Review Article

Cytogenet Genome Res 120:1125 (2008)

DOI: 10.1159/000118737

Veterinary cytogenetics: past and perspective

P.K. Basrur a G. Stranzinger b
a
b

Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON (Canada)
ETHZ and University Zrich, Vetsuisse Faculty, Zurich (Switzerland)

Accepted in revised form for publication by M. Schmid, 10 January 2008.

Abstract. Cytogenetics was conceived in the late 1800s

and nurtured through the early 1900s by discoveries pointing to the chromosomal basis of inheritance. The relevance
of chromosomes to human health and disease was realized
more than half a century later when improvements in techniques facilitated unequivocal chromosome delineation.
Veterinary cytogenetics has benefited from the information
generated in human cytogenetics which, in turn, owes its
theoretical and technical advancement to data gathered
from plants, insects and laboratory mammals. The scope of
this science has moved from the structure and number of
chromosomes to molecular cytogenetics for use in research
or for diagnostic and prognostic purposes including comparative genomic hybridization arrays, single nucleotide
polymorphism array-based karyotyping and automated
systems for counting the results of standard FISH preparations. Even though the counterparts to a variety of human
diseases and disorders are seen in domestic animals, clinical applications of veterinary cytogenetics will be less well
exploited mainly because of the cost-driven nature of de-

mand on diagnosis and treatment which often out-weigh

emotional and sentimental attachments. An area where the
potential of veterinary cytogenetics will be fully exploited
is reproduction since an inherited aberration that impacts
on reproductive efficiency can compromise the success
achieved over the years in animal breeding. It is gratifying
to note that such aberrations can now be tracked and tackled using sophisticated cytogenetic tools already commercially available for RNA expression analysis, chromatin immunoprecipitation, or comparative genomic hybridization
using custom-made microarray platforms that allow the
construction of microarrays that match veterinary cytogenetic needs, be it for research or for clinical applications.
Judging from the technical refinements already accomplished in veterinary cytogenetics since the 1960s, it is clear
that the importance of the achievements to date are bound
to be matched or out-weighed by what awaits to be accomplished in the not-too-far future.

Retracing the history of cytogenetics as a preamble to

reviewing the status of veterinary cytogenetics was a rewarding as well as a humbling experience. The invitation to
write this review for the volume in honor of our friend and
Swedish colleague, Ingmar Gustavsson, provided us with
the delightful opportunity to regain insight into the multifaceted interests and far-reaching visions of scientists of the

past centuries, who described, sketched, experimented and

theorized to nourish this area of science throughout its long
gestation. The exercise also brought home the truth that the
pioneers accomplished all these in the absence of much of
the currently available research tools and often without recognizing the significance of their discoveries. An exhaustive review befitting the events that contributed to the birth
of cytogenetics and its present day relevance to animal
breeding and production is a daunting task. Innovative
technologies and their applications to domestic animals
have led to an explosive growth of literature, albeit modest
relative to that in human cytogenetics. Even with the currently available privileges of computer access to scientific
literature, one can only hope to do a superficial job of con-

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Department of Biomedical Sciences, University of Guelph
Guelph, ON, N1G 2W1 (Canada)
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densing the developments pertinent to this field. This review is an attempt to touch up on the important chain of
events that led to the birth and growth of cytogenetics and
the current status of veterinary cytogenetics as an area of
scientific enquiry.
Concepts of heredity and the birth of genetics

The familial nature of traits and tendencies has fascinated and frightened human populations over the ages. Explored by philosophers and exploited by fiction-writers, this
feature also figures in various ancient scriptures including
the old testament. On the other hand, the recurrent trends
of traits have assisted breeders to perpetuate the desired and
discard the defective among animals and plants. However,
the physical basis of these traits and the cause of occasional
departure from the norm have remained matters of conjecture and controversy, even among evolutionary biologists.
The prevalent notion on inheritance in the premendelian
days was that organisms are molded by the environment
and that the environmentally acquired characters gradually
blend into their hereditary makeup. This belief received a
scientific boost in 1809 when the French Zoologist, JeanBaptiste Lamarck, a well-known comparative anatomist
and taxonomist, theorized that environmental conditions
determined the direction of species adaptation. Lamarck, a
major figure in the history of biology for envisioning evolutionary change for the first time, pointed out specific features (the long neck of the giraffe and the striped coat of the
zebra, among others) as appropriate examples of adaptive
modifications that gradually became transmissible to the
progeny (Burkhardt, 1970, 1972). Lamarcks line of argument was widely accepted and persisted for half a century
probably because it supported the then popular belief in the
superiority of the environment over heredity in determining the fate of organisms. Even Darwin who had recognized
the occasional variation among members of the same species, which he called sports, had not dealt with how inheritance operated at the time he wrote The Origin of Species
(Darwin, 1859). Although he maintained that the laws governing inheritance are mainly unknown, Darwins own theory of pangenesis (organogenesis by the participation of
gemmules produced by each organ of the parent), was not
incompatible with the inheritance of acquired characters
(Darwin, 1859, 1871; Dodson, 1955).
Two of the strongest opponents to the Lamarckian concept of inheritance of acquired traits were a German physician, Friedrich Leopold August Weismann (Churchill, 1970)
and Darwins cousin, Francis Galton. Weismann advocated
the germ plasm theory (Weismann, 1892) to explain heredity, according to which all multicellular organisms consist
of somato plasm (somatic cells) and germ plasm (germ cells).
The former does not participate in heredity while the latter,
which consists of hereditary determinants, is carried
through generations. The main tenet of this theory was that
the changes to the soma (body), acquired by mutilation or
training during the life time of an organism, is not inher-

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Cytogenet Genome Res 120:1125 (2008)

