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What is Biotechnology?

Biotechnology is a buzz-word in today's world, when everyone wants to use


biotechnology for betterment in their sphere of activity. The governments of
developed and developing countries are committing substantial funds for research
in biotechnology. Even though man has exploited biotechnological methods for
thousands of years in brewing' wine making, bread making and food preservation,
is industrial application in large scale for development of better products are
gaining momentum only recently. Application of genetic engineering techniques
via recombinant DNA technology is responsible for the current 'Biotechnology
boom' occurring world over. Modern biotechnology has played a substantial role
in the development of the health care and chemical industries. It has made possible
the availability of several. diagnostics, prophylactic and therapeutic products.
The term biotechnology was coined during late 1970s Biotechnology hag
been defined in various ways, mostly unsatisfactorily. (Biotechnology has been
defined as the application of biological organisms, systems or processes to
manufacturing and service industries.It it also defined as the application of
scientific and engineering principles to. the processing of materials by biological
agents to produce goods and services. Biotechnology is the commercial
exploitation of living organisms or their components. It is alst defined as the
industrial exploitation of biological systems or processes and it is largely based
upon the expertise d biological systems in recognition and catalysis.
The extraordinary multidisciplinary nature biotechnology is shown by an
anectode that the fruits o biotechnology are born on a tree whose roots are the
biological sciences, in particular microbiology, genetic: molecular biology and
biochemistry, whose trunk is chemical engineering in its widest sense.
Biotechnology: Past, Present and Future
Microorganisms have been used to produce beer vinegar, yogurt and cheese for
over eight millennia; The ancient Sumerians, produced beer and ale houses were
popular in Roman civilization. Wine was also popular with the Romans.
References to wine and vinegar are scattered throughout the Bible, which is an
indication that their production, dates back to early times.
Ethanol was the first chemical to be produced with the aid of
biotechnology. The origin of distillation are not clear but, by the fourteenth
century it was widely used to increase the alcoholic content of wines and beers.
Louis Pasteur was the first to observe that yeast converts the sugars to
alcohol in the absence of air. Such an anaerobic process is known as
'fermentation'. Souring and spoilage occur later and are due to the activities of a
group of bacteria, the acetic acid bacteria which convert alcohol into vinegar
(acetic acid). Pasteur's intention was to heat the alcohol just enough to kill most of
the microorganisms present, a process that does not mostly affect the flavour of
the wine and beer. This process is known as 'Pasteurization'.
Another major milestone in biotechnological generation of valuable
products was the development of the antibiotic industry; arising initially from the

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discovery of the chemotherapeutic properties of penicillin by Fleming, Flory and
Chain in 1940.
In the late 60's considerable excitement was generated by the prospect of
using microbial cells as a source proteins, the so called single cell proteins or
SCP. The dried cells of certain algae, bacteria, yeasts, moulds, and mushrooms
serve as food collectively called as single cell proteins. However the introduction
of SCP has not been successful. The developed countries did not need SCP as they
had a plentiful supply of proteins from conventional sources whereas the under
developed countries cannot afford to buy SCP or even to build and run SCP plants.
The treatment of waste products like manure from animals by anaerobic
digestion by mixed microflora, eventually generating biogas (mainly methane and
CO2) has become an increasingly important process. It is highly efficient in terms
of conserving and concentrating the energy available in the waste (over 80% of the
free energy is recovered from the gas).
Introduction
During 1980s, biotechnology became the major growth area. This change
came about through a single development; the ability to explain together in vitro,
DNA molecules derived from different sources. These gene splicing ability is
referred to as 'gene manipulation.' This is also known as recombinant DNA
technology. Recombinant DNA technology and genetic engineering techniques
found several useful applications in the area of vaccines, foods, antibiotics,
alcohols, hormones and monoclonal antibodies. It has been possible to diagnose
genetic defects by use of the restriction mapping technique. This is based on the
fact that the base sequence in defective gene differ from that in normal genes,
leading to the production of different-sized DNA fragments when a gene is cut
with a restriction endonuclease.
One of the important applications of genetic engineering is the production
of insulin and somatostatin using bacteria. It is also a new process to insert foreign
genes into cells and produce transgenic plants/animals with desired traits.
Genetically engineered vaccines against E. coli (pigs), Feline .leukemia
virus, Rinderpest virus, Rabies virus, Aujezsky's disease virus and against many
other microbes have been prepared.
The emergence of recombinant DNA technology has also given rise to
diagnostic DNA/RNA probes. Along with this technology, hybridoma technology
leads to the development of monoclonal antibodies against very many diseases,
which have enhanced diagnostic sensitivities and specificities in therapeutics.
Superovulation techniques with gonadotrophic hormones led to embryo
transfer on an increasing scale in 1980s when embryo freezing techniques became
available.
A new DNA amplification system, known as Polymerase chain reaction
(PCR) has greatly, facilitated DNA analysis and has found several applications.
This works with intact or broken DNA pieces (as small as 50 base pairs). It is
essentially an in vitro method for copying simultaneously the two corresponding

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DNA strands which make up a gene sequence. The specific DNA segment to be
amplified is selected by using primers (short segments of DNA which have
sequence complementary to the target DNA). By heating the DNA samples, the
two strands separate, allowing the primers to bind to the flanking sequences, one
on each strand. Thereafter, the primers initiate the synthesis of the daughter
strands, complementary to the parental strands in the presence of DNA
polymerase. This cycle of heating, annealing of primers and the synthesis of new
strand can be repeated several times. Roughly twenty cycles can amplify the. DNA
by a factor of about a million.
Biotechnology will, in the future, make an important contribution to the
quality of life. In medicine, many organizations are involved in the large scale
production of human interferon. Genes for human insulin and growth hormone
have been cloned and expressed in bacteria and the genes for many other human
proteins of value for diagnob - or treatment are being cloned to facilitate their
large scale production. At present, microbially derived hormone, insulin is already
being marketed and being used for therapy in diabetics.
Biotechnological innovations will produce efficient vaccines and antibiotics
for veterinary use. If vaccines from genetically engineered microorganisms live up
to expectations, we will witness the eventual eradication of deciminating diseases
such as foot and mouth disease and rinderpest. Growth hormones may be useful to
increase the meat and milk yield.
Many of our energy needs could be solved by conversion of light energy
from sun and waste materials. Electricity could be produced from these sources.
Biotechnology has a profound impact on food and drink industries. This
may be achieved by use of industrial enzymes to manufacture high fructose corn
syrups which are used as sweeteners in soft drinks. Recombinant DNA techniques
will be employed to make renin which is used to clot milk for making cheese. Till
recently, most of these renin have been extracted from the stomach of young
calves.
Biotechnology can provide methods for the improvement of whole crops,
both in terms of quality and yield. It can provide supplements or alternatives to
expensive chemical fertilizers and pesticides.
Activities of Biotechnologies
Recombinant DNA and genetic engineering
Recombinant DNA allows fragments of DNA from an
animal/Plant/Microbe to be transferred to a host bacterium, which in turn
incorporates the fragments into its own genome, thereby giving new capabilities
for synthesis or biochemical reactions. Genetic engineering refers to the isolation
of individual or group of genes and transforming them to other organisms. In the
host, the transformed gene replicates and expresses as part of their new host
genome.
The ability to extract a gene coding for a desired product and transfer it to
another organism has opened the way either to the more effective production of

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useful proteins or to the introduction of a novel characteristics in the host
organisms. Thus, large scale production of hormones, vaccines, blood clotting
factor or enzymes by friendly bacterium becomes possible. But why go to all these
trouble, why not just extract the desired protein from useful source? There are four
reasons.
First reason is, it's often not possible or practical to grow certain types of
cells, on large scale. For example, mammalian cells, particularly of human origin
may be difficult to obtain, grow slowly and are not amenable to the simple
culturing "techniques available for, the growth of microorganisms. It has been
speculated that the production of interferon from cultured human cell is likely in
the long term, to be supplanted by production of genetically engineered
microorganisms.
Secondly, availability of the natural source material may be strictly limited
and may not be available all the time. Thirdly, the natural supply may be
unavoidably contaminated. Haemophilics receiving factor VIII isolated from the
occupational groups are exposed to the risk of receiving hepatitis or the AIDS
virus. Fourth reason is its cost.- Since the natural source material availability is
limited, it is expensive.
In addition to these reasons, there is also additional possibility for the
production of truly novel proteins. Let us take the example of enzymes. The
specificity, catalytic activity and stability of enzymes are governed by the precise
structure of the enzyme molecule. By selectively modifying the gene coding for
the enzyme before it is introduced into the host organisms, the structure and hence
properties of enzymes, may be advantageously modified, hence a new brand of
super enzymes. Recombinant insulin is now available for use in man, who are
intolerant to pig insulin.
There is also a great potential for the modification of genome of
economically important plants. The introduction into crops of the ability to fix
nitrogen from the atmosphere would not only save the cost of applying
nitrogenous fertilizers, but would eliminate the potential problem of water
pollution from nitrates, washed off from agricultural land. It is estimated that the
nitrogen fixing brussal sprouts could be produced at half the cost of the traditional
variety. The levels of storage proteins in seeds could be increased to give high
protein wheat. It is also possible that crops could be engineered for a greater
resistance to herbicides or infections.
Diagnostic procedures based on nucleic acid probes
One of the significant impact of biotechnology on clinical medicine is the
development of various diagnostic procedures based on nucleic acid probes. The
test sample can be prepared either whole or sectioned and the DNA examined
directly from organisms using chosen species or subspecies specific or cross
reacting DNA probes, i.e., in sift hybridization. The DNA can also /be identified
from the sample either as whole DNA fixed on to a nitrocellulos membrane or first
digested with restriction enzymes, thus breaking it into variously sized small

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fragments which car be separated by electrophoresis on agarose or polyacrylamid
gel, transferred to nitrocellulose membrane and then probed by labeled DNA to
pick out possible restriction enzyme polymorphisms. The use of DNA probes for
taxonomis studies such as the identification of subspecies and variations within
species with geographical location is o epidemiological interest.
Hybridoma technology I
Hybridomas are hybrids between myeloma tumor cells and antigen
stimulated lymphocytes which can be cloned, grown in large quantities and for
indefinite periods of time and secrete high concentration of monoclonal antibodies
and hence mono-specific antibodies. A monoclonal antibody is an antibody
directed against one antigenic determinant or I epitope of an antigen. It is a single
isotype. By testing a I number of different clones it is possible to select those
producing monoclonal antibody of an appropriate specificity and isotype suitable
for a particular serological assay. The hes nogenicity, reproducibility and
permanent availability of monoclonal antibodies are the attributes arousing the
great interest in hybridoma technology.
Monoclonal antibodies have been used as immuno diagnostic reagents for
detection of the causative agents or antibodies produced by causative agents; for
experimental purposes for characterization of antigenic epitopes and as vaccine in
the case of monoclonal anti-idiotype antibody and for immunoprophylaxis or
immunotherapeutics to infectious diseases.
Recombinant vaccines
With the development of recombinant DNA technology novel vaccines like
recombinant vaccines, sub-unity synthetic vaccines and peptide vaccines have
been produced which are more antigenic than conventional vaccines and can be
produced in large amounts and stored at room temperature.
The first recombinant vaccine produced for animal diseases is against foot
and mouth disease virus (FMD). The FMD virus has got four viral proteins, viz.,
VP1, VP2, VPS and VP4. Of these proteins viral neutralizing property resides in
VP1 fraction. Even in this portion, the amino acid residues 141-160 and 200-213
are the portions which are responsible for virus neutralizing ability. Once these
specific amino acids have been identified, synthetic peptides could be produced
corresponding to the amino acid residues of 141-160 and used as vaccine against
FMD.
Vaccinia-recombinant rinderpest vaccine has been produced using
Haemagglutinin and/or Fusion protein genes. A recombinant vaccinia virus
expressing the immunogenic rabies glycoprotein has been produced and used
successfully to protect foxes against rabies in Europ. Fowl pox recombinant
Newcastle disease virus .vaccine expressing Haemagglutinin Neuraminidase (HN)
gene hsts been designed and used to protect chickens.
Mammalian cell culture
Some mammalian proteins can be produced from cultured mammalian
cells. The two most likely candidates in this category are monoclonal antibodies,

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for reasons of the complexity of the transcription and translation of their genetic
material and interferon, for reasons of cost and effectiveness. Animal cell culture
products include enzymes, hormones, immunobiologicals, monoclonal antibodies,
interleukins, lymphokines and insecticides. Human leukocyte cell lines are used
for production of interferon. Dog kidney cell lines are used for canine distemper
virus vaccine and cow kidney cell lines are used for FMD virus vaccine
production.
Reproductive techniques
The embryo manipulation techniques are purely biotechnological. This
include the splitting and freezing of embryo, in vitro fertilization and embryo
transfer. Embryo splitting is done to produce identical twins and is now routine in
cattle. Successful nuclear transplantation in sheep makes cloning (making large
groups of genetically identical animals) more likely. Splitting of embryos are also
done to increase the number of embryos.
In farm animals, in vitro fertilization combined with embryo transfer has
become a common technique. These methods make it possible to use valuable
animals repeatedly as donors to supply many oocytes for improvement and
manipulation in animal production.
The first successful embryo transfer in farm animals was done in 1934 in
sheep and goats. The first offspring after transfer of embryo in cattle and sheep
was reported in 1951. Embryo transfer includes a) selection of donors; b)
induction of. superovulation; c) embryo collection; d) evaluation of embryos; e)
selection of recipients and f) transfer of embryos. Since embryo transfer
techniques become available in livestock, embryos can be maintained in vitro and
this makes them accessible to further manipulation prior to transplantation.
Transgenic animals
Transgenic animals are produced by transfer of genetic materials from one
animal to another. Animals into which foreign DNA is integrated are called
'transgenic'. The foreign genetic material will generally consists of a structural
gene with regulatory sequences that are required for the transcription of the
introduced genes. The first direct evidence for transfer of traits from one
generation to another through genes was given by Gregor Mendel.
The classical example of genetic engineering was given by Palmiter and
colleagues (1982), when they showed that mice injected, with a fusion gene
containing the metallothionein promoter and rat growth hormone gene resulted in
a dramatic increase in the size of the animal that expressed this gene. Transgenic
pig was produced by microinjection of embryos with fusion genes consisting of
metallothionein promoter linked to human growth hormone expressed in vector
pBR 322.

Plants and plant cell culture


Plants, quite apart from their key role in producing foods, are also
important source of other raw materials. Most of the bulk plant products are starch

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and sugars; 90% of the cars in Brazil now use a mixture of petrol and alcohol, the
latter derived from fermentation of corn sugars. Sugar has also been used as feed
stock for its conversion into ethylene oxide.
Plants are also important source of high value drugs; some 25% of the
drugs are of plant origin.
Many plant cell culture can regenerate entire plants. Cell culture provides a
good way to extend studies in plant , pathology and somatic cell
hybridization. Some of the substances produced by plant cell suspension
cultures include- alkaloids, quinines, antibiotics, anti-viral agents, cardiac
glycosides, amylases, carotenoids, etc. The ability to culture plant cells on a large
scale, either for the production of biomass or in order to extract the desired
product from cell cultures is becoming a highly desirable technology.
Fuels
The world is running short of combustible fuels, in particular mineral oil.
Biotechnology might provide all i solutions; new fuels and alternative carbon feed
stocks. In r the economics of Brazilian fuel-alcohol process, sugarcane waste is
used as fuel for distillation of waste material such as pulp mill sludge for
producing alcohol in many countries.
Another potential biotechnological fuel is methane. All that is needed is
some pig slurry or animal waste material like dung and a hole in the ground with a
lid on top; nature does the rest. It is one example of biotechnology that can ; be
transferred to third world agricultural societies with on much difficulty.
The most aesthetically pleasing biotechnological fuel would be hydrogen
derived from the biophotolysis of water. Biophotolytic production of hydrogen
from water is based o: the photosynthesis of plants with bacteria-derive
hydrogenase enzymes and light. The great advantage wit a fuel of hydrogen
derived from water is that when burn it produces no pollution and regenerates its
source material.
Biocatalysis
Enzymes are nature's supreme catalysts, exhibiting great specificity and
enormous catalytic power. At preset about 150 of the 2000 known enzymes find
commercial applications and another 200 are available for use in genet:
engineering. The latter include restriction endonucleases an ligases. The enzymes
have been in use for centuries particularly in food processing (for example cheese
makir and hair removal from hide). Animals, plants and microb are the three
important biological sources of enzymes. Over one half of the industrial enzyme
market is accounted for by proteolytic enzymes, for example, milk clotting
enzyme for production of cheese, detergent proteases, animal rennets and
proteases of Aspergillus used in baking.

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Waste treatment and utilization
Sewage disposal is a problem long faced by man kin Today's sewage plants
are good examples of simple biotechnology-a fixed bed of microbes degrading the
sewage products trickling over them.
In cheese production, milk is curdled and liquid whey is a waste material.
This whey contains few proteirns, minerals and 4% lactose. Two thirds of world's
populate cannot digest it, However, it does find some application the manufacture
of ice creams, packet soups and desserts, but its use can be widely extended if the
lactose is split in its constituent sugars, glucose and galactose. Methods ha been
developed for splitting this with the enzyr β-D-galactosidase.
Cellulose is another waste material particularly in the form of straw from
cereal crops. This waste cellulose could be biologically degraded and used as a
feed stock for the production of microbial proteins. It has been estimated that
sufficient protein can be produced in this way from agricultural waste
alone to feed-the entire world population. The ability of some fungi to convert
cellulose has been commercially exploited for producing edible mushrooms for
human food.
Fermentation
Fermentation starts with biocatalysis, the distinction of being the oldest
form of biotechnology. Traditionally, fermentation has meant the production
of palatable alcohol from carbohydrates. "However, fermentation i.e., the
application of microbial metabolism to transform simple raw materials into
valuable products - can produce amazing range of useful substances eg. chemicals
such as citric acid, antibiotics, biopolymers and single cell proteins. What is i
needed is the knowledge of these microorganisms, the f control of their
metabolism and growth and the ability to i handle them on a large scale.
Current uses of fermentation process include a) f production of biomass
(Spirulina and yeast); b) production of enzymes and nucleic acids; c) production
of metabolites; I both primary (eg. ethanol, lactic acid) and secondary (eg.
antibiotics, " antimetabolites); d) enzymatic conversion of : specific substrates (eg.
glucose to fructose) and e) enzymatic conversion of multiple substrates (eg.
biological wastes).
Idlies and doshas which are the staple diet of South Indians are also the
products of fermentation. When the ground up rice and urid dhal (black gram) are
left overnight with salt, it becomes acidic and it is leavened by carbon dioxide,
primarily produced by Leuconostoc mesenteroids. The acidity is attributed to the
presence of Streptococcus faecalis. High salt content has an excellent preservative
action and is valuable as a condiment.
Process engineering
It is one thing for the laboratory scientist to clone a novel gene, discover a
new antibiotic or invent a new catalyzed process, but it is quite another to transfer
this technology to the scale of operation required to make useful product in a
significant quantity. This last is the responsibility of the engineer, be he chemical,

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biochemical, or whatever. Harvesting, pretreatment, filtration of the raw material,
reactor design and control, recovery/reuse of biocatalyst or organism, product
extraction and analysis, effluent treatment, water recycling - all these are his
concern. Chemical engineers have shown this to be very adept at handling
chemical processes on a large scale. However, biological processes differ from
chemical processes in many ways eg., the requirement for sterility productions or
extraction of edible materials diluted in large volume.

Cell Culture and Fermentation Technology


Introduction
One of the earliest workers in cell and tissue culture was who developed a
reproducible technique for tissue culture using frog Later in 1912 Carrel used
tissue embryo extracts as culture media. They successfully 'demonstrated that
'animal cells can be grown indefinitely in vitro just like other microorganisms.
Initially cold blooded animals like frogs were used, so that no incubation was
required, but later warm blooded animals like chicks and rodents were used and
eventually mammals became a favourte material.
Mammalian cells are grown in vitro for a number of reasons including the
production of viral vaccines. Under standard tissue culture techniques, cells are
immersed in a pool of medium containing essential nutrients and metabolic
products. The concentration of both constituents changes as the cell population
grows, thus influencing cell survival and function.
Culture Media
Culture medium is one single most important factor for culturing cells and
tissues. It provides the optimum conditions of factors like pH, osmotic pressure,
etc., and the chemical constituents, which the cells or tissues are incapable of
synthesizing (unlike microorganisms, which can synthesize them from simple
inorganic substances). Animal cells , need either a completely natural medium or
an artificial medium supplemented with some natural products. For some tissues,
the natural medium is preferred, since it is the cheapest and the most convenient.
Natural media'
The natural media used in cell culture systems fall in the following three
categories; i) coagula, such as plasma clots; ii) biological fluids, such as serum and
iii) tissue extracts of which embryo extract is the most common.
Plasma clots have been in use for a long time and are now available
commercially either i) as liquid plasma in siliconized ampoules or ii) as
lyophilized plasma, to be reconstituted by the addition of distilled water saturated
with carbon dioxide.
The most commonly used biological fluid is serum, which is commonly
obtained from adult human blood placental cord blood, horse blood or salf blood.
Of these, human placental cord serum and fetal calf serim seem to be particularly
satisfactory. The serum is tested for sterility and toxicity before use.

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Tissue extracts: Embryo extract is the most commonly used, although
effective substitutes have now been developed to replace the embryo extract as a
natural medium. The most important component of the substitutes is a mixture of
amino acids. Embryo extract is often prepared from chick or bovine embryos of
different ages (up to 10 days).
Defined media,
Media with serum: Although natural media are very useful and convenient
for a wide range of uses, they suffer from the disadvantage of poor reproducibility
due to lack of knowledge of the exact composition. For this reason, synthetic
media have been designed for a variety of uses and purposes, like the following: a)
media for immediate survival, b) media for prolonged survival, c) media for
indefinite growth and d) media for specialized functions.
For immediate survival, a "balanced salt solution' with defined osmotic
pressure and pH is added. This requirement is met by the combination of certain
inorganic ions, salts and glucose. For longer survival, serum may be used or the
balanced salt solution may be supplemented with amino acids, oxygen, vitamins
and serum proteins. One such medium was developed and modified by Eagle
(1955) and is described as 'minimum essential medium (MEM). MRM was
developed for mammalian cells gown on monolayers with more fastidious
requirements. This has been used for primary mammalian cells and established
cell line& This contains essential amino acids, vitamins and salts. Other more
complex synthetic media include the following: a) 199, b) CMRL 1066, c) RPMI
1640 and d) F12. All these media are supplemented, with 5-20% serum. RPMI
1640 (Roswell Park Memorial Institute 1640 medium) is commonly used and
marine. Normal and neoplastic white blood cells; these grow as suspension
cultures and have a reduced calcium requirements.
Role of serum in cell culture
Serum is an extremely complex mixture of many small and large
biomolecules with different physiologically balanced growth promoting and
growth-inhibiting activities.
Some of the major functions of serum are to provide the following:
a. basic nutrients, in solution and bound to proteins;
b. hormones and growth factors stimulating cell growth
and specialized functions;
c. attachment and spreading factors;
d. binding proteins (albumin, transferrin) carrying hormones,
vitamins, minerals, lipids, etc;
e. Non-specific protection factors against mechanical damage and
viscosity (shear forces during agitation of cell suspension);
f. protease inhibitors and
g. pH buffer.

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Disadvantages of using serum in cell culture
1. For most cells, serum is not a physiological fluid which they contain in the
original tissue except during wound healing and blood clotting processes,
where serum promotes fibroblast growth, but suppresses epidermal
keratinocyte growth.
2. The potential cytotoxicity of serum is often overlooked. Besides
selective inhibitors, bacterial ( toxins and lipids, serum may
contain polyamine oxidase, which upon reaction with polyamines forms
cytotoxic polyaminoaldehydes. In contrast to FCS and human pregnant
serum, horse serum shows relatively high level of this enzyme. This
may explain reports on the suppressor activity of these ; sera, and the
preference of some workers for horse serum.
3. Serum may contain inadequate levels of cell specific growth factors which
have been supplemented and an overabundance of others which may be
cytotoxic.
4. Risk of contamination with virus, fungi or mycoplasma.
Serum free media: The use of serum in culture media has several
disadvantages, which led to the development of many serum free media. Some of
the disadvantages include the following: a) serum varies from batch to batch and
deteriorates within one year; b) changing serum in a batch requires fresh testing,
so that the replacement is as close to the previous batch as possible; c) if more than
one cell types are used, which may require different serum concentrations, so that
a number of batches may need to be maintained and co-culturing may be difficult;
d) the demand for serum usually exceeds the supply for a variety of reasons; e)
when cell culture is used for downstream processing to recover cell products the
presence of serum is an obstacle to purification; f) serum increases the cost of the
medium, since its cost is ten times the cost of its constituents if used as substitute
and g) serum may stimulate undesirable growth and may even inhibit growth in
some cases.
The major advantage of serum free media is the ability to make medium
selective for a particular cell type, since each cell type appears to require a
different recipe. Examples of serum free medium include MCDB 110, MCDB
402, MCDB 153, Iscov's and LHC media.
Primary Culture
Primary cell culture can be obtained either a) by allowing cells to migrate
out from the tissue, which is adhering to a substrate or b) by disaggregating the
tissue mechanically or enzymatically to produce a suspension of cells. Most of the
normal untransferred, cells survive and proliferate to produce a primary culture
and when attached to a substrate, these cells need to be obtained by disintegration.
Primary cell cultures retain most of the characteristics of the cells from which they
originated. Primary cell cultures exhibit a phenomenon called contact inhibition
which results in the cells lining up in strongly oriented strands with regard
chromosomal number, primary cultures usually retain their diploid karyotype. the

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most commonly employed tissues for preparing primary cultures are kidney,
lungs, lier, thyroid and testes. Primary cells are often used in preference to
established cell lines and are regarded as a better representation of cells in vivo.
Primary cells are more likely to reflect the true activity and functions that they
display in their natural environment.
Disaggregation is achieved by any one of the following 3 methods: a)
physical disruption, b) enzymatic digestion and iii) treatment with chelating
agents. Physical disruption may often be combined with other methods. Of the
many enzymes that are used for disaggregation, trypsin and pronase are
the most commonly used. Similarly, tissues like epithelium (which needs Ca4+,
Mg++ ions for its integrity) can be treated with chelating agents, such as
citrate and ethylene-diamine tetra acetic acid (EDTA).
Enzymatic disaggregation
Trypsin is the most common enzyme used for disaggregation for
the following reasons: i) it is tolerated by a variety of cells, ii) it is effective for
many tissues and iii) its residual activity is neutralized by serum of the medium or
by a trypsin inhibitor in case of a serum free medium. Disaggregation by trypsin
can be damaging to epithelial cells or ineffective to fibrous tissues. Since
intracellular matrix contains collagen, collagenase has proved effective for
disaggregation of several normal and malignant tissues, which may be rather
insensitive to trypsin. Crude collagenase is often used with a finely chopped
tissue in complete medium.
Mechanical disaggregation
Enzymatic disaggregation is labour intensive and : involves
damage of cells. Therefore, mechanical disaggregation of cells is
sometimes preferred. In this method, tissue is carefully sliced and the cells that
spill out are collected. Alternatively, the cells are either a) pressed thfough
the sieves of a gradually reducing mesh, or ii) forced through a syringe and needle,
or even repeatedly pipetted. Although the method may cause mechanical
damage, the cell suspension is more quickly obtained than in the enzymatic
disaggregation.
Separation of viable and non-viable cells
The dissociated cells grow well when seeded on culture plates at high
density. In the adherent culture, non-viable cells will be removed at the first
change of medium. In the suspension culture, on the other hand, non-viable cells
are gradually diluted out, when cell proliferation starts. However, non-viable cells
can also be removed from primary disaggregate by centrifuging the cells in a
mixture of Ficoll and sodium metrizoate, when viable cells are collected from the
interface after centrifugation.
Cell Lines
Cell lines can be divided into two types: adherent (monolayer cells) and
non-adherent (suspension cells). Adherent cells attach to the plastic surface of a
flask or plate and therefore need to be detached from this surface before they can

