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TECHNOLOGIES FOR ASSI STED CONCEPTION/ EMBRYO TRANSFER
IN AGRIC=T~ AD VET=IN=Y PRACTICE
Neal L. First
INTRODUCTION
The natural reproductive processes of domestic animals have
been controlled or assisted largely for the purpose of producing
a larger number of offspring from individuals of superior meat or
milk production.
-For some technologies a secondary benefit has also been
increased reproductive efficiency. Thus, the purpose for
development of methods for assisting natural reproduction has
been different between animal agriculture and human medicine
where enhancement of reproduction has been the focus.
The developed and developing technologies to be discussed
here are artificial insemination, superovulation, embryo
transfer, freezing of embryos, sexing of embryos, multiplication
of embryos by bisection and by cloning, production of embryos in
vitro and modification of embryos by gene transfer.
Artificial Insemination
The oldest and thus far most used biotechnology is
artificial insemination. More than 60% of the 10 million United
States dairy cows and nearly all West European dairy cattle are
presently mated by artificial insemination (Betteridge, 1986~.
The strength of this tool comes from the ability to statistically
identify and select the very best bulls in the country in terms
of genes for milk production and the ability to extend each
ejaculate to produce more than 500 inseminations. Artificial
insemination has been standard practice in the dairy industry for
more than 30 years. During this period its use has contributed
heavily to a doubling of the milk production of each cow and
reduced the number of cows consuming the nation's grain and
forage resources by 50% (Reid, 19781. It has been available but
little used in other species except in limited economic
situations of low labor or high animal value.
Superovulation
Superovulation by injection of a follicle stimulating
hormone has been utilized for many years to increase by 5- to
10-fold the ovulation rate of cattle, sheep and swine (Pineda and
Bowen, 1980~. While this treatment is common practice in the
cattle embryo transfer industry as well as for human in vitro
fertilization, better methods are needed. The ovulatory response
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is unpredictable ranging from 1 to 20 or more in cattle (Massey
and Oden, 1984~. Ovulation does not always occur (Moor et al.,
1984) and an increase in frequency of oocytes with abnormal
chromatin occurs for all species superovulated and studied thus
far (First and Eyestone, 1987~. For cattle and sheep some
increase in yield of fertilizable oocytes has been achieved by
use of an early luteal priming dose of follicle stimulating
hormone in addition to the usual midiuteal treatments (Ware et
al., 1988~. A more uniform superovulation and increased
frequency of high quality transferable bovine embryos has also
been reported from the use of recombinant derived pure ESH
material (Chappel et al., 1988~.
Embryo Transfer
More recently superovulation and embryo transfer have been
used to multiply scarce exotic breeds of cattle and to accelerate
genetic improvement by expanding the maximum number of offspring
possible from genomic combinations of the best females and
progeny tested sires. The embryos are collected from cows
induced by the use of hormones to ovulate -5 to 15 eggs. The
embryos are then transferred nonsurgically to other recipient
cows whose estrous cycles are synchronous with the donor cow
(Seidel, 1981~. Bovine embryos are commonly stored frozen if
more embryos are harvested than the number of recipient cows
available (Massip et al., 1987) or bisected to double their
number if insufficient embryos are harvested (Baker and Shea,
1985; Leibo, 1988~. These latter procedures are currently in use
in the embryo bans f er industry. The use o f embryo bans f er has
resulted in collection of the best dairy cows into a small number
of herds designed to produce and sell embryos. In 1987 this
commercial embryo transfer industry performed approximately
150,000 transfers in the USA and 250,000 in the world, of which
approximately 30% were with frozen embryos (Seidel, personal
communication).
So far neither artificial insemination nor embryo transfer
is widely practiced in domestic species other than cattle. This
is either for reasons of low economic payback or technical
difficulties such as low semen extension, inability to freeze
semen effectively or absence of nonsurgical transfer methods.
