Download Sex determination: There are areas in which cattle management

Sex determination:
There are areas in which cattle management could be improved if calves of a
predetermined sex could be produced. Dairy farmers, for example, would prefer most
of their calves to be female and thus potentially become replacements for the milking
herd. Most Holstein male beef calves have a very low value in many countries. Faster
genetic gain can be expected, especially if the technique is used in conjunction with
schemes such as MOET (multiple ovulation and embryo transfer). Beef farmers may
often prefer their cows to produce bull calves,although the economic advantage may
be less pronounced. There are two main approaches to producing a calf of a particular
sex:(1) determination of the gender of embryos and selecting those of the desired sex
for transfer, and (2) using either X- or Y-bearing spermatozoa to produce female or
male embryos respectively.
Embryo sexing:
A number of methods have been used with some degree of success to determine the
sex of an embryo before it is transferred to a recipient.These include:
• Karyotyping.Individual cells are removed from the embryo,usually without
compromising its viability, and checked to see if they are XX (female) or XY (male)
(Winterberger-Torres & Popescu, 1980).
• Immunology. Antibodies are used to detect the presence of male-specific antigens
(White & Anderson, 1987; Utsumi & Iritani, 1993).
• Detection of metabolic differences (Rieger,1984),including the colorimetric assay
of an enzyme linked to the X chromosome (Williams, 1986).
• DNA analysis (Bredbacka et al., 1995). This approach has been facilitated by the use
of polymerase chain reaction to amplify specific stretches of DNA, such that the
technique is now available commercially (Shea, 1999).
The main drawback to embryo sexing is that embryos of the unwanted sex are usually
discarded, resulting in a waste of resources.
Semen sexing:
If semen were sexed,embryos of the required sex could be produced,either in vivo or
in vitro, thus avoiding the effort and wastage of having to determine the sex of an
embryo, and then possibly discarding it if it proves to be of the wrong gender. Sexed
semen is potentially of use (1) in practical management to inseminate cows with the
aim of producing calves of the required sex and (2) for the fertilization of oocytes in
vitro to produce embryos of the required sex.
For many decades there have been attempts to separate X- and Y-bearing
spermatozoa on the basis of their physical, biological and immunological properties.
The latter has shown some promise, with the possibility that sexspecific antigens
could be present on bovine spermatozoa (Howes et al.,1997). The ultimate aim would
be to immunize mice with the appropriate protein, use individual immune cells to
produce antibody in tissue culture and to grow the best cultures on an industrial scale
to produce pure monoclonal antibody.The appropriate antibody would then be used to
kill spermatozoa of the unwanted type. The existence and siting of such antigens is
still not well established, and this and other procedures have been overtaken by the
development of flow cytometry techniques, whose use has been reviewed by Seidel
(2003).
Flow cytometry is made possible by the fact that X-bearing sperm contains 3–4%
more DNA than Y-bearing sperm and the availability of a fluorescent dye which binds
specifically to nucleic acids. Details can be found in the literature (e.g.,Seidel,2002)
and only a simple overview of the procedures used will be provided here:
• Freshly collected semen is stored undiluted for up to 9 hours.
• Aliquots of semen are stained with a fluorescent dye (Hoechst 33342) which
permeates the cell membrane and binds specifically to adenine–thymine base pairs on
the DNA molecules of the chromosomes. Female spermatozoa would be expected to
take up more dye.
• Sperm are diluted in a sorting medium.
• Spermatozoa in the sorting medium are introduced into a flow cytometer within a
cylindrical wall of sheath fluid, which guides the flow of liquid through the sorter.
• An oscillating crystal divides the liquid flow into droplets containing individual
spermatozoa.
• As each spermatozoon passes a pair of detectors a laser beam causes fluorescence of
the dye,and the detectors measure the intensity of fluorescence so that a computer can
determine whether it is X or Y bearing.
• A positive or negative charge is applied to each droplet according to whether the
spermatozoon in it is X or Y bearing. Droplets failing to meet the determined criteria
are not charged.
• The droplets pass between a pair of continuously charged plates, which attract
oppositely charged particles, deflecting the flow towards a receptacle for the
appropriate type of sperm. Uncharged droplets pass undeflected into a waste
container.
The process is illustrated in Fig. 13.1 and components of the equipment used are
shown in Fig. 13.2. The percentage of sperm in the original ejaculate that actually
survive the sorting and subsequent freezing process is,not surprisingly,very low.
However, the final sample contains a very high proportion of healthy, viable
spermatozoa so that,using techniques such as ‘multi thermal gradient’ freezing
(Chapter 10), it has been possible to use sexed semen for live cow insemination as
well as for in vitro fertilization. The sale of sexed bovine sperm commenced in the
UK in September 2000.