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.E. Wrathall Livestock Production Science 62 2000 287 –316
of a cloned, transgenic animal transmitting TSE to enable more oocytes from preferred females to be
infectivity via recombinant products destined for utilised. Then in the third category is a range of
medical use is an alarming but not implausible techniques, some relatively new, such as sexing,
scenario if sanitary precautions are overlooked. This cloning and genetic modification, which select or
paper explains how TSE transmission risks might modify gametes and embryos to obtain offspring
arise in reproductive technologies and how they with specific characteristics. Taken together, these
should be avoided. Topics covered include the technologies not only supplement what can be
following. achieved by conventional breeding but also enable
the production of new foods, industrial products, and •
Brief summaries of the reproductive technologies drugs for use in human medicine.
in domesticated ruminants. •
Conventional disease transmission risks via re- 2.2. Selective breeding
productive technologies. •
Differences between conventional infectious dis- Although not strictly a reproductive technology it
eases and the TSEs. is apposite to mention selective breeding because
• Natural TSE transmission, especially during re-
susceptibility to some TSEs is strongly affected by production in ruminants.
genetic factors. Selective breeding enables promising •
Risks of TSE transmission via reproductive tech- individuals to be identified and genetic lines to be
nologies, and their control. selected and improved in respect of commercially
important characteristics. One of these characteristics is disease resistance, and selection for resistance to
2. The reproductive technologies conventional diseases, such as mastitis and lameness,
has aroused much interest. Discovery of strong 2.1. Background
correlations between certain genotypes and scrapie susceptibility in sheep and goats though apparently
Selective breeding and the science of genetics not in cattle was followed by development of blood
have been used to good effect over the last 100 years tests to identify those genotypes Dawson et al.,
to improve farm livestock, but the reproductive 1998; Goldmann et al., 1998. Combined with
technologies are now enabling even faster progress. selective breeding, these tests are now being used to
Some, such as artificial insemination AI and em- increase the proportions of scrapie-resistant animals,
bryo transfer in cattle, sheep and goats, are already particularly in sheep populations, and reproductive
well established, and others such as cloning and technologies such as AI and embryo transfer are
genetic modification are on the threshold of commer- enabling even faster progress. Conversely if selection
cial development. Use of AI and embryo transfer is is made for other desirable factors without due
also starting in other domesticated ruminants such as regard to TSE genetics, the result may be popula-
deer and water buffalo. tions of animals that are more susceptible to TSE
Much has been written elsewhere about the re- infections. This could also apply to production of
productive technologies, so the information given cloned and transgenic animals which, for example in
here is simply to help understand how TSE transmis- the case of individuals designed to produce recombi-
sion risks might arise. Attention is mainly confined nant proteins for medical use, should ideally carry
to use of the technologies in cattle, sheep, goats, and TSE-resistant genotypes.
deer, i.e. the ruminants reported to be susceptible to TSE infection. Essentially the technologies fall into
2.3. Semen collection and artificial insemination three main categories. Firstly there is AI which
AI enables effective use of the male in breeding pro-
grammes. The second category includes superovula- AI was developed in the 1930s as a technique to
tion, embryo transfer and in vitro fertilisation; tech- control venereal disease in cattle, and later to facili-
nologies developed mainly in the past 10 to 20 years tate selective breeding, so it is not a new reproduc-
A .E. Wrathall Livestock Production Science 62 2000 287 –316
289
tive technology. It is used extensively in cattle, ing new technology involves production of sexed
especially dairy breeds, but has also been developed semen by flow cytometry see below and for this
for many other species. Partly due to the physical and other situations where available sperm numbers
difficulty of insemination, seasonal breeding and are very low a modified AI technique has been
their tendency to extensive types of husbandry, AI is proposed in which the semen is deposited deep
not widely used in sheep, goats and deer. Disease within the ipsilateral uterine horn ovulation side to
agents can often be found in the semen of infected increase the chance of a functional sperm reservoir in
males, and others may contaminate during collection the active oviduct Hunter and Greve, 1998. Epidur-
and processing. Nevertheless it is well known that if al anaesthesia may be required in this case.
semen donors are properly selected and managed, and if the semen is properly processed, AI is seldom
2.3.2. Semen collection and AI in sheep and goats a cause of disease transmission Hare, 1985; Phil-
Rams and bucks, like bulls, can be trained to pott, 1993; Eaglesome and Garcia, 1997.
donate semen into an artificial vagina small version of the bovine one, and their semen can be frozen.