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be used. Suspended cells do not normally attach to the surface of the culture
vessel.
The major reason for the use of cell lines is that they are often easier to
handle than primary cells, grow continuously and a large number of cells can be
obtained. The cultured cells are of three types; a) Precursor or stem cells which are
capable of proliferation, but remain undifferentiated until the correct inducing
conditions are applied so that some or all of the cells mature to differentiated cells,
b) Undifferentiated cells also known as committed precursor cells, c) Mature
differentiated cells. This equilibrium may shift according to the environmental
conditions. High serum, growth factors and low cell density will promote cell
proliferation and low serum levels, appropriate hormones and high cell density
will promote differentiation.
The source of the culture will also determine which of the above three types
will be present in the culture. Cell lines derived from the embryo may contain
more stem cells (or precursor cells), which will be .capable of greater self renewal
than the cultures from adults; the cultures from tissues which are undergoing
continuous renewal in vivo (eg. epidermis, intestinal epithelium, hemopoietic
cells) will still contain stem cells and these may survive indefinitely and cultures
from tissues which renew only under stress (fibroblasts, muscles, glia) may only
contain committed precursor cells with a limited culture life span. From any of the
3 kinds of cells derived from the primary explant, cell lines may be developed,
After the first subculture, primary culture becomes a cell line and may be
propagated and subcultured several times. With each successive subculture, the
competent population with the ability to proliferate most rapidly will gradually
predominate and non-proliferating or slowly proliferating cells will be diluted out.
The established cell lines will have altered chromosome number, shorter
doubling time and should be subcultured indefinitely. Established cell lines do not
exhibit the property of contact inhibition. The cell lines show a great variation in
karyotype. Vero, BHK21 and MDCK are some of the cell lines commonly used in
the field of veterinary virology (Fig. 2).
The cell lines may be propagated in an unaltered form for a limited number
of cell generations, beyond which they may either die out or give rise to
'continuous cell lines'. The continuous cell lines are often aneuploid and have
larger variation in chromosome numbers, while the finite cell lines are often
euploid, with little variation in the chromosome numbers. The alteration in the
culture, giving rise to a continuous cell line is commonly called 'in
vitro transformation'.
Maintenance of cultures - cell lines
When a primary culture is grown in a medium, as they grow and increase in
number, the available medium is used up and there arises a need to
subculture it from a heterogeneous primary culture containing many types of
cells derived from the original tissues. During subculturing, a very homogeneous
'cell line' emerges. The culture now called a 'cell line', can be propagated,

13
characterized and stored. The term 'cell line' implies the presence of several cell
lineages either similar or distinct. A cell line may be finite or continuous,
depending upon whether it has limited culture life-span or it is immortal in culture.
Finite cell lines grow up to 20-80 population doubling before extinction. Some
commonly used cell lines are listed in Table 1.
Most of the primary cultures or continuous cell lines grow as monolayers.
These cultures will need a periodic change of medium, whether or not cells are
proliferating. In cultures, where cells are proliferating, the usual practice in
subculturing adherent cell line involves the following steps: i) removal of the
medium and ii) dissociation of the cells in the monolayer with trypsin or other
enzymes. Intervals between change of medium and subculturing vary from one
cell line to another depending upon the rate of growth or metabolism. Rapidly
growing cell lines like HeLa are subcultured once in a week and the medium
is changed 4 days later. More slowly growing cell lines are required to be
subcultured every 2, 3 and 4 .weeks and the medium j changed weekly between
subcultures.
Table 1. Cell lines in regular use
Name Morphyology origin
A. Finite Cell lines Human embryo lung
MRC 5 Fibroblast -do-
W 138 Fibroblast -do-
IMR 90
B. Continuous cell lines
BHK 21 Fibroblast Newbom Syrian hamster Kidney
CHOK 1 -do- cells Adult Chinese hamster ovary
HeLa epithelial Adult human
Vero Fibroblast Adult monkey kidney

Maintenance of aseptic condition is one of the most I difficult tasks in the


tissue culture technique. Bacteria, yeasts, fungi, moulds, protozoa and
mycoplasma appear as contaminants in tissue culture. To avoid contamination of
cultures, antibiotics should be included in the medium at the recommended
concentration. The commonly used antibiotics are given in the Table 2.
Large scale cell cultures
Availability of genetically modified cells for the production of recombinant
compounds has increased the ability of large scale cell cultures in industry.
Specific promoters like human metallothionein II A and baculovirus IE1
promoters have been utilized to provide high expression of foreign genes, so that
the large scale ceil cultures may become commercially profitable. In a large scale
cell cultures may unit, for completion of reaction, cell biomass must be separated
from supernatant, which usually contain the product. Cell separation is Usually
achieved by Cell Culture and Fermentation Technology

14
Table 2. Details of some of the most commonly used antibiotics
Name of Storage Active against Working cone. Inhibits bacterial
antibiotic temp so mg/l protein synthesis

Gentamycin 4°C Bacteria Gram ± 50 mg /l Inhibits bacterial


Mycoplasma protein synthesis
penicillin -2Q°C Bacteria Gram + 100.000U/I Interferes with
bacterial cell wall
synthesis
Streptomyci -20°C Bacteria Gram ± 50-loomg/l Interferes with protein
n synthesis

Tylosin -20°C Bacteria 6-10 mg/l -do-


Gram +
Mycoplasma
Nystatin -20°C Yeasts l0-200U/ml Permeability of cell
Molds membrane affected

The cell culture products are enzymes, hormones, animal vaccines,


monoclonal antibodies, interferons, etc. (Table 3). Some of the important enzymes
obtained from animal cells are asperginase, collagenase, hyaluronidase, pepsin,
renin, trypsin, hydroxylase and urokinase. Similarly four important hormones
obtained from cultured cells include luteinizing hormone, follicle stimulating
hormone, chorionic hormone and erythropoitin. Table 3. Cell lines and the
products obtained from them
Table 3. Cell line and the product obtained from them

Human leucocytes Interferons


Mouse fibroblasts Interferons
Human Kidney Urokinase
Dog Kidney Canine distemper vaccine
Chick embryo fluid FMD vaccine
Duck embryo fluid Vaccines for influenza, measles and mumps
Vaccines for rabies and rubella
Cell lines of mouse, rat or Monoclonal antibodies
human origin Tissue-type plasminogen activator (tPA), Beta-
Chinese hamster ovary cells (CHO) gamma interferons Factor VIII

Production of FMD vaccines is the most /important example of the use of


large scale cell culture. There are several other vaccines including polio vaccines,
bovine leukemia virus vaccines, rabies vaccines etc., which are produced at
commercial scale; using cell culture. Due to heavy demands for a variety of

15
interferons for cancer therapy, recently, interferons a and 0 have been made in
large quantities. For production of interferon a, 8000 litres of cell culture virus
have been used. Similarly interferon p was produced by the cells grown on micro
carriers at scales of greater than 1000 litres.
Production of monoclonal antibodies with the help of cultured cells has also
achieved significance in industry, due to the increasing utility of these antibodies
in i) in vitro diagnostics, ii) in vivo imaging, iii) therapy in humans and animals
and iv) industrial applications like immuno-precipitation. Two approaches have
been followed for the production of monoclonal antibodies; i) in vivo production
using ascitic tumours in mice or rats; ii) in vitro production through cell culture
techniques. The second method is economically viable in view of the increasing
demand. It has the following additional advantages: a) it can be scaled up, for
instance, one kg of antibody (which can be produced through cultured cells in
single fermenter), would require 20,000 mice if made by ascetic tumours and b)
the risk of contamination is greatly reduced.
Homogeneous bioreactor systems
a. Suspension cultures: The classical system is the suspension culture using
a stirred tank with different impeller types and installations, equipped with or
without a spin filter. In large scale bioreactors, slight modifications of several
internal parts of bioreactors used for bacterial fermentation are made in order to
adopt them for culturing animal cells. The modifications are in the agitation
system. Marine type impeller, vibromixer or rotating flexible sheets replace the
turbine type impeller widely used in microbial fermentation. Perfusion systems
were also developed for submerged cultivation of animal cells.
b. Microcarrier cultures: Among the several cell culture I systems
available, some of them are anchorage dependent } and require a surface for their
attachment and subsequent f growth. In microcarrier (MC) system, large
surface is provided for the cells to grow in an unlimited volume. MC culture is
the growth or maintenance of anchorage I dependent cells on the small
beads suspended in a stirred ! tank. The advantage of MC cultures are based on
the provision of a large surface area in relation to the volume of the vessel used.
This may be understood by comparing roller bottles to MCs in terms of the surface
area afforded. A standard 500 cm2 roller bottle requires 100 ml of nutrient medium
wherein 1 ml provides a minimum of 30cm2 of growth surface. One gram of 0.2
mm diameter beads will have a surface area of more than 6000 cm 3. MG beads
have been manufactured from different synthetic materials like dextran,
polyacrylide and polystyrene.
Heterogeneous systems
In this system, cells are immobilized and separated from the medium. This
system has been developed to accommodate the growth of surface adherent
cells. The following are some immobilization techniques employed widely.
a. Adsorption: This system consists of a ceramic 'cylinder with uniform
sequence channel along its length. It provides a surface area of 4.25 m2. There are

16
two types of materials, one with a smooth surface for anchorage dependent cells
and a rough surface for suspension grown cells. To achieve immobilization, cells
are injected into the ceramic cylinder and allowed to settle and attach -before the
circulation of f medium is started. This system is used for production of a protein
over a long period of time.
b. Entrapment: Entrapment of cells in various polymers is a gentle means of
immobilization which affords protection from mechanical stress. The cells are
mixed with an aqueous solution of sodium alginate which is then dropped into a
solution of calcium chloride. Calcium alginate is insoluble and forms beads in
which the cells are entrapped. The cells can also be mixed with an aqueous
solution of agarose, the temperature is decreased below the gel-forming
temperature and the block thus formed is dispersed in paraffin oil. The force with
which the dispersion is made controls the size of the beads. As with alginate,
agarose entrapment is more suitable for suspension grown cells.
Collagen beads are also used as matrix, cells penetrate the pores and
colonize the beads. Collagen promotes adhesion of anchorage dependent cells.
Fibrin is another bead which is used for entrapment. Fibrinogen is converted to
insoluble fibrin through the action of an enzyme thrombin. This reaction has been
used to entrap the animal cells in beaded fibrin. The matrix is suitable for both :
suspension grown and anchorage dependent cells.
c. Encapsulation: This involves two methods, one based on the membrane
formation by polyacrylate anions-polyacrylate cations and the other on the
membrane formation by cellulose sulphate (poly anion). Cells may be physically)
entrapped in different configuration of hollow fibres by simply injecting them in
the extra capillary space.
Mammalian cell technology demands more and more sophisticated,
effective and economical method and devices for cell production and
concentration. The trend in the large scale production system will be towards cell
retention systems, because of their large advantages over low density cultivation
systems.
Tissue and Organ Cultures
For development of primary culture and cell lines, a variety of tissues and
disaggregation methods are used to give a good yield of separated cells. For
obtaining cell cultures, the tissue is cultured using 'primary explantation'
technique'. This technique is used for cultivation of pieces of fresh tissues derived
from the organisms and this was almost the exclusive technique used for
animal tissue culture till about 1945. Different forms of primary explantation
techniques are still widely used. These techniques differ only in the type of vessel
(flasks, test, tubes, etc) used for growing tissues, but are uniform in in principle.
The primary explantation techniques is also used for gmbryo and organ culture.
The different explantation techniques are a) slide cultures, b) carrel falsk cultures
and c) roller test tube cultures.

17
Tissue culture
Slide or coverslip culture
In this technique, slides or coverslips are prepared by placing a fragment of
tissue (explanation) on to a cover slip, which is subsequently inverted over a
cavity of a depression slide. This is very useful for morphological studies through
"the use" of time lapse cine micrographic investigations. This .technique is simple
and relatively inexpensive .'Halls in living state are spread out in the manner
suitable for microscopy and photography. Cells grow directly on cover stos and
can be "fixed and stained to make permanent sll3es. Few of the limitations of this
technique are a) supply of oxygen and nutrients is rapidly exhausted, so that the
medium quickly becomes acidic and requires transfer for pidly growing tissues, b)
sterility cannot be maintained for Tlong period and c) only very small amounts of
tissue can be cultured.
Flask cultures
The main use of flask cultures is in the establishment of a strain from fresh
explants of tissue. A good carrel flask has excellent optical properties for
microscopic examination, even though Solystyrene culture fasks can also be used.
This method has few advantages; a) tissue can-be maintained in the same flask for
months or even years b) large numbers of cultures can be easily prepared and
amounts of tissue can be grown with large amount of medium.
Test tube cultures
Test tubes are cheap and convenient vessels for tissue i culture and can be
used for preparing a large number of/ cultures, which can be placed in stationary
racks or- roller j drums. However, this technique has disadvantages like poor
optical property for microscopy difficulty, in quantitation due to the curvature and
high risk of contamination.
Organ culture
Organ culture usually implies culturing pieces of an! organ in vitro and its
objectives to maintain those architecture of the tissue and direct it towards normal
development as occurs in vivo. Media used for growing organ culture are
generally same as those used for tissue culture. I Organ cultures are grown in both
solid medium and. medium.
Cryopreservation
Cryopreservation involves the storing of cells at a very low temperature
(- 180 C) as in liquid nitrogen, in a state of suspended animation until they are
needed. A large amount of time, effort and money would be wasted if cells would
not be preserved when they are not needed
Cryopreservatives
Healthy cells are suspended in a solution of either .glycerol or dimethyl
sulphoxide JDMSO), the cryo-preservatives with high concentration of serum,
cooled at a defined rate in liquid nitrogen vapour and then placed in liquid
nitrogen. The function of the cry preservative is to reduce the water content of the
cells. The DMPO enters cells extremely quickly by diffusion across the lipid layer

18
of the plasma membrane. In the presence of DMSO, ice crystals, which would
otherwise rupture cell membranes and cause them to lyse do not form. The high
serum concentration probably contributes to cell integrity by maintaining the
intracellylar protein concentration of cells rendered probably contributes to cell
integrity by maintaining the intracellular protein concentration of cells
rendered Cell Culture and Fermentation Technology 33 permeable by the DMSO.
Glycerol has much the same effect
Freezing mixture
A double concentrated freezing mixture consists of 40 (v/v) growth
medium (containing 10% sefum). 40% (v/v) FCS and/20%_ (v/v) UMSO (or
glycerol). The mixture should be made the order stated and mixed well by
inversion. The mixture can be filtered through a ,0.2 time sterile disposable filter
into a sterile universal tube and can be stored frozen at -20°C. When mixed with
an equal volume of cell suspension in complete medium, the effective freezing
mixture concentration is obtained.
Freezing down Cells
Cells should be cryopreserved only when they are healthy and growing in
exponential phase. Cultures should be checked carefully for contamination before
cryopresgr-vation. The cells are centrifuged at 150-200 g. at 4°C for 5 min. The
supernatant should be carefully discarded, taking care not to disturb the pellet. The
cells are then resuspended in the residual medium by gently tapping the side of the
tube near the pellet, until no cells remain stuck to the bottom and adjusted to
double final required concentration with fresh, ice-cold growth medium (antibiotic
free) containing 10% (v/v) FCS. Adherent cells should be resuspended to 2 x
106/ml and suspension cells to 1 x 10/Snl. The cell suspension should be placed on
ice and an equal volume of the freezing mixture is added and mixed thoroughly.
Aliquots (1 ml) of cell suspension should be put into cold cryotubes. A freezing
plug allows the cells to be cooled at a defined rate in the vapour that forms over
the liquid nitrogen within the storage tank; the cooling rate should be l°C/min. If a
freezing plug is not available, a polythene box is an acceptable alternative.
Thawing procedure
A waterbath should be maintained at 37°C and the complete medium is
warmed up to the temperature. The vials should be collected from the liquid
nitrogen storage with-care. The vial may be thawed as quickly as possible in the
37°C water bath. The contents of the vial should be pipetted into a 1C ml sterile
centrifuge tube containing 9 ml complete medium, the cap secured, and the
contents mixed by inversion. The cells must be removed from the freezing mixture
in which they were stored by centrifugation at 150-200 g for 5 min and then
resuspended in 10 ml of complete medium. A sample should be taken for counting
and to check the viability.

19
Usefulness of Cell Culture in Veterinary Research
In the field of veterinary research, cell culture system has become an
important tool in the diagnosis and control of viral diseases of animals and birds.
Several biological
products including those used in the diagnosis and control of viral diseases are
produced through cell culture systems, These include viral vaccines,
monoclonal antibodies, hormones, immunoregulators and tumour specific
antigens.
Disease diagnosis
Isolation and subsequent identification of the causative agent is the only
way to prove the etiology of any viral infection. Since viruses require a living
system for their replication, the virologists are left with only three options,
employing either experimental animals, embryonated eggs or cell culture systems.
Although experimental animals/birds are best suited for the isolation and
identification, cell culture system is cheap and easy to handle, while
embryonated eggs can carry adventitious agents and) contaminants. Cell
cultures can be made devoid of them, f Primary cultures like calf kidney
moholayer, pig kidney ' monolayer, sheep kidney monolayer and chicken embryo
fibroblast monolayer and cell lines like Vero, BHK21 and are routinely used
for virus isolation and identification.
Virus vaccines
Cell culture system also plays a vital role in the development of viral
vaccines. These viral vaccines seem to be the main animal cell product with high
market value. Animal cells are used with the substrate for vaccine production. The
best examples for cell culture vaccines are rinderpest vaccine, foot and mouth
disease vaccine, sheep pox vaccine, rabies vaccine and Marek's disease vaccine.
Monoclonal antibodies.
Somatic cell hybrids have become an important source of cellular products
which cannot be obtained from short primary cultures. The best example for such
a system is the
hybrid myeloma used in the production of MAbs against the antigen of choice.
The production of MAbs involves the inoculation of the antigen of choice into
BALB/c mice, the spleen cells could be removed from the mice and put into
culture. These cells in the culture will divide and produce '- antibodies directed
against the antigen, but will last for only a short period in culture. However, cell
hybridization makes it possible to combine the spleen cells producing antibodies
with mouse myeloma cells which can be cultured continuously. The
mouse myeloma cells viz., SP2/0 cells are fused with the spleen cells and the
resulting hybrids are screened for the production of desired antibodies. Such
hybrids will provide a continuous supply of MAbs raised against specific epitopes
and will be useful in differentiating several strains of viruses such as Newcastle
disease virus, Marek's disease virus, infectious bronchitis virus, etc. MAbs are
useful in differentiating vaccine virus from field viruses.

20
Hormones
Glycoprotein hormones are produced in cultured cells and have therapeutic
use. Several clones of pituitary tumor cells are available which synthesize and
secrete hormones like prolactin, growth hormone and adrenocorticotrophic
hormone. These include rat GH cells and mouse AtT20/Dl6 -cells. The GH cells,
previously known as MtTW5 were established from rat pituitary tumour. These
cells apart from producing growth hormone also synthesize prolactin. The
AtT20/D16 cells are clonal strains isolated from radiation induced pituitary
tumour in mouse and produce large amounts of ACTH during serial propagation in
culture. Islets of Langerhans of rat, pig and human foetuses are grown in vitro for
the production of insulin employing hollow fibre cell culture systems.
Immunoregulators
Animal cells are employed in the production of several non-antibody
immunoregulators like interferons, inter-leukins, colony stimulating factors, B-cell
growth factor, macrophage activating factor, T-cell replacing factor and migration
inhibition factor.
Tumour specific antigens
The best example of this is the carcino-embryonic antigen (CEA). This is a
tumour associated antigen produced by adenocarcinoma cells which are found in
sera or other body fluids indicating a state of malignancy. Production of CEA is
achieved by its extraction from cultured adenocarcinoma cell lines. This tumour
specific antigen is more useful in screening for anti-tumour agents and detecting
malignancy in patients using immuno assays.
Fermentation Technology
The term 'fermentation' is derived from the Latin word ferverq, meaning 'to
boil', which describes the action of yeast on extracts of fruit or malted grain during
the production of alcoholic beverages. However, 'fermentation' is interpreted
differently by microbiologists and biochemists. To a microbiologist, the
fermentation means 'any process for the production of a product by the mass
culture of microorganisms'. To a biochemist, it means 'an -generating process in
which organic compounds art electron donors and acceptors', that is, an anaerobic
rocess where energy is produced without Participation of oxygen or other
inorganic electron acceptors. In tins chapter, fermentation' is used in its broader,
microbiological context.
The fermentation process
The central component of the fermentation process is the fermenter in
which the organism is grown under conditions optimum for product formation.
Operation of upstream and downstream of the fermenter is also important. Before
the fermentation is started, the medium must be formulated and sterilized, the
fermenter sterilized and inoculated with a variable metabolically active culture
which is capable of producing the required product. Product recovery or
downstream processing involves the extraction and purification of the biological
products and further processing and treatment of effluents produced by

21
fermentation. The most widely used industrial scale fermenters are stirred, baffled,
aerated tanks provided with systems of temperature, pH and foam formation
control.
The growth of the microorganisms may result in the production of a range
of metabolites, but the predominant type of metabolite synthesized depends upon
the nature of the organism, the cultural conditions employed and the growth rate of
the culture. If a microorganism is introduced into a nutrient medium which
supports its growth, the inoculated culture will pass through a number of stages
and the system is termed as batch culture. Initially, growth does not occur and this
period is referred to as lag phase and may be considered as a period of adaptation.
Following an interval during which the growth rate of cells gradually increases,
the cells grow at a constant maximum rate and this period is referred to as the log
or exponential phase. As nutrients are exhausted or toxic metabolites accumulate,
the growth rate of the cells deviate from the maximum and eventually growth
ceases and the culture is said to enter the stationary phase. After a further period of
time, the like prolactin, growth hormone and adrenocorticotrophic f hormone.
These include rat GH cells and mouse AtT20/Dl6 I -cells. The GH cells,
previously known as MtTW5 were established from rat pituitary tumour. These
cells apart from producing growth hormone also synthesize prolactin. The
AtT20/D16 cells are clonal strains isolated from radiation induced pituitary
tumour in mouse and produce large amounts of ACTH during serial propagation in
culture. Islets of Langerhans of rat, pig and human foetuses are grown in vitro for
the production of insulin employing hollow fibre cell culture systems.
Immunoregulators
Animal cells are employed in the production of several non-antibody
immunoreguiators like interferons, inter leukins, colony stimulating factors, B-cell
growth factor, macrophage activating factor, T-cell replacing factor and migration
inhibition factor.
Tumour specific antigens
The best example of this is the carcino-embryonic antigen (CEA). This
is a tumour associated antigen produced by adenocarcinoma cells which are found
in sera or other body fluids indicating ^ state of malignancy. Production of CEA
is achieved by its extraction from cultured adenocarcinoma cell lines.
This tumour specific antigen is more useful in screening for anti-tumour
agents and detecting malignancy in patients using immuno assays.
Fermentation Technology
The term 'fermentation' is derived from the Latin word ferverq, meaning 'to
boil', which describes the action of yeast on extracts of fruit or malted grain during
the production of alcoholic beverages. However, fermentation' is interpreted
differently by microbiologists and biochemists. To a microbiologist, the
fermentation means 'any process for the production of a product by the mass
culture of microorganisms'. To a biochemist, it means an energy-generating
process in which organic compounds act as both electron donors and acceptors',

22
that is, an anaerobic process where energy is produced without the participation of
oxygen or other inorganic electron acceptors. In this chapter, fermentation' is used
in its broader, microbiological context.
The fermentation process
The central component of the fermentation process is the fermenter in
which the organism is grown under conditions optamum for product formation.
Operation of upstream and downstream of the fermenter is also
important Before the fermentation is started, the medium must be formulated and
sterilized, the hereafter steris and moculated with a variable metabolically active
cultre which is capable of producing the required product Product recovery or
downstream processing involves the extraction and purification of the
biological products processing and treatment of effluents produced by
fermentation. The most widely used industrial scale fermenters are starred,
baffled, aerated tanks provided with systems of temperature, PH and foam
formation control.
The growth of the microorganisms may result in the production of a range
of metabolites, but the predominant type of metabolite synthesized depends upon
the nature of the organism, the cultural conditions employed and the growth rate
of the culture. If a microorganism is introduced into a nutrient medium which
supports its growth the inoculated culture will pass through a number of stages
and the system is termed as batch culture. Initially growth does not occur and
this period is referred to as lag phase and may be considered as a period of
adaptation. Following an interval during which the growth rate of cells gradually
increases, the cells grow at a constant maximum rate and this period is referred to
as the log or exponential phase As nutrients are exhausted or toxic metabolites
accumulate the growth rate of the cells deviate from the maximum and eventually
growth ceases and the culture is said to enter the stationary phase. After a further
period of time the viable cell number begins to decline and the culture enter the
death phase.
The term tropophase was used to describe the log or exponential phase of a
culture during which the sole products of metabolism are either essential for
growth such as amino acids, nucleotides, proteins, nucleic acids, lipids,
carbohydrates, etc., or the byproducts of energy yielding catabolism such as
ethanol, acetone and butanol. The metabolites produced during the tropophase are
described as primary metabolites. The term idiophase was used to describe the
phase of a culture during which products other than primary metabolites are
produced and these are termed as secondary metabolites.
Secondary metabolites have been defined as compounds which are
synthesized by slow growing or non-growing cells, play no obvious role in cell
growth and are taxonomically limited in their distribution.
The microorganisms may be grown in batch, fed-batch or continuous
culture. In "batch culture', the microorganisms are grown on a limited amount of
medium until either one essential nutrient component is exhausted or toxic by-

23
products accumulate to growth inhibiting levels. Another method of culture is
continuous culture, wherein, there is a continuous addition of fresh medium to
the culture vessel. This procedure is repeated several times until the vessel is full.
However, if an overflow provision is installed in the side of the fermenter, the
addition of fresh medium displaced an equal volume of culture, then continuous
production of cells could be achieved. In 'fed-batch culture' batch culture are fed
continuously or sequentially with fresh medium without the'removal of culture
medium.
The earliest example of the commercial use of 'fed-batch culture' is the
production of baker's yeast. It was recognised as early as 1915 that an excess of
malt in the production medium would result in a high rate of biomass production
and an oxygen demand which could not be met by the fermenter. This resulted in
the development of anaerobic conditions arid the formation of ethanol at the
expense of biomass.
The penicillin fermentation provides a very good example of the use of fed-
batch culture for the production of a secondary metabolite. The penicillin process
is a 'two-stage' fermentation an initial growth phase is followed by the production
phase or idiophase. During the production phase, glucose is fed to the fermenter at
a rate which allows a relatively high growth rate (and therefore rapid accumulation
of biomass) yet maintains the oxygen demand of the culture within the aeration
capacity of the equipment. During the production phase, the biomass must be
maintained at a relatively low growth rate and thus, the glucose is fed at a slower
dilution rate. Phenylacetic acid is a precursor of the penicillin molecule, but it is
also toxic to the producer organism above a threshold concentration.
Thus, the precursor is also fed into the fermenter continuously, thereby
maintaining its concentration below the inhibitory level.
Continuous culture offers the best control over the growth of the cells.
However, the commercial adoption of continuous culture is confined to the
production of biomass and to a limited extent the production of potable and
industrial alcohol.. Fed batch culture method is commonly used in fermentation
technology.
Applications of fermentation
The technology utilized in fermentation processes are designed to obtain
maximum growth of an organism under the optimum physical conditions in a
specific medium for the production of desired end products of commercial value.
This includes the cultivation of microbes for the production of cells themselves,
eg. biomass or the production of useful metabolites or proteins. Many of these
processes are not new, but are currently undergoing rapid development as a result
of recombinant DNA technology.
Microbial biomass
Microbial biomass is produced commercially as a single cell protein (SCP)
by both uni- and multi-cellular bacteria, yeast, filamentous fungi or algae which
can be used as food or feed additives. SCP is of great nutritional value because of