Nevertheless these technical problems are being solved. For
example, the first method for nonsurgical transfer of swine
embryos was recently reported (Sims and First, 1987~. In spite
of their past impact on the dairy industry, the tools of
artificial insemination and embryo transfer as practiced are
slower than desired in effecting productivity changes, and are
restricted to the gene pool of existing livestock breeds.
Artificial insemination allows genetic change only through the
male side of the pedigree and embryo transfer results in a cohort
of embryos after superovulation, mating and recovery that are no
more alike than siblings. Because heritabilities are low for
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most desired production traits, methods are needed for producing
large numbers of duplicate copies of high performance individuals
or embryos.
Freezing of Embryos
Freezing embryos for storage, cryopreservation, is a
valuable technology important in facilitating embryo transfer and
in preserving rare genetic traits. Embryos of cattle can be
frozen and, after thawing and transfer, produce pregnancies with
nearly the same efficiency as fresh embryos (i.e., 45-60%; Massip
et al., 1987~. The embryo transfer industry often stores frozen
cattle embryos to preserve surplus; in 1986, for instance, it
froze 30% of the cattle embryos it later transferred (Seidel,
unpublished).
Frozen cattle embryos are routinely thawed by a one-step
method that allows nonsurgical embryo transfer directly from the
straw, as with artificial insemination (Leibo, 1982; Renard et
al., 1982; Chupin et al., 1984; Massip and van der Zwalmen,
1984~. Following freezing and thawing procedures, 50-80% of
livestock embryos survive, including cattle (Lehn-Jensen et al.,
1981; Kennedy et al., 1983; Renard et al., 1983; Pettit, 1985),
sheep (Ware and Boland, 1987), goat (Chemineau et al., 1986) and
horse
embryos (Slade et al., 1984, 1985; Takeda et al., 1984~.
Effectiveness of embryo freezing varies with mammalian
species. Pig embryonic blastomeres have a relatively high lipid
content (Edidin and Petit, 1977) that impedes freezing (Polge et
al., 1974~. Because each species possesses a physically unique
embryo, a universal cryoprotection scheme has not been possible.
Sexing of Embryos
Sexing of embryos before transfer is especially sought by
the dairy cattle industry where females are the desired milk
producing unit. To be useful sexing techniques must be accurate,
efficient, rapid and without detrimental effects on the embryos.
The three most successful approaches have been 1) cytogenetic
karyotyping of the cells of the blastocyst, 2) immunological
detection of a male specific antigen on male embryos and 3) use
of DNA hybridization probes to identify Y (male) chromosome
specific DNA. -
Sexing by Karyotyping. Cytogenetic methods are highly accurate
and also allow identification of aberrant chromosomal karyotypes.
However, karyotyping requires a large number of coicemid arrested
cells to insure a few readable metaphase chromosome displays
(King, 1984~. Embryonic biopsies of sufficient size to yield
sufficient cells may be damaging to the embryo. One approach to
providing sufficient cells has been to bisect the embryo followed
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by cytogenetic analysis of the smaller half and transfer of the
larger. In one experiment this resulted in a normal pregnancy
rate after transfer of the half embryo but sex could be
determined for only 60% of the half embryos subjected to
cytogenetic analysis. Sex when identified was predicted with
100% accuracy (Picard et al., 1984~. Recent research has
resulted in methods to increase the frequency of-metaphase
spreads (Kent and First, 1988). Nevertheless, a large number of
blastomeres, 15-20, is needed for accurate assay. Cytogenetic
analysis does provide an accurate standard for quick evaluation
(less than 15 hr) of other methods.
Sexing by Immunological Methods. The sex of mammalian embryos is
determined by the presence or absence of the Y chromosome derived
from the father since males are the heterogametic sex and can
produce either X or Y bearing sperm cells while oocytes from the
female contain only X chromosomes. Antigens which are coded from
Y specific genes are found in and on male but not female cells.
Male specific antigens are expressed as early as the 8-cell stare
in mice. One such antigen
Goldberg
expressed as early as the 8-cell
has been called H-Y (Krco and
, 1976; Epstein et al., 1980; reviewed by Haseltine,
1983~. Fluorescent labeled antibodies to one or more male
specific antigen provide a way to recognize individuals
expressing male specific genes. Except for cases of chromatin
translocations which often cause intersexuality, the presence of
a Y chromosome antigen specifically identifies a male.