ited because of the imperviousness of germ plasm (referred

to as Weismanns Barrier) to environmental and/or somatic
influence (Churchill, 1970). On the other hand, Galton, the
father of Biometry (and the inventor of fingerprinting, use
of questionnaires for data collection and the Galton Whistle for hearing test), believed that acquired traits are not
inherited and also refuted the role of circulating gemmules
in heredity (Galton, 1865, 1871). Galton introduced the theory of natural inheritance, which, in essence, was the
blending theory restated in mathematical terms (Galton,
1865, 1897; Bulmer, 2003). Based on the data on the inheritance of a variety of (quantitative) human traits including
stature and intelligence, Galton proposed a blending inheritance for such traits as skin color and a mutually exclusive inheritance for traits like eye color in humans (Galton,
1865).
Six years after Darwin published his concepts on the origin of species, Gregor Johann Mendel, an Austrian monk
at the Augustinian monastery of St. Thomas in Altbrunn
(and a teacher of mathematics and Greek), delineated his
discoveries on the inheritance of traits in garden peas, although the significance of his observations on factorial inheritance remained unknown to the scientific world during
his life time (Mendel, 1865; Dodson 1955). However, as
Mendel himself had predicted, his time came with the rediscovery of his findings at the beginning of the 20th century by prominent biologists in different parts of the world
(Correns, 1900; de Vries, 1900; Tschermak, 1900; Wilson,
1925). The popularity accorded to Mendels rules during the
first decade of the 20th century heralded the birth of a new
science, later named genetics by William Bateson who is
also credited to be the first to have applied Mendels rules to
domestic animals (Bateson, 1902; Johannson and Rendel,
1966). The renewed interest and activities in the area of inheritance soon pointed out that Mendels factors, renamed
genes by Wilhelm Johannsen, a Danish botanist, plant
breeder and statistician (Johannsen, 1909), are the blue
prints of inheritance and that mutations are the universal
tools for variations in living organisms. Contributions of
scientists in different aspects of biology and medicine during the 19th and early 20th century gradually led to the classification of genetics into different sub-disciplines as quantitative genetics, biochemical genetics, population genetics,
cytogenetics and combinations of these as medical genetics,
based on the objects studied and the tools used to define the
questions posed. However, the cornerstones for all these
sub-disciplines and for the recognition of the fundamental
nature of chromosomes as the gene-cargo passing from generation to generation were already laid out by investigators
during the preceding centuries (for review: Wilson, 1925;
Wilson and Morrison, 1966).
Cells, chromosomes and heredity

Cytogenetics is often regarded as an offshoot of genetics.

However, the recognition of the main ingredients of cytogenetics including chromosomes, cells and the field of cy-

tology predate the rediscovery of Mendels laws and the

birth of genetics. The name, cell itself was introduced over
300 years ago, by Robert Hooke, a biologist and chemist (inventor of meteorological instruments, and Hookes law of
elasticity) regarded as a virtuoso scientist in his days (Bennett et al., 2003). Using the compound microscope that he
himself had devised, Hooke recorded the porous microscopic texture of thin slices of cork and other objects and
described them in the book he authored (Micrographia) in
1665 (Bennett et al., 2003; Jardine, 2003). Since then, various
other investigators including his contemporary Leeuwenhoek are reported to have illustrated plant cells, microorganisms, blood cells, and spermatozoa in great detail some
of which were later confirmed by Hooke himself (Bennett
et al., 2003).
The cell received substantial attention in the 1800s from
various investigators. By 1830, the concept of cells and cellular autonomy in morphological and physiological terms,
were accepted by the notable biologists of that time. Robert
Brown, a Scottish physician, botanist, taxonomist and plant
geographer (and the discoverer of Brownian movement) reported the invariable presence of the prominent structure
within the cells of orchids as well as many other plants,
which he termed nucleus. His descriptions of the nucleus
as an integral part of plant and animal cells (Brown, 1833;
Ford, 1991; Harris, 1999) was a major break-through which
stimulated a flurry of activities including reports and
sketches on stages of cell division eventually leading to the
cell theory independently postulated by Schleiden and
Schwann. The basic tenet of the cell theory that the cell represents the unit of structure, function, and reproduction,
was accepted by the biologists of that time; however,
Schleidens erroneous views on cell division had to be corrected by later investigators (Hughes, 1959).
The latter half of the nineteenth century must have been
the most important period in the gestation of cytogenetics,
a period which witnessed great upheaval in experimental
analysis which replaced the ideologically driven notions of
preformation with explicable concepts for biological phenomena. During this period the pattern of cell reproduction
detailed in cell theory was disproved and the fact that every
cell is derived from a pre-existing cell was demonstrated
leading to the concept of cell lineage and to the search for
the mechanisms of cell and nuclear replication. The key
events that nourished the formative aspects of cytogenetics
and some of the scientists (among the many) who helped to
connect the mystery dots related to heredity are briefly described below.
One of the landmark discoveries of this period was that
of Walther Flemming, a German physician who was the first
to document and describe the process of nuclear division. A
professor of anatomy, Flemming pioneered the use of synthetic aniline dyes to visualize the changes taking place in
the nucleus during cell division. He observed that the red
dye was heavily absorbed by the granular structures in the
nucleus, and named them chromatin in 1882 (Paweletz,
2001). Flemming recorded his microscopic observations (on
stained cells of salamander larvae during cell division) in

hand drawings, and indicated that during cell division the

granular chromatin coalesced into thread-like structures,
split longitudinally, became condensed and moved to opposite poles and produced two nuclei identical to each other
and to the original nucleus. He named this process mitosis.
Flemming did not grasp the relationship between cell division and heredity since Mendels theories were not widely
known at the time he wrote the book Cell, Nucleus and Cytoplasm (Paweletz, 2001). The linear elements visualized as
prominently stained bodies during nuclear division were
named chromosomes by a fellow German anatomist, Heinrich Waldeyer (Waldeyer, 1888) who, along with the multitude of his discoveries also coined the name neuron and
proposed the neuron theory of the nervous system. A few
other discoveries of major importance in the context of cytogenetics had already occurred during the latter half of the
19th century. These concerned the nature of hereditary material during the vital process of reproduction: before, during and after fertilization.
Oskar Hertwig, a German anatomist and a leader at the
time in the field of causal factors in animal development,
was the first to define fertilization as the fusion of sperm
nucleus with egg nucleus. Working on Ascaris megalocephala, a horse nematode with low chromosome number, he
demonstrated in 1875 that the chromosome order of the
daughter cells after cleavage divisions is the same as that of
the parents (Churchill, 1970). Edouard van Beneden, a Belgian embryologist and marine biologist, working at the time
on another horse parasite, Ascaris lumbricoides, is credited
with the discovery that gametic nuclei contained half the
number of chromosomes as the parents and that gametogenesis must involve a mechanism by which maternal and
paternal chromosome numbers are halved. His studies also
pointed out, for the first time, that members of the same
species carried the same chromosome number (Dunn, 1965;
Churchill, 1970). Several biologists of the late 1800s are
credited with discovering the important features of meiosis.
Included among them is Weismann who worked on sea urchin eggs, and observed that meiosis involved different
kinds of cell divisions which he named equational division
and reductional division. Familiar with the findings of van
Beneden, Hertwig, Strasburger, Flemming, and other biologists of that time, Weismann reasoned that chromosomes,
based on their importance in cell division and in the process
of reproduction, must be the bearers of heredity (Weismann, 1892; Dunn, 1965; Churchill, 1970).
Hertwigs student, Theodor Boveri, working on the horse
round worm, Ascaris megalocephala, at that time at the
Wrzburg University (Germany), was able to demonstrate
that a maturing egg disposes of half of its nuclear material
through two divisions (involving the formation of the two
polar bodies), thus reducing the chromosome number by
half, prior to fertilization (Boveri, 1888; Baltzer, 1967). He
also noted that egg and sperm contribute similar numbers
of chromosomes which arrange themselves directly onto
the spindle of the first cleavage division. Boveri, switching
to sea urchins for his studies, was also the first to recognize
that the centriole of the sperm serves as a cell division or-