24
its high protein (45-50%), vitamins and lipid contents and the general presence of
a complete array of all essential amino acids. The advantages of using microbial
biomass are 1) microorganisms in general have a high rate of
multiplication; 2) they can utilize a large number of different carbon sources, some
of which are waste products; 3) the strains with high yield and good composition
can be selected and produced relatively easily and 4) microbial biomass
production is independent of seasonal acid climatic variation. ICI Pruteen process
for the production of bacterial SCP for animal feed was a milestone in the
development of the fermentation industry. The disadvantage is that many
microorganisms produce toxic substances and it has to be made sure that the
biomass does not contain any such substances. However, the economics of the
production of SCP as animal feed were marginal, which eventually led to the
discontinuation of the Pruteen process.
Microbial metabolites
The metabolites produced during the tropophase of the culture are referred
to as primary metabolites such as amino acids, nucleotides, proteins, nucleic
acids, lipids, carbohydrates, etc. and the by-products of energy-yielding
metabolism are ethanol, acetone and butanol.
The metabolites produced during the idiophase are referred to as secondary
metabolites. Secondary metabolites are synthesized from the intermediates and
end products of primary metabolism. Few of the secondary metabolites include
antibiotics (penicillin, cephalosporin, tetracycline, streptomycin and griseofulvin),
anti-tumor agents (actinomycin), anticancer agents (krestin and bestatin) and
immunosuppressent (cyclosporin A).
The production of microbial metabolites may be achieved in continuous, as
well as in batch systems. The secondary metabolites are produced by slow
growing organisms in continuous cultures.
Microbial enzymes
The major commercial utilization of microbial enzymes is in the food and
beverage industries in medicine, textile and leather industries, in analytical process
as well as in detergent and dairy industry. Most of these enzymes are synthesized
in the logarithmic phase of the batch culture and may, therefore, be considered as
primary metabolites. However, some, for example, amylases of Bacillus
steorothermophilus are produced by idiophase-type cultures and may be
considered as equivalent to secondary metabolites. Enzymes may be produced
from animals and plants as well as microbial sources, but the production by
microbial fermentation is the most economic and convenient method.
For example, microbial renin is being used instead of calf renin and
proteases used in detergents are produced by microbes. It is now possible to
engineer microbial cells to produce animal or plant enzymes.
Transformation process
The microbial cells can be used to catalyze the conversion of a compound
into a structurally similar, but functionally more valuable compound. Such

25
fermentations are termed transformation processes, biotransformations, or
bioconversions. The production of vinegar is the oldest and most well established
transformation process (the conversion of ethanol into acetic acid). Other
examples of transformation are sorbitol to sorbose and production of new
antibiotics that are more effective than the existing ones. The anomaly of the
transformation process is that a large biomass has to be produced to catalyze a
single reaction.
Recpmbinant products
The advent of recombinant DNA technology has extended the range of
potential microbial fermentation products. It is possible to introduce genes from
higher organisms into microbial cells such that the recipient cells are capable of
synthesizing 'foreign' (or heterologous) proteins. Examples of the "hosts' for such
foreign genes include E. coli, Saccharomyces cerevisiae and other yeasts and
fungi. Products produced by genetically manipulated organisms include interferon,
insulin, human serum albumin, factors VIII and IX, epidermal growth factor and
bovine somatostatin.
The genetic improvement of product formation
Microorganisms usually produce commercially important metabolities in
very low concentration. Thus, to improve the potential productivity, the organism's
genome must be modified and this may be achieved in two ways, by mutation or
by recombination.
Mutation
Each time a microbial cell divides, there is a small probability of an
inheritable change occurring. A strain exhibiting such a changed characteristics is
termed a mutant and the process giving rise to it, a mutation. The probability Of a
mutation occurring may be increased by exposing the culture to a mutagenic agent
such as UV light, ionizing radiation and various chemicals, for example,
nitrosoguanidine and nitrous acid. Such an exposure results in the death of a vast
majority of cells. The survivors of the mutagen exposure may then contain some
mutants, a very small proportion of which may be improved producers.
The synthesis of a primary microbial metabolite (such as an amino acid) is
controlled such that it is only produced at a level required by the organism. The
control mechanisms involved are the inhibition of enzyme activity and the
repression of enzyme synthesis by the end product, when it is present in the cell at
a sufficient concentration. Thus, these mechanisms are referred to as feed back
control. It is obvious that a good 'commercial' mutant should lack the control
system, so that 'over production' of the end product will result.
The isolation of mutants of Corynebacterium glutamicum capable of
producing lysine will be used to illustrate this approach. The first enzyme in the
lysine synthesis pathway is aspartokinase, which is inhibited only when both
lysine and threonine are synthesized above a threshold level referred as concerted
feed back control. A mutant which could not catalyze, the conversion of aspartic
semialdehyde into homoserine would be capable of growth only in homoserine-

26
supplemented medium. If such organisms are grown in the presence of very low
concentration of homoserine, the endogenous level of threonine would not reach
inhibitory level for aspartokinase control and thus, aspartate would be converted
into lysine which could accumulate in the medium.
Recombination
Recombination is a process which helps to generate new combination of
genes that were originally present in different individuals. Techniques are now
widely available which allow the use of recombination as a system improvement.
In vivo recombination may be achieved in asexual fungi (for example Penicillium
chrysogenum, used for the commercial production of penicillin) using the
parasexual cycle. The technique of protoplast fusion .has increased greatly the
prospects of combining together characteristics found in different production
strains. Protoplast fusion has been achieved with the filamentous fungi, yeast,
streptomyces and bacteria and is an increasingly used technique. improved the rate
of glucose consumption (and therefore lysine production) of a high lysine
producing strain of B, flavum by fusing it with another B. flavum strain which was
a non lysine producer, but consumed glucose at a higher rate. Among the fusants,
one strain exhibited high lysine production with rapid glucose utilization.
Threonine production by E. coli has been improved by incorporating the
entire threonine operon of a threonine analogue-resistant mutant into a plasmid
which was then introduced back into the bacterium. The plasmid copy number in
the cell was approximately 20 and the activity of the threonine operon enzymes
was increased 40-50 times. The organism produced 30 g dm+3 threonine compared
with the 2-3 g dm+3 of the non-manipulated strain. described the construction of
an E. coli strain capable of synthesizing commercial level of phenylalanine (an
important fermentation product as it is a precursor in the manufacture of the
sweetener, aspartame). Several of the phenylalanine genes are subjected to control
by the repressor protein of the tyrR gene. In vitro techniques were used to generate
tyrR mutations and introduce them into the production strain. The promoter of the
pheA gene was replaced to remove repression and attenuation control.

27
Biotechnological Approaches to Vaccine Production
Introduction
At present, the majority of veterinary vaccines are produced by
conventional methods similar to those implemented by Jenner or Pasteur. These
include conventional live, attenuated vaccines and killed or inactivated vaccines.
Both of these types of vaccines have proven to be effective particularly in
reducing the clinical manifestations following exposure to virulent field strains of
the pathogens. One of the important impediments in the case of live vaccines is to
ensure that the organism is attenuated sufficiently not to cause the disease, but still
replicate to a sufficient level to induce an appropriate immune response. However,
only a limited number of viral diseases can be prevented by live attenuated viral
vaccines Live viral vaccines carry the remote risk that they may revert to virulent
state and most DNA-containing viruses have the potential to establish persistent
(or latent infection. New viral strains may arise by recombination of the vaccine
virus with other viral strains in animal populations; pregnant animals or their off
springs may by adversely affected by the vaccine strain.
Certain apparently avirulent viral strains can revert to virulence, either by
host induced proteolysis or surface proteins of the phenotype, as in the activation
of the HN and F0 proteins of Newcastle disease virus and of the HA protein of
influenza virus or mutations in the genotype, as in the reversion of attenuated oral
Sabin poliovirus vaccines. Similarly, the so called inactivated foot and mouth
disease (FMD) viral vaccines are found to be infectious far too frequently. For
example, at least 44% of the outbreaks of FMD in Europe from 1968 to 1981 were
caused by incompletely inactivated vaccines or escape of virus from vaccine
manufacturing facilities (FAO, 1981). Similarly, outbreaks of FMD in three South
American countries in 1979 and 1980 were caused by three different vaccines that
contained infectious virus. In addition, conventional whole-agent vaccines,
particularly crude preparations, have been implicated hi post vaccinal pyrogenic
and allergic reactions, abortions, Guillain-Barre neurological syndromes and
adverse sequelae.
Conventional killed or inactivated viral vaccines also have potential risks,
for example, incompletely or improperly killed batches of virus could result in
contamination of the vaccines with active wild-type virus or with contaminating
virus in the vaccine. Therefore, elaborate safety testing is a crucial and costly part
of the production process.
Improvements in conventional biochemistry, recombinant DNA
technology, peptide synthesis, molecular genetics and protein purification had laid
the foundation for the development of new vaccines which should be more
efficacious, cost effective and lead to fewer side effects.
Animal vaccines must be cheap, easy to administer and effective. To
produce a vaccine, genetic engineers move genes around in the infectious
organisms to separate the pathogenic factors (components that cause the disease)
from the antigens (components that stimulate an immune response).

28
Five strategies are currently being applied to the generation of new
types of vaccines: 1) recombinant DNA cloning of immunogenic surface
protein; 2) chemical synthesis of polypeptide vaccines; 3) construction of
recombinant vaccines having guest genes for foreign surface proteins; 4) genetic
engineering of non-pathogenic mutant agents and 5) production of
monoclonal anti-idiotypic antibodies that mimic the major immunogenic
epitomes of the surface proteins of infectious agents.
One approach is to remove virulent genes from the infectious organisms.
Scours is a disease caused by E. coli that affects newborn calves and piglets.
The resulting diarrhoea and severe dehydration can cause heavy mortality.
Disease causing strains of the bacteria produce one protein which allows the
bacteria to adhere to the gut of the young animals and another which governs the
water loss. If the gene for the water loss protein is removed, scours can be
prevented. The organism sticks to the gut without causing diarrhoea and acts as a
vaccine, stimulating an immune response against the adhering protein. The
vaccine is often given to pregnant animals which pass immunity to their off
springs.
Recently, recombinant DNA technology has helped to develop new
generation vaccines, which are cheaper, safer and more effective. Some vaccines
are made not by disarming the pathogen, but by transforming the genes coding for
the antigens of pathogens into harmless organisms. This method has been used to
produce rabies vaccine. The gene for the surface glycoprotein of the rabies virus,
is inserted into the DNA of another virus, vaccinia. Vaccinia, virus causes cow
pox, .but relatively harmless to dogs. It acts as a vector, transporting the piece of
rabies virus RNA into a vaccinated individual and subsequently producing rabies
antibodies. This stimulates -a protective immune response against rabies.
Subunit vaccines are produced by genetic engineering. They are purified
single proteins from the surface of a pathogen which can be produced cheaply in
fermenters. The great advantage of subunit vaccines is that they contain no live,
potentially infectious organisms. The synthetic vaccines are advantageous because
the immune system of the animal is challenged with only one antigen, thereby
omitting other components of the virion that might adversely affect the immune
response. The major drawback with subunit / peptide vaccine is that the antigenic
mass cannot be greater than the, amount injected. There is no amplification
of the antigen. The conventional vaccine for FMD (killed vaccine) was responsible
for nearly half of the outbreaks of the disease in Europe between 1968 and
1981, because it contained a low percentage of live organisms. It may now be
replaced by a subunit vaccine.
Many important antigens are proteins and can be made in harmless
organisms through genetic engineering. Genetic engineering also provides a way
to produce vaccines even if the infectious organisms cannot be grown in animals
or in artificial cultures. Genetic engineers have taken a gene that codes for a
surface protein (hepatitis B surface antigen) and inserted it into E. coli or into

29
yeast. When the proteins are produced by gene expression in new hosts, they are
easy to purify for use as vaccines. It is not even necessary to produce the whole
protein to obtain a genetically engineered vaccine. Only small regions of the
proteins called epitopes are bound by antibodies.
Immunity to flu lasts for a year or so, not because the vaccines are no good,
but because the virus keeps changing its protein coat. Scientists have investigated
several strains of the flu virus, looking for regions that do not vary and were
exposed sufficiently to stimulate an immune reaction. They found a peptide of 18
amino acids which they knew, from the protein structure studies, was located in an
exposed part of a viral protein called hemagglutinin. This region was short enough
to be produced chemically in a peptide synthesizer. Joining this synthetic peptide
to a carrier protein provides a vaccine that protected mice from several different
strains of the influenza virus.
Protein and peptide preparations are 'dead' vaccines. 'Live' vaccines can
also be produced by genetic engineering. If genes from other organisms are
inserted into the DNA of vaccinia, the virus used as a vaccine, will produce the
corresponding proteins as it grows inside human cells. Experimental vaccines to
protect animals against infection from rabies, herpes, hepatitis B and influenza
have been produced in this way. Upto 25 genes can be inserted into vaccinia DNA
and there are plans to produce multiple vaccines to confer immunity to several
diseases simultaneously.
Anti-idiotype antibodies have been produced against very many antigens
and are being used as an antigen. One of the biggest advantages of this is that the
virulent/inactivated microbe need not be used, instead anti-idiotypic antibodies
could be used for development of immune response. Anti-idiotypic antibody
mimic the structure of the original antigen. Anti-idiotypic MAbs have also been
evaluated as immunogen.
Recombinant Subunit Vaccines
Recombinant DNA (rDNA) technology, first described in 1970, deals with
the splicing of the gene coding a known protein into a DNA vector, such as double
stranded DNA virus a bacterial plasmid, and subsequent transfer of the chimeric
DNA into an alternate host cell for its replication and regulated expression of the
guest gene. By means of recombinant DNA technology, genes (even those of RNA
viruses after making complement DNA with reverse transcriptase) can be
transplanted between plants, animals
and microorganisms. Modern advances in molecular biology and nucleic acid and
protein chemistry have contributed to the development of rDNA technology.
These advances include a) the elucidation of the structure and functions of
bacterial plasmids and viruses; b) the finding that retroviruses possess reverse
transcriptase, which permits them to replicate by a recombinant DNA pathway; c)
the discovery and isolation of restriction endonucleases that cleave DNA generally
where thymine, cytosine, adenine and guanine bases form palindromic sequences
on opposite strands; d) the isolation of DNA ligases, SI nuclease and terminal

30
deoxyribonucleotidyl transferases; e) the development of rapid protein and DNA
sequencing procedures; and f) the finding that microorganisms such as E. coli,
Bacillus subtilis and yeasts can serve as an alternate hosts for chimeric DNA
vectors containing guest genes encoding foreign proteins.
The first step in the production of recombinant subunit vaccine is the
isolation of immunogenic genes, which are amplified by cloning. The specific
genes of virions are purified from the preparation of DNA or cDNA in case of
RNA viruses. The DNA is amplified by cloning and cleaved with restriction
endonucleases to small fragments. The DNA fragments which code for
immunogenic proteins are identified and used for the preparation of recombinant
vaccines.
Vaccinia virus, a member of the pox virus family, is a large double-
stranded DNA virus that replicates in the cytoplasm of infected cells. Vaccinia
virus has a wide host range, including human, cattle, horses, swine, sheep, goats,
mice and monkeys. The difficulty of using, vaccinia virus as a vector include its
large genome, the non-infectious nature of the isolated DNA and the ability of
homologous sequences in plasmid and virus DNA to recombine withni infected
cells.
Initial experiments indicate that vaccinia virus vectors retain infectivity,
accommodate at least 25,000 bp of exogenous DNA. Heterologous genes
including hepatitis B virus surface antigens, influenza virus haemagglutinin,
herpes virus glycoprotein D, malaria sporozoiie surface antigen and rabies
glycoprotein have been expressed in this vector systems.

Recombinant vaccines against viral diseases


Foot and mouth disease was the first disease against which effective
vaccine was produced through gene splicing. The FMD virus genome is a single,
positive-sense strand of infectious RNA, made of four viral proteins, viz., VPI,
VP2, VPS and VP4. In this virus, the neutralizing immune response appears to be
directed primarily against VP1. VPi gene carries type and strain specificities,
receptor binding site, T cell epitopes and immunogenic sites. Studies have shown
that the peptides corresponding to residues 141-160 and 200-213 of VPI are
capable of neutralizing FMD virus. The neutralization elicited by residues 141-160
is much greater than that ef antiserum to residues 200-213. These data demonstrate
that VPI of FMDV is an ideal target antigen for the development of a subunit
vaccine against FMD
Rhabdoviridae possess an immunogenic surface glycoprotein G
(haemagglutinin). Rabies virus subunits or the isolated glycoprotein G alone
elicited neutralizing antibody and induced immunity in animals. The glycoprotein
G genes of rabies and vesicular stomatitis viruses have been cloned in E. coli. A
recombinant vaccinia virus expressing the immunizing rabies- G glycoprotein has
been developed.This vaccine has been successfully used to protect foxes against
rabies. The administration of recombinant rabies vaccine to adult foxes, raccoons

31
and skunks elicited high levels of rabies virus neutralizing antibodies and long
term protection against rabies.
Recombinant vaccines have been produced against rinderpest virus. Dr.
T.D. Yilma and his colleagues in USA were the first in the field to construct the
Wyeth. strain of vaccinia virus recombjnants expressing haemagglutinin(H) and
fusion (F) genes cloned from the original highly virulent Kabate '0' strain of
rinderpest virus. This vaccine was administered to cattle intradermally in two
doses, four weeks apart. All the vaccinated cattle developed neutralizing antibody,
but the antibody response in the cattle receiving flie recombinant expressing the
rinderpest F. gene were significantly lower than the response in cattle given the
recombinant expressing the rinderpest H gene. Solid immunity with recombinant
vaccine required two doses.
Dr. T. Barret and his colleagues, working at UK inserted the F-protein gene
from Plowright's cell cultured rinderpest vaccine virus into the vaccinia virus.
When this vaccine was injected intradermally into cattle and pigs, the antibody
response was higher in pigs than in cattle. Two doses were administered.
In Japan, Dr. Yamanouchi's group inserted the H-protein gene into the
tissue culture strain of vaccinia virus. The vaccine was given intradermally into
rabbits and they produced neutralizing antibodies.
Newcastle disease recombinant vaccine has been produced using different
virus vectors. The haemagglutinin-neuraminidase (HN) gene and the
phosphoprotein (P) gene of Newcastle disease virus (NDV) were inserted into an
avian leukosis virus vector. These recombinants were used to immunize four
weeks old chicken. Birds receiving the virus containing the HN gene developed
low levels of serum haemagglutination inhibition titres. Upon challenge, all birds
vaccinated with the HN gene containing virus were protected from the disease.
Birds immunized with the P gene containing retrovirus developed more severe
clinical signs. In another study, a cDNA copy of the RNA encoding the fusion(F)
protein of NDV was inserted into a fowl pox virus vector. Ocular or oral
administration of recombinant fowlytjox virus gave partial protection, whereas
both intramuscular or wing web routes of inoculation gave complete protection
after a single injection .
Three glycoproteins (gp 115/110, gp 63 and gp 51) from herpes virus of
turkeys and Marek's disease virus would protect chicks against Marek's disease.
The surface glycoprotein of infectious bovine rhinotracheitis and pseudorabies
virions have been reported to induce neutralizing antibodies and protective
immunity in cattle and swine, respectively. A p-galactoside pseudorabies
glycoprotein fusion protein from E. colt is reported to protect mice from challenge
with pseudorabies virus.
The genome of blue tongue virus is divided into 10 RNA segments. VP2
protein of type 10 blue tongue virus induce both neutralizing antibodies and
protective immunity in sheep. RNA segment 2 which represents the VP2
neutralizing antigen, has been inserted into an insect baculovirus (Autographa

32
californica, AcNPV). Infection of Spodoptera frugiperda cell lines (which
supports the baculovirus to grow in high titre) by the recombinant virus produced
VP2/ protein that was precipitated with BTV-10 and to a lesser extent BTV-
serotype 11 and 17 viruses .
Canine distemper virus (CDV) and measles virus (MV) are members of the
Morbillivirus genus of the family Paramyxoviridae and contain a non-segmented
single-stranded RNA genome. These viruses have membrane glycoprotein
haemagglutinin (HA) which is responsible for haemagglutination and attachment
of the virus to the host cell and the fusion glycoprotein (F) which causes
membrane fusion between the virus and the infected and adjacent cells. Vaccinia
virus-MV, HA and F recombinants were produced and inoculated into dogs. Of
these two recombinants, vaccinia virus-MVHA gave better neutralizing antibody
production than W-MVF protein .
VP-2 of canine parvovirus which is a major component of the cap side
protein was expressed in baculovirus. This recombinant virus was able to elicit
good protective response .
Recombinant vaccines against bacterial diseases
The first work of rDNA bacterial protein vaccines involved the cloning of
somatic pili of enterotoxigenic E. coli (ETEC) for use in preventing diarrhoea in
livestock. The ETEC have somatic pili composed of 14 to 22 KDa protein
molecules that adhere to the mucosal surface of the small intestines, initiating
colonization by the bacteria. Pili isolated from ETEC have been used as vaccines
to prevent diarrhoeal diseases in both humans and animals.
An alternate method for protecting domestic animals against ETEC has
been achieved through genetic engineering. Monoclonal antibody to K99 pilus
antigen produced in hybridomas and amplified in mice significantly reduced the
severity of scours and the mortality of calves so treated.This product was
successfully tested in Canada and had since been approved by the USDA for use
in the US.
A cloned pilus vaccine is being developed for use in humans against
penicillin-resistant strains of Neisseria gonorrhoeae. The genes for the protective
antigen of the tripartite protein toxin of Bacillus anthracis have been cloned and
expanded in an E. coZ//pBR 322 system for the purpose of developing a vaccine.
Production of rDNA protein vaccine against parasitic diseases is in
progress, including those for avian coccidiosis and malaria.
Peptide Vaccines
The recent interest in synthetic peptide vaccines is due primarily to the
remarkable advances in protein and nucleic acid chemistry and in
immunochemistry. It is now possible to define many biologically important
proteins 'in terms of their amino acid sequence. This is particularly true for the
disease-inducing viruses, since determination of the genome structure by direct
cDNA sequencing or after recombinant DNA cloning is relatively easy and the

33
nucleotide sequence so obtained can be used to predict the structure of
immunogenic virion proteins.
Synthetic peptide vaccines offer the advantage of safety, purity and low
cost as compared to live or inactivated vaccines. It is now possible to induce virus
neutralizing antibody response using peptides of specific amino acid sequences.
The peptides can be chemically synthesized in pure form or made by bacterial
expression using rDNA technology. In the latter case, the peptides may be fused
into other expressed proteins (fusion peptides).
Fusion protein vaccines
FMD virus particles contain a positive-strand genome of molecular weight
2.6 x 106 in association with 60 copies of each of 4 proteins; VP1, VP2, VPS
(molecular weight of around 24KDa) and VP4 (molecular weight of 7KDa). The
capsid proteins can be isolated from disrupted virus b(PAGE. There is
considerable evidence that neutralizing (activity is elicited primarily by VP1,
based on protection of I swine and cattle against virus challenge.
Results from studies have demonstrated that VP1 was located on the
surface of the FMD virion. The antigenic determinants which elicit virus
neutralizing antibodies as well as the virus attachment site for susceptible cells
were located on VP1. The finding that animals, injected with VPl or a fragment of
this protein, induced protection hi animals against infection when exposed to live
virus represented an important observation. As a result, several groups decided to
make peptides based on the structure of these fragments in large amounts by
rDNA technology. Identification of the DNA fragments encoding VPl was
established by sequence analysis of restriction endonuclease fragments and prior
knowledge of the amino acid sequences of portions of VPl. VPl has been
expressed in the K12 strain of E. coli. Such cells grown in culture express 1-2
million copies per cell of VPl fusion protein. The extracted and purified fusion
protein has been shown to elicit neutralizing antibodies 'and protective immunity
against type A12 FMD in both swine and cattle . Two vaccinations with fusion
protein vaccine were required before the immunity of the animals was challenged
after a month later. Cattle vaccinated on day one and again at 3.5 or 7.5 months
with a similarly cloned and expressed 26 KDa fusion protein vaccine containing
residues 1 to, 211 of VPl were protected against virus challenge at 7 and 10.5
months .
In most instances, two doses of the fusion protein vaccine have been
required to stimulate protection of cattle against contact exposure. In studies of a
29 KDa protein, consisting of a 190 amino acid fusion protein linked to amino
acids 137-168 of VPl of type A12 virus, protection was demonstrated in 5 of 6
cattle vaccinated with a single 250 ^g dose. The cattle showed high levels of
neutralizing antibodies 21 days after vaccination and on day 35 their immunity
was challenged by contact exposure to infected cattle. Five of the six vaccinated
animals were protected against infection.

34
Synthetic peptide vaccines
Few of the advantages of synthetic vaccines in terms of cost, ease of
storage and transport include:
a) the product would be stable indefinitely at ambient temperature, thus
obviating the need for cold storage and saving the cost of transporting
large volumes of vaccine under refrigerated conditions;
b) there would be no need for down stream processing;
c) the production facility would be very simple compared with that used for
biological products, and
d) the product would be defined in precise chemical terms and could be
altered readily to elicit the appropriate responses in different situations.
The first indication that a synthetic peptide can elicit an antitoxin activity
was reported by, who demonstrated that a tetradecapeptide of diphtheria toxin
induced protective antibodies when administered to guinea pigs.
Found out two regions of VPj protein of FMD virus was responsible for
eliciting immunogenic response. One of these included amino acids 146-154 and
other at the carboxy terminus of the molecule included amino acids 201 - 213.
Synthetic peptides were produced linked to a carrier protein, most commonly
keyhole limpet haemocyanin (KLH) and the conjugate was tested as an
immunogen. Usually, equal weights of peptides and carrier proteins have been
coupled, which is equivalent to about 30 copies of peptide per protein molecule,
when KLH is used as carrier. tested the immunogenicity in rabbits of 7 peptides
linked to KLH representing amino acids 9 to 24, 17 to 32, 25 to 41, 1 to 41, 141 to
160, 151 to 160 and 200 to 213 of VP1 from of strain of FMD virus. Antibodies
were generated against all 7 peptides and those made against peptides 141 to 160
and 200 to 213 were able to neutralize the homologous virus. When the peptide
141 to 160 was inoculated into guinea pigs, the animals were protected by a single
dose of 200 ug. Subsequently; it was shown that subtype serological specificity
could largely be minimized by sera made against the A10orA12 virus 141 to 160
peptide.
Cattle were protected against FMD type Ol by a single injection of an
organically synthesized peptide. A 4KDa peptide residue was prepared from the
two previously identified immunogenic regions (amino acids 141 to 160 and 200
to 213) connected by a diproline spacer and terminating on each end with cysteine
residues and was administered in Freund's complete adjuvant. The serum
neutralizing titre rose rapidly and by day 14 reached a plateau that was maintained
up to day 32. Two of the three cattle given a single dose (5 mg) of the peptide,
were protected when exposed .to live virus and three others given two doses of 1
mg each were similarly protected. Thus far, this 40 residue synthetic peptide is the
smallest agent in molecular size found capable of inducing protection in cattle.
Two peptides derived from highly conserved regions of bovine rotavirus
outer capsid proteins (VP7 and VP4) were tried as rotavirus vaccine. The VP4-
VP6 peptide conjugate provided better protection, than VP7-VP6 peptide

35
conjugate. A mixture of VP4 peptide, VP6 and VP7 peptide conjugates provided
better heterologous protection than immunization with bovine rotavirus.
Research is in progress for chemical synthesis of peptide vaccines for
bacterial diseases, for example, diphtheria and cholera. A synthetic
hexadecapeptide corresponding to amino acid residues 186-201 of the A chain of
diphtheria toxin covalently bound to both synthetic muramyl dipeptide adjuvant
and synthetic multichain poly-DL-alanine induced protective anti-toxic immunity
in guinea pigs. For cholera, several peptides ranging between 11 and 18 amino
acid residues in length corresponding to regions of the 103 amino acid chain of the
B subunit of Vibrio cholerae have been synthesized and linked either to tetanus
toxoid or to multichain poly-DL-alanine.
Genetically engineered reassortants and deletion mutant vaccines
The ability of genetic engineers to manipulate genes allows for the removal
of pathogenic genes from infectious agents without destroying their ability to
propagate and express immuno dominant epitopes.
Genes reasserting and deletion techniques are being applied to both viruses
and bacteria. Highly attenuated temperature-sensitive mutants of influenza virus or
avian strains (that do not replicate efficiently in mammals) are being used with
virulent wild-type virus to co-infect cell cultures. Some reassortants can be
isolated that contain the six internal genes from an avirulent parental virus and the
two genes for the surface proteins HA and NA of the wild-type virus. Humans
immunized with such rea'ssortants from temperature sensitive mutants exhibit
lower rates of infection and shed less virus than those who received inactivated
influenza vaccine.In human trials, an avian-human- influenza A reassortant virus
had the immunogenicity of wild virus and was not transmissible between
individuals.
The 11 genes of rotaviruses; which cause diarrhoeal disease in young
animals and humans, can also undergo reassortants in mixed infections. A
reassortant with its surface antigen VP1 "genes from a human rotavirus and its
other genes from bovine or rhesus rotavirus have diminished growth
characteristics in human and appear to have potential use as immunizing agent.
Because of this high degree of cross protection, an attenuated bovine rotavirus
vaccine is being considered as a vaccine for humans.
Deletion mutants of several bacteria have been engineered and their
potential usefulness as vaccines is being explored. Included in these studies are
deletion mutants of Salmonella, Shigella and Vibrio cholerae. The gal-E strain of
Salmonella typhi Ty 2 la (lacking UDP galactose-4-epimerase activity and highly
deficient in two additional enzyme activities as well), was able to proliferate long
enough following oral administration to confer immunity against wild type strains
of Salmonella typhi. Similarly, mutant strains of Salmonella defective in aromatic
biosynthesis (they require amino- and dihydroxybenzoate, which are not available
in host tissues) given in two doses either intramuscularly or orally to calves

36
resulted in induction of resistance to oral challenge with more than 1011 organisms
of a virulent UDC stain of S. typhi.
A hybrid strain of Salmonella typhi / Shigella sonei appears to have a
potential as a vaccine against both typhoid and dysentry. A plasmid encoding a
gene for Shigella sonei transformed by conjugation of the Ty 21a attenuated strain
of Salmonella typhi produces a hybrid bacterium which elicits antibodies in
animals to antigens of both organisms.

