14

Semen and embryo sexing

Ken Reed

Introduction
The importance of sex
One of the interesting aspects of reproduction in mammals is the intensive effort made by females in
nurturing their young and the distinctive nature of the sexual differences that result from this. In most
species of mammals the male is larger and displays aggressive behaviour, a consequence of competition
for females which have in turn been selected for nurturing a small number of progeny, both before and
after birth.
Production of livestock is defined by a number of biological demands. The female is rate-limiting in
reproduction, and hence in genetic improvement programs, so females are at a premium in nucleus
breeding schemes and in the expansion of production herds. The dairy industry demands an excess of
females but the needs of the meat industries are for high feed conversions and growth rates and for
meat quality, defined largely in terms of fat deposition. Specific industry sectors may find male traits
undesirable, as is the case with boar taint and excessive aggression.
Every industry sector has an inherent productivity bias in favour of one sex. Progeny of the undesired
sex are costly in terms of wasted reproduction and production potential.
Sex is an economic trait, but selection for progeny of one sex presents a unique challenge since it is
only useful before pregnancy.

Sex determination in mammals

The development of all living organisms is programmed by their genetic material called DNA
(deoxyribonucleic acid) which is packaged into long strands known as chromosomes (Figure 14.1). Every
cell of the body contains an identical set of chromosomes within its nucleus with the exception of a few
cell types, especially red blood cells, which do not contain a nucleus and therefore do not contain DNA.
Every time a cell divides, starting with the fertilised egg through embryo, foetal and adult development,
it makes two faithful copies of its chromosomes and passes them on to its two daughter cells.

DNA
Hair
Chromosomes
Cell (condensed)
Skin

Blood
Chromosomes
Nucleus (extended)
Sperm

Figure 14.1: The genetic material DNA is in chromosomes in the nucleus of every cell. All of the cells that comprise
an animal’s body contain a nucleus (except red blood cells). Within the nucleus is the genetic material DNA,
packaged in sixty separate chromosomes (cattle). In a normal cell the chromosomes are extended and cannot easily
be distinguished. When a cell divides its chromosomes condense and can be seen clearly with a microscope.

64 Cattle Breeding Technologies

 The nucleus of every cattle cell contains 60 Female: 60, XX Male: 60,XY
chromosomes which are arranged in 30 pairs.
These comprise 29 pairs called autosomes and one Germ cells
pair called the sex chromosomes. One member of (2n chromosomes: ‘diploid’)
each pair of autosomes is inherited from the
animal’s dam (egg) and the second inherited from Reduction
Ovary division Testis
its sire (sperm). The remaining two sex chromo-
(‘meiosis’)
somes are also inherited from dam and sire but
are paired only in females: we refer to these as X
chromosomes. The male, in contrast, has only one Gametes
(1n chromosomes: ‘haploid’)
X chromosome (inherited from his dam) and one
additional unique chromosome called the Y
chromosome (inherited from his sire) (see Figure Egg (oocyte):30, X Fertilisation Sperm: 30, X or 30,Y
14.2). The X and Y are together known as the sex
chromosomes. The Y chromosome is genetically
dominant: sex is determined solely by the presence
or absence of a Y chromosome. Zygote
The Y chromosome is very much smaller than
the X and contains only a handful of genes needed
for fertile male development. The Y chromosome
clearly contains no genes that are essential for life
Embryo (diploid):
as it is absent in females with no detrimental 60, XX (female), or
effects. The one critical gene it contains is a 60, XY (male)
‘trigger’ for testis differentiation, a gene known
as the Sex Determining region of the Y chromo-
some (SRY). This gene initiates the development
of testes, and hormones secreted by the testes then
induce all the characteristics of a male. In the
absence of SRY the embryonic gonad differentiates
as an ovary and female development follows.
In considering sex determination in mammals, Figure 14.2: During reproduction, one half of the genetic
our focus must therefore be the Y chromosome. material of each parent is passed on to progeny. Germ
All female gametes (eggs) are genetically similar, cells in the gonads (ovary and testis) undergo a process
with all containing 29 autosomes and a single X of reduction division (‘meiosis’) to produce oocytes and
chromosome. Males, however, produce two sperm containing half the number of chromosomes found
different classes of gametes (sperm), one of which in normal cells: one member of each of the 29 autosome
contains 29 autosomes and a single X chromosome pairs and an X chromosome (oocytes), or 29 autosomes
while the other contains 29 autosomes and a Y and either an X or a Y (sperm). Fusion of sperm and
chromosome. In this sense, males determine the oocyte haploid nuclei after fertilisation restores the total
sex of progeny since embryo sex depends on diploid number of 60 chromosomes in the embryo (see
which type of sperm succeeds in fertilising an egg. Chapter 1) .