Cytogenet Genome Res 120:1125 (2008)

13

ganelle and contributes to the cleavage of the egg. He showed

not only that the presence and integrity of the chromosomes
are essential for normal function of the cell and normal development of the individual, but that abnormal chromosome compositions lead to abnormal growth and development, including cancer (Wilson, 1925; Stern, 1950; Baltzer,
1964).
Among the prominent scientists of the late 19th century
who contributed to the genesis of cytogenetics, is the wellknown American biologist, Edmond Wilson who was an
early advocate of the chromosome theory of inheritance
(Wilson, 1893, 1896). Wilson, who worked on the inheritance of sex, and his student Clarence McClung, were able
to demonstrate the role of chromosomes in sex determination (McClung, 1902a). Working on testicular cells of locusts (and later on the long-horned grasshopper), McClung
described the behavior of accessory chromosomes in male
germ cells (McClung, 1902b) which he (and Wilson) considered to be the determinant of sex and called X chromosome
although the original identification of the specific chromosome was incorrect (McClung, 1902a, b, 1914; Wilson, 1905,
1910, 1914). McClungs student, Walter Sutton, also studying spermatogenesis at that time (in the Lubber grass hopper, Brachystola magna), observed that chromosomes occurred in distinct pairs and that during meiosis they maintained their integrity and the homologous chromosomes
paired, following which each chromosome moved to a
daughter cell (Sutton, 1902). Studies on Brachystola magna
led Wilson and McClung along with Sutton (who became a
well-known cardiac surgeon in his later life) to provide some
of the earliest evidence that a given chromosome carries a
definable set of hereditary value (Sutton, 1903; Wilson,
1905, 1910, 1914). These scientists and Boveri (Boveri, 1888,
1902; Stern, 1950) were among the earliest to describe the
main features of reduction division (meiosis) and, along
with Flemming, van Beneden, Strassburger and Montgomery, to demonstrate, almost simultaneously with the rediscovery of Mendels rules, that chromosomes are the carriers
of hereditary secrets (Wilson, 1925; for review: Hughes,
1959; Gilbert, 1978, 1987).
Cytogenetics

It is believed that Mendels rules had to wait in the wings

for over 30 years because of the lack, at that time, of a proper scientific back-drop on which they could be projected
(Dodson, 1955; Wilson and Morrison, 1966). The developments in nuclear cytology during the latter half of the 19th
century provided just that back-drop (Wilson, 1914). The
unmistakable parallels between the deduced behavior of
Mendels factors (genes) and the observed behavior of the
chromosomes during the vital processes of life, convinced
notable scientists including Morgan, Sturtevant, Muller and
Bridges (Morgan et al., 1915; Bridges, 1916) during the earlier decades of the 20th century to accept part of Weismanns
chromosome theory of heredity (Crow, 2000). The vigorous
growth of this field of studies during the first quarter of the

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20th century, when the tools available for genetic research

were mainly planned-breeding and the light microscope,
was mainly nourished by experimental findings on fruit
flies, yeast, corn and other organisms, and the questions
posed by investigators were mainly around details of transmission of traits in planned-breeding of plants and animals.
To this end, chromosomes of fruit flies, grass hoppers, lilies,
maize and other experimental systems served to demonstrate the mechanism of sex determination, sex linkage and
the phenomenon of criss-cross inheritance and to embellish
the concept of chromosomes as the physical basis of Mendelian inheritance, albeit with various modifications (Wilson and Morrison 1966; Gartler, 2006).
During this period, investigations based on staining procedures and the light microscopic observations on nuclear
elements were grouped under the umbrella of cytology
which, in essence, referred to nuclear cytology and served
to represent the physical presence of the hereditary material. The type of investigations in cytology during the first
half of the 1900s generally tended to be descriptive and
qualitative, and primarily concerned with the morphology
of the giant chromosomes of fruit flies, meiotic chromosomes of lilies, lampbrush chromosomes of egg-laying organisms and other features relevant to functional differentiation of chromosomes as physical evidences of gene expressions (Wilson and Morrison, 1966). This connection
was recognized by the scientists of that time including Wilson (1925) and further groundwork was accomplished by
Darlington (1937), DeRobertis et al. (1954), Dodson (1956),
Swanson (1957), Hughes (1959) and White (1973) among
others, to bring cytology into genetic thoughts and develop
the hybrid science of cytogenetics.
Veterinary cytogenetics

The initial steps in veterinary cytogenetics were substantially aided by the developments that preceded the 1960s. In
this regard, it is important to acknowledge that the theoretical backbone of this area of science was the wide array
of information gleaned from studies on giant chromosomes
of insects and lower organisms. Furthermore, developments
of domestic animal cytogenetics were achieved with tools
borrowed from human cytogenetics which had already undergone a major break-through by the 60s. These included
a series of independent refinements in approaches to expand the scope of technical achievements and a variety of
key findings in humans and experimental animals. Notable
developments before the early 60s were the rediscovery and
refinement of in vitro culture techniques for mammalian
cells, the discovery of a plant alkaloid (colchicine) to arrest
cells in metaphase (Levan, 1938) followed by the synthesis
of its analogue (colcemide) with less toxicity and more potency as a spindle inhibitor (Dustin, 1947) to increase the
number of dividing cells retained in mid-mitosis, the adaptation of a hypotonic treatment of cells prior to fixation
(Hsu, 1952; Makino and Nishimura, 1952) to give mitotic
cells more room for the distribution of their chromosomes

