. The test groups underwent
sensitization treatment and elicitation, while the control groups underwent elicitation only.
32
Appendix 2
Current Status of Cloning in Animal Production in Australia and New Zealand
Table 5. Feed study of milk and meat by formula feed supply
using rats
(1) Changes in rat body weight by milk formula feed supply (mean±standard deviation)
Female
Male
Study group
Number
of
animals
Amount of
body
weight (g)
increase in
1-14 weeks
Feeding period (weeks)
1
2
3
4
5
6
8
10
12
14
Basal diet
10
146±6 189±20 255±34 299±45 344±59 373±58 428±56 448±58 516±47 547±77 401±76
Ordinary cattle
High concentration (10%)
10
146±6 194±11 260±13 304±17 350±19 378±22 426±36 475±43 515±43 544±48 398±48
BNT cloned cattle
High concentration (10%)
10
146±6 175±19 245±15 295±14 333±18 367±16 425±22 462±34 503±41 530±44 384±41
SCNT cloned cattle
High concentration (10%)
10
146±7 196±10 261±17 310±23 353±27 379±30 432±41 473±41 519±43 545±48 399±43
Basal diet
10
117±5 150±6 184±12 208±9 229±10 242±10 267±16 283±15 304±18 310±20 193±18
Ordinary cattle
High concentration (10%)
10
118±5 152±7 181±7 209±12 234±16 247±15 273±20 298±21 316±28 329±40 211±42
BNT cloned cattle
High concentration (10%)
10
119±7 157±11 186±12 208±22 223±27 244±26 272±19 292±21 313±26 326±30 207±27
SCNT cloned cattle
High concentration (10%)
10
118±7 151±12 181±12 209±13 229±16 247±19 274±18 293±19 317±21 330±33 213±33
Note: Aside from studying a 10% milk powder concentration, 10 cattle each were also fed a low concentration (2.5%) and
a medium concentration (5%), but no significant differences were noted.
(2) Changes in rat body weight by meat formula feed supply (mean±standard deviation)
Female
Male
Study group
Number
of
animals
Feeding period (weeks)
1
2
3
4
5
6
8
10
12
14
Amount of
body
weight (g)
increase in
1-14 weeks
Basal diet
10
143±5 216±12 279±14 336±16 382±18 426±22 489±31 534±37 560±42 590±50 447±51
Ordinary cattle
High concentration (5%)
10
143±5 221±9 284±17 337±24 386±30 432±36 492±44 541±55 575±62 604±65 462±65
BNT cloned cattle
High concentration (5%)
10
143±5 219±10 286±14 343±18 392±21 431±26 488±34 535±39 564±43 591±49 448±51
SCNT cloned cattle
High concentration (5%)
10
143±6 215±9 278±14 336±20 392±29 435±35 499±48 551±60 581±70 613±80 469±76
Basal diet
10
120±4 167±14 198±15 228±18 253±26 272±26 290±26 313±33 330±38 338±35 218±38
Ordinary cattle
High concentration (5%)
10
120±4 169±11 200±12 230±15 254±19 274±18 297±23 316±28 331±31 341±30 221±30
BNT cloned cattle
High concentration (5%)
10
120±5 171±8 201±10 236±15 260±20 280±23 311±29 330±27 347±35 361±40 241±39
SCNT cloned cattle
High concentration (5%)
10
120±4 167±8 195±9 227±11 250±12 268±12 292±16 310±19 329±20 336±20 216±17
Note: Aside from studying a 5% meat powder concentration, 10 cattle each were also fed a low concentration (1.0%) and
a medium concentration (2.5%), but no significant differences were noted.