2.3.1. Semen collection and AI in cattle Sometimes, however, semen is obtained by electroe-
Semen is routinely collected by stimulating the jaculation, which entails placing the instrument into
bull to ejaculate into an artificial vagina, a device the rectum, and sedation or even general anaesthesia
consisting of a strong rubber or plastic cylinder with is usually required. Insemination presents more
a softer latex liner, and filled with warm water problems in sheep and goats than in cattle, partly
Parkinson, 1996a. The inner surface of the liner in because of seasonal breeding, and consequently
contact with the penis is lubricated with petroleum oestrus synchronisation is often used in these
jelly or liquid paraffin, and a rubber extension cone species. Further problems arise due to their smaller
leads to a receptacle tube for the semen. Most bulls size and anatomical differences of the cervix. In-
donate semen voluntarily but occasionally electroe- travaginal insemination with fresh semen can be
jaculation is used, especially with untrained farm successful but pregnancy rates with frozen–thawed
bulls. Electroejaculators the electrodes are inserted semen deposited into the vagina are low. The
by the operator with gloved hand into the bull’s transcervical route is technically difficult and can
rectum. Whatever the collection method, the semen is cause injury, so AI often involves surgery under
diluted with a liquid extender and stored in plastic general anaesthetic or sedation and a local anaes-
straws, usually in a frozen state. Extenders contain thetic McKelvey, 1999. With the animal inverted in
animal origin substances such as skimmed milk and a restraining cradle small amounts of frozen–thawed
or egg yolk in buffered saline with antibiotics, and semen are injected directly into the uterus or
cryoprotectants such as glycerol are also used if the oviduct via laporotomy, or by laporoscopy using a
semen is to be frozen. It should be noted that grasping probe and inseminating pipette.
although the semen is diluted with extender, the spermatozoa are not separated from the seminal
2.3.3. Semen collection and AI in other fluids.
domesticated ruminants Except perhaps in beef cattle where farmers may
AI in domesticated deer, especially the red deer wish to breed within a narrow time period, it is not
Cervus elaphus scotticus, the larger North Ameri- normal practice to synchronise oestrus for AI. For
can sub-species known as wapiti or Rocky Mountain insemination the straw is warmed if necessary to
elk Cervus e . nelsoni, the fallow deer Dama
thaw the semen, then fitted into a metal catheter-like dama, and various hybrids, is being developed along
device AI ‘gun’ and covered with a disposable similar lines to sheep Asher and Dixon, 1994.
plastic sheath Parkinson, 1996b. With a hand in the Semen is collected by electroejaculation under seda-
animal’s rectum to guide the ‘gun’ through the tion, and extenders such as egg yolk glycerol are
vagina and cervix, the technician deposits the semen used for freezing. In larger deer types, e.g. wapiti
into the uterus. This is normally a quick procedure elk, AI is possible by per-rectal manipulation, as in
and no sedation or anaesthetic is required. A promis- cattle, but in others laparoscopic insemination is the
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.E. Wrathall Livestock Production Science 62 2000 287 –316
routine, especially with frozen semen. Semen collec- FSH, and LH, and uterine prostaglandins long
tion and AI have been practised for many years in chain fatty acid compounds such as PGF2a. Apart
domestic buffalo
Bubalus bubalis
Jainudeen, from the gonadotrophins these are all available as
1996, but are still in the development stages in synthetic analogues, so use of tissue extracts is
bison Bison bison Dorn, 1995. The techniques unnecessary, thus avoiding any associated disease
resemble those used for cattle. risks. The gonadotrophins, however, are a different
matter because they cannot be synthesised, and FSH 2.4. Oestrus synchronisation and superovulation
has a key role, especially for superovulation. Several FSH preparations are commercially available Gor-
Oestrus synchronisation facilitates the supervision don, 1994; Christie, 1996 but the most effective and
of breeding programmes and efficient use of sires by widely used are extracts from the pituitary glands of
natural mating or AI. It is especially important in pigs or sheep. Equine chorionic gonadotrophin
species such as sheep and buffalo which show little eCG, extracted from pregnant horse blood, is
sign of oestrus in the absence of a male. It is also a sometimes used, as also is the human menopausal
vital component of embryo transfer programmes. gonadotrophin hMG which is extracted from urine,
Synchronisation is commonly achieved by the use of but whilst their origins suggest they should be safer
intravaginal devices coils, sponges or other soft from a disease perspective than the pituitary-derived
polyurethane plastic appliances impregnated with FSH products, both have disadvantages. Some years
slow release synthetic progestagens, with or without ago it was reported Looney and Bondioli, 1988 that
oestrogen, which inhibit ovarian activity. Sometimes an effective bovine FSH had been produced by
progestagen is given as a subcutaneous implant recombinant DNA technology but this has not been
instead of by intravaginal device. Alternatively pros- marketed commercially. Due to their disease risks,
taglandin injections spaced 10 to 12 days apart are cadaver-derived pituitary hormones have now been
used to re-schedule ovarian activity. To overcome replaced by recombinant products in human medi-
limitations caused by seasonal breeding in sheep, cine.