with a minimum of overlap, the introduction of the air-drying procedure to retain intact cells on the microscopic slide
without the need to use physical force to ensure their adherence to glass slides (squash technique) prior to microscopy
(Rothfels and Siminovitch, 1958) and the discovery of the
mitogenic properties of phytolectins (Nowell, 1960) to enable the use of blood cells for chromosome delineation. Other impetus to veterinary cytogenetic research are the discoveries of a sex-related dimorphism in human interphase
nuclei (Barr and Bertram, 1949) which was also noted to be
a feature of domestic animal nuclei (Moore, 1966) and the
identification of the correct number of human chromosomes (Tjio and Levan, 1956) which was an area of error and
controversy since the late 1800s through to the mid 1900s
(Gartler, 2006). Advances in human cytogenetics are
thought to have been substantially delayed because of the
mis-identification of the human chromosome number
(Gartler, 2006) although the activities in this area subsequent to 1956 have been phenomenal including the recognition of chromosome abnormalities as major factors in malformations, mental retardation, infertility and other reproductive disorders (Ford et al., 1959; Jacobs and Strong, 1959;
Lejeune et al., 1959).
Unlike the situation in human cytogenetics which had a
30 years hiatus due to the general acceptance of the incorrect chromosome number, the correct diploid chromosome
numbers (2n) of some of the common domestic animals
were already reported before 1956 (Krallinger, 1931; Oguma
and Makino, 1937) and included in Makinos mammalian
chromosome atlas even though the information presented
was mainly gathered from stained paraffin sections or
squash preparations (Makino, 1951). The advent of newer
technologies in human cytogenetics provided new impetus
to re-examine the chromosomes and to construct the normal karyotypes of common domestic animals which were
till then considered difficult, if not impossible to work with.
Combinations of these procedures and the methods for culturing whole blood (Basrur and Gilman, 1964) and solid
tissues (Basrur et al., 1963) from mammals were routinely
used for the delineation of chromosomes during the early
days of veterinary cytogenetics.
Although cytogenetic findings in human subjects had
helped to recognize the causal relationship of chromosome
anomalies to malformations, subfertility, and ill-health, the
relevance of cytogenetics to farm animal breeding was not
realized till Ingemar Gustavsson pointed it out through a
series of seminal work initiated in the 60s (Gustavsson and
Rockborn, 1964; Gustavsson, 1969, 1971a, 1975, 1980). It is
unlikely that there is any veterinary cytogeneticist today
who is not familiar with Gustavssons work. His name is
synonymous with the 1/29 Robertsonian translocation in
cattle that illustrated for the first time the relevance of cytogenetics in terms of animal breeding. The younger scientists can see that Gustavsson continues to explore important
aspects of cytogenetics even today; however, many young
scientists may not know the extent of his influence on his
contemporaries and colleagues over the years. His contribution to veterinary cytogenetics which has spanned over 40

years and covered various territories is being extended by

his innumerable students. Although no review of veterinary
cytogenetics can proceed without citing Gustavssons work,
we will leave the pleasure of dealing with his older findings
which paved way for further work by other cytogeneticists,
to those who continue to maintain personal connection
with him through their work (see dedication in this volume).
Cytogenetics and animal reproduction

Domestic animal cytogenetics during the period from

the 60s to mid 70s mainly involved the construction of
karyotypes based on chromosome number and morphological criteria including length, arm ratio of each chromosome
based on the position of its centromere, and the presence of
the nucleolar organizer. By the late 1970s, the scope of cytogenetics had widened considerably and laboratories were
established in various countries under the direction of investigators predominantly known for their cytogenetic interest in specific domestic animal species including cattle
(Gustavsson and Rockborn, 1964; Popescu, 1971, 1972;
Logue, 1977; Rieck, 1984; King, 1990), pigs (McFee et al.,
1966; McFeely, 1967, 1990; Gustavsson, 1969, 1971a, b, 1975),
sheep (Bruere, 1969, 1974), goats (Basrur and Coubrough,
1964; Padeh et al., 1965; Soller et al., 1966; Basrur and Kanagawa, 1969; Padeh et al., 1971) and domestic fowl (Fechheimer and Jaap, 1978), with considerable overlap in the
species investigated (Herschler and Fechheimer, 1966, 1967;
Fechheimer, 1973).
It is not surprising that cattle and pigs which are among
the most important food animals under domestication attracted the greatest share of scientific attention during this
period. In cattle alone, over 13,000 animals belonging to 80
different breeds were karyotyped by the middle of the 70s
(Popescu, 1977). The largest contribution to this line of
study came from Sweden, Germany and France although
significant contributions, based on smaller numbers of animals, were also made by scientists in other parts of Europe,
New Zealand, Russia, North America and South America.
As stated before, the Swedish group under the stewardship
of Gustavsson has the distinction of being the first to report
on the Robertsonian translocation involving the longest
and the shortest chromosomes in bovine karyotype, now
well-known as the 1/29 translocation (Gustavsson and
Rockborn, 1964). This chromosome rearrangement, also referred to as Gustavssons anomaly by his friends and colleagues (Basrur, 1980) has a negative impact on the fertility
of the carriers, varying in degrees depending upon the breed
of the carrier and the breeding pattern (Refsdal, 1976;
Dyrendahl and Gustavsson, 1979). This translocation has
since then been observed in over 50 breeds of cattle studied
by cytogeneticists all over the world. A conservative estimate would put the number of investigators having been
involved with Gustavssons anomaly as over 40, spread over
30 different countries including Canada (Schmutz and
Moker, 1989; Schmutz et al., 1990), France (Darre et al.,

Cytogenet Genome Res 120:1125 (2008)

15

1972; Popescu, 1990a, b), Portugal (Rangel-Figueiredo and

Iannuzzi, 1990, 1993), Spain (Arruga and Zarazaga, 1984;
Arruga et al., 1984; Arruga, 1987), Hungary (Kovacs, 1978;
Kovacs and Csukly, 1980; Kovacs et al., 1992), Poland (Slota
et al., 1988, 2004), Romania (Nicolae and Popescu, 2001),
Switzerland (Stranzinger and Forster, 1976; Stranzinger et
al., 1981), Italy (Di Meo et al., 2006), United Kingdom (Harvey, 1971; Logue, 1977; Logue and Harvey, 1978), and Brazil
(Pinheiro et al., 1979). It is gratifying to learn that Gustavssons anomaly which was once wide-spread in cattle populations of various countries has not been detected in some
European countries to day. Up-to-date results of cytogenetic studies on this and other anomalies from different countries in Europe are summarized by Ducos et al. (2008).
Studies aimed at testing the capacity of spermatozoa
from 1/29 translocation carrier bulls to fertilize bovine oocytes in vitro showed that they are comparable to normal
bovine spermatozoa in their ability to initiate cleavage and
early post-fertilization growth. Earlier studies on embryos
derived using semen from 1/29 translocation carrier bulls
had also shown that the incidence of embryos with chromosome anomalies (related to the translocation) correlates well
with the fertility reduction generally detected in 1/29 translocation carrier bulls (Popescu, 1980; King et al., 1981;
Schmutz et al., 1991, 1996). More recent investigations using
a molecular marker for the 1/29 translocation (Joerg et al.,
2001) on oocytes fertilized in vitro with spermatozoa from
a carrier bull, showed no significant deviation from the expected 50:50 ratio for normal and translocation carrier blastocysts (Menetrey et al., 2003).
The main achievement during the period following the
reports on the 1/29 translocation was the discovery of a variety of chromosome anomalies related to infertility and
poor viability in farm animals, using the Giessener System
in Germany (Rieck, 1984) and other chromosome errors including Robertsonian type translocations in cattle (Popescu, 1971, 1977), pigs (McFee et al., 1966; McFee and Banner,
1969; Masuda et al., 1975; Miyake et al., 1977; Alonso and
Cantu, 1982), goats (Padeh et al., 1965, 1971; Soller et al.,
1966; Hulot, 1969, 1970; Sohrab et al., 1973) and sheep (Bruere, 1969, 1974; Bruere et al., 1976, 1978). Since these translocations were detected mainly on the basis of the overt appearance of the altered chromosomes, the identity of the
chromosomes involved in some of these translocations reported earlier was not unequivocally established. Their
identification awaited the arrival of refinements in chromosomal techniques involving various banding procedures.
Some of these refinements were introduced originally for
the delineation of human chromosomes although they were
even more essential for veterinary cytogeneticists since
chromosomes of domestic animals including cattle, goats
and (to some extent) sheep carry mainly one morphologic
(acrocentric) type of chromosomes and lacked other distinguishing criteria. Adaptations of the major banding techniques introduced by various cytogeneticists at different
times have helped to uncover the identity of over 45 Robertsonian translocations in cattle alone (Fries and Popescu,
1999; Joerg et al., 2001). Furthermore, the specific chromo-