Control of progeny sex in livestock

Realistic intervention in sex selection can occur at two stages: 1. during embryo transfer and 2. at
fertilisation. The former involves determining the sex of a fertilised embryo without impairing its viability;
the latter requires separation of X- and Y-bearing sperm which are normally present in semen in equal
proportions, without impairing their ability to fertilise eggs.
Many futile attempts have been made to separate semen into fractions enriched for X- or Y-bearing
sperm – centrifugation, filtration, chromatography, electrophoresis, immunoseparation etc. – and some,
unfortunately, have been commercialised. Two approaches have finally proven successful and these will
be discussed in some detail.
Attempts at embryo sexing are necessarily more recent and have been more limited, partly because
of restricted analytical options but in part due to the significant cost of experimentation – embryos are
intrinsically far more valuable than sperm. Nevertheless, embryo sexing has been available commercially
in Australia for some years.

Practical aspects of reproductive technologies for cattle breeding 65

 Small pieces of DNA (or genes) present on the Y chromosome but not on the X chromosome can be
multiplied many times in the laboratory by a process called the Polymerase Chain Reaction (PCR). After
such multiplication, these specific DNA sequences can be seen on a special backing material called a gel.
Invention of the PCR provides an analytical method which is sensitive and accurate enough to determine
sex rapidly from a small number of cells. Combined with robust technology for removing a few cells
from the embryo (embryo biopsy), PCR is the basis of the widely used system for embryo sexing that is
described in this article.

Embryo sexing
From the time a sperm and egg fuse to form a diploid zygote (i.e. containing 30 pairs of chromosomes in
cattle; see Figure 14.2), every cell of the embryo and the developing foetus, neonate and mature animal
contains an identical complete set of chromosomes within its nucleus. The essential material of
chromosomes is DNA (deoxyribonucleic acid) — molecular software that encodes information for the
60–70,000 genes on the chromosomes that specify the animal. Cells of all individuals of a species contain
the same chromosomes and hence the same genes, with one important difference: the cells of a male
contain a chromosome, known as the Y chromosome, that females lack. A single gene on the Y
chromosome triggers testis differentiation which in turn initiates development that results in male
phenotype.
Since maleness is determined genetically by a unique (Y) chromosome, an assay for the genetic
information within that chromosome — for Y-specific DNA — in any cell at any stage in the life cycle
will establish whether that cell is male. Detection of DNA sequences unique to the Y chromosome
identifies a male; the absence of such sequences identifies a female. This is the basis of embryo sexing.

Embryo biopsy (and splitting: ‘artificial twinning’)

Application of an assay to sex embryos demands that a small number of cells be removed from the
embryos without compromising their viability (i.e. their ability to form a normal pregnancy).
Many procedures have been described that allow a minute biopsy of just a few cells to be removed
from an embryo. The approach developed in Australia by Dr. Charles Herr is unique for its speed and
simplicity and is described in Figure 14.3.

