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.
Hair Cell Skin Chromosomes (condensed) DNA

Blood Nucleus Sperm Chromosomes (extended)

Figure 14.1: The genetic material DNA is in chromosomes in the nucleus of every cell. All of the cells that comprise an animals 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.

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The nucleus of every cattle cell contains 60 chromosomes which are arranged in 30 pairs. These comprise 29 pairs called autosomes and one pair called the sex chromosomes. One member of each pair of autosomes is inherited from the animals dam (egg) and the second inherited from its sire (sperm). The remaining two sex chromosomes 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 X chromosome (inherited from his dam) and one additional unique chromosome called the Y chromosome (inherited from his sire) (see Figure 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. 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 as it is absent in females with no detrimental effects. The one critical gene it contains is a trigger for testis differentiation, a gene known as the Sex Determining region of the Y chromosome (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, our focus must therefore be the Y chromosome. All female gametes (eggs) are genetically similar, with all containing 29 autosomes and a single X chromosome. Males, however, produce two different classes of gametes (sperm), one of which contains 29 autosomes and a single X chromosome while the other contains 29 autosomes and a Y chromosome. In this sense, males determine the sex of progeny since embryo sex depends on which type of sperm succeeds in fertilising an egg.

Female: 60, XX Germ cells (2n chromosomes: diploid) Reduction division (meiosis)

Male: 60,XY

Ovary

Testis

Gametes (1n chromosomes: haploid) Egg (oocyte):30, X Sperm: 30, X or 30,Y

Fertilisation

Zygote

Embryo (diploid): 60, XX (female), or 60, XY (male)

Figure 14.2: During reproduction, one half of the genetic material of each parent is passed on to progeny. Germ cells in the gonads (ovary and testis) undergo a process of reduction division (meiosis) to produce oocytes and sperm containing half the number of chromosomes found in normal cells: one member of each of the 29 autosome pairs and an X chromosome (oocytes), or 29 autosomes and either an X or a Y (sperm). Fusion of sperm and oocyte haploid nuclei after fertilisation restores the total diploid number of 60 chromosomes in the embryo (see 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.
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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 6070,000 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 Blastocoel (fluid-filled cavity) Zona pellucida (shell) Trophoblast (develops into placenta) Inner cell mass (ICM) (develops into foetus) Splitting (artificial twinning) Cutting blade ICM Biopsy removal Trophoblast biopsy DNA assay

Biopsied embryo

Demi-embryos

Embryo transfer

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.

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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 sperms 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
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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 1224 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 suspension

Y-bearing sperm

MEA protein

Y X
X-bearing sperm

Vibrating flow nozzle Fluorescence detector Laser beam Charged microdrop containing single sperm Electrostatic deflection plates

Anti-MEA antibodies

Y X
Second antibody (anti-chicken)

Solid matrix

Y
X-bearing sperm Y-bearing sperm

Collection vessels

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.
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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 sexspecific proteins, where the females 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.

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