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somes involved in other types of rearrangements including

tandem rearrangements, reciprocal translocations, pericentric inversions and deletions have also been identified in this
species, using the banding techniques (Iannuzzi et al., 1993;
Fries and Popescu, 1999; Iannuzzi et al., 2001, 2003). These
categories of rearrangements are not easy to detect in cattle
with conventional methods unless they involve grossly detectable changes in length of the derived chromosomes. In
this regard, cytogeneticists using banding methods on
swine chromosomes have benefited the most and have identified over 65 different translocations in this species by the
end of the 20th century (Chowdhary, 1998). This number
has increased considerably since then because of the efforts
of a laboratory in France which has identified 78 new rearrangements during the past five years of their swine testing
program (Ducos et al., 2008).
Refinements in chromosome techniques have also helped
to identify that all chromosomes of the domestic pig are susceptible to rearrangements (Chowdhary, 1998). It would appear that the chromosome repair mechanism in pigs is different from that in other mammals and in some unknown
way this difference allows most of the aberrations to survive
in living animals (Fries et al., 1986, 1990a, b). To some extent, the hypothesis that an altered repair mechanism may
exist in pigs has been proved by using irradiated semen to
inseminate normal sows and examining the viable progeny
for their chromosome status (Fries and Stranzinger, 1982;
Fries et al., 1990a). Another interesting spin-off of these
studies on pigs is the finding that some of the chromosomes,
including chromosomes 1, 7, 14 and 15 of the swine karyotype, are more prone to be involved in reciprocal translocations while chromosomes 9, 10, 12, 8 and Y are among those
least often involved in such rearrangements. The non-randomness of distribution also extends to the specific sites on
these chromosomes, including band 21 of the long arm of
chromosome 1 (1q21) which is involved in reciprocal translocations with six different chromosomes (5, 7, 14, 15, 17 and
18). It has also been noted that in specific chromosomes
some sites are more prone to breakage and to display fragile
sites when subjected to different treatments (Yang and
Long, 1991; Ronne et al., 1995; Chowdhary, 1998). Since
translocations, regardless of the type involved, lead to the
production of unbalanced gametes (Henricson and Backstrom, 1964; Ford and Clegg, 1969; Akesson and Henricson,
1972; King, 1981), they lead to fertility reduction in carriers
due to early elimination of chromosomally unbalanced zygotes sometimes even before implantation (Hageltorn et
al., 1976; King , 1981; Popescu and Boscher, 1982; Gustavsson, 1990, 1991; Yang et al., 1992). A survey of pig populations in Sweden has shown that approximately 50% of the
breeding boars, removed from the population due to reduced fertility, are reciprocal translocation carriers, even
though they display normal phenotype and semen profile
(Gustavsson, 1990). It has been proposed that reciprocal
translocations in males might sometimes induce degenerative changes in mammalian testicles (Chandley et al., 1972).
This observation has, to some extent, been supported in a
few investigations over the years (Gustavsson, 1990, 1991;

Villagomez, 1993). In spite of the fertility reduction and the

potential for damaged germ cells in carriers of chromosome
rearrangements, carriers of such rearrangements in France
have been estimated to be alarmingly high (1 in 200) among
the phenotypically normal pigs (Ducos et al., 2008).
High resolution banding

Persistent attempts to gain insights into the structure

and organization of chromosomes have resulted in the refinement of procedures to generate elongated and finely
banded chromosomes (Caspersson et al., 1968; Ikeuchi,
1984; Ronne, 1984; Di Berardino et al., 1985). In this regard,
synchronization of cell cultures using a single or double
block with thymidine or methotrexate, or using ethidium
bromide to prevent chromosome coiling has made it possible to obtain vast quantities of cells in prometaphase stage
of mitosis. These elongated chromosomes are now routinely used to produce high resolution bands for the study of
chromosomal break points in translocations as well as in
gene mapping ventures for the assignment of gene loci. Using different combinations of these procedures, the main
domestic species of Bovidae (cattle, river buffalo, goats and
sheep) have been examined for the identification of homologies between them (Iannuzzi et al., 2000; Iannuzzi, 2007).
Thus, the standards established for G-banded karyotypes of
domestic animals at the Reading Conference (Ford et al.,
1980) and modified in details for R-banded karyotypes at
the second International Conference for Standardization
held in Jouy-en-Josas in 1989 (ISCNDA, 1990) and further
refined by Di Berardino and associates (ISCNDB, 2001), illustrate the incremental advance in supplementing and
strengthening the task of chromosome identification in domestic animals over the years. These studies have demonstrated a high degree of chromosome conservation in bovids and related species based on RBG-banding patterns of
elongated chromosomes (Hayes et al., 1991; Iannuzzi, 1996;
Iannuzzi et al., 2000). These workers noted only minor differences in autosomes and the X chromosomes of cattle
compared to that of the other domestic species including
water buffaloes, goats and sheep. Similarly, G- and R-banding patterns and a landmark system were established for the
pig karyotype in 1988 (Ronne et al., 1988). A further refinement in segmental delineation of chromosomes was introduced by Dutrillauxs group with a simple method that reveals hybridized sites and R or G bands on chromosomes
simultaneously (Dutrillaux and Lejeune, 1971; Dutrillaux,
1973; Dutrillaux et al., 1973; Lemieux et al., 1992). With the
use of p-phenylenediamine antifade solution at alkaline
conditions (pH 11), the chromosomes stained with propidium iodide display R or G bands based on the timing of 5bromodeoxyuridine incorporation, with banded chromosomes appearing red and the hybridization spots appearing
green (Ronne et al., 1985, 1987).

