Research in Assisted Reproductive
Technologies
CSREES-supported research in assisted reproductive
technologies includes:
Assisted Reproductive
Technologies
Various techniques have been developed and
refined to obtain a large number of offspring
from genetically superior animals or obtain
offspring from infertile (or subfertile)
animals. These techniques include: artificial
insemination, cryopreservation (freezing)
of gametes or embryos, induction of multiple
ovulations, embryo transfer, in vitro fertilization,
sex determination of sperm or embryos, nuclear
transfer, cloning, etc. To take full advantage
of the benefits of assisted reproductive
technologies, one must understand the basic
physiology of the female and male reproductive
systems as well as various methods to synchronize
reproductive cycles.
Artificial Insemination
and Cryopreservation
Artificial insemination (AI) has been used
to obtain offspring from genetically superior
males for more than 200 years. Improvements
in methods to cryopreserve (freeze) and store
semen have made AI accessible to more livestock
producers. In the same manner as cryopreservation
of semen, embryo freezing allowed for the
global commercialization of animals with
high genetic qualities. Semen from bulls
is especially amenable to freezing and long-term
storage. In the dairy industry, where large
numbers of dairy cows are managed intensely,
AI is simple, economical, and successful.
More than 60 percent of dairy cows in the
United States are bred by AI. However, the
situation is different for beef cattle, where
breeding populations are usually maintained
on range or pasture conditions. In the United
States beef industry, AI accounts for less
than 5 percent of inseminations. For reasons
that are not yet well understood, it is more
difficult to freeze and store semen from
other livestock species, including horses,
pigs, and poultry, than it is to freeze cattle
semen. CSREES has supported research
projects to understand the physiological
processes associated with cryopreservation
of semen or embryos and to develop improved
methods of cryopreservation for gametes (eggs
and sperm) and embryos from several livestock
species.
Multiple Ovulation
and Embryo Transfer
Development of embryo transfer technology
allows producers to obtain multiple progeny
from genetically superior females. Depending
on the species, fertilized embryos can be
recovered from females (also called embryo
donors) of superior genetic merit by surgical
or nonsurgical techniques. The genetically
superior embryos are then transferred to
females (also called embryo recipients) of
lesser genetic merit. In cattle and horses,
efficient techniques recover fertilized embryos
without surgery, but only one or sometimes
two embryos are produced during each normal
reproductive cycle. In swine and sheep, embryos
must be recovered by surgical techniques.
To increase the number of embryos that can
be recovered from genetically superior females,
the embryo donor is treated with a hormone
regimen to induce multiple ovulations, or
superovulation. CSREES has supported basic
and applied research to enhance the efficiency
of superovulation and embryo transfer in
livestock species.
In Vitro Fertilization
As an alternative to collecting embryos
from donor animals, methods have been developed
recently to produce embryos in vitro (in
the laboratory). The methods are also called in
vitro embryo production or IVP. Immature
oocytes (female eggs) can be obtained from
ovaries of infertile or aged females, or
from regular embryo donors (described above).
Ovum (egg) pick up is a nonsurgical technique
that uses ultrasound and a guided needle
to aspirate immature oocytes from the ovaries.
Once the immature oocytes have been removed
from the ovary, they are matured, fertilized,
and cultured in vitro for up to
seven days until they develop to a stage
that is suitable for transfer or freezing.
CSREES has supported a number of basic
research projects to understand the physiological
mechanisms associated with production of
embryos in vitro.
Sex Determination
of Sperm or Embryos
The beef industry in the United States prefers
male calves, which tend to have higher body
weights and higher feed efficiency (compared
to female or heifer calves) when placed in
feedlots for the growing and finishing stages
of meat production. In contrast, the dairy
industry prefers heifer calves, which will
ultimately produce offspring and milk for
human consumption. Thus, methods are needed
to determine the sex of sperm or embryos
so producers can control the sex of the offspring
of their livestock. Using a specific dye
that binds to DNA (the Hoechst 33342 stain)
and a flow cytometer/cell sorter, the DNA
content of individual sperm is measured.
In cattle, the X-bearing sperm contain 3.8
percent more DNA than the Y-bearing sperm.
In mammals, the presence of a Y chromosome
(and one X chromosome) determines that the
individual will be a male. Female mammals
contain 2 X chromosomes. Although the process
to sort the X and Y bearing sperm is slow
(approximately 10 million live sperm of each
sex can be obtained per hour—this is
about the number of live sperm required for
one conventional dose of frozen semen for
artificial insemination), this procedure
determines the sex with higher than 95 percent
accuracy.
The ability to sex semen has a large potential
for commercialization; thus, much of the
research to develop and refine sperm sexing
technology has been conducted in the private
sector. XY,
Inc., a small company in Ft. Collins,
CO, has been the leader in developing sperm
sexing technology in cattle, horses, and
pigs. Researchers at the USDA
Agricultural Research Service have also
played a major role in developing sperm sexing
technology for poultry and swine.
CSREES has supported basic and applied studies
on male reproductive physiology that have
laid the groundwork for many of the advances
made by private industry. In addition, CSREES
has supported basic research to develop novel
methods to determine the sex of bovine embryos:
Nuclear Transfer or
Cloning
Since the mid 1980s, technology has been
developed to transfer the nucleus from either
a blastomere (cells from early, and presumably
undifferentiated cleavage stage embryos)
or a somatic cell (fibroblast, skin, heart,
nerve, or other body cell) to an enucleated
oocyte (unfertilized female egg cell with
the nucleus removed). This “nuclear
transfer” produces multiple copies
of animals that are themselves nearly identical
copies of other animals (transgenic animals,
genetically superior animals, or animals
that produce high quantities of milk or have
some other desirable trait, etc.). This process
is also referred to as cloning. To date,
somatic cell nuclear transfer has been used
to clone cattle, sheep, pigs, goats, horses,
mules, cats, rabbits, rats, and mice.
The technique involves culturing somatic
cells from an appropriate tissue (fibroblasts)
from the animal to be cloned. Nuclei from
the cultured somatic cells are then microinjected
into an enucleated oocyte obtained from another
individual of the same or a closely related
species. Through a process that is not yet
understood, the nucleus from the somatic
cell is reprogrammed to a pattern of gene
expression suitable for directing normal
development of the embryo. After further
culture and development in vitro, the embryos
are transferred to a recipient female and
ultimately result in the birth of live offspring.
The success rate for propagating animals
by nuclear transfer is often less than 10
percent and depends on many factors, including
the species, source of the recipient ova,
cell type of the donor nuclei, treatment
of donor cells prior to nuclear transfer,
the techniques used for nuclear transfer,
etc. CSREES has supported research
projects to obtain a better understanding
of the basic cellular mechanisms associated
with nuclear reprogramming.
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