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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 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.
Cattle Breeding Technologies
or 60. in contrast. All female gametes (eggs) are genetically similar. The one critical gene it contains is a ‘trigger’ for testis differentiation. XY (male) Figure 14. or 29 autosomes and either an X or a Y (sperm). partly because of restricted analytical options but in part due to the significant cost of experimentation – embryos are intrinsically far more valuable than sperm. our focus must therefore be the Y chromosome. The former involves determining the sex of a fertilised embryo without impairing its viability. The Y chromosome is very much smaller than the X and contains only a handful of genes needed for fertile male development. These comprise 29 pairs called autosomes and one pair called the sex chromosomes. The Y chromosome clearly contains no genes that are essential for life as it is absent in females with no detrimental effects. at fertilisation. One member of each pair of autosomes is inherited from the animal’s dam (egg) and the second inherited from its sire (sperm). one half of the genetic material of each parent is passed on to progeny. have been commercialised. In the absence of SRY the embryonic gonad differentiates as an ovary and female development follows. however. immunoseparation etc. In this sense. and hormones secreted by the testes then induce all the characteristics of a male. Practical aspects of reproductive technologies for cattle breeding 65 . The X and Y are together known as the sex chromosomes. embryo sexing has been available commercially in Australia for some years. unfortunately.2).Y Fertilisation Zygote Embryo (diploid): 60. the latter requires separation of X. X or 30.2: During reproduction. The male. with all containing 29 autosomes and a single X chromosome. chromatography. during embryo transfer and 2. – and some. 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. Female: 60. Nevertheless. produce two different classes of gametes (sperm). Two approaches have finally proven successful and these will be discussed in some detail. XX Germ cells (2n chromosomes: ‘diploid’) Reduction division (‘meiosis’) Male: 60. one of which contains 29 autosomes and a single X chromosome while the other contains 29 autosomes and a Y chromosome. 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). The Y chromosome is genetically dominant: sex is determined solely by the presence or absence of a Y chromosome. Males. This gene initiates the development of testes. filtration. XX (female). without impairing their ability to fertilise eggs. X Sperm: 30. a gene known as the Sex Determining region of the Y chromosome (SRY). Control of progeny sex in livestock Realistic intervention in sex selection can occur at two stages: 1. In considering sex determination in mammals. 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.or Y-bearing sperm – centrifugation. electrophoresis.and Y-bearing sperm which are normally present in semen in equal proportions. Attempts at embryo sexing are necessarily more recent and have been more limited.XY Ovary Testis Gametes (1n chromosomes: ‘haploid’) Egg (oocyte):30. males determine the sex of progeny since embryo sex depends on which type of sperm succeeds in fertilising an egg. Fusion of sperm and oocyte haploid nuclei after fertilisation restores the total diploid number of 60 chromosomes in the embryo (see Chapter 1) .The nucleus of every cattle cell contains 60 chromosomes which are arranged in 30 pairs. Many futile attempts have been made to separate semen into fractions enriched for X.
see Figure 14. containing 30 pairs of chromosomes in cattle. 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. the absence of such sequences identifies a female. The approach developed in Australia by Dr. Since maleness is determined genetically by a unique (Y) chromosome. neonate and mature animal contains an identical complete set of chromosomes within its nucleus. A fine surgical blade is then used to remove some cells (6 to 12 cells). PCR is the basis of the widely used system for embryo sexing that is described in this article. A single gene on the Y chromosome triggers testis differentiation which in turn initiates development that results in male phenotype.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). This is the basis of embryo sexing. 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. Detection of DNA sequences unique to the Y chromosome identifies a male.3: Biopsy and splitting of embryos.e. 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.3. every cell of the embryo and the developing foetus. 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. Many procedures have been described that allow a minute biopsy of just a few cells to be removed from an embryo. Embryo sexing From the time a sperm and egg fuse to form a diploid zygote (i. Charles Herr is unique for its speed and simplicity and is described in Figure 14. After such multiplication. As indicated. Combined with robust technology for removing a few cells from the embryo (embryo biopsy). their ability to form a normal pregnancy). and it must be borne in mind that sperm remain embedded in the zona. 66 Cattle Breeding Technologies . Invention of the PCR provides an analytical method which is sensitive and accurate enough to determine sex rapidly from a small number of cells. these specific DNA sequences can be seen on a special backing material called a gel. 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. 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. 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. The embryo can be transferred immediately into a recipient and the biopsy may be frozen or assayed immediately.2). that females lack. The zona pellucida (shell) is sectioned during biopsy. 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. known as the Y chromosome.e.