Anti-idiotype antibody vaccines


Anti-idiotype antibody has the potential to act as vaccine for animals and
humans against infectious diseases.
Animals modulate their response to a foreign antigen by the, production
of a cascade of idiotype anti-anti-idiotype and higher anti-
idiotype antibiodies. However, they have shown generally only the ablity to
prime the animals for anamnestic-like response to infection agents. The anti-
idiotype antibody of particular anti-paratope antibody. The paratope is the binding
Ste in the Fab protein of the antibody and the antibody to wm be the mirror
image of the epitope of the original The advent of the monoclonal antibodies and
recombinant DNA techniques have made the development of anti-idiot™ antibody
vaccines a realistic goal. Thus, anti-idiotype antibodies can now be
produced in large quantities in hybndomas or by cloning and expression of
DNA transcribed from anti-idiotype antibody specific mRNA.
Anti-idiotypic antibodies that have been -examined as potential
immunogens include those mimicking epitopes of HbsAg, pohovirus,
reovirus, rabies virus, bacteria trypanosomes and cancer antigens. Anti-
idiotype antibodies have been prepared against five monoclonal antibodies to
rabies glycoprotein G. Two of the five antiMdiotype antibodies
injected into mice elicited specific £rus-neutralizing antibody
response Protective immunity against African trypanosomiasis have been
produced in mice using anti-idiotype antibodies . Anti-idiotype antibodies have
been produced against parvovirus Newcastle disease virus foot and mouth disease
virus and listeria .

37
Hybridoma Technology
Introduction
Increased research and accumulation, of knawledge in the fields of somatic
cell genetics, tissue culture technology and immunology have resulted in the
development of a technique for in vitro production of antibodies. The construction
of antibody secreting cell lines-hybridomas are produced by fusing antibody-
secreting lymphocytes with appropriate tumour cell lines - myelomas. Monoclonal
antibodies (MAbs) are the first products to move rapidly from the research
laboratories through industry to the market place. MAbs will find use chiefly as
research reagents, diagnostics in vitro and in vivo, as therapeutic agents and in the
purification process and vaccine production.
Hybridoma technology is a form of genetic engineering resulting in the
production of specific antibodies by specialized tissue culture lines. MAb is an
antibody directed against one antigenic determinant or epitope of an antigen. It is a
single isotype.

Early Research
Hybridoma technology, like recombinant DNA is rooted from basic biology
and is the result of years of basic research in cell fusion. In 1973, Jerold Schwaber
and Ed Cohen working at the University of Chicago's Liribida Institute were the
first to produce antibody secreting hybridomas by fusing normal antibody
producing human cells (B cells) to jnyeloma cells.
But it was working at the Medical Research Council Laboratories in
Cambridge, UK, devised and demonstrated a deliberate and rationale strategy for
the construction of continuous cell lines, which secrete monoclonal antibodies of a
desired specificity. That success, which has been widely reported, revolutionized
immunology and created an industry. These workers were awarded Nobel Prize
jointly with Niels Jerne of Denmark in Physiology and for this work.
The mechanism of antibody production was understood in 1957, when
Bernet proposed the clonal selection hypothesis. In 1960s, it became well
established that antibody diversity was a reflection of the diversification of
lymphocyte clones than an individual lymphocyte which produces only a single
type of antibody. Even homologous antigens induce a heterologous antibody
response because of the many different lymphocyte clones with appropriate
specificity for antigen proliferation and secretion of antibody.
Polyclonal Antiserum Vs. Monoclonal Antibody
Animals respond to an antigenic stimulus by producing a large variety of
antibody structures directed against the immunogen. Even a single antigenic
determinant is likely to be recognized by a variety of different antibody structures.
Thus, heterologous mixtures of antibody structures are produced in a polyclonal
response, and the populations of antibodies continuously change within the same

38
animal, since each antibody is secreted by all such individual cells into a common
blood pool.
The composition of polyclonal antibodies will change from day to day and
from animal to animal. Thus, polyclonal antisera, even when prepared employing
well standardized procedures tend to differ from batch to batch. But MAbs
produced by a single clone of cells which react with a single antigenic determinant
are the most specific biological probes available, making possible finer and more
sophisticated differences.
In 1975, Kohler and Milstein gave the concept of one lymphocyte-one
antibody in the form of hybridomas. Hybridomas are hybrids between myeloma
tumour cells and antigen stimulated lymphocytes which can be cloned, grown in
large quantities and for indefinite periods of time, and secrete high concentrations
of monoclonal and hence monospecific antibodies. Each lymphocyte produces
only one type of antibody, although the isotype may change. The homogeneity,
reproducibility and permanent availability of MAbs are the attributes arousing the
greatest interest in this quickly developing field.

Advantages of Monoclonal Antibodies


1. Single MAbs are chemically defined and single specificity molecules,
they can be used easily for standardization of specific assays.
2. MAbs provide a perpetual source of well defined homogeneous reagents.^
3. All the antibodies produced (100%) are active antibodies. Therefore, high
specific activity of labeling is possible in Radioimmuno assay, Enzyme
linked immunosorbent assay and Fluorescent immunoassay.
4. MAbs could be advantageously used as immunosorbents for antigen
purification using immunosorbent column chromatography.
5. Large amounts of MAbs (1-20 mg/ml of ascitic fluid) could be obtained
with modest investments.
6. MAbs specific for a particular target antigen can be obtained even without
prior purification of antigens.
7. MAbs are easily manipulatable. For example, bispecific antibodies can be
prepared from 2 MAbs and used in novel assays, antigenic targeting in
Electron microscopy and cytological studies.
8. Distinct antigenic cross reactivities can be easily defined and hence most
useful in diagnosis.
Disadvantages of Monoclonal Antibodies
1. MAbs are too specific. Limited MAb might miss important cross
reactive determmants. This could relate to the animals' immune
response to the antigenic stimulus. Polyclona1 sera may offer advantage
of broad reactivity.
2. Biological effects of MAbs on binding do not represent the situation in
ani^1

39
3. Single MAb is a single chemical and physical or chemical treatment
that affects one molecule will affect all the MAbs in that population.
Treatments like freeze thawing, proteolytic enzyme contamination and
ammonium sulphate precipitation may destroy all MAb activity. .
4. It is time consuming to produce MAbs. The entire process of producing
MAbs takes 3-4 months for each fusion experiment.

Monoclonal Antibody Production Strategy


The one cell - one antibody theory Provided the conceptual basis for the
practical advantage offered by the MAb technique. Each clone of lymphocyte8
produce a single antibody specificity. Nevertheless, the immune response to most
antigens are polyclonal; it involves hundreds of clones of lymphocytes, each
secreting a different antibody. These antibodies are markedly heterogeneous. They
usually consists of antibodies directed at different antigenic determinants. MAb
production involves the creation of hybrid cells derived from 2 parent cell types:
normal antibody producing cells and neoplastic myeloma cells. The hybrids are
formed by fusing the normal and neoplastic cells. Hybrids have characters of both
parents, viz., the infinite life of the myeloma cells and antibody secreting functions
of primed cells. Hybridization of antibody forming cells with malignant myeloma
cells results in hybridomas in which virtues of specific antibody secretion and
continuous growth are combined. Further, selection and cloning of the hybrid cells
allow the derivation of monoclonal antibodies of predefined specificity and
desired activity.
The method of establishing permanent cell lines capable of producing
antibodies directed to predefined immunogen is based on the fusion of immune
lymphocytes (usually spleen cells from mice previously immunized with antigen)
with myeloma cells adapted for growth in tissue culture conditions .The most
common fusion agent is polyethylene glycol (PEG). The selection for growth of
hybrid cells is based on the facts that a) spleen cells die in the tissue culture
medium and b) the myeloma lines that serve for fusions are mutants lacking either
the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT)
(azaguanine resistant) or thymidine kinase (bromodeoxyuridine resistant). Such
mutants die in the presence of aminopterin which blocks the main pathway of
DNA synthesis because they cannot use the salvage pathway. Hypoxanthine,
aminopterin and thymidine (HAT) allows the selective growth of hybrids. , The
parental myeloma cells that have not fused will not grow in the presence of HAT
medium. Normal lymphocyte die after a short period in culture. Therefore, only
the hybrid cells between the spleen cells and the myeloma cells will survive in the
presence of aminopterin, but for their growth, they require hypoxanthine and
thymidine that are utilized by salvage pathway.
The hybrid cells growing in HAT are then screened for their ability to
produce and secrete the desired antibody. The positive hybridomas obtained after
cloning are the source for monoclonal antibodies. Such cells can be cryopreserved

40
and for the production of large amounts of MAbs they can be grown in mass
cultures, where they secrete 10-50 μg antibody/ml or propagated as ascitic tumour,
where they usually produce 1-20 mg/ml of MAbs into the ascitic fluid.
Though the methodology involved in the production of MAbs appears
simple, there are some crucial steps, especially in the screening and selection of
positive hybridomas, that require preplanned, sensitive, simple and fast assays. In
addition, several precautions and devotion have to be practised with regard to the
tissue culture conditions, especially during the first phase of the hybridoma
establishment. . .
Purity and form of immunogen
The purity of the antigen to be used as immunogen is crucial for generation
of monoclonal antibodies. Molecules of low molecular weight (1000 daltons) are
poor immunogens and have to be coupled to larger immunogenic molecules.
Aggregated and particulate antigens elicit stronger responses. Adjuvants help to
achieve stronger responses. The most common carriers are albumins, keyhole
limpet haemocyanin, fowl gamma globulin and synthetic polypeptides.

Choice of animals
The choice of animal species and strain as immune spleen donor for fusion
is largely dependent on the myeloma cells available and the origin of the
immunogen. Mice are the most common species used for immunization primarily
because there are more murine myeloma cell lines available. The BALB/c strain of
mice is preferred since the myeloma cell lines available for fusion were derived
from this strain.
Immunization procedures
For most proteins, a dose of 1-50 μg per injection per mouse repeated two
or three times is sufficient to evoke a strong antibody response. For soluble
antigens, the first injection is given in the presence of adjuvant and the final
booster in aqueous solutions. Freunds' adjuvants (Difco Lab, USA) are commonly
used for preparing antigen emulsion.
For most antigens, groups of 5-10 animals are sufficient. About 50 ul of the
antigen-Freund's adjuvant emulsion (50ug) is injected intraperitoneally (i/p) into
each mouse. Two to 3 weeks later, same amount of the emulsion is injected i/p.
Ten to 14 days afterwards, individual animals are bled and the titre of the
antibodies are determined. Animals that show the highest antibody titre are
selected and atleast 3-4 weeks after the last booster, they are injected i/p with 20-
100 μg of soluble antigen on the first day and injected intravenously (i/v) with the
same dose on the following day. Three days after the i/v booster, spleens are
removed from two animals and their cells are used for fusion.
Choice of myeloma
Most of the mouse myeloma cell lines are available from the American
Type Culture Collection (ATCC), 12301 Parklawn drive, Roskville, Maryland

41
20852, USA. The most common mouse myeloma cell line which serve as parental
cells for generation of B cell hybridomas is Sp 2/0 Ag-14. This is a non-secreting
cell line and it is preferred since all of the antibodies produced and secreted will be
dictated only by the immune cell. Other cell lines used include NS1/1, Ag 4.1,
NSO, P3/X63, Ag8, FOX-NY from mouse and 43Ag 1.2.3 and YB 2/0 from rats.
Growth of myeloma cell lines
The myeloma cells are generally grown in Dulbeccp's modified Eagle's
medium (DMEM) with high glucose (4.58 g/1) or in RPMI both supplemented
with glutamine (2mM final concentration), antibiotics (usually 100 U/ml
penicillin; 10 μg/ml streptomycin) and 10% foetal calf serum. Cells are maintained
at 37°C in a humid atmosphere of 7-10% CO2. The optimum conditions of a
culture to be used for fusion are high viability and logarithmic growth for atleast
one week before fusion (not exceeding 3-8 x 105 cells/ml). A good practice is to
grow the cells in the presence of 8-azaguanine (30 mg/ml) and to check whether
they all die in HAT. These HGPRT negative cells in the log phase of growth are
used for fusion.

Spleen cells
The spleen is removed from the immunized mice under sterile conditions
and transferred to a petridish containing DMEM. Single cell suspension of
splenocytes is prepared and B lymphocytes are separated by layering the
suspension over Ficoll Hypaque gradient.
Cell fusion
The spleen lymphocytes and myeloma cells at a ratio of 2:1 are mixed and
placed in a centrifuge tube. After mild centrifugation, the supernatant is removed
leaving the cell button. One ml of 50% prewarmed PEG (4000 mol. wt) is added
dropwise with a wide-mouthed pipette for 90 sec. After resuspending the pellet
gently for 1 min, prewarmed DMEM without serum is added drop wise to dilute
PEG with gentle mixing. The supernatant is removed after centrifugation and 5 ml
of DMEM with HAT is added with gentle mixing and the final volume is made
upto 50 ml, keeping the concentration of cells at 1.5 x 10 viable cells and
distributed 0.1 ml aliquots in the wells of 96 well flat bottom micro culture plates
(5 plates can be prepared from 50 ml) and incubated at 37°CJn 5-10% CO9
incubator. After 3-4 days hybrid clones are checked. 0.1 ml of DMEM with HAT
is added and is repeated on the 7th day. When vigorous growth and change of
colour to yellow are observed, usually 10-14 days following fusion, supernatants
can be removed aseptically for screening for antibody activity. Wells are refilled
with HAT medium. Positive wells are transferred into 24 well plates and then to
small 25 cm3 culture flasks. Thus, the positive cultures are expanded by repeated
determination of antibody activity. Hybrid cells secreting the desired antibodies
should be frozen in liquid nitrogen (LN2) at each stage. The positive hybrid clones
are again cloned for MAb production.

42
Cloning of hybrids
Cloning and recloning of hybrid cells is essential to ensure the
monoclonality of the antibodies and to get rid of non-producer cells. Cloning
under limiting dilution conditions is prepared when the fraction of specific
antibody secreting hybridoma is lower than one in 103 cells. Limiting dilution is a
dilution in which hybrid cells are distributed at a concentration of 1 cell/well.
Within 2 weeks, the 'one clone' wells reach a cell density that allows screening
MAb. To ensure monoclonality, the cloning procedure have to be repeated for a
few cycles until all the subclones .detected appear to secrete the specific antibody.
Cryopreservation of cells
Hybridoma cells can be kept frozen in liquid nitrogen for many years in
freezing medium containing 25-50% FCS and 10% DMSO. Ampoules containing
hybrids are frozen in a programmable freezer at the rate of 1°C drop/min from 4°C
to - 80°C and then the tubes are transferred into liquid nitrogen, where they can be
kept for a long time.
Large scale production of monoclonals
For large scale production, hybridomas can be grown either in tissue
culture, where they secrete upto 100 Hg/ml (usually 10-50 ug/ml) or in vivo, as
tumours in the peritoneal cavity of Balb/c mice, where they produce upto 40 mg of
MAb/ml (usually 2-20 mg/ml). For large scale growth, spinner bottles or roller
bottles are useful. For maximal yield of MAbs, the supernatant is to be harvested
at least one day after the cells have reached the stationary phase of growth. Recent
development in biotechnology has led the industry to use hollow fibre technology
for in vitro /targe scale production of MAbs.
Ascitic fluid preparation and purification
Balb/c mice of 6 weeks old are injected Lp with mineral oil, pristane (2, 6,
10, 14-tetramethyl pentadecane) for priming. Two weeks later, hybridoma cells
from selected clones are injected i/p into pristane primed-mice. Seven to ten days
after injection, the peritoneal cavities will appear swollen and an 18-gauge needle
is inserted into the cavity to collect ascitic fluid in heparin. Ascitic fluid is
collected 3 times in a 6 day period.
The ascitic fluid is centrifuged (1000 g for 15 min) to remove cells and
fibrin clots. The supernatant is centrifuged again at 52,000 g for 10 min to remove
lipids and small fibrin clots. Antibodies are then precipitated from the supernatants
by the addition of an equal volume of saturated ammonium sulphate to the
clarified ascitic fluid. The resulting precipitate is dissolved in normal saline
(0.85% sodium chloride) and dialyzed extensively against normal saline for 12 to
16 hrs in cold. The dialysate contains about 80% of antibodies.
Screening assays for MAb activity
The assay system used for the screening of antibody activity in the
hybridoma culture supernatants is a key factor that determines success in obtaining
the desired MAbs. The main factors to be considered are sensitivity, simplicity,
ability to test many samples, speed and reliability. Solid phase radioimmunoassay

43
(RIA) and enzyme linked immunosorbent assay (ELISA) are the frequently used
techniques for screening antibody activity in MAbs. ELISA is used commonly for
both soluble and cellular antigens. The main principle of the ELISA is that in the
final step of the assay, the anti-immunoglobulin is enzyme linked, instead of
radioiodinated. The degree of antibody binding is evaluated by colour
development that follows the addition of substrate to the system. The amount of
colour developed is proportional to the level of enzyme bound antibody present.
Although ELISA is somewhat less sensitive than RIA, the handling of
samples, colour development and its measurements are faster, especially with
automatic scanner that enable the reading, typing and computing of the results of
the 96 wells within 1-2 min. Another practical advantage of ELISA, especially for
the screening procedure, is that, it allows faster evaluation of results and selection
of positive wells based on visual observation.
Uses of Monoclonal Antibodies
There are three main areas in which MAbs are used human and veterinary
medicine. These areas are: 1) in human and diagnostic reagents, either for
detection of the causative agents directly in tissues or body fluids or as a reagent
used in indirect diagnostics such as serological detection of antibodies to the
causative agent; 2) for experimental purposes ranging from molecular dissection
of getigenic epitopes to monoclonal anti-idiotype antibody utilized as a vaccine to
induce protective-immunity and 3) immunoprophylaxis or immunotherapeutics
applied to infectious diseases or as a vehicle for delivering toxic substances to, for
example, tumours or as a tool to identify and locate target tumours.
Immune diagnostic reagents
Antigen detection
With the development of MAbs of various specificities, advances have been
made in the detection of infectious agents (or their antigens) directly in tissue or
body fluid specimens. More amount of work have been done on viruses because of
the difficulties of viral culture.
MAbs have been developed against a large number of viruses including
rabies virus foot and mouth disease virus, Newcastle disease virus feline
leukemia virus , bovine leukemia virus bovine enteric coronavirus and rotavirus .
MAbs have been produced against various parasites, including Trichinella
spiralis, Babesia bovis , Dirofilaria immitis and Trypanosoma cruzi. MAbs have
also been raised against bacterial species, including Mycobacterium spp. and
Vibrio cholerae .
For detection of antigen, the following are the for commonly used methods using
MAbs:
1. MAbs are attached to particles like latex Or erythrocytes, which then
detect the presence of 35 anfygen in fluids by visible agglutination,
usually miriutes. Disadvantage of this technique is the inability of small
antigens to cause agglutination.

44
2. Direct demonstration of antigen in tissue sections cultured cells or
smears by the use of a MAbl conjugated to a fluorochrome,
enzyme (used in I conjunction with a substrate that has an insoluble
cleavage product) or radioisotope as a detecting agent.
3. A competitive-binding type of assay in which MAb is attached to a solid
matrix such, as the well of a polystyrene microtitre plate. Antigen
conjugated with a fluorochrome, enzyme or radio-isotope is added only
in sufficient quantity to bind all the available antigen-binding sites of the
antibody, then premixed with the known sample to be tested for the
presence of an antigen. Decrease in binding of the known antigen
indicates the presence and quantity of antigenic material in the
unknown sample.
4. Antibody immobilized in polystyrene wells can 'trap' antigen in fluid
preparations. The trapped antigen may then be detected using another
antibody to the antigen. This second antibody may be conjugated with a
fluorescent, radioactive or enzymic agent for easy detection.
Alternatively, layers of anti-antibody can be added to increase sensitivity
or an amplified I system may be used. For this type of assay, MAbs may
be used for both the trapping and detection, but they must have different
specificities for the test to function properly unless the antigen is a
single repeating unit such as the o-chain of some bacteria.
Although MAbs are very useful in the detection of antigens or their
components, caution should be exercised in that some MAbs may be too specific
for general diagnostic use. Van Zaane (1983) reported that two MAbs were
capable of distinguishing some strains of hog cholera virus from bovine viral
diarrhoea virus; however, both antibody preparations failed to react with some
strains of hog cholera yirus by an immunofluorescence test. It is therefore essential
to ascertain the reactivity of the MAb with an antigenic determinant present on all
antigens to be tested for, before diagnostic use.
On the other hand, the exquisite specificity of MAbs allows the
differentiation of antigens previously thought to be indistinguishable. For
example, using a panel of MAbs, it is possible to differentiate various street strains
(i.e., non-laboratory strains isolated from natural sources) and two vaccine strains
of rabies virus using immunofluorescence technique . Similarly, Mab to Marek's
disease tumour associated antigen can be used to identify Marek's disease in
chicken. As this antibody does not cross react with antigen expressed by lymphoid
leukosis virus, a differential diagnosis is possible. Because specific MAbs could
be produced to rare and minor antigens, they have the potential to distinguish
closely related strains of viruses, which cannot be differentiated by 'conventional
serological methods. For example, MAbs that distinguish canine parvovirus from
feline panleukopenia virus have recently been produced.

45
MAbs were produced against 42 KDa major structural proteins of rotavirus,
which could be used for preliminary serotyping of strains from different animal
species.
Anderson (1984) described an enzyme immunoassay in which a blocking
MAb was used to detect group-specific antibodies against blue tongue virus. This
assay was capable of detecting antibodies against all 22 serotypes of blue tongue
virus. The use of MAbs against bovine diarrhoea virus in immunofluorescent
antibody techniques has aided preliminary serological characterization of strains
(Peters et al., 1986) and both indirect and competitive ELISA techniques utilizing
MAbs have been used to detect antibodies against the virus in bovine sera.
Accurate and rapid diagnosis of infectious agents is of i considerable
importance in veterinary medicine, particularly I when dealing with highly
infectious agents such as foot and mouth disease virus. MAbs have an important
role in the diagnostic laboratory; however, if the antibody is not carefully
selected, erroneous diagnosis may result. The following criteria should be
followed for selection of MAb for diagnostic purpose.
1. Antibody must have sufficient affinity for the antigen to permit efficient
combination with low antigen concentrations and to displace efficiently
host antibody already attached to the antigen.
2. The antibody should ideally be directed against an epitope of the antigen
not recognized by host antibodies and therefore not masked by them.
3. The antibody should be directed against a repeating / epitope which is
readily accessible such as I lipopolysaccharides of Gram-negative
bacteria or virus coat proteins.
4. Multivalency of the antibody may increase test sensitivity. For example,
IgM may be more effective than IgG.
In addition to these criteria, the antibody should be stable, should accept
conjugation procedures without loss of ' antigen binding capacity and should be of
the appropriate specificity for its purpose. Care should be taken that MAb is not
directed against a host cell or contaminant present in virus prepared from tissue
culture.
Antibody detection
The second use of MAbs in disease diagnosis is in the detection of antibody
most commonly used as an antiglobulin reagent conjugated with a detection
system (fluorochrome, enzyme or isotope) in a primary binding assay. The most
commonly used method is an indirect assay in which the antigen is passively
adsorbed to a plastic (polystyrene) surface by hydrophbbic interaction. The
sample-serum or other body fluid suitably diluted is applied to the immobilized
antigen. After an incubation period, the monoclonal anti-species antibody
conjugate is added. Each step is followed by a thorough washing cycle. These are
essential steps in which measures are usually taken to eliminate non-specifically
bound proteins to reduce background' activity in negative samples. In the final step
if an enzyme conjugate is used, then a chromogenic substrate is added followed by

46
spectroscopy, while radio-isotope or fluorochrome conjugate binding may be
assessed directly.
Primary binding assays are currently employed only to a small extent in
veterinary medicine. However, with suitable apparatus becoming more common,
primary binding assays and, in particular, enzyme immunoassay will replace
current serological tests (complement fixation test, serum neutralization test,
precipitation test and agglutination test). Standardized reagents are essential for
conduct of these assays for obtaining consistent results. In this respect, MAb has
an advantage that antibody of identical specificity can be prepared at will and the
enzyme conjugation procedure results in no discernible differences from batch to
batch.