goats and
deer, photoperiodic
conditioning or
courses of melatonin treatment may be used. 2.5. Embryo collection and transfer
Withdrawal of progestagen, or injection of prosta- glandin, is normally followed by synchronous
Embryo transfer began in cattle and sheep in the growth of follicles and ‘rebound’ into oestrus
1950s and initially involved full-scale surgical inter- within 2 to 4 days. If, however, instead of natural
vention. It was not until non-surgical techniques rebound, a series of injections of follicular stimulat-
were developed for cattle in the 1970s that the ing hormone FSH is given to coincide with the
technology began to be widely used commercially. decline of progestagen, this stimulates growth of
Data from the International Embryo Transfer Society extra follicles and ‘superovulation’. Small FSH doses
IETS Thibier, 1998 show that almost 400,000 may lead to twins or triplets whereas high doses can
bovine embryo transfers took place world-wide in potentially give many more embryos for collection
1997. Most were in-vivo-derived embryos i.e. and transfer. Some programmes incorporate gonado-
flushed from the uterus about a week after con- trophin releasing hormone GnRH injections to
ception but | 30,000 were produced in vitro. Annu- ensure timely release of endogenous FSH and or
al transfers in sheep and goats number at least 6000 luteinising hormone LH from the animal’s own
and 10,000, respectively unpublished data, with pituitary gland.
smaller numbers in buffalo and deer. In view of their potential for carriage of TSE
infectivity see later it is pertinent to consider the 2.5.1. Embryo collection and transfer in cattle
origin of hormonal preparations used in reproductive Collection is usually preceded by synchronisation
technologies. The natural hormones include ovarian and superovulation, and by natural mating or AI at
steroids progesterone and oestrogen, hypothalamic the synchronised oestrus. About 7 days after insemi-
peptides e.g. GnRH, pituitary glycoproteins e.g. nation, when embryos have descended into the uterus
A .E. Wrathall Livestock Production Science 62 2000 287 –316
291
and developed to the morula or blastocyst stage, but ethylene glycol as cryoprotectant, have been de-
are as yet unhatched from the zona pellucida, they veloped which give good results and have the
are collected from the uterine cavity by the non- advantage that they enable direct transfer of frozen–
surgical flushing technique Christie, 1996. This has thawed embryos without cryoprotectant removal
similarities to AI, but requires more complex equip- Voelkel and Hu, 1992. Fresh i.e. unfrozen em-
ment and greater skill. Various types of ‘Foley’ bryos can be loaded directly into straws in the
catheter of silicone rubber, plastic, etc. are used, original collection medium and transferred into
some of which are stiffened with an inner metal recipients within a few hours of their collection.
stylet during manipulation through the cervix into the Recipients are usually oestrus synchronised to
uterus, whilst for others a metal tube introducer ensure synchrony between the maternal reproductive
with inner rod trochar is passed through first, then cycle and the developmental stage of the embryo.