Male specific antigens such as H-Y antigen are known to be
highly conserved across species and present on cells of at least
70 species of vertebrates including mice, rats, cattle, dogs,
goats, donkeys, horses, pigs and humans as well as on cells of
the female, the heterogametic sex, of birds (Wachtel, 1984a).
Both polyclonal (Krco and Goldberg, 1976; Epstein et al., 1980;
White et al., 1982) and monoclonal (Koo and Varano, 1981)
antibodies have been prepared against the serological H-Y
antigen.
The H-Y antigen has been detected on preimplantation murine
embryos by incubation with H-Y antiserum and complement (Krco and
Goldberg, 1976; Epstein et al., 1980; White et al., 1982;
reviewed by Wachtel, 1984a,b). Cell lysis occurred in
approximately 50% of the embryos which were exposed to murine H-Y
antiserum and complement from guinea pigs; but embryos cultured
in medium with H-Y antiserum or complement alone were not
damaged. Karyotypes of embryos that were unaffected by culture
with antiserum and complement showed that 92% were female
(Epstein et al., 1980~. When unaffected embryos were transferred
to pseudopregnant recipients, 86% of the pups born were female
(White et al., 1983~. The obvious disadvantage of this
complement dependent cytolytic method for detection of male
specific antigen is that survival of male embryos is reduced.
When a male specific antibody and fluorescent labeled second
antibody were utilized together the male specific antigen on male
embryos was identified without toxicity to embryos of either sex
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(White et al., 1983~. Removal of complement and use of a
fluorescent labeled second antibody for detection of binding of
the first antibody have resulted in identification of both male
and female embryos without cytotoxic death of the male embryos
(White et al., 1984~. These studies in mice suggest that it
should be possible to sex embryos of bovine and other species by
use of an antibody to one or more male specific antigens.
The accuracy of White et al. (1984, 1987a,b,c) in predicting
each sex from application of this assay to several species is
shown in Table 1. As indicated in Table 1 for the bovine the
assay was nearly perfect for identification of female embryos
(89%~. However, only 80% of male embryos were identified. This
reduced efficiency for males appeared to occur because dead cells
of some embryos exhibited an autofluorescence thus when embryos
with dead cells were female the accuracy of male identification
was reduced. It is likely that this problem could be eliminated
by choosing a fluorescent label of a different wave length. A
perfect prediction of sex may not occur from this method since
females with a fragment of Y chromosome transiocated to an X
chromosome may test as males by this test or by DNA
hybridization. Overall, the accuracy for bovine was 86%. Using
a similar method Wachtel et al. (1988) report an accuracy in
bovine embryos of approximately 85%. While large scale testing
of this antisera and second antibody system will be essential it
would appear that with minor adjustments in the method such as
indicated above, a method for determining the sex of embryos of
domestic animals is at hand and ready for use. Its wide scale
use will depend on the availability of antisera recognizing male
cells.
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Table 1. Accuracy of immunological detection of embryonic H-Y
antigen in various speciesa
"b~so,~-.e
~ -. , Sexed
Species Male Female References
Murine 78% 83% White et al., '983
67% 80% Piedrahita and Anderson,
1985
Bovine 80% 89% White et al., 1987a
Ovine 88% 82% White et al., 1987b
Porcine 77% 86% White et al., 1987c
aFrom White, 1988.