Anti-idiotype antibody
Anti-idiotype antibody is an antibody that recognizes the antigen combining
sites of other antibodies. Anti-idiotype antibody (Ab2) has antibody activity to the
antigen-combining amino acid epitope sequence (of the folded molecule) of the
hypervariable region of the antibody (Abl) molecule. Anti-idiotype antibody can
be prepared by immunization of a heterologous species or a homologous species
'with affinity purified Abl. The resulting Ab2 should be able to compete with the
original antigen for the antigen binding sites of Abl. If a polyclonal Ab2 is made,
even in a homologous species, it generally has to be absorbed extensively to
remove anti-species or anti-allotype activity.
If Ab2 is monoclonal, it would have to be tested exhaustively with
immunoglobulins with or without Abl activity and with or without the Fc portion
to eliminate non-specific reactions that often occur between immunoglobulins.
The monoclonal Ab2 can be used in a primary binding assay as a capture agent for
Abl. If Ab2 was raised in a homologous species, Fab and Ab2 should be used for
capture, while an anti-Pc reagent conjugate should be used for detection. An
alternative approach is to attach Ab2 chemically to particles such as erythrocytes
or latex beads and passive agglutination should take place in the presence of Abl.
By either method, detection of antibody may be accomplished in the absence of an
antigen, a technique which may prove useful to serology that otherwise involves
antigens that may be very expensive, toxic or difficult to obtain. Anti-idiotype
antibodies have'been produced against Staphylococcus spp. and Streptococcus spp.
and Brucella abortus unpublished work).
Experimental uses of monoclonal antibodies
Cells of immune system
MAbs have been used extensively to characterize various cell populations
involved in immune responses of mice and man. The Lyt system of antigens have
been developed for functional differentiation of T lymphocytes. Lyb antigens have
been used for differentiation of B lymphocytes. Similar studies in domestic
animals are in infancy. Krawiec and Muscoplet (1984) developed MAbs specific

47
for canine T lymphocytes and others capable of distinguishing medullary and
cortical thymbcytes. Equine lymphocyte surface antigens have been studied..
MAbs against bovine lymphocytes have been produced. Three MAbs that
distinguished T and B lymphocytes of cattle in peripheral blood were produced
and used in a study of a bovine leukemia virus-infected herd to show that no
consistent differences in B cell number resulted from the virus infection.
catalogued about 100 MAbs for their reactivity patterns with bovine, ovine,
caprine, porcine, equine and human lymphocytes. Reactivity of the MAbs based
on immunofluorescence analysed by a fluorescent antibody cell sorter resulted in
patterns suggesting specificity with various T lymphocyte subsets, including
suppressor/cytotoxic cells, as well as T lymphocyte reagents and both T and B
cells, class I and class II MHC antigens, monocytes, granulocytes and
erythrocytes, and IgM (B cells) and light chains. Interestingly, interspecies cross
reaction was extensive with some reagents and limited with others, with
monospecificity being relatively rare to some antigens, such as MHC-class I
antigen. Lunney (1982) used monoclonal antibody to swine lymphocyte antigen
complex to identify and purify la antigens and to study the immune response to
various antigens including a synthetic peptide.
Pregnancy and Sex determination
MAbs have been used to detect pregnancy by the presence of progesterone
in milk. Using a direct double antibody solid phase enzyme immunoassay it was
found that 1-2 pg of progesterone per ml of milk could be detected which was an
excellent assay without extraction or centrifugation of the milk.
MAbs have been prepared to a variety of other hormones including
somatotropin, human chorionic gonadotropin and bovine and porcine insulin.
An interesting and at present, controversial application is in the area of
embryo sexing. Antibody to H-Y antigen, the male specific transplantation antigen
has been sought over the years to establish definitely the sex of an embryo, ^n
general, polyclonal antisera were unsatisfactory because of their inconsistencies in
detecting H-Y antigen and their low titres after absorption.
Molecular structure of antigens
Antigens are defined as a substance capable of reacting with antibodies;
however, the reactivity with antibody is confined to the antigenic determinants or
epitopes present on the antigen molecule. The epitope complements the antigen-
binding site of the variable region of the antibody molecule in its configuration.
Epitopes of antigens fall into two broad categories of 'sequential' and
'conformational' determinants. Sequential determinants are sequences of amino
acids of an unfolded or randomly folded peptide whereas in confonriational
epitopes the amino acids are brought into close proximity by the conformation of
the antigen; as a consequence, antibody to a true conformational determinant
rarely reacts with the unfolded molecule.
A considerable number of protein antigens and viruses have been studied in
detail with MAbs to determine their epitbpa structures. The molecular dissection

48
approach has been used with MAbs to select antigenic variants of several viruses
of veterinary importance. A single amino acid change - arginine at position 333 of
a glycoprotein in the virulent form of the rabies virus was fbjuid to be the only
change in non-pathogenic variants. Neutralizing antibodies to antigens such as
VP2 antigens is bluetongue virus and foot and mouth disease virus VP1 antigen
and its peptides have been described.
MAbs have been found to be useful in dissecting, mapping and identifying
the antigenic epitopes of virus proteins that elicit the production of neutralizing
antibodies. Two distinct serotypes of vesicular stomatitis virus (VSV) have been
identified and characterized: VSV-New Jersey (VSV-NJ) and VSV-Indiana (VSV-
IN). VSV-NJ possesses 4 major non-overlapping epitopes, whereas VSV-IN
appears to have 5 epitopes, which exhibit varying degrees of antigenic similarities
as detected by MAb. One MAb identified in these studies detected an epitope
common to both VSV-IN and VSV-NJ, but neutralized only VSV-IN. This
particular antibody has proven useful for distinguishing VSV from other vesicular
diseases causing viruses such as foot and mouth disease, vesicular exhanthema and
swine vesicular disease viruses.
MAbs have been extensively used in the study of tumour-causing viruses
and the survival and function of tumour virus proteins in host cells. MAbs have
also been developed against the transformation-specific glycoproteins coded for
by feline leukemia oncogene v-fms, avian retrovirus reverse transcriptase, bovine
leukemia virus transformed sheep cells and Marek's disease virus tumour
associated antigens.
Affinity chromatography
Because of their exquisite specificity, MAbs are suitable for use in
purification of antigens. This usually entails the immobilization of the MAb on an
insoluble matrix, such as agarose beads, by a covalent linking agent, for example
cyanogen bromide. The beads with MAb attached can be used either in a batch
procedure or in a column chromatography for antigen purification. Antigen
purification by affinity has been applied to substances ranging from la antigens of
the swine lymphocyte ' antigen complex, glycoproteins of herpes viruses, and to
antigens of Trichinella spiralis. The technique has great potential, especially for
purification of antigens from complex mixtures containing related or physico-
chemically similar substances, thus, isotypes of immunoglobulin for
standardizations and immunochemical studies would be the prime candidates.
MAbs are useful in enzyme purification, which cannot be purified by
biochemical methods. By coupling MAb to cyanogen bromide activated Sepharose
column, highly purified enzyme preparations with good yield can be obtained.
Immunochemical applications of MAbs
The studies on molecular structure of antigens have been greatly facilitated
by application of MAbs to immunoblotting analysis. Digested, degraded or native
antigen mixtures are separated by standard procedures such as one or two
dimensional polyacrylamide gel electrophoresis. Usually more than one replicate

49
is run, one gel is stained directly, while the other may be subjected to
electroblotting, thereby transferring the separated materials onto the nitrocellulose.
The nitrocellulose membrane is then blocked with a non-reactive protein and
incubated with the MAb of choice. Mouse MAb binding to antigen may be
detected by the antibody being conjugated with its own detection system, for
example, an isotope or enzyme; alternatively, an indirect system using an anti-
mouse immunoglobulin conjugated with a detection system as the second layer
may be employed. Several methods are available for either purpose; however the
immunoperoxidase technique has advantages in that it provides very low
background activity and highly sensitive and eliminates the use of isotopes.
Western blotting technique has been widely applied to the study of protein
antigens.
MAbs to animal immunoglobulins have been prepared. Antibodies to
bovine IgG, IgG1; IgG2 and IgA have been prepared. MAb to porcine IgM and to
canine IgG subtypes have also been produced.
There are three ways by which MAb specificity can be assayed; firstly, with
the isotypes passively adsorbed on to polystyrene; secondly, with a panel of 500
normal sera (also passively adsorbed to polystyrene) and thirdly, with an antigen-
antibody complex in which the antigen is immobilized and the antibody
presumably in its native state to allow the MAb to react with the Fc end of the
molecule exposed. The latter assay invariably yielded the highest 'titres' with the
MAbs; however, it is unfortunately the most expensive in terms of reagents.
Bifunctional antibodies have been produced by Milstein and Cuello (1984)
by fusing two hybridoma cell lines, one producing antibody to peroxidase and one
to somatostatin, resulting in a single cell that produces antibodies to peroxidase
and somatostatin, resulting in a single cell of dual specificity. They found that
some of the resulting hybrid hydridoma products were very useful for immuno-
histochemical staining when applied to sections containing somatostatin in the
presence of peroxidase.
MAbs have been useful in several other immunochemical areas. The study
of the complement cascade has been enhanced by the use of MAb of various
isotypes in a haemolytic system. It was shown that mixture of IgG2a IgG2b and IgG3
could effectively cause lysis, whereas IgD could not. IgG preparations resulted in
weak haemolysis and IgM was highly haemolytic.
Monoclonals as in vivo reagents for animals
The current use of monoclonals in domestic animals can be categorized as
follows:
1. Passive antibody administered prophylactieally or therapeutically for
infectious diseases;
2. Passive antibody to target on particular cell markers; either alone or
coupled to cytotoxic agents;
3. Passive antibody to enhance the clearance of toxic compounds;

50
4. Passive antibody to modulate the cellular or messenger
components of the in vivo immune responses;
5. Anti-idiotype antibody administered as an immunogen.
The oral application of a MAb directed to K99 pilus antigen of
enterotoxigenic Escherichia coli (ETEC) prevented severe fatal enteric disease in
colostrum-fed and colostrum-deprived calves experimentally challenged with
ETEC. The intravenous administration in sheep of a MAb to blue tongue virus that
neutralized the challenge virus, prevented viraemia, the development of
precipitating antibodies and clinical disease. The authors speculated that vaccines
containing a single antigenic determinant might generate a similar neutralizing
antibody and may both prevent blue tongue disease and perhaps interrupt virus
transmission.
In rabies, antigenic variants, previously undetected with conventional
polyclonal antisera, have been detected with MAb with specificity and either the
nucleocapsid or glycoproteins of several fixed and street strains of the virus. These
findings have been quite revealing in the sense, they have proved an explanation
as to why current vaccines are not universally protective. They have also shown
which additional variants must be included in the development of an effective
vaccine.
Anti-idiotype (Ab2) has been used as an antigen to prepare anti-anti-
idiotype (Ab3) which has the same specificity as Abl. In other words, Ab2 may be
used as a vaccine to induce an antibody response to the original antigen. Thus,
Ab2 was used to induce a response to transplantation antigens in mice and to
produce protective immunity in mice against trypanosomes. Ab2 has been made to
rabies glycoprotein G and some of the Ab2s, when used to immunize mice,
resulted in a specific neutralizing antibody response. These data clearly indicate
that Ab2 can mimic an antigen epitope and cause the production of an immune
response to it.
MAbs can be used as guided missiles in tumour therapy. The antibodies
when coupled to a toxic molecule and administered, can selectively kill the
targeted tumour cell on virus infected cell. This makes such therapy economically
feasible in the animal world. Doctors hope to be able to use MAbs for purposes
other than diagnostic tests, in particular, to attach to them toxic drugs that will
bind cancer cells. Injected into a cancer patient, the combined molecule would
circulate around the body in the blood until it finds the tumour and then deliver the
drug to its target. This treatment could greatly reduce the very traumatic side
effects of the present cancer therapy. MAbs directed against various inducer and
helper T cells might be desirable in controlling the autoimmune disease.

51
Biotechnology in Animal production
Manipulation of Growth
Growth means increase of cell number, cell size and/or deposition of
substances within cells. Individual growth of the neural system, bones, muscles
and fat. Animla production are anabolic processes requiring nutrients above
maintenance level, which may be provided by feed intake and by body resources
such as lipid stores, glycogen and unstable proteins.
Insulin
Insulin is an anabolic hormone and it enhances glucose uptake in muscle
and adipose tissues, fat synthesis either by provision of glucose and/or acetate
utilization ruminants and protein synthesis by facilitating uptake of amino acids
and inhibiting proteolysis in. muscle Insulin . also regulates somatotropin (STHT
receptors in liver, hence it may act presumably for release of somatomedins.
Insulin exerts a short feedback with the blood sugar level. Feed intake provokes
insulin release, restricted feeding — the simplest measure to avoid fattening-
depresses it, while starvation produces diabetes-like symptoms. On the contrary,
glucagon induces hyperglycaemia by glycogenolysis in the liver and
gluconeogenesis. It is postprandially released concomitantly with insulin,
obviously to prevent the insulin induced hypoglycaemia.
Growth hormone releasing hormone (GHRH), Somatotropin (STH), Growth
hormone (GH) and Somatomedins
Somatomedins are hormones produced in the liver when stimulated by
STH. High levels of somatomedin have been found in faster growing breeds of
pigs, sheep cattle and chickens: in bulls a parallel increase in STH concentration
was shown during pubertal growth between 8-11 months of age. Contrary to
insulin, STH concentration are elevated during restricted feeding resulting in
lipolysis and glucose mobilization. The STH supplementation will have a
beneficial effect to compensate for shortage of feed.
GHRH administration stimulates insulin secretion in vitro and releases STH
in cattle. Frequent administration of STH preparations have produced anabolic
responses, accompanied by reduced adipose tissue. The human pancreatic hp
GHRH has been demonstrated to release STH in cattle. Depression of STH
concentration has been observed following an excess of administration of
somatostat released from the hypothalamus recombinant porcine STH was given at
a but, there was a doubling increase in M. longissimus dorsi. The fat
content was decreased lineraly to a minimum of 25% with increasing doses of
STH.
Thyroid hormones
Thyroid hormones exert a direct effect on the cell nucleus; they are
essential growth hormones for several tissues including the CNS and bone.s
Thryoid function - supporting feed additives are iodinated casein and thryoprotein.
Some positive results hafve been reported in veal calves, pigs, chickens and lambs.
In mature cattle, the depression of the thryroid function by thyrostatics (methyul -

52
or propyl - thiouracil) resulted in remarkable improvement of body weight gain
four week period.
Reproductive steriod (androgens, oestrogens, gastagens)
Steriod hormones exert their biological effects atg the target tissues by
binding to specific cytosolic hormones receptos. The hormone - receptor complex
then interacts with DNA in the cell nucleus initiating increased RNA synthesis
followed by an increased protein synthesis and specific hormonal effects.
Castration changes the growth and fattening performance entirely. Weight gain
and feed conversion ratios are reduced. Growth rates in bulls, especially around ft-
in months of age were positively correlated to the response in, testosterone to
choriogonadotropin injection/ Exogenously administered reproductive hormones
act anabolically. The mode of action of androgens directly on muscle cells is due
to their having, specific receptors within the cytoplasm. A similar mode of action
is suggested fop bestrogens. Indirect anabolic effects are probably mediated by
stimulation of STH.
Use of anabolics have brought out improvement in the range of 10-15%
true anabolic weight gain. A positive effect on feed conversion and in general no
adverse effect on meat quality was shown.
The anabolic agents have two possible mode of action:
a) they enter the muscle cells and have a direct action on protein synthesis and/or
degradation;
b) they enter other endocrine organs such as hypothalamus, anteriof
pituitary, gonads, pancrease and thyroir pituitary, gonads, pancrease and thyriods
and alter the synthesis, metabolism-or secretion of other hormones. These
hormones may then produce other anabolic effect in muscle and affect
intermediary metabolism in other parts of the body
The administration of anabolic agents have been found to bring about a
change in the concentration of circulating endogenous hormones. For example
oestradoil injection increases, the concentration of circulating plasma growth
hormone.
Probiotics as growth promoters
The term 'probiotic' originated from two Greek words meaning 'for life' and
contrasted with the term antibiotic means 'againg life'. It is defined as a live
microbial feed supplement which beneficially affects the host animals by
improving its intestinal microbial balance. The live cultures of natural and
beneficial bacilli can be introduced into animal gut hi massive numbers to
supplement those Already present. The increasing level of beneficial organisms in
the gut crowd out the existing pathogens proliferate, ensure that there is no room
for others to be established.
The probiotic preparations available in the market are generally composed
of organisms of Lactobaciui and/or Streptococci? species, few may contain
Bifidobacterium and yeast cultures. These products are viable, freeze dried or

53
microeneapsulated which proliferate and establish in large numbers in the
gastrointestinal tract particularly in the small intestine.
Probiotic preparations benefit the host by:
a) having direct antagonistic effect against -specific group of undesirable or
harmful organisms through production of antibacterial compounds, eliminating or
minimizing their competition for nutrients;
b) altering the pattern of microbial metabolism in gastro-intestinal tract;
c) stimulation of immunity and
d) neutralization of enterotoxin formed by pathogenic organisms. Probiotics help
to favourably influence, the growth rate, feed efficiency and prevent intestinal
infections. The use of bacterial cultures is increasingly becoming popular in
poultry and pig feeds, while the use of yeast cultures is gaining importance in
dairy cattle rations.
The normal bacterial flora in the alimentary tract of pigs and birds are
species of Lactobacitli, Streptococci, E. coli and Clostridium welchii, in the
descending order of concentration. The Lactobacilli group of organisms are the
most common ones which include L. acidophilus, L. bifidus, L. bulgaric'us, L.
plantarum, L. casei and Streptococcus cremoris and S. lactis. The yeast cultures
used in dairy cattle ration is Saccharomyces cerevisiae.
Ideal characteristics of a probiotic
1. It should be non-pathogenic to animals and man.
2. It should be gram-positive since they are more resistance to destruction
by
digestive enzymes.
3. It should have high tolerance for bile and low pH.
4. It should be capable of producing lactic acid.
5. It should easily proliferate under in vivo 'and in vitro conditions.
6. It should have high survival rate after passing through manufacturing
processes.
7. It should possess high stability at room .temperature and long life when mixed

with feed.
Mode of action of probiotics
The normal gut microflora has a supportive role in disease protection and
digestion of food. 'However, stressful conditions such as introduction of artificial
rearing, castration, weaning, parturition, change of diet and many more increase
gut pH which may favour growth of pathogenic organisms. The probiotics bring
about its beneficial effects through one of the following mechanisms.
a. Neutralization of toxin
b. Suppression of i) viable numbers of specific bacteria
ii) antibacterial agents and
iii) competition for adhesion sites
c. Alteration of microbial metabolism

54
d. Stimulation of immunity
Uses of probiotics
Use of probiotics in cattle ration have improved weight gain and better feed
efficiency. The most striking observation made so far is with 'yea-secc' a yeast
culture containing Saccharomyces cerevisiae. In different studies, increase in milk
yield from 3.5 to 8.4% have been observed. In addition, the milk production was
more economical with a 13% reduction in feed cost. The yeast culture was found
to influence rumen metabolism by altering the microbial fermentation rates, the
protein turn over, of the microbes, the hydrogen ion exchange and the metabolism
of easily soluble carbohydrates. Thus, there is an improvement in the digestibility
of structural carbohydrates thereby increasing the energy supply to the microbes,
inhibition of methane build up, stimulating the formation of propionic acid,
increased flow of proteins of microbial origin resulting in better feed conversion
for milk yield.
Probiotics are extensively used in poultry industry. In a broiler trial, birds
receiving probiotics supplementation (100 mg/kg feed) gained 1268 g as against
1204 g in control group at the end of 8 weeks; There was also a marginal
improvement in feed efficiency (2.30 Vs 2.26). In a short term feeding trial with
layers, there was a 5.6% increase in egg production; a reduction in the incidence of
thin shelled eggs (6.7% Vs 19.7%); a significant reduction in serum cholesterol
(114.3 gm/dl Vs 176.5 mg/dl) and a significant reduction in yolk cholesterol
(11.28 mg Vs. 14.69 mg) (Mohan, 1991).
Probiotics have proved to have potential to improve broiler growth, milk
yield in cattle and help to reduce cholesterol in eggs.
Manipulation of Lactation
Lactation may be improved by manipulation of mammogenesis,
lactogenesis and galactopoiesis. Each stage is regulated by a multifactorial
hormone complex. Hormone treatment to induce mammogenesis artificially (i.e.,
without pregnancy) in dairy ruminants employ steroid hormones (oestrogens,
gestagens and glucocorticoids). Somatotropin has been shown to enhance
mammary development. Hormpnal manipulations of lactogenesis and especially
galactopoiesis are focussed on somatotropic hormone (STH).
STH regulates the partitioning of nutrients within the organisms
preferentially to the mammary gland and mobilizes them by its lipolytic action
precursors for milk synthesis.
Mammogenesis
Mammogenesis is defined as a growth and differentiation of the mammary
gland to the stage prior to active secretion. Generally in cows, little or no lobule-
alveolar development occurs before first pregnancy but appears to be a continuous
exponential process through gestation and terminates at the onset of lactation; in
cows a gradual involution is observed that becomes more pronounced during the
dry period. Mammary development is largely stimulated by hormones. Steroid and
thyroid hormones and proteohormones of the prolactin and growth hormone

55
family play a crucial role. Oestrogens and growth are responsible for ductial
development, whereas progesterone and prolactin regulate lobule-
alveolar .proliferation. Maximal growth, however, was achieved by additional
application of glucocorticoids. Oestrogen progesterone treatment stimulated
alveolar development.
Prolactin is important for the formation of alveoli in cows goats and ewes,
in goats and sheep, foetal numbers, concentration of placental lactogen during late
pregnancy and milk yield have been correlated. STH treatment of heifers resulted
in increased mammary parenchyma.
Lactogenesis and galactopoiesis
Induction and maintenance of lactation are dependent on reproductive
steroids, prolactin (PRL), thyroid hormones -STP, insulin, corticoids and possibly
placental lactogen. In ruminants, PRL seems to be essential for lactogenesis, but
once lactation is established, it is not alimiting factor for milk production. thyroid
hormones play an important role in lactational performance of cattle. T3 and T4
(T3 -tri-iodothyronine, T4-thyroxin) play a central role in metabolic regulations.
increase in milk and milk fat yields after treating cows with T4 have been
reported. STH has been found in higher concentration in high yielding cows. Milk
yield increased from 8-41% when treated with STH in cattle. Increase in milk
production due to SIH application were also observed in
Glucocorticoids were used to improve lactation after artificially stimulated
mammogenesis. Milk yields of more than 10 kg/day have been obtained by use of
glucorticoids.
Manipulation of Woo! Growth in Sheep
Wool production in sheep is essentially a protein output. Wool growth is
dependent on the availability of S-amino acid and total amino acids available in
the blood supply to the wool follicle. About half the cysteine derived form
intestinal absorption in sheep is converted into wool. there are three major
possibilities for increasing the availability of S-amino acids and hence production
of wool. these include.
a) manipulating the microbes of the microbial fermentation digestive system of
sheep to improve microbial growth efficiency,
b) manipulating the microbes in the rumen to increase the utilization of dietary
protein 1 that escapes rumen fermentation and
c) fusing recombinant DNA processes to create animals that are able to synthesize

S-amino acids the primary limiting amino acids for wool growth.
Manipulation of the rumen microbial digestive system
To increase wool production, manipulation of the rumen microbial
ecosystem must be primarily aimed at increasing the availability of essential
amino acids to the. animal. This can be achieved by-
a. increasing the efficiency of microbial growth in the rumen;

56
b. increasing the amount of intestinally-digestible dietary protein that escapes
rumen fermentation;
c. increasing the digestibility of fibrous feed in the rumen which increases amino
acids (and also volatile fatty acid production) and possibly encourages the growth
of more efficient organisms;
d. producing, by recombinant means, bacteria that synthesizes proteins with high
S-amino acids content or that secrete S-amino acids and
e. decreasing the amino acid utilization for other metabolic functions including
deamination to supply glycogenic precursors and substrates for oxidative
metabolism.
Manipulating the rumen ecosystem to achieve maximum microbial and
dietary protein outflow from the rumen is the major strategy when attempting to
selectively increase wool growth in the mature animal. A continuous supply of
fermentable carbohydrates, animonia, peptides, amino acids and other nutrients is
needed to promote efficient utilization of ATP for microbial protein yield.
Increased microbial synthesis of proteins was observed in the rumen of sheep on
diets with casein as the N-source compared to urea based diets.
`Wool growth is increased by extra amino acids absorbed from the small intestine.
Manipulations that increases the rate of fermentative digestion will increase
microbial protein relative to energy. These include:
1. treatment of the forage with ammonia which increases digestibility;
2. addition of small amounts of readily digestible fibre (which stimulates
digestion of the basal dietary fibres);
3. maintaining a protozoa-free rumen and
4. optimizing nutrient availability by balancing microbial nutrients in the
rumen.
The ruminant DNA material can be inserted into sheep embryos at the 4-8
cell stage by micromanipulation. Scientists are attempting to introduce bacterial
DNA fragments into the ova of sheep coding for the synthesis of cysteine, which
is the primary limiting amino acid for wool growth. The biosynthetic pathway of
cysteine synthesis is:
serine + acetyl CoA -------------------------- 0-acetylserine + CoA
(serine transacetylase)
0-acetylserine + HgO -------------------------- cysteine + acetate
(O-acetylserine sulphydtylase)
H2S is generated from sulphur compounds in the rumen and the
S2~ is not used in microbial growth but is absorbed and converted to sulphate in
the rumen epithelium or eructed. Eructed gases are inhaled into the lungs before
the gases are finally exhaled. Thus, the lungs and the rumen epithelium are only
tissues receiving quantities of H2S and therefore the only ones likely to
synthesize significant quantities of cysteine in transgenic animal with
the necessary enzymes in its genome. When protein intake is high, the availability
of non-essential amino acid serine may not be limiting; however, serine is a

57
glucogenic amino acid and if a drain of serine into cysteine creates a serine
deficiency, then the animal must be capable of increasing the synthesis of this
amino acid. The carbon fragments have to arise from the glucogenic precursors,
mainly propionate or other glucogenic amino acids.
If sheep are given diets deficient in bypass protein and/or glucogenic
precursors then, this is likely to be a detrimental manipulation. The process could
result in serine becoming rare and limiting.

58
Manipulations of Rumen Microorganisms
Introduction
Genetic engineering of microorganisms to increase rumen
productivity in developing countries is investagated m recent times.
Fibrous feeds and residues of low digestibility comprise the major proportion of
feed available to most ruminants under small conditions developing countries.
In this context the primary objective of the manipulation of rumen organisms
must be to increase the rate and extent of digestibility of these fibrous feeds as
this is a primary limiting factor Because of feeding of ruminants with the feed of
low digestible fibrous feeds, productivity of these livestocks have been extremely
low typified by low growth rates, late maturity maturity, low reproduction rates,
low milk yield and stunted mature body size. The low productivity can be
overcome to some extent by "supplementation to ensure that:
a. the nutrients for the growth of ruminal organisms are balanced, ensuring an
efficient fermentative digestion
b. small quantities of bypass nutrients are supplied to balance the nutrients that
arise from fermentative digestion. This ensures an efficient utilization of nutrients
by the animal.
A 5-10 units increase in digestibility of fibrous forage can lead to 100%
increase in productivity: Supplementation strategies, combined with technology
for increasing forage digestibility could potentially increase the productivity of
each ruminants by approximately 5-fold.
The Target Organisms
Fermentative digestion of forages occurs in ruminants in the well developed
forestomach (the retieulo-rumen). This organ is maintained in a relatively stable
continuous anaerobic culture of mixed population of protozoa, bacteria, fungi and
bacteriophage. The rumen fluid pH is maintained at close to neutrality by
continuous saliva which balances the production of acidic end products of
fermentation. The short chain fatty acids are either absorbed directly or pass out of
the rumen in the digesta to the intestines where they are absorbed.
The anaerobic bacteria are the major organisms in the rumen which help in
fibre digestion. The information on their nutrition and biology is lacking. The
widely distributed anaerobic fungi which grow on fibrous feed particles in the
rumen have only recently been discovered. The fungi are mostly fibrolytic with
the major substrates being cellulose and hemicellulose. Their major role may be
weakening of feed particle structure as most species have radii which penetrates
plant tissues and therefore tend to digest plant materials from the inside, whereas
bacteria attach to and attack damaged parts of plants and digestion occurs from the
outside towards the centre. As the rate of fibre breakdown in the rumen is often a
primary limitation to 'intake of low digestibility feeds, the possibility of
developing fungi with enhanced ability to grow or secrete cellulase enzyme could
be highly important. A number of species of ciliate protozoa occur in the rumen.
They are targeted for elimination from the rumen. Improved digestibility and

59
improved efficiency of microbial growth can be achieved in the rumen of sheep on
straw based diets from which protozoa can be eliminated. Bacteriophage can be
found in large numbers in the rumen of sheep and cattle. The phage outnumbers
bacteria as much as 10:1.
Application of Recombinant DMA to Rumen Organisms
The practicability of inserting novel genes into bacteria has been proven by
the widespread use of biochemical products derived from genetically engineered
microorganisms. The type of alteration that is most desirable is an increased
ability by rumen organisms to digest coarse plant fibre, by the increased
production of cellulolytic and xylanolytic enzymes. While some species of
Tbacteria (eg. Ruminococcus albus). possess both types of enzymes, other
dominant rumen species produce one or the other (eg many strains of B.
succinogenes are cellulolytic and are unable to digest xylan, while B. ruminicola
has only xylanolytic activity. By endowing the dominant populations of bacteria
with digestive capability for multiple cell wall constituents, it may be possible to
enhance fibre digestion by a sort of mixed populations that occur naturally.
Using familiar bacterial species, the transfer of functional genes is
now a routine procedure. Many bacterial genes have inducible promoters and such
switches may be useful for the timed induction of nutrient synthesis
antibiotic production antihelminthic synthesis, etc. The initial step in this
process is to identify inducible genes in the ruminal anaerobes and isolate
promoter sequences responsible for control functions.
The genetics of ruminal anaerobes and their involvement with epigenetic
factors such as plasmids and bacteriophage are at a very early stage of
investigation. A highly significant increase in cellulolytic activity Of
Ruminococcus albus by mutation and selectio Ruminococcus albus by mutation
and selection. has been reported. The introduction of completely novel enzymes
into dominant species of rumen bacteria and the addition of highly manipulable
control mechanisms to specific genes are two clear examples.
Gene libraries have been constructed and used to isolate" the genes for
cellulase enzymes which can be expressed as active molecules in the E. coli host
cells. The occurrence of plasmids and bacteriophage in rumen bacteria has been
demonstrated. It is clear, therefore that essential requirements for producing
genetically altered rumen bacteria already exist.
The genetic manipulation of rumen microorganisms by altering the
inherited characteristic is the most powerful potential tool for enhancing the rate
and extent of digestion of norou's feeds within the rumen. Its success depends on
the identification of the specific genetic information, the development of
appropriate genetic techniques and creation of new organisms with high
probability of survivial in the harsh environment of the rumen of animals fed on
highly fibrous feeds.