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. The suspension is passed through an extremely fine nozzle that vibrates at high frequency. Sperm is treated with a fluorescent dye that binds to their DNA. ultimately. if it lies within parameters that correspond to Y-bearing sperm. Nevertheless. The instrument used is a Fluorescence Activated Cell Sorter (FACS). 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. extreme care is the only way to ensure accurate results. If the signal strength lies within the range selected for X-bearing sperm. conditions are adjusted to ensure that each droplet contains on average just one sperm. 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. 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 . and individual droplets are deflected towards one or the other.or Y. Contamination may come from airborne dust. according to their DNA content. 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). one at a time.or Y. 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. only sperm bearing the Y chromosome are used. X-bearing sperm are used. The pregnancy rate for each demi-embryo is about 50%. the procedure is equally applicable to embryo bisection (splitting) — the generation of two identical half blastocysts (demi-embryos) that yield identical twins. if females are desired. but it yields enough sperm for use in In Vitro Fertilisation (IVF). depending on the charge they carry (see Figure 14. Semen sexing Sex of progeny can be selected before fertilisation if X. Fluorescence Activated Cell Sorting (FACS) Separating X. since the assay can amplify and detect a single molecule of DNA. The stream of minute droplets passes through an ultraviolet laser beam. or it may arise from degraded sperm adhering to the embryo biopsy.4). causing the sperm’s DNA to fluoresce (glow).carrying sperm can be separated. causing the liquid stream to break up into microdrops. as described above. The combination of a reasonable biopsy size can reduce the impact of contamination but. this minute difference can be detected by a sophisticated instrument and used to separate individual sperm. The strength of the fluorescence signal is measured and. making this an effective means of improving pregnancy rate. The combination of sexing and twinning is a potent breeding tool. Assay for Y-chromosomal DNA The assay for Y-chromosomal DNA is done by the Polymerise Chain Reaction (PCR). If male progeny are desired. the microdrop is given an electrostatic charge. a straw in a haystack becomes as easily visible as a hay bale. one positive the other negative. While it was designed for embryo biopsy. In this case the entire blastocyst must be bisected through the inner cell mass (see Figure 14. The procedure is very slow and extremely expensive so it is not at all suitable for commercial application. contamination by as little as a single molecule can give a false positive.carrying sperm has proven extremely difficult. More importantly. resulting in an overall rate of some 100%. The stream of microdrops then falls between two charged plates.3) and each demi-embryo is transferred into a recipient dam (an intact zona is not needed). PCR in embryo sexing The main source of error lies in the extreme sensitivity of PCR. the microdrop is given the opposite electrostatic charge. it may come from the products of previous assays. In other words. Only top-quality embryos are suitable for this treatment (see Chapter 10).
A high proportion of sperm with a Y chromosome have a protein known as MEA on their cell surface. If this is the case.4: Separation of sperm with a Fluorescence Activated Cell Sorter (FACS). A serious drawback is that 95% of sperm is wasted. expert operator and it takes 12–24 hours to process a single ejaculate. However. A third vessel catches the vast majority of drops where sex cannot be determined. However. Sperm separated by FACS have been used in IVF and pre-sexed calves and lambs have been born. Cattle Breeding Technologies 68 . There is a distinct possibility that some or even one of these genes may code for a protein that is expressed by sperm. 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.X-bearing sperm) are collected in the second vessel. It requires a full-time. this approach is only useful for experimental purposes. Antibodies directed against MEA can be prepared by immunising chickens with the protein. being capable of yielding fractions up to 95% purity. Recent studies suggest a real possibility of finding and using sex-specific proteins on sperm. such a protein could be identified and used as the basis of a simple separation method. The most promising candidate seems to be a protein known as Male-Enhanced Antigen (MEA). 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. Separation of sperm using antibodies (Immunoseparation) The only certain. the X chromosome contains a few thousand genes and even the degenerate Y chromosome contains a few dozen in addition to SRY.5: Immunochemical separation of sperm. The FACS costs about $500. its value in supplying separated sperm for further analysis is yet to be realised. both overseas and in Australia. But the system has the undisputed advantage that it works.000 and has high operating costs. identifiable difference between the two classes of sperm is their different sex chromosome complement. Figure 14. Antibodies have been prepared against MEA by injecting a fragment of the protein into chickens.
it may be possible to genetically modify breeding males and/or females to ensure that they throw progeny of disproportionate sex ratio. This work has recently been continued at the Queensland Department of Primary Industries where the goal is to develop a consistent. Conclusion Embryo sexing has been in full commercial use for seven years with a procedure that was developed entirely in Australia. quality-assured technology that will allow routine on-site enrichment of sperm. (1994). It involves detection of Y-chromosomal DNA in a few cells that have been removed from an embryo. Wallingford. UK. Laboratory production of cattle embryos. kits to provide rapid. Practical aspects of reproductive technologies for cattle breeding 69 . As far as sperm separation is concerned. high volume enrichment for X. allowing selection for sex with natural mating. where the female’s immune response may kill or incapacitate sperm expressing that protein. low cost.or Y-bearing sperm (to a level of 80 to 85% purity) may become a reality. An immunised female would produce progeny with a heavy bias towards one sex. This in vitro separation of sperm will be based on recognition of sex-specific proteins by antibodies. Further reading Gordon I. CAB International. It may eventually be possible to extend this to in vivo sex selection by immunising females with sexspecific proteins. Beyond the immediate horizon.sperm.
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