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Sexing by Y Chromosome Specific DNA Hybridization
The third method is based on the isolation, cloning and
subsequent labeling of unique and repetitive DNA sequences from
the Y chromosome. Most of the unit e and transcribed sequences
coding for critical male specific substances such as H-Y antigens
are located at or near the pseudoautosomal (X pairing region) of
the Y whereas repetitive sequences such as GATA-GATA or GACA-GACA
tend to be dispersed over the length of the y'/2. These Y
specific fragments are used as probes to locate homologous
sequences present in DNA from blastomeres, trophoblasts,
amniocytes and other cell types (Gosden et al., 1984; Moyzis et
al., 1987; West et al., 1987~. As fewer three to five
blastomeres can be biopsied from embryos and using an
oligonucleotide pol~erase chain reaction for signal
amplification, embryonic sex can be determined in from 1 to 3 hr
(Grey and Langlois, 1987; Ou et al., 1987~. Numerous Y specific
probes are currently available for sex selection in humans (Page
et al., 1987; Kent and First, 1988), bovine (Leonard et al.,
1987; Ellis et al., 1988; Popescu et al., 1988) and equine (Kent
et al., 1988a,b).
Multiplication of Embryos
The ability to produce multiple copies of an individual or
embryo is of interest not only to researchers, but also to the
livestock industry. Genetic identicals provide the perfect
control for experimental conditions thus reducing the genetic
variation in experiments to zero. A large number of genetically
identical embryos provides a means for embryo phenotypic
selection wherein clonal lines descendent from one embryo are
selected by progeny test for clonal multiplication to large
numbers. This system approaches phenotypic selection and could
permit rapid change in selected characteristics such as meat or
milk production. Two methods of embryo multiplication will be
discussed here. They are embryo bisection and nuclear
transplantation.
Embryo bisection is a procedure whereby an early embryo
(2-cell through the blastocyst stage) is bisected to yield either
2 cells as with a 2-cell embryo, or 2 or more cell masses as with
a morula or blastocyst stage embryo. This procedure results in
identical offspring in sheep (Willadsen, 1982), pigs (Willadsen,
1982; Rorie et al., 1985) and cattle (Ozil et al., 1982; Baker
and Shea, 1985; Deibo, 1988~. Since this procedure is successful
it can be concluder] that these cells are totipotent. However,
the number of identicals produced by this method is limited. If
the embryo is divided more than twice survival to offspring is
reduced. This is likely due to the requirement of a minimum cell ~
number at the time of blastulation. In the mouse this minimum is
8-16 cells. If blastulation occurs with fewer cells, a
trophoblastic vesicle will form without an inner cell mass
~ 102 -
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(Tarkowski and Wroblewolsa, 1967). Therefore, the limit to the
number of identicals produced by splitting is maximally four and
efficiently two (Robl and First, 1985). This procedure is
commonly used in the cattle embryo transfer industry and results
in a pregnancy rate nearly equivalent to the whole embryo with
the number of offspring nearly doubled (Leibo' 1988).
Nuclear Transfer
The second method for producing multiple copies of an embryo
is by nuclear transplantation. A nuclear transplantation
procedure has recently been shown successful in producing cloned
embryos and offspring in cattle (Prather et al., 1987), sheep
(Willadsen, 1986; Smith and Wilmut, 1988) and rabbits (Stice and
Robl, IgSS). This procedure is a modification of a procedure
developed for the frog in 1952 (Briggs and King, 1952).
As shown in Fig. 1 the procedure involves transfer of a
blastomere or nucleus from a valuable embryo of a multicellular
stage into an enucleated metaphase II oocyte with subsequent
development to a multiple cell stage and use as a donor in a
serial recloning. This procedure is being developed in private
industry as well as by the cited authors. Collectively in the
USA and Canada several hundred pregnancies have been produced in
cattle and recloning has been performed. Thus far the largest
number of calves cloned from one embryo has been seven. These
were born at Granada Genetics in 1987.
A system for cloning of embryos useful to the livestock
industry depends on the ability to produce offspring from donor
embryos of large cell number and the ability to reclone as the
clones develop to advanced cell number or to multiply donor ceils
in culture.
Studies with sheep at Edinburgh, Scotland, suggest this
should be possible. The frequency of development to blastocyst
after use of donor cells from blastocyst inner cell mass was 56%
and pregnancies resulted (Smith and Wilmut, 1988~.