Synaptonemal complex analysis

Synaptonemal complexes (SCs) are core protein structures which appear at the early stage of meiosis and are assembled and disassembled during the prophase stage. They
are thought to be physically related to the process of chromosome pairing and recombination events which are the
essential features of meiosis. The use of whole mounts of
microspread germ cell suspensions for electron microscopic delineation of pachytene stage meiocytes (Counce and
Meyer, 1973) was an avenue to explore the behavior of sex
complements and the form and fate of autosomes involved
in a variety of structural aberrations in domestic animals.
One of the most interesting findings generated by the
microspread technique is the specific pairing pattern of the
sex chromosomes in meiocytes (Solari, 1989). Homology of
the X and Y chromosome, predicted on a theoretical basis
previously (Burgoyne, 1982), has been noted to be restricted
to the distal tips of the sex complements called the pseudoautosomal region which undergoes pairing and crossingover as autosomal homologues do. Abnormal crossing-over
in this region has been noted to result in cases of XX males
with testis, probably due to the transfer of testis inducing
segment from the Y chromosome to the X chromosome.
Synaptonemal complex analyses on several reciprocal and
Robertsonian translocation carriers have shown in various
cases that non-homologous associations are established between translocation multivalents and the XY pair. Non-homologous pairing and autosomal contact of sex bivalents are
thought to be one of the factors responsible for the degeneration of pachytene spermatocytes since this association
can cause interference with the process of X inactivation
which is thought to be an essential step for normal spermatogenesis (Koykul and Basrur, 1994). Synaptonemal
complex analysis in pigs carrying different reciprocal translocations has demonstrated such associations between the
translocation quadrivalents and sex bivalents (Gustavsson,
1990). This phenomenon leads to the production of chromosomally unbalanced gametes and, in turn, to increased
embryonic mortality expressed as hypoprolificacy in translocation carriers (Gustavsson, 1990). Meiocytes of domestic
animals have been examined by various investigators using
molecular approaches to study synaptonemal complex formation in fetal germ cells of normal and translocation carrier bovine females (Koykul and Basrur, 1994; Rho et al.,
2007), meiotic processes in X-autosome translocation carrier bulls and boars (Basrur et al., 2001a, b), and cross over
frequency analysis in meiotic germ cells of normal and
cloned bulls (Hart et al., 2008).
Fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) technique has

become an integral part of the modern cytogenetic laboratories. Compared to other methods, FISH requires relatively few metaphase spreads for analysis and the spatial resolution and sensitivity provided by FISH greatly enhance the

Cytogenet Genome Res 120:1125 (2008)

17

detection of structural aberrations in chromosomes. Today,

the chromosome constitution of the nucleus of any domestic animal can be determined and the incidence of aneuploidy recorded with relative ease using repetitive sequences specific for the X chromosome or the centromere regions
of specific chromosomes to generate fluorescence signals
(Bugno et al., 2005, 2006). Another valuable tool for the
analysis of rearrangements such as reciprocal translocations is chromosome painting with whole chromosome (or
whole arm) specific probes after suppression of repetitive
sequences (Cremer et al., 1980; Basrur et al., 2001b). These
approaches, in combination with flow cytometry, are also
used to sort-out cells carrying specific chromosome segments or gene loci of interest (Cremer et al., 1989). Localization of DNA sequences in the chromosomes by FISH has
also accelerated the speed of gene mapping in man and domestic animals. Since several cosmid probes, each labeled
with different fluorochromes, may be used in this method,
identification of the position of different sequences on the
same chromosome and their spatial relationship to the centromere and telomeres becomes relatively easy (Trask,
1991).
Application of different versions of these techniques has
already helped to resolve some points of conjecture and resolve areas of cytogenetic contention in the past. For example, using the currently popular FISH approach on decondensed sperm nuclei (Sperm-FISH technique) to investigate
the meiotic segregation pattern of the chromosomes involved in 1/29 translocation carrier bulls, Bonnet-Garnier
et al. (2006) found that the proportions of normal and balanced spermatozoa were similar to the theoretical expectation from alternate segregation alone (97.42% and 96.78%,
respectively). However, the observed proportions of spermatozoa missing chromosome 1 or 29 did not follow the
expected 1:1 ratio. Furthermore, the proportions of nullisomic spermatozoa were significantly higher than disomics
for chromosomes 1 and 29, while disomic and diploid spermatozoa detected in samples of normal bulls were significantly lower compared to those of translocation carriers
(Bonnet-Garnier et al., 2007). These observations confirm
the assumptions based on the proportions of normal and
translocation carrier calves among the progeny of Robertsonian translocation carriers and also point out that disomic segregants may be more vulnerable to elimination (during spermiogenesis or in transit through the duct system).
Another aspect that has been resolved (to some extent) is
the issues related to freemartinism in cattle. This syndrome,
representing the most frequently detected form of intersexuality in cattle and other ungulates, is caused by vascular
connections established in fetal membranes during the early stages of development of heterosexual twin fetuses. Such
vascular communications in shared fetal membranes lead
to the exchange of circulating fetal cells, which in the case
of heterosexual twins cause XX/XY cell chimerism in blood
and gonads, and the hormonal exchange between developing fetuses resulting in varying degrees of masculinization
of the female reproductive tract (Basrur and Stoltz, 1966;
Herschler and Fechheimer, 1967; Fechheimer, 1973, 1979).

18

Cytogenet Genome Res 120:1125 (2008)

Diagnosis of this condition in heifers (long before they reach

the age of sexual maturity) was made possible by chromosome analysis of blood cells since it was discovered that the
presence of XY cells among the heifers leukocytes indicate
vascular anastomosis and the resultant interchange of circulating cells and hormones between fetuses (Ohno and
Gropp, 1965; Ohno, 1967; Kanagawa and Basrur, 1968).
More recently, molecular approaches were substituted for
conventional chromosome analysis and the results have
shown that the use of Y-chromosome DNA segments in
polymerase chain reaction (PCR) or FISH offers improved
assay sensitivity and efficiency over karyotyping (Padula,
2005; Sohn et al., 2007). Taking advantage of the efficacy of
these approaches, Padula (2005) studied male and female
heterosexual twin sets in cattle, with emphasis on the male
twin. Male co-twins of freemartins, although phenotypically normal, have long been known to display chimerism
in blood and gonads (Ohno et al., 1962; Ohno and Gropp,
1965; Teplitz et al., 1967; Ohno, 1969a) and to exhibit compromised fertility (Teplitz et al., 1967; Ohno, 1969a; Weiss
and Hoffman, 1969; Stafford, 1972; Vale-Filho et al., 1984).
However, since the bull calves born twin to heifers are not
invariably sterile, and the impact on their fertility is variable, this aspect has generated controversies and the topic
itself has been periodically visited by scientists from various
angles. The questions posed had included whether their fertility is affected at all, whether germ cell chimerism exists
at all in the testes of these bull calves and whether the sex
ratio is distorted among the offspring of bull co-twins of
freemartins (Ford and Evans, 1977; Gustavsson, 1977; ValeFilho et al., 1983, 1984). Contradictory results obtained
from such studies had only served to generate more controversies till recently. Using the FISH approach on heterosexual twins in cattle and sheep, Padula (2005) and Brace et al.
(2008) have found answers to some of these questions and
have confirmed that chimerism does indeed extend to the
gonads of the male co-twin. Furthermore, various other organs which hitherto have been thought to be free of chimerism in both twins were also noted to exhibit chimerism
(Brace et al., 2008). These results point to the refinements
currently in place in veterinary cytogenetics to serve as diagnostic and prognostic tools.
Cytogeneticists using FISH approaches continue to concentrate on increasing the resolution by modifying and defining the target and by increasing the specificity of the
probes used (Speicher and Carter, 2005). Procedures for the
hybridization of multiple probes, labeled in different colors
to target metaphase spreads or interphase nuclei, have increased the use of cytogenetics for clinical application and
basic research. For example, Molteni et al. (2007) recently
identified the chromosome status of an 18-month-old bull
that was being entered for semen donor tests. Using a combination of procedures (RBA-banding, Ag-NOR techniques,
and FISH with specific molecular markers of BTA11 and
BTA21), the bull was identified as a carrier of a reciprocal
translocation with break points at 11q28 and 21q12. Without the use of refined FISH approaches, precise identification of such exchanges in cattle would have been impossible.