60
Non-genetic Manipulation of Rumen Microbes
Rumen fermentation has to be optimized to improve feed utilization in
ruminants. This means to maximize the digestion of lignocellulosic compounds
and the use of nutrients (i.e., proteins, starch and lipids) to escape rumen
fermentation. There are three steps to optimize fermentation.
a. Supplying the nutritional requirements of rumen microflora to ensure
maximum microbial activity and microbial growth yield.
b. Modifying microbial population , (bacteria, protozoa and fungi) to select the
species that are most apt to supply biomass and metabolites that the host animal
requires.
c. Altering the physiology of the digestive tract (i.e., salivary secretion, rumen
motility rates of liquid and particle phase outflow
The first step requires that the known quantitative and qualitative needs of
the ruminal microbial populations be met. This can be done by controlling. the diet
(nature, amounts, physiclil form of feed stuffs, balancing nutrients). The other two
ways require means and methods that will bring lasting and significant
modifications. These two aspects corresponds to the manipulation of the rumen
microbes.
Addition of antibiotics
The antibiotics used as feed additives in the ruminants are frequently presented as
a model to study the modes of action in manipulation of rumen function. Among
the antibiotic ionophors (monensin and lasalocid) have been have been studied
more frequently. Their mode of action on cellular ion transport induces important
changes in rumen microbes and consequently in the modification of rumen
fermentation.
Ionophors are potent against Gram-positive bacteria and some Gram-
positive bacteria which have a Gram-positive like cell wall structure such as
Ruminococcus albus, R. flavefqciejis and Butyrivibrio fibrisolvens. By contrast,
Bacteroids ruminicola and R. succinogenes become resistant to this antibiotic after
2-4 days growth in the presence of monensin. Selenomonas ruminantium is not
affected by adding this antibiotic. That is why the enzyme activity in the rumen is
modified by an increase in the succinate dehydrogenase activity, which leads
to higher succinate production and to the conversion of succinate into propionate.
Similarly, acetate kinase activity is greately reduced (Wallace et al., 1981),
which expresses a drop in the number of acetate producing microorganisms.
These antibiotics also have a negative effect on the lactate producers,
which are, for the most part, ,1 Gram-positive bacteria, without inhibiting
ruminal lactate Jpitilizers which produce propionic acid. These are the main
reasons for modifications observed on improvements in molar
proportions of propionate and decreases in acetate or butyrate, when
animals are fed monensin.
In vitro calcimycin induces a decrease in propionic acid to the advantage of
acetic and especially butyric acids and favours methane production. In vitro

61
aberixine has effects similar to monensin, from which it is derived, but contrary to
this molecule, it has no negative, effects as regards microbial synthesis.
Elimination of rumen ciliate protozoa (Defaunation)
Protozoa accounts for upto50% of the biomas of rumen microorganisms
and their elimination causes modifications in the bacterial population and in
organic matter digestion in the rumen. Defaunation increases the total number of
bacteria in the rumen, because ciliates prey on bacteria and compete with them for
nutrient utilization. The composition of microflora is modified towards more small
Gram-negative rods and selenomonas-like -bacteria in defaunated animals.
Protozoa host methanofrpnir bacteria that are closely associated with their
surface. These bacteria capture the hydrogen released by protozoa to convert it
into methane-by reaction with CO2. The number of fungi in the rumen increases
after defaunation. The strongest effects of defaunation were observed on cell-wall
carbohydrate digestion. The microbial nitrogen yield is largely enhanced by
defaunation and results in the net microbial growth and a depressive effect on the
organic matter fermented in the rumen of treated animal.
The defaunation can be considered to be an advantaged only for animals
fed low protein diets, in which bacterial nitrogen constitutes the main supply of
intestinal proteins. Defaunation can be used for saving dietary proteins form being
degraded in the ruman in low-protein diets, but it lowes cell-wall degradation in
roughages - poor diets. under conditions of defaunatin, new strains of cellolytic
bacteria or ganetically modified rumen bacteria could be developed to improve
cell-wall carbohydrate digestion.
Addition of fats
when large amounts (5-10% of dry matter) of non-protected
triglycerides or freee polynsaturated fatty acids from oils are added to the diets, a
large decreae in ciliated protozoa in the rumen is induced (tamminga et a., 1983),
which probably leads to modififcations of poplations. Adding these substances
also provokes a drop in orgnaic matter digestion, especially in cell wall
carbohydrates, ammonia and methane production and in the ammonia and
methane production and on the acetate/propionate ratio. The microbial nitrogen
yield in the rumen is increased by adding oil rich in polyunsaturated fatty acids.
Addition of protein degradation protectors
Chalupa (1977) showed that diphenyloidonium chemicals inhibit rumen
acids degradauion, which results in large amounts of amino acids in the rumen.
Amino acid ddegradation may be prevented by inhibiting their intracellular
transport in bacteria. These molecules also have an effect on rimen fermentiatn
products. They bring on a decrease in methanogenesis, but without modifying the
H2 concentration in the meidum.

Addition of buffer substances


The incorporation of buffer substances into ruminant feeds (bicarbonate alone
or mired with magnesium or calcium bicarbonate) prevents a drop in pH with

62
diets rich in easily fermentable sugars and limits the risk of acidosis Massive
intake of salts cause rise in osmotic pressure in the rumen and disturb development
of microorganisms.
Addition of bacteria or rumen content
Adding rumen content (in fresh or in freeze-dried form) can be useful if
rumen metabolism has been impaired (e.g acidosis, bloating, per os antibiotic
treatment). For example when animals are defaunated, it is advisable to introduce
freeze-dried and thawed rumen contents, to provide all species of bacteria, which
can develop in this new condition.
Addition of branched chain volatile fatty acids
In vitro addition of branched chain volatile fatty acids (isobutyric and
isovaleric acid) as 1% of the feed stuff increased both me apparent dry matter
digestibility and microbial growth in the rumen. The positive effects on microbial
nitrogen synthesis are greater when protein sources have a low ruminal
degradability. In such conditions, branched chain volatile fatty acids supply
br,anched-chain carbon skeletons which are lacking in baceria because amino acid.
(leucine and valine) cow have shown that the maximal dose was around 89g per
animal per day, which amounts to 0.45% of the diet. Investigations on these type
of 'probiotic' compounds might be worthwhile, because as they are usually
produced in the rumen and metabolized by bacteria.
Feed distribution method
When the nature of the ration is known, the efficiency of its utilization can
be improved by modifying the conditions of its distribution so as to obtain regular
fermentations in the rumen. Several frequent feedings lessened the drop in milk fat
content generally observed in dairy cow with restricted diets. With mixed diets
containing upto 60% concentration the flow rate.of microbial nitrogen in the
intestine doubled when the feeding schedule was changed from 1 to 2 meals per
day to continuous distribution.

63
Manipulation of Reproduction
Introduction
Artificial insemination has contributed considerably to the improvement
of cattle industry. In recent years, tremendous progress has been made in the
manipulation of reproduction in cattle by using techniques like control of oestrus
cycle. The embryo transfer techniques seem to be the technique of choice to
increase the number of off springs from excellent parent stock. The non-surgical
embryo recovery and transfer into cattle has allowed transfer of this technology
from the laboratory to the farm so that increased number of off springs from a
superior cow is possible. The embryo transfer technology in cattle includes
techniques like super ovulation and breeding of donors, embryos collection and
evaluation, selection of recipients and transfer of embryos.
Embryo Transfer Technology
The first successful transfer of fertilized rabbit eggs was reported in 1931
by Waltefr-Heape at Cambridge (UK). In farm animals embryo transfer was
successfully done for the first time in 1934 in sheep and goats. irtie first offspring
after transfer of embryo in cattle and swine were reported in 1951.
Embryo transfer includes a sequence of various steps which are a) selection
of donors, • b) induction of superovulation, c) embryos collection, d) evaluation of
embryos, e) selection of recipients and f) transfer of embryos. Until now, embryo
transfer techniques have been extensively used in cattle and more sporadically in
sheep, goats and pigs.
Selection of donor
Regularly cycling cows or heifers can successfully respond to
superovulatory treatment and be used for embryo transfer. The following selection
criteria for donors will ensure a good probability of producing embryos of high
quality.
1. Between the ages of 3 and 10 years.
2. Free of genetic diseases and conformational abnormalities.
3. Exhibit regular oestrus cycle.
4. Show superior production traits of economic importance.
5. Free from infectious diseases and
6. Previous sound reproductive performances including no more than two
inseminations per conception.
Induction of superovulation
Superovulation means the induction of multiple ovulation by application of
exogenous hormones (PMSG-pregnant mare serum gonadotrophin; FSH - follicle
stutmlating hormone; HMG - human menopausal gonadotrophin) in the early
follicular or in the luteal phase of thte oestrus cycle, in order to collect a large
number of fertilized eggs. Most frequently PMSG or FSH is used. Only a single
injection of PMSG (2000 to 3000 IU) is sufficient as the substance has a long half
life time. In contrast, multiple injections of FSH (35-50 nag) are required to induce

64
superovulatory responses (twice daily injections over four days). HMG (1000-
1500 IU) also requires multiple injections; however, the drug is more expensive
and only rarely used.
Forty eight to sixty hrs after beginning the gonadotrophin treatment,
prostaglandins (PGF2oc) are administered to induce oestrus. Inseminations are
performed at oestrus and the embryos are recovered 6-8 days after insemination.
On an average, only one third of the superovulated bovine donors show a good
ovarian response.
Embryo collection
In bovines, embryos are recovered by non-surgical methods. Special
catheters are introduced via the cervix into the uterine horn and embryos are
flushed with 250 to 300 ml of flushing medium (phosphate buffered solution with
1-2% fetal calf serum). In the cow, the embryos usually enter the uterus on day
four after oestrus. Non-surgical techniques avoid the potential damage of the
reproductive tract by adhesions, are repeatable and do not require elaborate
facilities. Embryos are collected by non-surgical methods 6-8 days after the onset
of oestrus. Prior to day 5, the embryos jnay be in the oviduct and after day eight
they may be hatched (escape from the zona pellucida), difficult to visualize and
fragile. At day 7, bovine embryos are about 150-190 fi in diameter, are still within
the zona pellucida and at late morulla or blastocyst stage of development.
In case of other farm animals like sheep, pigs and goats, embryo recovery is
performed by surgical methods. The abdomen of the donors is opened by midline
cut and embryos are recovered by flushing oviduct and/or uterine horns. Embryos
are collected 4-5 days after insemination or mating.
Evaluation of embryos
After recovering ova and embryos from the flushing medium, their further
developmental capacity has to be evaluated. Morphological criteria in embryo
evaluation are shape, colour, number and compactness of cells, size of the
perivitelline space, number and size of vesicles and the status of the zona
pellucida. Embryos are classified into 4 groups:
a. Excellent embryos - embryos in the appropriate developmental stage with a
perfect morphology.
b. Good embryos - embryos in the appropriate stage of development with slight
morphological deviations.
c. Degenerated and/or retarded embryos.
d. Unfertilized ova.
Selection of recipients
The normal physiological and health conditions, the reproductive status,
lack of any reproductive disorders, compatibility of the donor with respect to the
size of the foetus and oestrus synchronization are of considerable importance. In
bovine, it has been proved that heifers and young cows are best suited as
recipients. The oestrus of donors and recipients should be synchronized within 24
hrs.

65
Transfer of embryos
Special catheters are available to perform non-surgical transfer in cattle. It
is important to transfer the embryos into the tip of the uterine horn without
damaging the endometrium. In cattle, after superovulatory treatment 8-15 follicles
are detected at oestrus and about 6-11 ovulations on the day of embryo recovery.
The average number of embryos recovered is 5-10 representing 60-70% of the
number of corpora lutea. Pregnancy rate after transfer of freshly .'collected
embryos is 55-65%, and of frozen/thawed embryos is 50-60%. An average
of 5-7 embryos per successful flushing can be recovered in dairy cattle. About
60-70% of these embryos are considered to be capable of further development.
Statistically 2.5 to 3 calves per successful flushing can be produced.
The oestrus cycle/ of the donor and recipient should be closely
synchronized if transferred embryos are to survive. Pregnancy rates are generally
reduced unless the embryo is placed in the lumen of the uterine horn on the same
side of the corpus luteum.
Cryopreservation of embryos
A positive breakthrough in embryo transfer of a larger scale was due to the
development of Cryopreservation method. Modifications and adaptations of
different procedures have made it more feasible to freeze and store bovine
embryos at - 196°C for varying time intervals. An optimal method for bovine
embryos includes a one-step addition of 1.4M glycerol as a cryoprotectant, 20 min
equilibration period, 0.25 ml straws as embryo containers, slow cooling
(0.3°C to 0.1°C/min) down to -35°C and subsequent plunging into liquid
nitrogen (- 196°C). Embryos are thawed by placing the straw directly into warm
water. Glycerol is removed using sucrose in two steps. Using this
procedure, the percentage of morphologically intact embryos was 95.3% and the
pregnancy rate after non-surgicla transfer was 51%.
Embryo Splitting
Mammalian eggs are more tough and can sustain extensive damage and still
develop normally. Early cleavage-stage embryos are favoured for manipulation to
produce identicals. Embryos from the late morullae upto late blastocyst stage can
be collected easily by non-surgical procedures and after splitting into two halves,
they can be returned directly to recipients non-surgically.
Embryos to be bisected are collected non-surgically 6-7 days after oestrus.
At this time embryos are compact morullae or blastocysts. After collection,
the embryos are washed in modified PBS supplemented with 20% heat-
inactivated FCS and classified morphologically. Only embryos of good quality
should be selected for splitting. The embryo of good quality is fixed on the
holding pipette and the zoiia pellucida is cut by a vertical motion of the micro
knife. The crack in the zona pellucida is opened by moving the glass needle and
the hook, the embryo is either split S inside the zona or after moving it out of the
zona. Bisection I can be done by cutting with the micro knife or by separating the

66
two halves with the glass needle. One half remains in and will be put back into the
original zona. The second half is transferred to a surrogate zona (made by
preparing a zona of a degenerated embryo). Bisection of bovine embryos
results in two genetically identical demi-embryos. The demi-embryos can
be cultured for several hrs or transferred immediately after splitting. The transfer
can be done in one of the following ways:
a) each half in one recipient into the ipsilateral horn (unilateral transfer);
b) both halves in one recipient into both horns (bilateral transfer) and
c) both halves in one recipient into the ipsilateral horn.
Alternatives two and three are similar or identical to naturally arising twin
pregnancies. Alternative one will be used in commercial programmes, because it
will result in the highest number of calves per collected embryos.
Cryopreservation of micro surgically split bovine embryos allows the
production of identical twins of different ages. Survival rates are significantly
lower, only about 25% of frozen/thawed embryos will survive.
Embryo Sexing
The sex of an individual is determined at fertilization and depends on
whether the X-bearing ovum is fertilized by a X or Y-bearing spermatozoa,. There
are several methods for diagnosing the sex of embryos.
a. Sex chromosome identification
b. Demonstration of H-Y antigen
c. Assay of sex-linked enzyme
d. Blotting DNA probes
Sex chromosomal analysis
Oocysts fertilized with X-bearing spermatozoa develop into phenotypic
females and those fertilized by Y-bearing sperm into males. Karyotypes of each of
the domestic animal differ in the number of chromsomes and each have
morphologically characteristic differences in X and Y chromosomes. The X
chromosomes contain approximately 4% more DNA than the Y chromosomes.
Sex chromosomal analysis is done using cells in the metaphase. Cells have
to be removed from an embryo to be sexed and cultured in vitro until they reach
mitosis. Sex diagnosis was possible in 68% of embryos. Splitting of bovine
morullae and blastocysts allow an experimental approach to the cytogenetic sexing
of embryos collected on days 6 to 7. The procedure starts with splitting of embryo.
If the two parts do not look exactly the same, the good half is used for transfer or
freezing, the poor half serves for cytogenetic study. Poor halves are cultured in
medium with colcemid for 4 hrs. Cells are fixed, stained and examined under the
microscope for identification of sex chromosomes. After sexing the first
half, the second half can be transformed or frozen for any desired period.
The disadvantages of the karyotyping method for sexing are a) only 60%
accuracy; b) embryo viability may be reduced by some degree; c) time-consuming
procedure; d) requires well-trained personnel for cytokaryology typing and e)
additional expense for splitting.

67
Demonstration of H-Y antigen
A new approach for identification of embryonic sex utilizes immunological
detection of a male specific proteins, referred to as either histocompatibility-Y (H-
Y) antigen or serologically detectable male (SDM) antigen. Many workers suggest
that these two factors (viz., H-Y and SDM antigens) are the same or had parts of
the same molecule.
H-Y antigen is present at the 8-16 cell stage of mammalian embryos. The
detection of the H-Y antigen by antibodies may be an effective method for 'sex
selection' prior to transfer of embryos. Zona pellucida-free embryos are incubated
in diluted H-Y antiserum in the presence of guinea pig complement and
scored for lysis. Surviving embryos were karyotyped and transferred to
show the genetical sex. Highly specific monoclonal antibodies have been
prepared against H-Y antigen which can be used in the fluorescent system
(Kohler and Milstein, 1975). The advantages of sex selection using MAbs against
the H-Y antigen are: a) accuracy is about 85%; b) the test needs about 2 hrs and
does not affect the viability of embryos and c) the test can be applied by
inexperienced people.
Enzyme immunoassay eliminates the need for the fluorescent microscope.
The principle is based on the reaction of an enzyme (carried by monoclonal H-Y
antibody) with a substrate (added to solution) to form a coloured product
signifying the presence of H-Y antigen.
Metabolic activity of X-linked enzymes
This is another method to identify the sex of embryos by enzymes that are
associated with the number of active X-chromosomes. In mice, levels of X-linked
enzymes such as glucose-6-phosphate dehydrogenase, hypoxanthine guanine
phosphoribosyl transferase, phosphoglycerate kinase and a-galactosidase have
been shown to be dependent upon the number of X-chromosomes. Female
embryos contain two functional X-chromosomes compared to the male single X
chromosome. Therefore, female embryos theoretically produce greater levels of
enzymes intrinsic to X chromosomes expression.
One of the two X-chromosomes of a normal female is inactivated in early
embryonic development. Prior to X-inactivation, the levels of X-linked enzymes
are known to be dependent on the number of X-chromosomes. Such enzymes are
glucoses-phosphate dehydrogenase (G6PD) phosphoglycerate kinase, p-
galactosidase and hypoxanthine guanine phosphoribosyl transferase. Williams
(1986) devised an effective method of visual colorimetric in situ assay system for
the X-linked enzyme G6PD in predicting the sex of mouse embryos.
DNA probes
DNA probes have been used for sexing embryos. Foetal sex can be
determined with DNA samples of as little as 20 ng using a repetitive Y probe pS4
(Disteche et al., 1984). A cDNA sequence (probe) is produced which binds to a
gene present only on the Y-chromosome. This probe can be labeled to identify the

68
presence of Y-chromosome in cell preparations. In another study, DNA sample
was prepared from blastomeres of bovine embryos and dot blotted with Y-specific
radiolabeled probe.identified the sex of blastocyst stage bovine embryos within 30
hrs. A total of 150 biopsies were evaluated. Definite results were obtained in 57%
of these biopsies.
In vitro Fertilization
In the last two decades, rapid progress has been made in the development of
new technologies of reproduction for genetic improvement in farm animals. The
recombination of artificial insemination and embryo transfer has completely
transformed the breeding scheme in the bovine. Recent developments in the
oocyte maturation and in vitro fertilization in farm animals is likely to offer a new
dimension in research and development for future application in the improvement
of animal reproduction.
Birth of live offspring following in vitro fertilization plus embryo transfer
has been reported in several experimental animals. In humans, satisfactory results
of in vitro fertilization and transfer has a means to treat infertility and been
reported from several countries since 1978. However, there have been very few
reports of successful in vitro fertilization in farm animals. In 1982 the first in vitro
fertilized calf was born in the United States and since then more calves were born
by this technique in other countries.
Preparation of the oocyst
The duration of capacity for fertilization of the ovtijlated oocyte in the
oviduct is estimated to be 12-24 hrs in the cow. The time for the penetration of
spermatozoa into the reproductive tract is estimated as 3.5 hrs after ovulation in
the cow. To induce superovulation for in vitro fertilization experiments,
gonadotrophin is usually used in animals. The combination of the pregnant mare's
serum gonadotrophin (PMSG) or follicle stimulating hormone (FSH) both in
combination with prostaglandin (PG) has been used to induce superovulation. The
advantages of this method are that many oocysts are retrieved at the same time and
the timing of ovulation can be adjusted. Usually follicular oocysts are collected
from the Graffian follicle.
The immature oocyte is collected from the ovary and cultured 'in vitro to
induce maturation. Cow ovaries are collected at the slaughter house and
maintained in PBS at 30-32°C for transportation to the laboratory. Following
aspiration of follicles, the oocytes are then observed under a dissecting
microscope. The oocytes are graded according to their morphology, washed twice
with HERBES and once with BMOC-3. After washing, 5-10 oocytes are
transferred to 0.2 ml of BMOC-3 drops under paraffin oil at 37°C with 5% CO2 in
95% air for 12-32 hrs.
Preparation of the spermatozoa
The capacitated spermatozoa is essential for in vitro fertilization. After
capacitation, the acrosome reaction m the head of the spermatozoa allows the
release of enzymes for penetration into the zona pellucida of the oocytes. The

69
capacitation process is completed during the movemen of the spermatozoa from
the oviduct to the uterus. Capacitation involves the removal probably by the
enzyme action of macromolecular material located on the surface of the
spermatozoa. Re-exposure of the spermatozoa to seminal plasma leads to loss of
capacitation and the process may well involve the restoration of the surface layer
or 'decapacitation factor' which normally coats the spermatozoa as they pass
through the male reproductive tract, Experimental procedures to induce the
capacitation or acrosome reaction of the spermatozoa involve m vttro incubation
with follicular fluid and serum.
In vitro fertilization in farm animals
Among farm animals in vitro fertilization has been achieved in the cow, pig
and goat; but the number of newborn animals were limited. The oocytes are
recovered from ovarian follicles or from oviducts near the time of m vitro
ovulation following treatment of donors with PMSG and PG For in vitro
capacitation semen is incubated, treated with high ionic strength medium and
subsequently incubated in defined medium prior to insemmation of the oocytes.
Produced six calves from a combination of in vitro fertilization and
incubation in the rabbit oviduct before transfer. Laparoscopy was used to recover
in vivo matured follicular oocytes from superovulated heifers. The oocytes
enclosed in the expanded cumulus wer then mixed in vitro for fertilization with
fresh semen in a single medium and used at a concentration of 1x10 sperms/ml.
Following the fertilization period (16-20 hrs), the embryos were cultured in vitro
upto 2 or 8 cell stage. Following in vitro development, the 2 or 8-cell embryos
were either transformed into the cow oviduct or the rabbit oviduct. The
embryo (8-cell to blastocyts) obtained after incubation in the rabbit were
transformed to the uterus of virgin heifers either surgically or non-
surgically. The transfer of in vitro fertilized embryos is now practicable, but the
laboratory methods of fertilization still need development.

70
SELECTION METHODS
There is only one way to select and that is to "keep the best and cull the
poorest. The various selection methods are techniques for identifying or estimating
the genetic values of individual candidates for selection. The procedure discussed
here apply to selection for quantitative traits.
A. Performance Testing
A performance test is a measure of the phehotypic value of the individual
candidate for selection. Since the phenotypic value is determined by both genetic
and environmental influences' the performance test is an estimate, not a measure
of the genetic value. The accuracy of this estimate depends on the heritabiliry of
the trait, i.e., on the degree to which the genetic value is modified by the
environmental influences. If heritability is 1.00 or if environmental effect are
completely removed (usually impossible) the performance test is an exact measure
of the genetic value. If non-random environmental effects are removed the
phenotype is an unbiased estimate of the genetic value because the random
environmental effects are equally likely to .be positive or negative.
Advantages of Performance Test
a. High accuracy. Among simple procedures the performance test is the most
accurate. Among more complex procedures performance records .must
usually be included to obtain near maximum accuracy.
b. Environmental influences can be minimised by testing candidates for
selection in the same pen or in similar environmental conditions.
c. The measure is direct, not on a relative basis.
d. All candidates for selection can be tested in contrast to progeny testing
where only a few parents can be tested, i.e., it allows a high selection
differential, e. Generation intervals are usually short
e. Testing can usually be done .on the farm under normal management
conditions.
Disadvantages of Performance Test
a. Accuracy become low when heritability is low
b. Phenotypes are not available for one sex in sex - limited traits such as milk yield

or egg production .
c. Traits which are not expressed until maturity may become expensive or difficult

to manage by performance tests since most selection decisions must be made


before maturity.
Performance tests should be the backbone of most selection programmes.
Although much publicity has been given to other selection methods, it remains a
fact that most of the progress in livestock improvement to date has been due to
selection on the individual's own phenotype (i.e., performance test).
B. Pedigree Selection

71
Pedigree selection is useful when inadequate information is available about
the individual, such as when selection decisions must be made before the
individual expresses the trait (carcass merit or mature weight) and when dealing
with sex-limited traits (yield nr maternal ability). If a performance record is
available on the individual the addition of pedigree information usually adds little
to the accuracy of estimation of the breeding value of the individual.
Pedigree selection is based on the fact that relatives possess many of the
same genes, thus an estimate of the breeding value of one animal provides some
information about the breeding talue of his relatives. In fact, the accuracy of
pedigree selection from single relatives is equal to the accuracy of the
performance test on the relative multiplied by the fraction of genes this
relative has in common (i.e.relationship) with the candidate for selection.
Essentially a pedigree evaluation is only an extension of performance testing since
each Individual is performance tested and performance records are used to
evaluate related" animals.
In general, estimation of the breeding value of an individual for quantitative
traits has low accuracy when based on the records of ancestors. This low accuracy
is due to both incomplete heritability and to segregation of genes for traits with
polygenic inheritance. In addition, ancestor records .were usually made several
year ago under environmental conditions quite different from those now existing.
The value of the pedigree Vas greatly exaggerated by breeders of the past,
although it is very useful when used properly.
Since each progeny obtains one half of its genes from the, sire and the other
half .from the dam, the sire and dam sides of the pedigree should be accorded
equal weight. This holds true if there is equal information, but if many more close
relatives on one side of the pedigree have records this side should receive more
emphasis. This is particularly true if the sire has a large number of progeny with
records.
If we evaluate the genetic variation, we find that 25per cent of the variation
among breeding values of full-sibs is determined" by each parent and the
remaining 50 per cent is attributable of the segregation of the genes of the parents
during gamete formation. The contribution of each partn't to the phenotypic
variation, however, is .25 h2 because the genetic values are modified by the
environment (h2 denotes heritability). If we carry this partitioning back additional
generations we find that the fraction of phenotypic variance accounted for by each
grand rent is .0625 ha, by each great-grandparent is .0156 h1, etc., each generation
decreasing, to of the preceding generation From this rapid reduction in
accountable genetic variation as we go furthes back in the pedigree, we see that,
although all of the progeny genes are-descended from the distant ancestors, the
segregation of genes during each generation of gamete formation has so mixed up
the genes that most genetic variation is due to recombination, and that the
descendants closely resemble only very close relatives.