the trochar is removed and the catheter is passed The straw containing the fresh or frozen–thawed
through the introducer. Once in position a balloon- embryo is loaded into a transfer ‘gun’ of similar
cuff on the catheter is inflated to prevent leakage; the construction, although longer, than an AI gun; the
collection fluid medium is then injected through into embryo is then transferred non-surgically in a man-
the uterine lumen and the embryos are flushed back ner similar to AI. The need to manipulate the cervix,
via plastic tubing into a collection flask. As with AI, ovaries and uterus per rectum, and to deposit the
the operator keeps a hand within the cow’s rectum to embryo well down the uterine horn on the side of the
manipulate the reproductive tract, catheter, etc. by active corpus luteum, means that an epidural anaes-
palpation through the rectal wall. At least one thetic is usually required prior to transfer.
assistant is also needed to handle the equipment and to instill the flushing fluid. Donors are almost
2.5.2. Embryo collection and transfer in sheep and invariably given an epidural anaesthetic during em-
goats bryo collection.
As with
cattle, oestrus
synchronisation and
Embryos are collected and processed in a fluid superovulation
precede embryo
collection from
medium which essentially consists of buffered saline sheep and goats, and the hormonal regimes are
with low levels of blood protein e.g. fetal calf serum broadly similar. Insemination, especially if frozen
or bovine serum albumen to maintain embryo semen is used, is by laporoscopy, as described
viability and prevent them sticking together. Anti- above. Embryos are usually collected 5 or 6 days
biotics are also added to control bacterial contamina- post-insemination when at the unhatched morula or
tion. Using a microscope, the embryos are picked out blastocyst stage, and this entails full surgical
from the uterine flushings and examined to establish laporotomy with exteriorization of ovaries and
their developmental stage and viability. For purposes uterus, laporoscopy using similar instruments to
of disease control embryos are usually washed 10 those used for AI McKelvey et al., 1986; McK-
times, as specified in the Manual of the International elvey, 1999, or a combined approach. Small Foley
Embryo Transfer Society IETS, and are sometimes catheters, similar in design to the bovine type, are
also treated with trypsin a proteolytic enzyme from used, and flushing media tend to be of similar
porcine or bovine pancreas to ensure certain viruses composition to those for bovine embryos. Sanitary
will be removed, if present Stringfellow, 1998. processing and freezing of in-vivo-derived sheep and
Embryos for freezing are passed through solutions of goat embryos are basically the same too. Embryos
a cryoprotectant e.g. glycerol, aspirated into plastic are transferred via laparotomy under general anaes-
straws 0.25 ml, cooled in a freezing apparatus, then thesia, or by the laparoscopic method which resem-
stored in a liquid nitrogen refrigeration tank. Eventu- bles that used for AI in sheep and goats.
ally, when thawed, the embryo is passed through dilutions of glycerol or sucrose in buffered saline to
2.5.3. Embryo collection and transfer in other remove the cryoprotectant; it is then loaded into
species another straw for transfer into the recipient. Alter-
Oestrus synchronisation and superovulation can be native freezing methods such as ‘vitrification’, using
achieved in deer, buffalo and bison in essentially the
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.E. Wrathall Livestock Production Science 62 2000 287 –316
same way as for other ruminants Asher and Dixon, blastocyst, without having to recover them surgic-
1994; Fennessy et al., 1994; Dorn, 1995. Embryos ally. Reasons why IVP embryos have proved less
are collected surgically in smaller species of deer, as popular for commercial transfer include their lower
in sheep and goats, but in larger species e.g. wapiti survival after cryopreservation, higher disease trans-
elk, and in buffalo, non-surgical embryo collection mission potential, and a tendency for the resulting
and transfer can be done under epidural anaesthesia, offspring to develop a foetal oversize problem with
as in cattle Misra et al., 1998. Deer and buffalo high mortality Walker et al., 1996; Kruip and den
embryos are amenable to freezing in the same way as Daas, 1997.