In Vitro Production of Embryos
The production of embryos in vitro from abattoir recovered
oocytes is best developed for cattle (Lu et al., 1987; Eyestone
and First, 1988~; although offspring have been produced from in
vitro fertilization of in vitro developed oocytes in sheep
(Crozet et al., 1987) and swine (Cheng et al., 1986~. There are
at least three reasons for producing embryos of cattle in vitro.
First, this technique provides large numbers of embryos for
commercial transfer and calf production. The value of dairy
calves is sufficiently low relative to beef calves in Europe and
Japan that there are economic incentives for transfer of in vitro
produced beef embryos into dairy cow recipients, particularly
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Representative terms from entire chapter:
bovine embryos
Figure 1
^1-
~ —
.
_- ~
I,.'
LN
~ it'' ~
DONOR EMBRYO
I
RECLONE
CATTLE EMBRYO CLONING MODEL
Transfer each donor cell
to separate egg
RECIPIENT EGG ~ ~
(~
with the goal of inducing twinning. In Ireland and Japan large
commercial ventures have been established for in vitro production
of cattle embryos. -Second, in vitro produced embryos are highly
valuable for research where large numbers or precise timing of
fertilization and development are needed. Third, the economic
feasibility of embryo cloning by nuclear transfer requires that
the enucleated oocytes be produced in vitro from abattoir
recovered ovaries and that the new zygote be developed in vitro
to a stage suitable for recloning.
In vitro production of embryos requires development of
technology in three areas, oocyte development and maturation, in
vitro fertilization and in vitro embryo development.
Oocyte Maturation
In domestic species oocytes recovered from follicles matured
in viva either with or without superovulation can be fertilized
and proceed through embryo development with good success (cattle:
Leibfried-Rutledge et al., 1987; swine: Cheng et al., 1986~.
However, oocytes recovered from small follicles (1-5 mm) many of
which have not completed growth and development produce zygotes
which fail to complete embryo development (sheep: Moor and
Trounson, 1977; Crosby et al., 1981; cattle: Leibfried-Rutiedge
et al., 1987~. Development is enhanced when the underdeveloped
oocytes undergo in vitro maturation in the presence of hormone
stimulated granulosa cells (Staigmiller and Moor, 1984; Critser
et al., 1986a; Lu et al., 1987) and to a lesser extent with
cumulus cells confined in a small volume of medium (Critser et -
al., 1986b; Sirard et al., 1988~. The embryo developmental
signals developed during this co-culture or the beneficial
material from the granulosa cells are unknown. At present the
frequency of bovine blastocysts developing from in vitro
fertilization of in viva matured oocytes is approximately >45
(Brackett et al., 1982; Sirard et al., 1985, 1986;
Leitfried-Rutledge et al., 1987), from immature oocytes
co-cultured with granulosa cells it is approximately 23 to 63%
(Critser et al., 1986a; Lu et al., 1987; Xu et al., 1987; Fukui
and Ono, 1988), from culture with significant cumulus
contribution per volume of medium it is 20 to 30% (Critser et
al., 1986b; Goto et al., lg88; Sirard et al., lg88) and from
immature oocytes cultured so a "helper cell" effect is lost <20%
(Critser et al., 1986b and unpublished; Sirard et al., 1988~.
In Vitro Fertilization
The second part of production of embryos in vitro is the
sperm capacitation and fertilization system. Here numerous
capacitation systems have been used including high ionic strength
media and glycosaminoglycans such as heparin-sulfate and fucose
sulfate, aging, pH shift, calcium ionophores and caffeine
- iOS
(reviewed by First and Parrish, 1987, 1988). In general any
agent which causes Ca++ entry into the sperm acrosome and causes
a pH increase within the sperm causes capacitation (reviewed by
First and Parrish, 1988~. From this and with incubation in
serum-free medium at body temperature for a given species in
vitro fertilization has been successful in cattle, sheep, swine
and goats (First and Parrish, 1987~.