72
When unequal information is available on the various candidates for
selection, it becomes difficult to know how much emphasis is to give to each kind
of record. For example, we v choose one herd replacement from two candidates;
candidate A has been performance sire. The best available method is to compute
the index.
I = b1 (X1 - X) + b2 (X2 - X)+ .... +b1 (X1 - X)
where b1 is a weight for the first type of record, b2 is a weight for the second
type of record X1 is a phenotypic record on the first type of relative, X2 is a
phenotypic record on the se type of record, and X is the population (or herd)
phenotypic average. By this method animals with the highest indexes should be
selected since they have the best chance t genetically ''best". The weights (b 1)
depend on:
1. Heritability
2. The relatives which have records available,
When one record on the relative is available the weight for this record is:
where h3 is heritability and R is the relationship between the relative that made the
record the candidate of selection. Thus it candidate A has a record (X A) on his dam
and candidate has a record (XB) on his granddam their respective indexes would
be:
IA=.5h2(XA-X)
IB=.25h2(XB-X)
Candidate A would be chosen if TA is greater that IB and candidate B would
be chosen if is greater than IA. Note, that the weights are directly proportional to
the relationships, thus giving much more emphasis to1 close relatives. Also,
performance tests fit directly into this index since the relationship of an individual
with itself is 1.0.
When there are several sources of information about the same candidate,
the weights combine information are more difficult to compute because some
information is "duplicated' For example, if we have-a record on the dam and a
record on the maternal granddam, we must reduce the weights somewhat because
the maternal granddam transmits her genes only through the dam'. if heritability
were 1.0 the weight for the granddam would be zero, but if heritability is less than
1.0 the granddara- does contribute some information about the genetic value of the
candidate in addition to that from the dam's phenbtype. In general, however, the
weights for ancestors are considerably reduced if a closer relative also has a record
available. The weight for sortie frequent combinations of records are listed in
Table 7.4. To illustrate the use o Table 28, suppose we wish to choose the
prospective herd sire which will best improve weaning weight from two
candidates (A and B) in the same herd. The pedigrees for "the two candidates-are:
The herd average weaning weight (H) for this herd has remained constant at
400 pounds for several years. The heritability of weaning weight is about .35, thus
the index equations would be:
IA=bc (CA-H)+bs (SA-H)+bD (DA-H)+b1 (GAl-H)

73
+b,(GM-H)+b,(GA,-H)+b4(GAl-H)
=.26 (420-400)+ .10 (30)+ .10(10)+ .04(180)+ .04(60)+ .04(50) + .04(130)
=26.0
IB=bc (CB-H) + bs(SB-H) bD (DB-H)
=.27(480-400) + .11 (520-400) +.11 (400-400)
=34.8
Thus, candidate B would be chosen on the basis of fewer records because
his own record and his sire's record are high whereas the high records behind
candidate A were made by more distant relatives. If the records were made in
different herds it would be necessary to substitute the appropriate contemporary
herd average for H for each record.
Several principles will be clear from examination
a. The emphasis given to relatives falls off rapidly as the relationship becomes
less.
b. Distant relatives are much more important when records on close relatives
are not available.
c. Distant relatives add little information when close relatives have records.
d. Selection accuracy increases as heritability increases.
e. Information on relatives is more important when heritability is low.
f. Adding information on additional relatives always increases accuracy,
provided they are weighted properly.
Advantages of Pedigree Selection
a. It provides information when performance tests are not available for the
candidates.
b. It provides information to supplement performance test information.
c. It allows selection to be completed at a young age. Pedigree records may
be used to select animals for performance or progeny testing in a multi-
stage selection scheme.
d. It allows selection of both sexes for sex limited traits; i.e., bulls can be
selected on the milk records of their female relatives.
Disadvantages of Pedigree Selection
a. Accuracy, relative to alternative selection procedures, is usually low.
b. Too much emphasis on relatives, especially remote relative, greatly reduces
genetic progresss.
c. Progeny of favoured parents are often environmentally favoured.
d. Relatives often make records under quite different environments, thus
introducing non-random bases into the selection system.
C. Progeny Testing.
Progeny testing is a special form of pedigree evaluation where the parents
are chosen on the basis of phenotype performance of their progeny. Of course, this
is a two stage selection system because some preliminary selection determines
which animals first produce progeny followed by further culling of those which
produce poor progeny.

74
. The great appeal of progeny testing is due to the high accuracy which can
be obtained when many progeny are obtained. However, a record on one progeny
provides equivalent information to that from a record on one parent or a full-sib. It
takes 2 to 3 progeny to provide accuracy equivalent to one record on each parent
and as heritability increases from zero to, 90 it takes /from 4 to 31 progeny, to
provide selection accuracy equivalent to one performance test record on the
candidate for selection. However, the selection accuracy increases as more
progeny are obtained until perfect accuracy is reached with a large enough number
of progeny as shown in Table.7.5. The selection index weights and accuracy are
computed from the following equations:
I=b(X-H)
2nh2
Selection index weight = b=
4+(n-)h2

nh 2
Accuracy of index =
4 + ( n − 1) h 2
=correlation between index value and true genetic merit of the candidate
where, n progeny each have one performance test record
X is the average for the n progeny records.
H is the average of contemporary herdmates of progeny
h2 is heritability
These values are tabulated in Table 7.5
Selection indexes computed as described in this section are directly
comparable to those described for performance testing and pedigree evaluation.
The weights of 2.0 may seem strange but they result because the average value of
progeny is the same as the average of their parents. This means that each parent
transmits one-half of its superiority to the progeny, thus twice the progeny
superiority equal's this parent's superiority if the other parent is of average value.

To be successful, progeny testing requires a high reproductive rate because :


a. If heritability is high, pedigree and performance testing each produce faster
genetic progress,
b. If heritability is low:
1. Many progeny are needed to obtain an accurate progeny test.
2. After completing progeny tests large numbers of progeny ^must be
produced from superior parents to offset the progeny of inferior parents
obtained during the progeny testing stage.
Advantage of Progeny Testing,
a. High accuracy when many progeny are obtained.
Disadvantages of Property Testing
a. Long generation interval. ,

75
b. Requires high reproductive rate.
c. Few candidates can be tasted, thus low selection intensity. To be successful
many more parents must be tested than will eventually be used heavily.
3.- Requirement for Successes in a Trogeny Test Programme
a. Mates of the tested candidates must be of equal merit.
b. The average environment of all progeny groups must be the same. This is-best
done by:
1. One progeny of each sire per herd
2. Progeny from the candidates randomly distributed among pens or herds. .
Definitely do not put all progeny of a parent in the same pen or herd or in
adjacent stalls in the stable.
c. High reductive rate.
d. Several to many tested candidates for each one selected for heavy use.
Alternative Methods of Progeny Testing
The selection index method of progeny testing is presented because:
1. It is more accurate.
2. The values are directly comparable with indexes from performance and
pedigree records.
Alternative forms of progeny tests have been widely used and will be
discussed with a reference to the selection index procedure.
1. The progeny (or daughter) average. In this method the candidate whose
progeny have the highest average is selected. This assumes that:
a. All parents have an equal number of progeny.
b. Equivalent environments were provided for all progeny groups.
c. The mates of each candidate for selection are equal.
When these conditions are met the progeny average is equally as good as
the selection index for comparison of progeny tested candidates, but does not
allow a progeny tested candidate to be compared with a performance tested
candidate. The selection index procedure becomes distinctly superior when some
candidates have many more progeny than other candidates.
2. The daughter-dam comparison. In this method the sire whose progeny excel
their dams by thee largest amount are selected. This assumes equal merit of mates
of the sires and equal environmental conditions for all records of dams and
daughters. Two factors usually make this procedure very ineffective:
a. Environmental changes—the dams usually make their records at a different
time than their daughters, thus large non-random environmental effects usually
remain in the daughter dam difference.
b. The importance of the dam is overemphasised relative to including her in a
selection index. Also, when one candidates has many more daughter-dam pairs
than another, the procedure is very inefficient. Accurate comparisons of daughter-
dam proofs with performance and pedigree records are difficult.
3. The equal parent index. This is an extension of the daughter-dam comparison
to n correct the merit of-mates by adding twice the daughter-dam difference to the

76
dam average. This usually provides slight improvement since the major drawbacks
of the daughter-dam procedure remain.
4. The contemporary comparison. This is more a principle than a selection
method. A contemporary comparison is. a comparison of two individuals of the
same age which are raised together in the same pen or the same environment. In
this situation, differences in per-1 formance are due to genetic differences and
random environmental influence, thus providing the most accurate possible
comparison of genetic values. The selection index uses the prince pie when all
records are expressed as deviations from the contemporary herd or environmental
average. Comparisons within a contemporary group are very accurate, but
usually we want to combine information on animals from one contemporary group
with information on animals in a different contemporary group. When heritability
is high it is better to compare e records directly, but when heritability is low 9less
than. 50) difference between express all records as deviations form the
contemporary group means. For example, from the data given below if we wish to
compare the milk production of the following cows by contemporary comparison
the cows would rank C, A, B, D, but on actual records, they would rank C, D, A,
B. Obviously the better ranking procedure for herd comparisons depends on how
much of the difference between herd averages is due to genetic differences and
how much of this difference due to feeding and management. Many studies have
shown that most differences between herd 'averages are environmental if
heritability is low.
D. Show Ring Selection
Selection on the basis of show ring performance has had considerable
vogue in the past Essentially this selection has been directed toward bringing the
conformation of the animals: to some ideal conformation. This improvement has
been based on two goals:
1. Improvement of conformation. 2. Correlated response.
Improvement of conformation has economic value because a part of the
sale price is deter mined by the conformation of, the individual. The ideal type was
chosen so that, in the opinion of the judges, the animal possessing this
conformation was most likely to be a profitabk .producer. In other words, the
judges were attempting to stress traits of conformation which are correlated with
productive ability.
With the advent of record keeping it was found that direct selection for
performance trato resulted in much faster progress than selection via correlated
conformation traits. Also, when subjected to intensive study, many of the
correlations between performance and show ring traits were found to be of non-
genetic' origin. If the correlations are of genetic origin, direct selection for
performance should improve conformation as well as the reverse situation. The
show ring has beensa good forum for discussion of what constitutes ideal type and
good management and has produced dramatic changes in the conformation of
some species. This has resulted primarily from education of the breeders, however,

77
for most animals, which are presented in the ring are good and the selection
differential among these animals is usually so small as to produce little change.
Advantages
1. It enables breeders to exchange ideas and experiences.
2. It allows comparisons among superior animals both within and between
breeds. 3. It allows new breeders to make contact with established breeders.
Disadvantages
1 . Emphasis is usually placed on traits of little economic importance.
2. Clever fitting and showmanship can mask defects of various kinds.
3. Differences between exhibited animals are usually small.
4 Conformation and production traits usually have low genetic correlations.
Summary
Performance test should be the basis of selection programme. Pedigree records,
when weighted appropriately improve the accuracy of selection and are useful for
preliminary selection of candidates to be performance of the progeny tested.
Progeny tests are useful when the population size is large, the reproductive rate is
high and heritability is low.
Choosing Breeding Animals
The future of the herds depends upon the animals saved for breeding. It is
often said that the sire is half of the herd because each sire contributes half of his
genes to many progeny whereas each cow in the herd transmits half of her genes
to only a few progeny. Thus, the following considerations are especially
important in selecting herd ;sires:
A. Choose healthy individuals free from serious genetic defects.
B. Check the reproductive organs. Be sure the testicles are descended. Have
your veterinary check semen quality. Buy pregnant females and sires which have
been successfully mated if possible. Infertile breeding animals leave few
progeny. C. Choose mature animals if good ones are available.
1 Top progeny tested parents are the best bet if available.
2. Parents of demonstrated top performance are next best.
3. Progeny of outstanding proven parents are next.
4. offsprings with poor parents but above average grandparents and other
relatives , are usually no good prospects.
D. Choose unproven young animals from good parents in preference -to below
herd average candidates, since those below herd average have been proven to be
poor risks as - breeding animals.
E. Good proven sires are often available by artificial insemination,
FERTILITY AND BREEDING EFFICIENCY
In animal husbandry there is no more important economic problem than
fertility of the animals. The profit that is obtainable through meat, milk, eggs,
wool, etc., is dependent on.
ARTIFICIAL INSEMINATION

78
Artificial insemination is the introduction of male reproductive cells into
the female reproductive tract by an artificial means. It is commonly abbreviated as
AI when associated with domestic animals'. In humans, artificial insemination is
abbreviated as AIH when the husband's semen is used and AID when the semen is
that of a donor.
The foremost value of AI in farm animals lies in its use as a tool for the
rapid improvement of quality of genes in future generations by maximum possible
use of best series on a mass basis.
Thus artificial insemination is primarily an economical measure in that
fewer bulls are required and maximum use can be made of the best sires.

History and Development of AI


An old Arabian document dated 700 of the Hegira (1300 A.D.), records that
an Arab chief of Darfur, introduced a wad of wool into the vagina of a mare (a
female horse) recently bred to an excellent stallion belonged to an enemy
chieftain. After 24 hours he then hurried home and introduced the cotton into the
vaginjhof his own mare. The mare then came in foal.
In 1777, Lazzaro Spallazani; priest of Modena and a professor of
physiology at the University of Eavia, begart a series of successful experiments
using AI on reptiles. In 1780, he used AI successfully to inseminate a Spanish
bitch.
In 1899, Elias I Invnoff Russian researcher, began a series of studies using
AI and was successful in pioneering the method in, birds, horses, cattle and sheep.
Mass breeding of cows through AI was first accomplished in Russia, where 19,800
cows (an average of 100 cowsper bull) were bred in 1931.j-.The Russians were
thus the first to aggregate the potential of fltfmey were also giving training to many
technicians and by 1938 many thousands of cattle and horses and millions of
sheep were being successfully bred. Cooperative Cattle AI associations were es-
tablished in Denmark inl936 and in the U.S.A. in 1938 and in Britain in 1942.
Since the Second World War the practice of AI in dairy cattle has grown
tremendously in all milk producing countries of the world, in Denmark, Japan and
Israel over 90% of cattle are artificially bred, in the U.S.A. over half the dairy
cattle population are artificially inseminated.
Artificial insemination was first attempted in India in 1939 by Dr. Sampath
Kumar at the palace Diary Farm, Mysore, and some healthy calves were obtained.
Comprehensive studies on problems of artificial insemination with special
reference to Indian conditions began in 1942 at the Indian veterinary Research
Institute (I.V.R.I.) Izatngar, under a scheme sponsored by the Indian Council of
Agricultural Research (I.C.A.R.). It was soon found that the technique could
successfully be used under Indian conditions but that its successful application
depended largely on organisation. The field work started at Izatnagar showed that
there were a number of problems to be solved, and in 1945-47 the Government of
India opened four regional centres at Calcutta, Bangalore,

79
Patna and Montgomery (now in West Pakistan), to study these problems
and to prepare for the development of artificial breeding on an all-India basis. The
work carried out at these centres demonstrated the feasibility of this method of
breeding in India, under both urban and rural -conditions, in spite of many
difficulties. The Montogomery centre was closed on August 15, 1947, v in the
partition of the country and the Bangalore centre closed in 1951. The State
Governments West Bengal and Bihar took over the centres at Calcutta and Pallia
respectively by the end of f 1950 and the Government of Mysore had by then
started artificial insemination work in Bangalore city.
During the First Five-Year Plan (April, 1951 to March, 1956), a master
project, the Key Village Scheme, was launched, which provides for the all-round
improvement of cattle and buffaloes in the country. To bring about rapid genetic
improvement in the stock, artificial insemination, was accepted as a major activity
of the scheme. Under this Scheme 600 .key villages and 150 artificial insemination
centers were established during the period 1952 to 1956. One centre was attached
to a group of four key villages, back key village had 3500 cows and/or she-
buffaloes, so that one artificial insemination centre was responsible for 2000
animals.
Under the Second Five-Year Plan (April, 1956 to March, 1961) the scope
of work has been further extended and by 1957, 400 artificial insemination centres
were operating. Besides the artificial insemination centres in the Key Village
Scheme almost all the states had additional artificial insemination units working in
areas outside the Key Village units. Some private agencies or cooperative
organisations dealing with livestock have also adopted artificial insemination for
breeding work.
A semen bank has been established at the National Dairy Research
Institute at Bangalore with a view to supplying semen from Jersey bulls for cross -
breeding work and also from bulls of superior Indian dairy breeds for selective
breeding or upgrading work. Semen from this bank is being flown to different
parts of the country.
The use of AI in other species has generally lagged behind its use in cattle
largely because of the problem of the satisfactory storage of semen. In the United
Kingdom, France and Germany 5% or less of the total number of matings of sows
and gilts are by AI, in Holland it is 20% and in some Scandinavian countries it is
over 30%.
According to present status AI is currently used in dairy and beef, cattle,
goats, buffalo, sheep, swine, horses, turkeys, bees, dogs, red fox, fish, mink,
humans and many other species in many countries of the world.
The first buffalo calf through AI was born in 1943 at the Allahabad
Agricultural Institute in U.P. India.
Advantages of AI over Natural Breeding
1. The main advantage of A.I. is that it increases the usefulness of superior
sire to an extra-ordinary degree. It makes available sires of inheritance for

80
milk and butter fat production to all dairymen within a limited area.
Previously only a few could get the advantage of good bulls.
2. The services of superior sires are greatly extended. By natural services, a
bull can be bred to 59 to 60 cows/aer year; on the contrary New York
Artificial Breeders Co-operative have sired 10,000 intone year by one bull.
It would have taken about 200' years to accomplish this by natural service
of that bull.
3. The breeder does not need to maintain a herd sire and thus can avoid the
botherations accompanied with the management of a bull. It helps to
regulate the breeding programme and the space between successive
calviugs without unnecessarily prolonging the dry period.
4. The dairyman does not have the problem of searching and purchasing a
new herd sire every two years to avoid in-breeding.
5. The technique of A.I. can be made use of in cross breeding for hybrid
vigour by quickly transporting the semen by air to different continents.
6. The intensity of the spread of genital diseases are lessened if A. I. is
conducted under complete sanitary conditions by the specially trained
persons.
7. Overcomes the difficulty of size and weight.
8. Increases rate of conception.
9. Outstanding animals located apart can be mated.
10. Helps in better record keeping.
11. Old heavy and injured sires can be used with advantages.
Limitations of A.I.
1. Requires well trained operations and special equipments.
2. Requires more time than the natural services.
3. Necessitates the knowledge of structure and function of reproduction, on
the part of the operator.
4. Improper cleaning of the instruments and .insanitary conditions may lead to
lower fertility.
5. Market for the bulls is reduced while that for the superior germ plasm is
increased.
6. Selection of the sire should be very rigid in all respect
Problems Under Indian Conditions
1. The sentimental views of people do not relish castration of icrub bulls
which are very essential.
2. The lack of understanding of A. I. some say that it produces weaker calves,
etc.
3. It hampers the prospects of the breeders in the disposal of their bull calves.
4. Severe climatic conditions are detrimental for preservation and
transportation of semen.
THE TWO COMPONENTS OF SEMEN Spermatozoa

81
Whole semen as ejaculated, generally appears as viscous, creamy, slightly
yellowish or greyish fluid and consists of spermatozoa or sperm suspended in the
fluid medium, called seminal plasma. Its composition depends, in the first place,
on the proportion of sperm and plasms and is further determined by the size,
storage capacity and secretory output of several different organs which comprise
the male reproductive tract. The volume of the ejaculate and concentration of
spermatozoa or the sperm density in ejaculated semen, vary widely from one
species to another.
In the majority of species, including man, mature spermatozoa have a
filiform 'structure owing to the presence of a-flagellate appendage, although non-
flagellar forms of sperm cells are not uncommon in certain lower animals, e.g.,
among Crustacea nematodes. The peculiar filiform structure determines, to a
considerable extent, the remarkable permeability of the sperm cell, which is
/perhaps best illustrated by the so called 'leakage* phenomenon, i.e., the
remarkable case with which even large molecules such as cytochrome or
hyaluronidase can detach themselves from the sperm structure and pass with the
extracellular environment. The high degree of permeability explains the cellular
speed with which exchange reactions can take place between the spermatozoa and
the surrounding medium, whether this be the seminal plasma or an artificial
pabulum.
In a typical flagellar spermatozoon it is usually possible to distinguish three
regions, viz., Yperm head, middle piece and tail; but even among closely related
species, one encounters an extraordinary diversity of form, size and structure.
Moreover, on examination of the semen from angle individual, one often finds in
addition to the normally shaped, spermatozoa, a variety" 'of , degenerated,
abnormal or miniature forms which represent every conceivable deviation from
""the normal structure, from tapering and double cells with a double head or tail to
giant (Fig. 8') and monster with containing several nuclei and several tails in a
mass of cytoplasm.
Although a high degree of sperm abnormality is undoubtedly associated
with sub-fertility, normal semen is seldom completely uniform and human semen
for example, is 'reckoned to contain as a rule, at least 20 per dent of abribrmal
forms. In the bull and in stallion the percentage of abnormal, forms in semen is
similarly high; in the ram, on the other hand, it appears, to be much less.
The shape of the head in normal spermatozoon varies greatly. It is ovoid-
in the bull, ram, boar acid rabbit; it resembles an elongated cylinder in the fowl
and has the form of a hook, in the mouse and rat.
In the bull the spermatozoon is about 68 ±3μ length. Of this length the tail
makes, up about 50 μ, the head about 8 - 10 μ , tie neck 1 μ and the midpiece 8-10
μ.
The main part of the, head is occupied by the nucleus, filled by closely
packed chromatin which consists largely of , desoxyribonucleoprotein. (Fig. sTi).

82
The anterior part of the nucleus is covered by a cap-like structure known as
the Acrosome Several investigations have revealed that in spermatozoa, yet other
cap, a loose protoplasmin structure named Galea capitals which envelops the
apical part of the sperm head and can break away spontaneously to form the so-
called spermatic'veil or floatin cap. However, whereas most authors including
Williams (1950) regard the acrosome proper and the galea capitis as two-distinct
structural entities, some consider them to be identical and Hancock (1952) for
instance is convinced that there is only one acrosomal structure and that the
detachable cap arises through postmortem changes and is the result of swelling
and loosening of the acrosome itself.
The narrow region which connect the sperm head with the middle piece is
known as the Neck (or Neck-piece) which is the most vulnerable and-fragile part
of the spermatozoon.
In the neck, close to the base of the sperm nucleus, is situated the
centrosome which marks the beginning of the axial filament, the central core of
both the middle piece and tail: The axial filament consists of 20 fibrils which run
uninterruptedly through the whole length of the middle piece and tail. The fibrils
are arranged in two rings of 9 fibrils each. The nine members of the inner ring are
surrounding the other 2 central fibres: Hence for bull spermatozoa, the numerical
pattern of the fibrils is 9 + 9+2, also present in. other mammals. These fibrils are
responsible for the whip-like lashing of the tail.
In the middle piece (or mid-piece) which is about the length of the sperm
head, though only one-tenth wide, the axial filament is surrounded by the 'Broad
helix', also called 'Spiral Body' or mitochondria sheath. The junction between the
mid-piece and tail is marked by the presence of a ring centriole.
In addition to the various fibrous cortical systems, the sperm cell of many
species including man and higher mammals, is protected externally around the tail.
The terminal tailpiece which is about 3μ in length is a solid bundle of fibrils.
Seminal plasma
The seminal plasma is a composite mixture of fluids secreted by organs
which in the higher species comprise the Epididymis, the Vas deferens, Ampullae,
Prostate, Seminal Vesicles, Cowpers glands and contain other glands located in
the wall of the urethral canal.

Prostatic Secretion
This differs in many ways from other, secretions of the mammalian body
and its composition shows considerable species variations. Much study has been
devoted to human and bovine prostatic fluids; both are colourless, and are usually
slightly acidic about 6.5). It contains several strong proteolytic enzymes. The
human fluid contains a fibrinolysiruso powerful that 2 ml of the same can liquefy
100 ml o/ clotted human blood in 18 hours at 37°C.

83
The prostatic secretion represents the main source of eitric acid and acid
phosphate for whole human semen. In men the prostatic secretion also provides
the main source of calcium.
Among the chemical peculiarities, the prostatic secretion is rather high in content
of zinc.
Seminal Vesicle Secretion
In several species including the rat, guinea-pig and bull, boar, stallion, etc.,
the seminal tesicles alone contribute more fluid than the rest of the accessory
glands together.
Compared with the prostatic fluid the seminal vesicle secretion is usually
less acidic and is sometimes distinctly alkaline. It has a higher dry weight, and
contains more potassium bicarbonate, acid soluble phosphate and protein.
In certain animals, e.g., the dog and the cat, the seminal vesicles are
altogether absent.
The normal seminal vesicle secretion is usually slightly yellowish but
occasionally, specially in man and bull, it can be deeply pigmented. The yellow
pigmentation is probably of composite origin but much of it is duer. to flavins.
The reducing power of the vesicular secretion is one of its most characteristic
chemical properties.
It is also important to note that the seminal vesicles are the main sources of
fructose in higher mammals. The identification, of the seminal sugar as fructose by
Mann (194 5) opened the way for detailed studies of the fructose generating
capacity of the accessory tissues.

84
COLLECTION OF SEMEN
Collection from the Bull
The primary objective in semen collection is to obtain maximum output of
high-quality spermatozoa per ejaculate. Factors known to influence semen output
are; 1. Age of the bull (maximum output occurs between 5 and 8 years of age).
2. Season of %e year (winter months are best for —total-sperm production).
3.Erequency~of ejaculation (increased frequency of ejaculation results in more
total sperm being obtained, but with fewer sperin per collection.) 4. Pre-
ejaculation sexual preparation, (This consists, essentially .of exciting the bull by
allowing him to see, approach, sniff at the teaser and by 'false mounting!lin which
ejaculation is prevented). 5. Correct techniques of semen collection methods
(proper conditioning of Artificial vagina or Electrical stimulatory equipment). 6.
Several studies indicate" that oxytion in and certain other hormones of the anterior
pituitary influence sperm output. Piolactin, growth hormone and testosterone
creases immediately after-ejaculation. 7. Recent research at Colorado State
University in U.S.A. also showed that circumference 9SC) or testicular size is
correlated positively with fertility. (r=0.58). Since the correlation of SC with
actuat testicular weight is r = 0.95, this means that Su is a reliable predictor of the
amount of sperm-producing tissue/within the testes. Therefore, the larger the testes
the greater, the sperm production potential.
Several methods of obtaining bull semen for AI have been developed, the
most common of which are: 1) Artificial/vagina (A V) and. 2) Electro-ejaculation
methods.
Before discussing the details of AV and .Electro-ejaculation, the pre-
requisites required for semen collection by either of these methods are. discussed
below:
Selection of Breeding Bulls
Bulls should be so selected whose pedigree is known and particularly
should have born of a high yielding dam (mother).
Preparation of Young Bull
Quality bull calves should be procured at 6-12 months of age. At 9 months
of age a nose ring made of copper is fixed. They may be stationed at centrally
located bull farms and reared under best of environment, feeding and
management. At 12 months of age the bulls will be screened for freedom from
vibriosis, trichomoniasis, brucellosis and other chronic diseases like Johrie's and
tuberculosis.
The bull calves so selected thust be finally screened Karyologically to
detect any chromosomal abnormality. It has/been reported that screening for
chromosomal abnormality alone improves fertility by 15%. The technology is now
available at Tamil Nadu Veterinary and Animal Sciences University, Madres.
At l8 months, of age, the bulls should be trained for semen collection. At 24
months of age the'' semen. with be collected and evaluated, .before final selection.
Once selected, bulls should be on balanced .ration comprising sufficient greens.