cattle embryos. 2.7.2. Oocyte collection and IVF IVP methods in
2.6. Ultrasound scanning cattle
Essentially oocytes are aspirated from immature Ultrasound scanning, especially with the versatile
ovarian follicles, then matured, fertilised and cul- and expensive B-mode apparatus, is a key element
tured in vitro to the morula or blastocyst stage. They in some reproductive technologies Taverne and
are aspirated either from excised ovaries or whilst Willemse, 1989. In large ruminants a probe trans-
still in situ. In the former case the ovaries may have ducer is held and directed by the operator from
been removed surgically although more often they within the rectum whilst in small species it is applied
are taken from batches of abattoir-slaughtered cattle to the external abdominal wall. The transducer is
to a laboratory where follicular aspiration is done connected by a rubber ensheathed electrical cable to
with hypodermic needle and syringe. The other the monitor where tissues of interest are imaged on
method involves aspiration from the ovaries of live the screen. Apart from its obvious use for pregnancy
cows by trans-vaginal oocyte recovery TVOR diagnosis, ultrasound can provide detailed images of
using a purpose-built ultrasound transducer which ovarian follicles and corpora lutea. Also, as de-
houses and guides the aspiration needle Kruip et al., scribed later, a specifically designed transducer can
1991; Looney et al., 1994. TVOR is a skilled be positioned in the vagina of a cow to guide needles
technique, and epidural anaesthesia plus a sedative for the aspiration of oocytes from ovarian follicles.
are required. With the transducer inserted into the Sanitary aspects of scanning should not be over-
vagina and the cow’s pelvic contents visualised on looked, particularly when transducers are placed
the monitor screen, a long e.g. 60 cm single or within the rectum or vagina.
double lumen needle with echo-reflective tip is guided through the anterior vaginal wall and across
2.7. In vitro fertilisation IVF and in vitro the peritoneal cavity into the ovary, the latter being
production IVP of embryos
held adjacent to the transducer by the operator’s hand within the cow’s rectum. Ovarian follicles are
2.7.1. Background to IVP technologies penetrated by the needle and fluids plus oocytes are
Collection of oocytes, IVF, then culture of the aspirated via plastic tubing into a receptacle. The
resulting zygotes, is principally used to produce transducer unit may be covered with a sanitary latex
embryos in cattle Gordon, 1994, but the technology cover when in use but has to be taken apart for
is also being developed for sheep and goats Alvarez cleaning afterwards, and needles must be sterilised if
et al., 1999; Graff et al., 1999, deer Pollard et al., re-used. TVOR can be used to collect oocytes
1995 and buffalo Chauhan et al., 1996; Galli et al., repeatedly from the same animals, including those
1998. IVP embryos are less used in routine com- with reproductive problems and in early pregnancy,
mercial embryo transfer than the in-vivo-derived thus enabling an almost unlimited supply of known
type, but are widely used in research. The technology lineage Garcia and Salaheddine, 1998. Oocytes
is also the foundation for other more advanced from batches of abattoir ovaries, on the other hand,
technologies such as cloning and transgenics since it are difficult to trace back to the donors, so disease
makes available a range of developmental stages, risks are higher.
from the oocyte and pronuclear zygote through to the Whatever method is used to obtain the oocytes,
A .E. Wrathall Livestock Production Science 62 2000 287 –316
293
their subsequent in vitro maturation, IVF and culture straightforward, but aspiration during life by TVOR
require controlled laboratory conditions, a variety of from smaller species such as sheep and goats is
equipment, sophisticated media, and strict sanitary usually impractical, so surgery laporotomy or
standards to prevent contamination. Media tend to be laporoscopy is the method of choice Smith, 1994;
similar to those for in-vivo-derived embryos, but Alvarez et al., 1999; Graff et al., 1999. Production
with higher levels of serum and antibiotics, and of good quality embryos by IVF IVP appears to be
gonadotrophins, steroids, heparin and transferrin may less efficient than in cattle.