Development of Embryos In Vitro
Embryos of each domestic species can be developed with good
efficiency to the blastocyst stage or later by transfer at the
1-cell or 2-cell stage into the oviduct of the respective
species. For cattle the embryos can also be successfully
developed in the oviduct of the sheep or rabbit (Sirard et al.,
~ 9 8 6; Eyestone et a ~ . , 19 ~ 7 ~ . Embryos of a ~ ~ Moment i c spec i es do
not develop to morulae or blastocysts when cultured in any of the
common culture
media (Wright and Bondioli, 1981~. Their development is blocked
at the transition from maternal to zygotic control of development
(Barnes, 1988; First and Barnes, 1988~. The embryos remain alive
but with cleavage arrested and in the resting phase (Eyestone and
First, 1989b).
Recently bovine (Eyestone and First, 1987, 1988) and ovine
embryos (Gandolfi and Moor, 1987) have been cultured through the
period of blocked development and to the blastocyst stage with
good efficiency by co-culture with oviduct epithelial cells or
media conditioned by cultured oviduct cells (Eyestone and First,
1988~. In the bovine the essential oviduct material is in the
protein fraction but its identity is unknown. In the sheep the
essential oviduct component is believed to be a protein of 92.5
KO or its combination with a 46 KO protein. The 92.5 KO fucose
rich glycoprotein increases greatly in the oviduct just before
the block period, transiocates to the zone and embryo and
disappears by the blastocyst stage (Gandolfi and Moor, 1988~.
Whether the bovine oviduct factor is the same is unknown. A
protein fraction from trophoblast cells also has similar activity
in enhancing embryo development (Heyman et al., 1987~. The
nature of these embryotrophic compounds needs elucidation as well
as their mode of action and the way in which the blocked
development relates to initiation of embryonic transcription and
the transition from short to long cell cycles (Barnes, 1988;
First and Barnes, 19887. In spite of these gaps in our .
knowledge, supplementation of embryo cultures with frozen oviduct
cell conditioned media has provided an in vitro method for
development of bovine embryos which has resulted in an
approximately normal pregnancy rate (50%) after transfer into
cows (Eyestone and First, 1989a).
- 106
Gene Transfer
The first production of transgenic mice by Gordon and Ruddle
(1981) and the evidence that mice transgenic for rat (Palmiter et
al., 1982) or human (Palmiter et al., 1983) growth hormone grew
to nearly twice normal size greatly excited animal scientists
with hopes of producing transgenic livestock. In the ensuing
years more than 400 strains of transgenic mice have been produced
for use in studying problems of biology, medicine and animal
agriculture.
However, only a few new transgenic lines of domestic animals
have been produced and in many cases the expected performance has
not been achieved. The slow progress has been largely due to the
low efficiency of production of transgenics by microinjection of
DNA into pronuclei and to the high economic value of each egg
microinjected.
At present transgenic swine have been produced in at least
five different laboratories, transgenic sheep in at least three
and transgenic cattle embryos, fetuses or offspring in three
laboratories (Rexroad and Pursel, 1988; Murray et al., 1988~.
A second problem has been failure of expression of the
desired response (i.e., growth) or failure of expression at the
desired time or in the desired tissue (Rexroad and Pursel, 1988~.
These problems are being resolved as more is learned about the
promoter and enhancer sequences used with the gene of interest
Especially exciting are the possibilities for targeting gene
expression exclusively to skeletal muscle for alteration of the
meat product (Shani et al., 1987) or to the mammary gland for
alteration of the composition of milk or for production of
pharmaceutical proteins in milk (Simons et al., 1987~.
Conclusions
A large array of gamete and embryo biotechnologies have been
developed for use in animal agriculture. Older technologies with
high economic value such as artificial insemination, embryo
transfer, freezing and splitting of embryos have over time
developed to high efficiency. Newer technologies such as embryo
sexing, in vitro production of embryos, embryo cloning and gene
transfer show promise for commercial use but require research to
become more efficient. Rapid development of each technology is
enhanced in a given species by availability of gametes and
embryos as well as the existence of supporting technology such as
nonsurgical embryo transfer and economic
incentives.
- 107
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