Cutting blade Trophoblast

biopsy DNA assay
Blastocoel
(fluid-filled cavity)
Zona pellucida Biopsy Biopsied embryo
(shell) removal

Trophoblast Embryo
(develops into Demi-embryos transfer
placenta)
Splitting
Inner cell mass (artificial
(ICM) (develops twinning)
into foetus)
Cutting blade ICM

Figure 14.3: Biopsy and splitting of embryos. The embryo is placed in suitable culture medium (refer to text) that
causes it to sink and adhere to the bottom of a plastic dish. A fine surgical blade is then used to remove some cells
(6 to 12 cells). The embryo can be transferred immediately into a recipient and the biopsy may be frozen or
assayed immediately. As indicated, the same procedure can be used for embryo splitting (‘artificial twinning’) but
in this case it is essential that the inner cell mass is split since these are the cells that give rise to the foetus.

The zona pellucida (shell) is sectioned during biopsy, and it must be borne in mind that sperm remain
embedded in the zona. Contamination of the sample with a single Y-bearing sperm would give a false
positive result — it is essential that zona fragments are not transferred with the sample.

66 Cattle Breeding Technologies

 A second source of contamination is the biopsy tool. This must be cleaned thoroughly after each use
to ensure that DNA is not carried over from one sample to the next.
While it was designed for embryo biopsy, the procedure is equally applicable to embryo bisection
(splitting) — the generation of two identical half blastocysts (demi-embryos) that yield identical twins.
In this case the entire blastocyst must be bisected through the inner cell mass (see Figure 14.3) and each
demi-embryo is transferred into a recipient dam (an intact zona is not needed). The pregnancy rate for
each demi-embryo is about 50%, resulting in an overall rate of some 100%, making this an effective
means of improving pregnancy rate. The combination of sexing and twinning is a potent breeding tool.
Only top-quality embryos are suitable for this treatment (see Chapter 10).

Assay for Y-chromosomal DNA

The assay for Y-chromosomal DNA is done by the Polymerise Chain Reaction (PCR), as described above.
PCR occupies a unique place in analytical practice — it is the only technique in any field of science that
is able to detect a single molecule.

The Polymerase Chain Reaction (PCR)

The PCR involves multiplying up the small piece of DNA under examination (the SRY region on the Y
chromosome – see earlier). In other words, a straw in a haystack becomes as easily visible as a hay bale.

PCR in embryo sexing

The main source of error lies in the extreme sensitivity of PCR; since the assay can amplify and detect a
single molecule of DNA, contamination by as little as a single molecule can give a false positive.
Contamination may come from airborne dust, it may come from the products of previous assays, or it
may arise from degraded sperm adhering to the embryo biopsy. The combination of a reasonable biopsy
size can reduce the impact of contamination but, ultimately, extreme care is the only way to ensure
accurate results.

Semen sexing
Sex of progeny can be selected before fertilisation if X- or Y- carrying sperm can be separated. If male
progeny are desired, only sperm bearing the Y chromosome are used; if females are desired, X-bearing
sperm are used.

Fluorescence Activated Cell Sorting (FACS)

Separating X- or Y- carrying sperm has proven extremely difficult. They clearly have different DNA
contents due to the difference in size between the X and Y chromosomes but the difference amounts to
less than 1% of total sperm DNA. Nevertheless, this minute difference can be detected by a sophisticated
instrument and used to separate individual sperm, one at a time, according to their DNA content. The
procedure is very slow and extremely expensive so it is not at all suitable for commercial application,
but it yields enough sperm for use in In Vitro Fertilisation (IVF). More importantly, for the first time it
provides separate fractions that can be studied for other differences that might be exploited to develop
a more useable separation procedure.
The instrument used is a Fluorescence Activated Cell Sorter (FACS). Sperm is treated with a fluorescent
dye that binds to their DNA. The suspension is passed through an extremely fine nozzle that vibrates at
high frequency, causing the liquid stream to break up into microdrops; conditions are adjusted to ensure
that each droplet contains on average just one sperm.
The stream of minute droplets passes through an ultraviolet laser beam, causing the sperm’s DNA to
fluoresce (glow). The strength of the fluorescence signal is measured and, if it lies within parameters
that correspond to Y-bearing sperm, the microdrop is given an electrostatic charge. If the signal strength
lies within the range selected for X-bearing sperm, the microdrop is given the opposite electrostatic
charge. The stream of microdrops then falls between two charged plates, one positive the other negative,
and individual droplets are deflected towards one or the other, depending on the charge they carry (see
Figure 14.4).
Collection vessels are located beneath the deflection paths so that all microdrops with a positive
charge (say Y-bearing sperm) are collected in one vessel and all drops with a negative charge (in this case