be added at different stages of the culture. A straw of frozen semen is usually used for the
2.8. Semen and embryo sexing IVF. Prior to its addition to the oocytes the semen is
washed to remove cryoprotectant and seminal plas- Production of sexed offspring is of value in many
ma, then motile spermatozoa are selected by a situations and can be achieved either by semen
method such as filtration through glass wool, or sexing or by embryo sexing. In the case of bovine
separation on discontinuous gradients of bovine semen, separation of male and female spermatozoa is
serum albumen BSA or ‘Percoll’ silica particles possible because their DNA content differs; those
bound with polyvinylpyrrolidone Gordon, 1994. In with an X chromosome being about 4 heavier
contrast to AI of live cattle, only the sperm fraction Cran et al., 1993; Johnson et al., 1994. The
is used for IVF, and, as described below, it is now technology involves addition of a fluorescent dye to
possible to use sexed sperm to produce IVF embryos the diluted semen which is then passed through a
of known gender. It is also possible to microsurgical- sophisticated flow cytometer. A laser beam activates
ly inject a single sperm into the oocyte to initiate the dye-stained DNA which enables male and female
apparently normal development, a technique known sperm to be identified and sorted within an electrical
as intracytoplasmic sperm injection ICSI. ICSI is field into separate collection tubes. Although | 95
often used in human IVF to enable infertile men to accurate, the sorting rate about 1000 live sperm s is
conceive Kurinczuk and Bower, 1997 but has had too slow to provide enough sexed sperm to use for
limited success in ruminants Catt et al., 1996; Chen conventional AI in cattle; in fact what many would
and Seidel, 1997; Keskintepe et al., 1997; Keskin- consider a minimum dose of 2.5 million live sperm
tepe and Brackett, 1999. Moller et al., 1972 would take almost an hour,
The embryos, once fertilised, are usually co-cul- whilst a typical dose for frozen semen 25 million —
tured for 7 to 9 days with somatic cells such as Parkinson, 1996b would take up to 10 h. Sorted
granulosa cells from ovarian follicles, oviductal sperm, however, can be used for IVF where the low
epithelial cells, or continuous cell lines from other numbers are adequate Cran et al., 1993. Encourag-
species, e.g. buffalo rat liver BRL cells, or monkey ing results have also been reported when small doses
5
kidney Vero cells. In the early days of IVF IVP, of unfrozen, sexed semen 3 3 10 sperm, obtained
newly fertilised bovine embryos were often trans- in minutes by a new sorting process, were given by
ferred surgically into ligated oviducts of live sheep deep intrauterine AI to oestrus-synchronised heifers
and ‘cultured’ there for a week before retrieval and Seidel et al., 1998. Semen sexing as used for cattle
transfer into recipients of the intended species. is possible in other ruminants e.g. Cran et al., 1997
Temporary culture in sheep oviducts is still used but has not been developed commercially. Unfor-
occasionally e.g. Campbell et al., 1996; Wilmut et tunately the complexity of the flow cytometry equip-
al., 1997; Galli et al., 1998 although because there ment means that its effective sterilisation is impracti-
are some potential disease risks this is not a wise cal.
practice. Embryo sexing has been possible for several years
in cattle, but its cost and the fact that half the 2.7.3. Oocyte collection and IVF IVP in other
embryos are of the ‘wrong’ sex have restricted its species
commercial use. One method involves microsurgical In both large and small ruminants, oocyte aspira-
removal of a few cells a ‘biopsy’ from the embryo tion from the ovaries of slaughtered females is
which are cultured then karyotyped to reveal which
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.E. Wrathall Livestock Production Science 62 2000 287 –316
sex chromosome is present Seidel and Seidel, Kato et al., 1998; Stice et al., 1998. The technique
1991. Unfortunately this is slow and rather inaccu- for removing the chromosomes from an oocyte no
rate, and the biopsied embryos tend not to survive distinct nucleus at this stage and replacing with a
well after freezing. Damage to the zona pellucida donor cell nucleus resembles embryo biopsy, and
also breaches the protective barrier against patho- requires similar microsurgical equipment. Whatever
gens, so embryos for export must undergo washing their type, the donor cells are usually co-cultured
and other necessary treatments before microsurgery. initially on rodent cell feeder layers in a medium
A second sexing method also involves a biopsy but containing a high level e.g. 10 of foetal bovine
in this case the sex Y chromosome is detected by serum. They are then dissociated with trypsin and
the polymerase chain reaction PCR Herr and Reid, replication is arrested by further culture in a medium
1991. Although faster and more accurate than with little serum e.g. 0.5. A whole cell is inserted
karyotyping, the PCR method is quite expensive. The beneath the zona pellucida of the recipient oocyte
microsurgical equipment required to hold the em- and, to provoke integration of the donor nucleus into
bryo, cut through the zona pellucida and remove a the surrogate cytoplasm, the oocyte is placed be-
biopsy is complex, expensive, and difficult to steril- tween metal wire electrodes on a glass microscope
ise. Fortunately, only the holding and cutting instru- slide and subjected to electrical pulses with ‘elec-
ments have contact with the embryo and, being trofusion’ equipment.