This finding also suggests that such rearrangements in cattle can be expected to turn up more frequently in the future (Molteni et al., 2007). Such approaches on metaphase
spreads or on interphase nuclei are now available for the delineation of complex chromosome rearrangements such as
those seen in some human malignancies (Speicher and
Carter, 2005).
Comparative gene mapping

Gene mapping is an area that has seen a spate of activity

in domestic animal cytogenetics recently although the total
number of genes mapped in all domestic animals still falls
far short of that achieved for the human genome. Even
among the domestic animal species some have been left far
behind the others, because of technical difficulties, lack of
interest on the part of breeders, and lack of funds for investigators to overcome these difficulties. Notwithstanding, a
large volume of gene mapping data on domestic ruminants,
including river buffaloes, pigs and cattle, appeared in scientific journals during the past few decades. Among the laboratories most actively involved in these ventures was the one
under the direction of Gustavsson and his associates who
had analyzed 70 loci including 32 structural genes by the
end of 1994 which constituted a lions share of the total number of loci assigned in pigs at that time (Chowdhary et al.,
1994). Other well known contributors to this area are
Womack and associates in Texas, Stranzinger and Fries in
Switzerland, Popescu and associates in France, Archibald
and associates in Scotland and Iannuzzi and collaborators
in Italy, among others (Fries et al., 1990a, b; Womack, 1990,
2005; Barendse et al., 1993; Fries, 1993; Harlizius et al., 1995;
Womack et al., 1997; El Nahas et al., 2001; Iannuzzi et al.,
2003; Everts-van der Wind et al., 2005; Iannuzzi, 2007; Molteni et al., 2007). The main strategies used to map unknown
loci include linkage analysis, in situ hybridization and somatic cell genetics. Mapping the genes of different species
of mammals has revealed that the syntenic relationship of
genes is strikingly conserved during the evolution of bovid
species (Hediger et al., 1989, 1991). Furthermore, genes on
human chromosome 11p are conserved as a part of bovine
chromosome 15 (Womack, 1990). Other examples of the
high degree of conservation are observed for the genes on
other human chromosomes, which are almost completely
conserved in cattle and other bovids, although the specific
chromosomes in cattle carrying these two groups are awaiting identification (Zhang and Womack, 1992; Zhang et al.,
1992).
By the end of the last century the bovine gene map contained over 300 loci (Fries and Popescu, 1999; Womack,
2005), most of which represent coding genes. Since they are
evolutionarily conserved, they are mapped by the use of human or murine sequences as probes. These loci, designated
as Type I loci, are not immediately useful for the mapping
of trait loci, because they are often not polymorphic. However, the Type I map allows us to identify the boundaries of
conserved synteny segments which are useful to direct

marker spacing and eventually to the identification of candidate genes once a trait locus has been assigned to a chromosome region. While the evolutionarily conserved Type I
loci will be indispensable for comparative mapping, highly
polymorphic species-specific DNA markers (the Type II
loci) will be useful to map trait loci (Willard, 1989). Microsatellites are among these Type II markers of choice. Using
this approach, the polled locus in cattle was noted to be
linked to the microsatellite markers assigned to bovine
chromosome 1 close to the centromere (Georges et al., 1993).
In the context of domestic animals, work of this nature can
be anticipated ultimately to lead to the unraveling of the
molecular basis of allelic variation at the loci controlling
traits of economic importance.
Chromosomal assignment of gene loci by means of in
situ hybridization is progressing rapidly in the domestic pig
(Yang et al., 1992). Comparative mapping in this species has
demonstrated that syntenic relationships of loci found on
porcine chromosomes 6 and 7 have largely been conserved
across the species. Furthermore, the region around the locus for halothane sensitivity has been physically mapped by
the assignment of the closely linked marker glucose phosphate isomerase (GPI ) to the centromeric region of chromosome 6 and the gene for hormone sensitive lipase (LIPE) is
assigned to pig 6cen]6q1.2 in the halothane region (Davies
et al., 1988). It was noted that a single amino acid substitution in the Ryanodine receptor gene (RYR1), also known as
the skeletal muscle calcium release channel (CRC) gene, is
responsible for the porcine malignant hyperthermia syndrome (MHS). This gene is now localized in chromosome 6
at 6p11]6q21 and the gene LIPE is now declared not to be
a candidate for this syndrome (Harbitz et al., 1990; Fujii et
al., 1991; Gu et al., 1992). The association of this gene to E.
coli type F18 infection leading to oedema disease was documented and patented by Vgeli et al. (1996).
Heterochromatin, epigenetics and chromatin
re-modeling

In recent years, research activities in the cytogenetic arena have shifted from gross changes in the number and
structure of chromosomes to regulation and epigenetic control of gene function. Thanks to the availability of refined
FISH approaches, investigators have started to probe the
mechanisms of gene regulation through histone modifications in whole or parts of chromosomes and heterochromatin assembly (Nakayama et al., 2001; Cheung and Lau, 2005).
It has long been known that histones are integral parts of
chromatin and that heterochromatin is associated with
gene silencing (Heitz, 1929; Ohno, 1967; Cattanach, 1975;
Jeppesen and Turner, 1993). In light of the information generated recently, the fundamental unit of chromatin, the nucleosome, is defined as a structure consisting of a short
length of double-stranded DNA wrapped around an octamer, made up of two copies each of histones H2A, H2B, H3
and H4 (Luger and Richmond, 1998). It is believed that histones play a role not only in DNA packaging during mitosis,

Cytogenet Genome Res 120:1125 (2008)