33
Appendix 2
Current Status of Cloning in Animal Production in Australia and New Zealand
Table 6. Mutagenicity by the supply (14 days) of milk and meat
using mice (micronucleus test)
(1) Milk
Test group
Negative control
group (basal diet)
Ordinary cattle
2.5% group
5% group
10% group
BNT cloned cattle
2.5% group
5% group
10% group
SCNT cloned cattle
2.5% group
5% group
10% group
Positive control
group
(Mitomycin C)
Number
of
animals
Incidence (%) of micronucleus
appearance
(Min – max)
Polychromatic erythrocyte rate (%)
(Min – max)
Assessment
6
0.27±0.10
(0.1 – 0.4)
49.2±6.6
(42.2 – 57.1)
6
6
6
0.22±0.17
0.20±0.14
0.12±0.10
(0.0 – 0.4)
(0.0 – 0.4)
(0.0 – 0.2)
49.4±3.8
45.7±5.0
44.5±7.5
(43.1 – 53.1)
(36.8 – 50.3)
(35.4 – 56.9)
Negative
Negative
Negative
6
6
6
0.30±0.14
0.25±0.12
0.17±0.08
(0.1 – 0.5)
(0.1 – 0.4)
(0.1 – 0.3)
44.0±6.7
47.4±8.1
44.7±8.4
(36.5 – 55.2)
(36.5 – 56.3)
(32.3 – 56.8)
Negative
Negative
Negative
6
6
6
0.22±0.13
0.28±0.15
0.25±0.05
(0.0 – 0.3)
(0.1 – 0.5)
(0.2 – 0.3)
49.7±7.4
49.5±7.8
44.0±6.2
(35.8 – 56.3)
(41.2 – 60.9)
(34.2 – 52.8)
Negative
Negative
Negative
6
6.02±1.03**
(4.6 – 7.6)
34.6±5.5
(26.3 – 40.3)
Positive
(2) Meat
Test group
Negative control
group (basal diet)
Ordinary cattle
1% group
2.5% group
5% group
Number
of
animals
Incidence (%) of micronucleus
appearance
(Min – max)
Polychromatic erythrocyte rate (%)
(Min – max)
6
0.20±0.18
(0.0 – 0.5)
47.7±9.7
(30.2 – 59.7)
6
6
6
0.17±0.12
0.13±0.08
0.12±0.15
(0.1 – 0.4)
(0.0 – 0.2)
(0.0 – 0.3)
50.0±9.1
47.3±13.1
46.8±10.5
(37.9 – 61.3)
(22.3 – 60.2)
(37.2 – 63.5)
Assessment
Negative
Negative
Negative
BNT cloned cattle
1% group
6
0.20±0.06
(0.1 – 0.3)
51.0±7.3
(41.3 – 59.3)
Negative
2.5% group
6
0.23±0.14
(0.0 – 0.4)
47.1±4.3
(40.6 – 51.1)
Negative
5% group
6
0.12±0.08
(0.1 – 0.2)
49.6±9.6
(37.9 – 61.9)
Negative
SCNT cloned cattle
1% group
6
0.18±0.10
(0.1 – 0.3)
48.3±8.4
(35.6 – 55.1)
Negative
2.5% group
6
0.22±0.10
(0.1 – 0.4)
51.7±7.3
(44.3 – 63.9)
Negative
5% group
6
0.22±0.08
(0.1 – 0.3)
48.4±8.1
(38.4 – 58.1)
Negative
Positive control
group
6
6.95±1.56**
(4.1 – 8.4)
25.7±6.5
(19.4 – 36.4)
Positive
(Mitomycin C)
Note: Milk and meat were freeze-dried and powdered and combined in feed. The positive control group was administered
a single dose of 2 mg/kg of mitomycin C intraperitoneally. Values were shown as mean±standard deviation.
**
denotes a significant difference at p<0.01 against the positive control group
34
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
List Tables
Table 1 Current Efficiency of Somatic Nuclear Cloning (Niemann &Lucas-Hahn, 2003) .....6
Table 2 Current status of somatic cell cloning technology by NT .........................................10
Table 3 Current Status of Livestock Cloning in Australia and New Zealand .......................17
Table 4 Criteria used in selecting animals for cloning ..........................................................19
Table 5 Suzi Milk Test Results (Updated 6 February 2003) ..................................................21
List of Figures
Figure 1 Animal Cloning: Diagramatic Representation ...........................................................5
35
Current Status of Cloning in Animal Production in Australia and New Zealand
Professor R. F. Seamark
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