cheaply fashioned from glass tubing or rod using a Subsequent
to nuclear
transfer the
resultant microforge, these can be discarded after a single use.
zygotes are cultured to morula or blastocyst stage There appear to be few reports of embryo sexing in
before transferring into recipients. Sometimes they ruminant species other than cattle.
are cultured in ligated oviducts of sheep e.g. Camp- bell et al., 1996, although this is not ideal due to
2.9. Cloning infection risks. Culture with high levels of serum and
co-culture cells can be successful e.g. Bourhis et al., Cloning, the production of genetically identical
1998; Kato et al., 1998 but, as with IVP embryos, animals, can be achieved in various ways, the
fetal oversize, congenital abnormalities and neonatal simplest being embryo division or disaggregation to
death have been linked to culture conditions and produce identical twins, triplets, etc. Triplet calves
exposure to serum during these very early stages and quadruplet sheep have been produced by sepa-
Lees et al., 1998. ration and transfer of individual blastomeres from
Probably the best known clone, ‘Dolly’, born in four- and eight-cell stage embryos Willadsen, 1981;
July 1996, was the result of transplanting a nucleus Willadsen and Polge, 1981, but a more practical
from a culture of mammary gland cells from a method for producing identical twins is to split
6-year-old sheep Wilmut et al., 1997. Since then morulae or early blastocysts and to transfer each half
cloned calves have been produced using nuclei from into one or two synchronised recipients Willadsen
cultured oviductal epithelium and ovarian follicle and Godke, 1984; Seidel and Seidel, 1991. Split
cumulus cells from an adult cow Kato et al., 1998. embryos, like biopsied ones, have the problem of
These results are significant because previous at- lowered viability after freezing, but pregnancy rates
tempts to clone mammals by nuclear transfer using over 50 can be obtained with singly transferred,
any cells other than those from very early embryos, fresh demi-embryos, which means over 100 per
or cell lines derived from them, had failed Stice et original embryo.
al., 1998, so it had been assumed that nuclear Identical animals can also be produced by nuclear
totipotency is lost early in development. transfer which involves removing the chromosomes
An original aim of nuclear transfer technology was from a mature usually metaphase I oocyte and
to multiply genetically superior animals for tradition- replacing them with the nucleus from a donor cell.
al farming, but factors such as the low success rate, The latter may be a blastomere from an early
poor viability after freezing, and welfare concerns embryo, or a cell from cultured fetal or even adult
about foetal oversize problems, have tended to tissue Campbell et al., 1996; Bourhis et al., 1998;
dampen enthusiasm. Abnormalities may also arise in
A .E. Wrathall Livestock Production Science 62 2000 287 –316
295
post-natal life, possibly as a consequence of defec- only about 5 of injected zygotes survive to be
tive nucleocytoplasmic interactions and genetic dis- transferred and, of these, only a tiny proportion
ease Morris, 1999; Renard et al., 1999. Adult cell incorporate the transgene in a balanced way to
nuclear transfer can, however, be of value for become productive transgenic adults Eyestone et al.,
preserving endangered breeds and species, as shown 1998. A higher rate of transgenic offspring has
by Wells et al. 1999a who produced a healthy calf recently been reported in cattle by using a re-
from the last surviving Enderby Island cow by plication-defective retroviral vector to introduce
transplanting granulosa cell nuclei into enucleated genes into metaphase II arrest phase oocytes Chan
oocytes from a conventional cow. Interest in the et al., 1998.