19

but also in the transmission of transcription-regulating epigenetic information from one cell generation to the next
(Struhl, 1998; Strahl and Allis, 2000).
The N-terminal tails of the histones that protrude from
the nucleosomes core particle can be modified by a variety
of post-translational covalent modifications including acetylation and methylation of lysines and arginines, phosphorylation of serines and threonines, ubiquitination and sumoylation of lysine residues, and ADP-ribosylation (Nakayama et al., 2001; Peterson and Laniel, 2004). In addition,
each lysine residue may carry one, two or even three methyl groups, and an arginine residue can be mono- or dimethylated. Specific patterns of such histone modifications on
one or more tails are correlated with global changes in chromatin structure which provides binding sites for interaction
with other effector proteins (Jenuwein and Allis, 2001). In
addition to covalent modifications, substitutions of a number of non-allelic histone variants for their core histone
counterparts may also provide structural diversity that may
be used to modulate the patterns of gene expression or gene
silencing (Peterson and Laniel, 2004).
Such changes in the pattern and distribution of histones,
albeit subtle, occur throughout mammalian development.
However, the most consistent change that can be visualized
is X-chromosome inactivation during the development of
female embryos, when one of the two X chromosomes becomes tightly packed into transcriptionally inactive chromatin (Lyon, 1961; Ohno, 1967). X inactivation, with few
exceptions, is random in that either the paternal or maternal
X may be chosen for inactivation. One of the exceptions is
the case of heterozygotes for structural changes involving
one of the X chromosomes. One such case is a family of
cross-bred beef cattle carrying a reciprocal translocation
between an X chromosome and chromosome 23, rcp(Xp+;
23q). In this family, the translocation is maternally inherited and females invariably show non-random patterns of X
inactivation, with the paternal (normal) X preferentially inactivated (Basrur et al., 1992, 2001a). Taking advantage of
the morphological mark provided by this translocation to
distinguish inactive from active X chromosome, an examination was carried out on the spatial distribution of a variety of histone isoforms including the histone H3, trimethylated at lysine-9 (H3K9me3) and at lysine-27 (H3K27me3),
and two macro histones (macroH2A1 and macroH2A2).
Comparison of the active and inactivated Xs in normal and
translocation carrier bovine immunostained cells revealed
that the histone variants H3K9me3, H3K27me3 and macroH2A1 are preferentially concentrated and consistently located in bands on the inactivated X in both types of bovine
cells, as described for the human X chromosome (Brinkman et al., 2006) whereas H3K27me3 was identified in two
intense bands on the inactivated X, at Xp22 and Xq31, representing the early replication regions of the X chromosome
(Coppola et al., 2008). H3K27me3 and macroH2A1 overlapped in the Xq31 region, while macroH2A1 and macroH2A2 were localized in the pericentric regions of all bovine
autosomes. However, contrary to the situation noted in the
human X chromosome, macroH2A2 was not detected as a

20

Cytogenet Genome Res 120:1125 (2008)

marker for inactivated bovine X (Coppola et al., 2008). The

potential to use similar approaches on prometaphase or interphase nuclei provides hopes and optimism for future
studies on the histone modification events in X inactivation.
Further refinements of FISH could widen our understanding on epigenetic control of specific gene function, alterations imposed on this process by environmental changes,
and the dynamic interplay between the genome and the regulatory factors in cells.
Perspectives

Cytogenetics connotes the visualized account of the

gene content (packaged as chromosomes) in the context of
a Mendelian phenotype. Over the years the phenotypes that
attracted chromosome examination had varied depending
upon the state-of-the-art tools available at specific stages in
the development of cytogenetics. Visual features explored
and exploited had included the level of intensity and affinity for stains (for unraveling inheritance of sex in grasshoppers (McClung, 1902b; Sutton, 1903) and later for studying
patterns of X inactivation in mammals (Lyon, 1961; Ohno,
1967, 1969a, b)), size and distribution patterns of chromomeres in early meiotic chromosomes of plants and invertebrates (Darlington, 1937), size and band distribution in giant chromosomes of dipteran insects (White, 1973), normal
chromosome morphology (for karytypic comparison of related and distant mammalian species, see Hsu and Benirschke, 1967), alteration in number and structure of chromosomes in relation to birth defects, metabolic errors and
reproductive problems and biochemical features (enzyme
phenotypes in starch gel electrophoresis (Smithies, 1955)),
lateral loop modifications of lampbrush chromosomes in
the primary oocytes of vertebrates and synaptic behavior of
homologous chromosomes and synaptonemal complex formation in primary oocytes and primary spermatocytes
(Swanson, 1957; Switonski et al., 1990; Switonski and Stanzinger, 1998), and chromosome constitution of gametes
(Creighton and Houghton, 1987). Among all these, the introduction in the 1970s of somatic cell genetic methods and
their refinements and application based on biochemical
phenotypes of specific enzymes, stand out as one of the major events (Ruddle, 2001) which has helped to recognize syntenic associations and segments of chromosomes carrying
homologues of human chromosomes in domestic animals
(Womack, 1990, 2005; Zhang and Womack, 1992; Zhang et
al., 1992; Womack et al., 1997; Barendse and Fries, 1999).
Introduction of array technologies for genome analysis
has augmented the potential of FISH approaches for testing
and treating clinical cases and for allowing higher reaches
in research. According to Speicher and Carter (2005), recent
developments in technology have effectively diminished the
distinction between cytogenetics and molecular genetics
since FISH technology can now identify even single-nucleotide changes in the genome. The potential of three-dimensional interphase cytogenetics in combination with refinements in mapping of DNA on microarrays enriched by

chromatin immunoprecipitation also promises to identify

epigenetic modifications and to provide new insights into
the functional organization of the genome.
The possibility of chemical and radiation toxicity affecting the genetic well-being of future generations of man and
domestic animals is very high today. Cytogenetic procedures have shown that a majority of health and reproduction-related problems in man and other mammals are the
results of de novo mutations or balanced translocations
(Pellestor, 1991; Ansari et al., 1993; Crow, 2000; Basrur,
2006). Testing for these defects in domestic animals has
been time consuming or impossible in the past although
various innovations including sex-sorting of sperm for
breeding farm animals are currently available for commercial use (Joerg et al., 2004; Basrur and King, 2005). Since
FISH can be applied to conventional metaphase spreads, interphase nuclei or sperm samples visualization of specific
defects and assessing the potential for its dissemination
have become a practical reality. Aneuploidy, which is the
most frequent cause of de novo genetic diseases in mammals and man (Bugno et al., 2005, 2006), is now being assessed in bovine sperm using FISH (Coppola et al., 2008). A

wide variety of array platforms have been developed including genome-wide scanning for polymorphisms and sub-microscopic copy number changes (Speicher and Carter, 2005).
The potential of testing for specific locus mutations in
sperm in combination with flow cytometry appears good
today. The possibility of using the loss of gene products
(e.g. human sperm protamines) by measuring the amount
of fluorescent monoclonal antibody bound to each protamine in single sperm, has already been tested over 20 years
ago (Mendelsohn, 1989). Since the loss of one or more fluorescent signals from a sperm is an indication of gene loss
mutations or loss of a specific chromosome resulting in its
being sorted out, it is not far fetched to expect the arrival of
a day in the near future when pre-selected semen samples
are routinely FISH-tested for sire evaluation.
Acknowledgements
We are grateful to Dr. W.A. King for reviewing the draft and Mr.
Ed R. Reyes for his help in preparing this manuscript.

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