Practical aspects of reproductive technologies for cattle breeding 67

 X-bearing sperm) are collected in the second vessel. A third vessel catches the vast majority of drops
where sex cannot be determined.
The FACS costs about $500,000 and has high operating costs. It requires a full-time, expert operator
and it takes 12–24 hours to process a single ejaculate. But the system has the undisputed advantage that
it works, being capable of yielding fractions up to 95% purity. Sperm separated by FACS have been used
in IVF and pre-sexed calves and lambs have been born, both overseas and in Australia. However, this
approach is only useful for experimental purposes, its value in supplying separated sperm for further
analysis is yet to be realised. A serious drawback is that 95% of sperm is wasted.

Separation of sperm using antibodies (Immunoseparation)

The only certain, identifiable difference between the two classes of sperm is their different sex
chromosome complement. However, the X chromosome contains a few thousand genes and even the
degenerate Y chromosome contains a few dozen in addition to SRY. There is a distinct possibility that
some or even one of these genes may code for a protein that is expressed by sperm. If this is the case,
such a protein could be identified and used as the basis of a simple separation method. Recent studies
suggest a real possibility of finding and using sex-specific proteins on sperm.
The most promising candidate seems to be a protein known as Male-Enhanced Antigen (MEA).
Antibodies have been prepared against MEA by injecting a fragment of the protein into chickens. These
chicken antibodies were used to separate sperm into a fraction containing 80 to 85% X-bearing (female)

Stained sperm Y-bearing sperm

suspension
MEA protein
Y

Vibrating X
flow nozzle
X-bearing sperm
Fluorescence
detector

Laser beam Anti-MEA

Charged microdrop antibodies
containing
single sperm Y

Electrostatic
deflection plates X
Second antibody
(anti-chicken)

Solid matrix

X-bearing Y-bearing
sperm Collection vessels sperm

Figure 14.4: Separation of sperm with a Fluorescence
Activated Cell Sorter (FACS).
Figure 14.5: Immunochemical separation of sperm. A
high proportion of sperm with a Y chromosome have a
protein known as MEA on their cell surface. Antibodies
directed against MEA can be prepared by immunising
chickens with the protein. The antibodies will bind to
sperm that contain MEA and these sperm can be removed
from semen by passing them over a solid support to which
is attached anti-chicken antibodies.

68 Cattle Breeding Technologies

sperm. This work has recently been continued at the Queensland Department of Primary Industries
where the goal is to develop a consistent, quality-assured technology that will allow routine on-site
enrichment of sperm.

Conclusion
Embryo sexing has been in full commercial use for seven years with a procedure that was developed
entirely in Australia. It involves detection of Y-chromosomal DNA in a few cells that have been removed
from an embryo.
As far as sperm separation is concerned, kits to provide rapid, low cost, high volume enrichment for
X- or Y-bearing sperm (to a level of 80 to 85% purity) may become a reality.
This in vitro separation of sperm will be based on recognition of sex-specific proteins by antibodies.
It may eventually be possible to extend this to in vivo sex selection by immunising females with sex-
specific proteins, where the female’s immune response may kill or incapacitate sperm expressing that
protein. An immunised female would produce progeny with a heavy bias towards one sex, allowing
selection for sex with natural mating.
Beyond the immediate horizon, it may be possible to genetically modify breeding males and/or
females to ensure that they throw progeny of disproportionate sex ratio.

Further reading
Gordon I. (1994), Laboratory production of cattle embryos, CAB International, Wallingford, UK.

Practical aspects of reproductive technologies for cattle breeding 69