nuclear transfer technology is also reviving with Another way of producing transgenic animals
prospects of combining it with genetic modification utilises nuclear transfer technology plus a process
technologies to create cloned transgenic animals. known as ‘transfection’. This has a major advantage
over microinjection in that potential nuclear donor 2.10. Genetic modification transgenesis
cells can be checked to ensure the desired gene is technologies in cattle and sheep
incorporated before they are used Cibelli et al., 1998. Cell lines, usually of embryonic or foetal
Genetic modification involves taking a gene from origin, are initially propagated in culture, as for
one organism the donor and inserting it into the cloning by nuclear transfer. The desired gene con-
genome of another. If successful, the modified struct is linked to a marker gene e.g. one conferring
‘transgenic’ individual has a new gene which resistance to a specified cell toxin and these genes
functions to produce a protein characteristic of the are together inserted into the cultured cells by
donor organism. Traditional techniques for gene transfection, a process usually achieved by concur-
insertion such as pronuclear injection and transfec- rent exposure of the cells and the construct to a
tion see below achieve rather random integration cationic lipid transfection reagent Schnieke et al.,
into the genome, but more precise ‘gene targeting’ 1997. After further culture in a medium containing
methods are being developed which will enable them levels of the toxin lethal to non-transfected cells,
to be inserted or deleted at specific locations on colonies of the resistant cells i.e. transgenic ones
particular chromosomes Wilmut, 1998. Gene dele- with marker gene are selected for further propaga-
tion, for example, can be used to delete the prion tion. These are then used for nuclear transfer in the
protein PrP gene, and individuals modified in this sure knowledge that the resulting embryos, if they
way should not then succumb to TSE. In ruminants, survive, will develop into transgenic offspring.
however, precision gene targeting is a relatively Aliquots of the cells can be frozen down and used
futuristic technology. again and again as nuclear transfer donors.
The primary steps in traditional genetic modi- Since only cells expressing the transgene are used,
fication are to identify and extract the DNA sequence fewer nuclear transfers and pregnancies are needed
of the specified gene; this is then used to produce the to obtain transgenic individuals. Moreover, because
desired gene ‘construct’. Next, many copies usually multiple cloned transgenic individuals are obtained
hundreds of the prepared construct are inserted into in the first generation, testing a few individuals for
the genome of an individual embryo. Typically the health and production is predictive for all present and
constructs are injected microsurgically into one of future clones of that type. Donor cell populations can
the pronuclei of a single cell embryo zygote, also be karyotyped beforehand to ensure that trans-
whereupon the latter is cultured for some days in genic animals will be of the desired sex. Thus, if
vitro or, for example, in a sheep’s ligated oviduct. transgenic females are needed to produce specific
Embryos surviving this culture period are transferred milk proteins e.g. for pharmaceutical use this is
into recipient females, hopefully to develop into possible in the first generation.
transgenic individuals Simons et al., 1988; Gordon, Although a variety of transgenic cattle, sheep and
1994; Velander et al., 1997. Unfortunately pronu- goats have been produced successfully by zygote
clear microinjection is not an efficient technique: injection and by nuclear transfer, adverse effects
296 A
.E. Wrathall Livestock Production Science 62 2000 287 –316
have been reported too, and it is evident that these and often require substantial use of biological materi-
technologies do carry risks as well as benefits. For als. Disease risks associated with oestrus synchroni-
example, transgenic goats and pigs intended for sation and superovulation arise mainly because
production of valuable proteins in milk for human donors and recipients may be treated with potentially
therapy were found eventually to develop peculiar contaminated hormonal products.
mammary lesions Ebert and Schindler, 1993. The IVP embryos are the foundation for most ad-
foetal oversize problems, already mentioned, which vanced reproductive technologies, including cloning
seem to be associated with in vitro culture are also and transgenics, but due to the properties of their
impeding progress with genetic modification tech- zonae pellucidae, which seem to make them ‘sticky’,
nologies. Another more hypothetical risk is that they are less amenable to pathogen removal by
carriage of or susceptibility to infection, including washing than in vivo derived embryos Stringfellow
infection with TSE agents, might permeate a narrow- and Wrathall, 1995; Marquant-Le Guienne et al.,
ly based, genetically modified population and remain 1998; Trachte et al., 1998; Booth et al., 1999;
in it undetected, only to manifest itself many years Langston et al., 1999 The potential for pathogen
later. exposure during oocyte collection, IVF and culture is
further increased by batch production methods, and by the many substances of animal origin, including
3. Overview of the risks of disease transmission cell cultures, which are routinely used Bielanski,