85
Maintenance ration is enough since semen production does not require any extra
ration.
Time for Collection
Semen collections should be made during early morning hours before
feeding when bulls are alert and fresh. Another advantage is that if the semen is
collected during early hours, fresh-Jots can be used on the very day which may
raise the fertility rate to a certain extent.
The Collection Yard
The semen collection yard should be located nearer to the laboratory. If the
place is windy, protective walls should be erected. The ground should be covered
with rough concrete flooring which should, remain dustless during collection. If
the collection is made on earthen flooring, water may be sprinkled to avoid dust in
the air. It is also desirable to have some sort of cover on top of; the, yard to
prevent direct sun shine or rain. The teaser animal is tied up inside a service crate
which will restrict the movement .and at the same time, it provides safety to the
teaser, bull and personnel. The wooden crate should be so constructed that, it
should not prevent the fare limbs of the bull from embracing of the teaser .and thus
hinder normal thrust. The dummy should never be taller than the bull to be,
collected. The angle between the back of the teaser and that of the bull should not
exceed 45° during ejaculation.
Cleanliness of Bull
The bull from-which semen is to be collected should be clean and free from
dirt, to avoid contamination of semen. Grooming with a brush or wiping the
underline with a damp towel a few minutes prior to collection could be done. If the
underline is extremely dirty, washing may be necessary and in that case it should
be done long before the collection so that the animal-is-dry at the time of
collection. Wet underline is more conducive to the transmission of microbes than
a dry one. The long hairs at the sheath of the penis should be cut to cm. Too short
hairs do not protect the opening of sheath and also aid in developing a tendency to
masturbate.
Selection of Teaser Cow .
As far as possible the-Teaser cow should be of the same breed, size and
colour of the bull. It should also be physically strong and docile so that she is able
to bear the weight of the bull and does not show irritable temperament .when she
has to stand in the service crate for long time, for collection of semen from a
number of bulls. It is better to select a cow which has gone through 2-3 calvings.
The teaser cow should neither be in oestrus nor be pregnant. It is better to tie her
tail with arope on one side.
Pre-ejaculation Sexual Preparation
Sexual preparation of bulls may be defined as prolonging the period of
stimulation beyond that
adequate for mounting and ejaculation. It has been noted that; motile sperm output
per ejaculation in bulls can be increased as much as two fold when two or three

86
false mounts (and active restraint) are' given as rapidly as possible (preferably by
10 minites) before ejaculation.
Stimuli other than false punting mayalsobe used to increase sperm output in
bulls. These stimuli include moving the stimulus animal, exchanging stimulus
animals, changing locations of preparations, changing the personnel handling the
bulls, and combination of these. Additionally undefined stimuli from a teaser
animal near a "Bull cause the bull to ejaculate more sperm. The .., undefined
stimuli are not visual since blinded bulls also respond to sexual preparation with
increased ' sperm output. Part of the stimuli must involve olfactory (sense of
smell) mechanisms since bulls mount estrous cows twice as fast as non-estrous
cows and yield larger volumes of semen and greater sperm numbers.

Tying of Aprons to the Bull


There are some advantages in tying of aprons behind the elbows which will
hang so much so that it does not touch ground while the bull is in standing
position. The apron cloth should be of coated rexin on one side facing penis of the
bull.
I The size of the apron is about 65 x 62-cm with semicircular cut at one end. For
keeping the apron straight and without any folding if should have two metallic
rings on each corner fixed up and rubber strap going round the heart girth of a
bull. The advantages of using bull apron are as follows:
1. It prevents cross infection from bull to bull, which-otherwise may take
place through contact of penis to the rump of the teaser.
2. It prevents losses of semen ejaculates which might happen in certain bulls
by tactile stimulation (due to rubbing the penis against the skin of the rump
of teaser) and thereby throwing semen outside before penis is directed in
AV.
3. The apron prevents the direct 'contact with the skin of teaser and thereby
more hygienic - ejaculates can be obtained.
Artificial Vagina (AV) for Bull
The first AV for bulls was designed in 1930 by Russian scientists. Several
modifications of the early model have been devised by English, Americans and
Danish scientists. The Danish model is now most commonly used in U.S.A., India
and many other countries. There are different kinds of AV for different kinds of
animals conditions of which are almost aiming to that of natural vagina.
The AV is simple in: construction and stimulates natural copulation. It
consists of: (a) A strong outer outer rubber having an inner diameter of 6~cTn-
with a length between 30 cm for young 'Bulls and 40 cm for adult bulls. The
cylinder has rims on either side. A valves fixed at about 5 chi from-one end to
admit water and air in the preparation of AV. The outer edges of. the cylinder arc
upturned and refilled so as to have the firm grip by the inner sleeve, (b) A thin
inner latex or of PVC liner of a diameter between 7.0 to 8 cm with a length of 50
to 55 cm, which is, turned back over each rim of the outer cylinder and tightly held

87
by thick rubber bands, (c) A latex rubber cone between 20 to 25 cm in length is
mounted on the cylinder to the end where water and air inlet valves are closer, (d)
A graduated glass collection tube of 10 ml capacity is fixed to the conical end
of-'rubber cone, (e) An insulation bag covering the glass tube and rubber cone is
fixed on the rubber cylinder with strap for protecting semen in tube from cold and
direct sun rays.
Preparation of AV
The AV for particular bull should be chosen depending upon the length of
its penis. For maximum recovery of good quality semen the bull should ejaculate
directly in the cone. If a longer AV is used for a bull having shorter penis, semen
will be deposited in the cylinder resulting loss of sperm and also admixture of
vaseline or other lubricant as used. On the other hand if too shorter AV is used, the
peniswill throw off the collection vial or get hurt by hitting the collection tube.
The liner should have appropriate thickness, too thin liner will bulge
excessively at the ends of the cylinder, if too thick liner is used due to inflexibility
some bulls might show reluctance to ejaculate. Since even a small quantity of
water is toxic to the spermatozoa, the liner should be constantly checked for
minute crevices. For easy detection, water mixed with ethylene blue solution may
be used for testing leakage of water droplets.
The parts of AV should be clean, stenle and dry before assembling. Before
semen collection, warm water between 45° to 50°C is filled through the filling
aperture into the space between outer cylinder and inner latex to provide an inner
temperature between 42°C to 46°C. Due to season and individual liking the inside
temperature varies. An inside temperature above 47°C may kill sperm in the
ejaculate.
Sterile soft paraffin is now smeared over the surface of the latex liner by
means of a stout glass rod. The warm water apart from providing natural warm
condition of the natural vagina, also facilitates the even distribution of the paraffin.
The pressure inside the vagina-should again match with the natural vagina
(45 to 55 /mm Hg). Younger bull requires higher pressure. AV is always provided
with an air screw along with water. Screw which can be used for blowing the air
between the two layers of outer cylinder and inner sieves to create the desired
pressure. At right pressure the inner lining of the AV should give a star like
appearance if seen from front. If the amount of water or pressure is excessive' it
may act as hindrance for the thrust resulting in no ejaculation.
Semen Collection
During collection the collector should hold the AV at 45° angle to the
ground, so that the long axis parallels the line of the penis. Excessive bending of
the penis before or during the thrust can distract and injure the .bull. The AV
should be handled in such a way that the panis just touches the lining. This
stimulus, arouses the ejaculation. Semen collections can take place twice a week
(one ejaculate each time) or once a week (two ejaculates per collection).
Electro-ejaculation in Bulls

88
The technique was first adopted in 1922 by Battelli for collection of semen
from guinea pigs.
In 1948 French investigators obtained bull semen by introducing a
rnultiringed, bipolar electrode into the rectum and applying upto 30 volts (700
miliamperes, alternating currents). Later models of electro-ejaculators employ a
single rectal probe with bipolar electrodes which is introduced into the bull's
rectum. Rhythmic application of electrical stimulation causes erection and
protrusion of the penis followed by automatic discharge of semen.
Semen samples collected by this method are more in volome and less in
concentration but there is no difference in total sperm concentration or fertility.
The salient points of the method are as follows:
1. At the beginning the rectum is washed with 6 per cent sodium chloride
solution.
2. The probe is then inserted up to about 12 inches and held in a position of rectal
floor.
3. Alternate current increasing in voltage gradually from zero to 5 volts and
returning again to zero within every 5 to 10 seconds is initially passed.
4. The subsequent stimulations made progressively higher so that at about fifth
stimulus a maximum of 10-15 volts is reached. Erection and ejaculation occur at
10 to 15 volts when.0.5 to I ampere current is flowed. The source of electric
current is AC/220-250 volts/single phase / 50 cycles.
The home consumption of electrical current in India is between 200-250
volts, 50 cycles. voltage is reduced by a step down transformer between 10-15
volts and low voltage at when passed through the rheostat, the changes in voltage
can further be made as desired by the operator. This is done by varying the
resistance Without opening the circuit.
Advantages
1. Semen can be collected from males that are too young or old or unable to mount

due to weak or injured legs.


2. No female or dummy is required for collection.
3. Less chance of contamination.
Disadvantages
1. The methods are highly technical and need considerable skill and practice.
2. The semen generally gets contaminated with urine.
3. Some Biales resist too much to, this method and refuse collection.
4. Sciatic nerves are temporarily affected .during the operation but is relatively
minor if the electrodes are kept over the ampullar region.
Collection of Semen from Buffalo Bull
ByAV Method
Semen from buffalo bulls are easily collected in a short AV measuring 20
to 30 cm. in length. Other aspects of collection is to be followed as in the cow bull.
Buffalo bulls are most 'susceptible' lo temperature variations.

89
Collection of semen from Sheep and Goat
Artificial vagina and electrical stimulation methods are the only methods
employed for collection of semen from rams and bucks.
It has already been discussed that the AV method is preferred since it is
quick and simple, is not .stressful to the male, and results in collection of better
quality semen. Semen can be collected ' several times per day by artificial vagina.
Electrical stimulation only be used when semen is required from a male which can
not be trained for collection by AV. ft has the' disadvantages that it causes;
considerable discomfort to the animal, frequent collections can not be made, and
semen may be contaminated with urine during collection. Semen collected by this
method is generally of larger volume but lower sperm concentration than that
collected by AV Electrical stimulation is however,', useful for testing large
numbers of males for fertility, including teaser rains or buck prepared, by surgical
intervention.
Collection by AV
The AV as used for rams and bucks is similar to that used for bulls. It
consists of an outer casings (20 cm x 5.5 cm of ram, 15 cm x 5.5 cm for buck)
made from heavy rubber and an inner liner made of rubber or synthetic material.
The shorter AV corresponds to the length of the buck's penis. The liner should
extend at least 5-8 cm. beyond the ends of the outer casing so that it may be folded
back and secured at the ends with rubber bands to form a water tight jacket.
Before use the liner is rinsed with 70% alcohol in distilled water and
allowed to dry. Next the jacket is half-filled with water at.48-50°C.
One end of the inner liner is then lightly lubricated with vaseline to a depth
of not more than 3 cm using a sterile glass rod. At the other end a sterile calibrated
semen collecting glass is inserted to a depth of 1.5-2.0 cm. While holding the glass
in position the vagina is inflated by blowing air through the air outlet. The
temperature of AV just before semen collection should be 42-45°C and can be
checked by insertion of a clean thermometer. In order to prevent cold shock to the
spermatozoa, the semen collecting glasses should be worried to 30-37°C. Before
collection the prepuce of the male should be/wiped, cleaned to prevent semen
contamination.
The teaser female have long wool or hair, or be soiled at the rear end. The
operator takes a kneeling position at the right side of the teaser and holds the AV
in the right hand along its flank and with the open end facing towards the male and
downwards at an angle of 45°.
When the male enters the pen he may engage in courtship behaviour, but
the operator should be prepared for the ram or buck suddenly mounting the teaser
female. When the male mounts, the erect penis is directed into the open end of the
AI. The left hand is used to gently grasp the sheath and deflect the penis into the
open end of the AV. A vigorous upward and forward thrust signifies that

90
ejaculation has occurred. The male should be allowed to withdraw his penis before
an attempt is made to remove the vagina. The semen collecting glass can then be
removed, labelled, covered and placed in a water bath at 30°C.
The frequency at which semen may be collected depends on the age,
condition and temperament of the animal. Rams-may-mount and ejaculate 20-
25Urmes or more a day. At such frequency the volume and concentration of
semen (and consequently the number of spermatozoa per ejaculate) decreases with
successive ejaculates. However, a regime of 3-5 collections daily for 4-5 day
periods separated by 2-3, day rest periods should not cause a marked reduction in
semen quality or quantity.
For goat bucks 2-3 collections daily on alternate days can be regarded as a
normal regime. Intervals of 0.5-1.0 hour between successive daily collections are
advisable to obtain ejaculates of good volume and concentration.
Collection by Electrical Stimulation
There are different types of electrical stimulators. Those in current use have
a bipolar rectal electrode like the one of Ruakura/Ram Probe. This is a self-
contained stimulator operated by batteries giving a 10 or .13-roJt output. When the
rectum of the male is dry the 15 volt output is recommended.
For collection, the male should be restrained in a lateral position on a
suitable table or clean floor. Long wool or hair should be clipped from around the
sheath, and the prepuce should be wiped with cotton wool. The rectal probe is
moistened or lubricated with vaseline and inserted into the rectum to a depth of
15-20 cm, taking care to avoid injury. The penis should be extended by
straightening the sigmoid flexture so that the glans penis can be grasped with a
clean hand and the penis withdrawn from the prepuce. A piece of gauze is then
secured behind the glans penis and the glans and the urethral process are inserted
into a clean, sterile test tube. It is best to hold
THE penis and test tube with one band so that other hand is free to massage
the penis in a forward direction between electrical stimuli. The rectal probe is
pressed towards the floor of, the pelvis assistant and a short stimuli (3-8 seconds)
are applied at 15-20 second intervals. After several stimuli accessory gland
secretions flow, followed, by semen. When a large .amount of clear fluid is
obtained initially this should be discarded to avoid diluting the semen. There is
considerable variation between males in the amount of stimulation required to
produce a satisfactory ejaculate. , However, apart from the discomfort and
muscular contractions occurring during treatment (herd, are no permanent ill
effects.
Collection of Semen from Boar
Pressure is especially important for collecting semen from the boar. The
boar ejaculates when the curled tip of the penis is firmly engaged in the sow's
cervix or in AV specially made to suit male boar or the operators gloved hand and
pressure is exerted on the coiled distal end of the penis throughout ejaculation.
The ejaculate from the boar consists of the following four fractions:

91
1. Pre - sperm fraction - Very thin like water, having no colour. The quantity is
about 10 ml and is sperm free. The deflection of this secretion is over at the height
of excitement. Its importance is in rinsing the urethra! tract which usually has a
high bacterial 'count. This fraction is always preferred to discard.
2. Sperm - rich fraction - The volume of second fraction is about 40-80 ml, is
dense and milky and contains about 80% of spermatozoa.
3. Low-sperm fraction - The third fraction consists of 150 to 200 ml of thin fluid
with low-sperm concentration. 1 his fluid originates from the secretions of seminal
vesicles and prostate gland which is known as seminal plasma.
4. Post - sperm fraction - The fourth fraction is gelatinous and secreted by
bulbourethral glands. It tenos to seal cervix of the sow during mating, preventing
loss of semen.
Semen Collection by Ay in Boar
Mating is a prolonged process in boar, varying from 3-25 minutes, and
waves of high and low sperm concentrations exist in the flow. The tip of the penis
locks into the entrance of the uterus, cervix and ejaculation occurs directly into the
uterus. For boar, the Norwegian type of artificial vagina is more common, the
length of which is about 18 cm while the diameter is like that of bull.
It contains two inner tubes, one for air and the other for water. When the boar
mounts the dummy, the collector guides the penis into the AV through the Y-
shaped hole in foam rubber piece. The collector then tries to catch and hold the
penis through the vagina with his right hand . Ejaculation is produced by digital
pressure on the spiral portion of the glans penis.
Latest design of a very simple AV for boar consists of only one simple
smooth rubber liner where by applying contiguous pressure by hand near distal
end of penis. semen ii collected.
Some 200 cc of semen is produced, but of this about 50 cc consists of-a
gelatinous material F sperm, and this is strained off before insemination or storage.
Collection of Semen from Stallion
Methods in current use employ either AV or latex condom. The Cambridge
or Russian model and the Missouri or American model arc considered suitable.
The AV technique is similar to that employed for the bull. It is essential in practice
that the mare shall be in full oestrus. The AV is, of course, larger than that used for
bulls and in the case of larger stallions it requires to be held 1; by two operators at
the time of collection. As the stallion mounts the marc, the operator nearer-tic
stallion guides the penis into the AV with his left hand which he then transfers to
the under surface of the penis in order to detect the peristaltic ejaculatory .waves..
With .his right hand he the other operator to hold the AV at a slightly inclined,
angle, until organism is detected en the collecting end is lowered so that the semen
flows into the attached collecting vessel.
The Missouri and Mississippi models arc most popular in USA. Mississippi
model „ is made of one piece .of rubber tubing 91.5 to 122 cm long and 18 to 20
cm in flat diameter. Water temperature is between 41-45°C inside the artificial

92
vagina. Many of the semen collectors prefer to collect stallion semen by placing a
condom over the penis just prior to intromission. Ejaculation occurs into the
condom which is stripped from the penis as the stallion dismounts.
Prior to ejaculation, the penis should be washed with warm soapy water
and-finsed with clean water to remove smegma and other debris on the surface of
the ,penis.
Unlike with bovine or ovine spermatozoa, the poor viability of equine
spermatozoa is partly due to poor initial sugar content of semen and its early
exhaustion during metabolism.
The first fraction of the ejaculate is of watery consistency and is devoid of
spermatozoa. In next phase the ejaculate comes from the testes and possess high
concentration of spermatozoa and thereby milky white in appearance. During the
third and final phase a glairy, viscous material resembling white of an egg is
expelled. This is the secretion obtained from seminal vesicles and Cowper's
glands.
However, using semen fresh, or stored upto two hours on normal mares, it
has beem found, that an inseminating dose of 100 million sperms is satisfactory.
The minimal number of frozen sperm required for high conception rates has not
been established.
DEEP FREEZING OF SEMEN
In India Artificial Insemination (AI) utilizing liquid (chilled) semen from
exotic dairy breeds has been playing vital role for nearly four decades. The advent
of "Frozen semen technology" has by this lime become remarkable by any
measure in turning this century to the gateway of white revolution.
As early as 1938 a German scientist, Jahnel found that by deep freezing
testicular tissue of rabbits, sperms could survive freezing to -192°C. During 1949
Polge. Smith and Parkes accidentally added 15-20% glycerol to a fowl sperm
solution and observed very good motility after- freezing. Thus the remarkable
property of glycerol in maintaining life of deep frozen sperms were established.
Later on it was observed that the resistance against deep freezing is quite different
in different species. It is known that bull semen cannot withstand a sudden drop in
temperature so called temperature shock. For that reason Smith and roige (1950)
cooled the glymul lieated bull in slowly to -79°C and got a much better survival of
the semen than that by rapid cooling. The first insemination with deep frozen
semen were performed by Steward (1951) in cooperation with Polge and Rowson.
Out of 5 inseminated cows one became pregnant.
Subsequent developments have been the introduction of new freezing
techniques, improved semen dilutors and additives.
Principle of Deep Freezing
The general principle of deep freezing of semen is to dilute the semen with
an extender con-tainine glycerol. After equilibration the suspension is frozen
according to special rules and thereafter stored in liquid nitrogen (-196°C) as the
cooling medium.

93
As intra and extra cellular water of media crystalised as ice, the salts get
concentrated in the residual fluid which becomes progressively more hypertonic.
Thus the sperm cells become subject-to severe osmotic stress and in particular to
increase concentration of electrolytes when the water is freezing out. Glycerol has
the ability to attach itself a considerable quantity of water, which is then not
available to form ice but can still act as a solvent. The use of glycerol therefore
enables freezing to be carried out slowly enough to avoid thermal shock without
exposing the sperm cells to lethal concentrations of electrolytes.
Merits and Limitations of Frozen Semen
Merits
1. Frozen semen permits, maximum utilization of semen from a given sire. No
semen need to be .discarded because of age, as with liquid semen.
2. Long term preservation removes barrier on time and distance-in AI
International semen transport, has come into existence today
3. Can be undertaken for:
a) Selective breeding
b) Grading
c) Cross breeding
d) Progeny testing of bulls
4. Transportation costs of semen are minimised compared to-liquid semen
usage, as large consignments can be delivered at infrequent intervals and
full utilization can be made at each bull's semen producing capacity.
5. Minimises requirement of breeding bulls to be maintained at breeding farm.
6. Provides round the clock breeding facility even at the villages.
7. Frozen can safely be used in an outbreak of disease (foot and mouth)
without dislocation in semen transport.
8. Complies with highest hygienic standards in artificial breeding.
9. Comparative studies on cost factor between liquid and frozen semen has
been done at National Dairy Research Institute, Bangalore. It has .been
observed that the average cost of A.I. with liquid semen came to Rs. 60.85
as compared to frozen semen which is Rs. 18.63. Further the cost involved
per calf born was Rs. 235.47 and Rs. 53.13 respectively in using liquid -and
frozen semen.
10. Frozen semen is extremely valuable in carrying on the influence of superior
sires even after 50-100 years of their death.
Limitations of using Frozen Semen
1. About 30 per cent of bull's semen does not withstand the rigors of freezing.
2. The cost involved initially is high.
3. Frozen semen limits number of sires used, if our selection methods are not
perfect. This may lead to wider damage among future progenies.
4. Requires sound technical experts for handling the frozen semen.

94
Dilutors for Deep Freezing
The idea that the sperm cell will remain in living condition without
expenditure of any significant metabolic energy for an indefinite period inspired
the scientists to preserve living cell in the frozen state. Further it was the
speotacular - discovery of Polge in 1949, which showed that the ideath of
spermatozoa on freezing could be avoided by suspending the cells in a medium
containing glycerol. Thus a practical answer to freezing technique was evolved.
At least 50 per cent of the bull sperm from an unselected" population
remains inactive or dead during freezing and thawing due to (1) internal ice-crystal
formation which affects the structure of the spermatozoa, (2) the increase in solute
concentration as water is withdrawn from the suspension medium- (3) by
interaction of these two physical factors, an ideal extender should qualify for
giving cryoprotection during freezing. For bovine spermatozoa the basic
components for an ideal extender are:
Buffers
Out of varieties of inorganic and organic buffers which have been tried for
deep freezing of bovine semen, an organic buffer known as 'Tris 9Hydroxymethyl
amino methane) first used by Graham (1972) at pH 7.0 which has been" found to
be most popular even to-day. The buffer can be successfully used for bull,
buffalo, ram, dog, poultry and even for human. It (i) prolongs life of sperm at
room temperature, (ii) penetrates cells (iii) acts as intracellular buffer, (iv) less
toxic
during freezing and (v) provides better clarity under microscope.
Fructose
Addition of fructose provides (i) glycolysable substrate for the sperim (ii)
prevents sperm agglutination, (iii) maintains required osmotic tension and
electrolyte balance, (iv) aids extra f c cryoprotection during deep freezing.
Glycerol
The possible ways' by which it functions are: (i) modifies size and shape of
ice crystals of the media, (ii) bind water and decreases freezing point we solution
resulting reduction of ice crystals, through salt buffering mechanism, (iv.) reduces
solute concentration, (v) prevents denaturation of proteins and thereby protects
plasma membrane.
Egg Yolk
(l)-When mammalian sperms are cooled to 5°C they are subjected to 'cold
shock' which causes leakage of intracellular enzymes, minerals, lipoproteins, AT?
from inside the cell. Lecithin, protein lipoproteins and other similar compounds
present in yolk provides effective means of guard ainst cold shock. (2) In addition
to this, the valuable nutrients of egg yolk are very slowly utilised sperms. (3) Egg
yolk also protects the enzymes having disulphide bridges (SF as of antiagglutinic
factor presents in seminal plasma.
Antibiotics

95
Semen under normal condition generally gets contaminated with both
pathogenic and non-pathogenic , micro-organisms. Organisms which are
contagious .and can infect cow through pathogens are: Brucella abortus, Vibrio
fetus, Trichomonas fetus, Leptospira pomona, Mycobac-terium tuberculosis, M.
paratuberculosis (Johnes disease) and viral agents that cause infectious bovine
rhinotracheitis (I.B.R.), Foot and Mouth disease.
Refrigeration greatly suppresses the multiplication of organisms, hut does
not necessarily stop it. Organisms which survive freezing temperature of -196oC
are Pseudomonas aeruginosa, Trichomonas fetus, Leptospira pomona, Foot and
Mouth virus and infectious Pustular Vul-vovaginitis virus.
Penicillin and Streptomycin are the two antibiotics widely used since 1950
and are still in use. They are found to be relatively harmless to sperm cells and
particularly in combination, inhibit broad spectrum microorganisms. 500 to 1000
I.V. of crystallin penicillin-G and 500 to 1000 micrograms of dihydro-
streptomycin per ml of extended semen is enough for routine use. procain
penicillin is toxic to the spermatozoa.
Antibiotics will nto completely eliminate corynebacterium phogenes,
Brucella, Trichnonas fetus, Mycobacterium, Pseudomonas and they will have no
effect on viruses, Rickettsie and on fungi.
Hence addition of antibiotics is not a 100% safety from pathogenic
contamination of semen. Semen from healthy bulls managed under full hygienic
environment and handled by an experienced hand is always preferred.
Methodology for Freezing Semen
1. Preparation of Diluent
Diluent is always prepared fresh, generally an hour before collection of
semen so that the medium gets stabilised. As has been mentioned earlier, Tris yolk
glycerol dilutor is the choice of preference for frozen semen. It is prepared by
mixing.
The diluent is kept ready in water bath at 30o C for further dilution. As far
as possible eggs should be fresh (not more than 7 days old) and stored in
refrigerator. Eggs should never be water washed since egg is covered by a thin
protective membrane which disappears on washing and thereby allows microbes to
enter into egg through the micro-pores on the shell.
2. Collection of Semen
The bull center should have a schedule to prescribe days for collection from
each bull. The usual practice is to collect semen from a healthy bull once a week
and twice from young bulls all of which should be sound, having good libido and
sexual behaviour.
Semen collections should be made during early morning hours before
feeding is done. The teaser cow should be well cleaned before she is brought to the
service crate. It is better to tie her tail with a rope on one side so that swinging of
the tail does not cause any obstruction in proper .mounting. The dummy should
not be taller than the bull to be collected.

96
The artificial vagina prewarmed with hot water and refilled to bring- the
inner temperature of 40o to 46°C_when ready for use. Half to two third capacity of
the A.V. should be filled with I warm water. The inflation of air should be done in
such a way that the opening of the A.V. should close, resulting in building out of
the inner lining ta form a. cushion. Insulation bag may be fixed on the graduated
glass tube to avoid temperature shock. .
During collection, A.V. should be held at 45° angle to the ground, so that
the long axis parallels ; the line of the penis. Excessive bending of the penis before
or during the thrust can distract and injure the bull. The A.V. should be handled in
such a way that the penis just touches the lining. . The condition inside AV should
equal to that of inside the natural vagina and this condition stimulates the
ejaculation. The vagina must never be pressed on the penis. Generally 15 minutes
of teasing and 1 or 2 false mounts will suffice.
3. Evaluation of Semen
Immediately after collection semen is evaluated for volume, colour,
consistency and presence of foreign matter. One drop of semen is placed on glass
slide and is evaluated under phase contrast microscope for initial forward motility.
Samples showing more than +3 are taken for further processing. From this, 0.1 ml
of semen is taken for estimating concentration of spermatozoa either by
photoelectric colgfimeter method or by haemocytometer connts. Till the final
dilution rate is decided, semen is pre-diluted to a known low volume of extender
and stored in water bath. The individual motility of the sperm is assessed with the
diluted semen using phase contrast microscope. Sample showing more than 60
percent of motility is taken for further processing.
4. Dilution of Semen
The basic idea of dilution of semen is (1) to preserve fertilising capacity of
the spermatozoa for a long period and (2) to increase services to a number of
females.
The dilution rate is decided based on the- motility rate sperm concentration
and capacity, of straw used. In general a minimum of 30 million motile sperms in
each dose is recommended. As-soon as the final volume of diluted Semen has
been determined the remainder of the diluent is added and the flask containing
diluted semen is gradually cooled to +5°C in a refrigerator over a period of 45 to
60 minutes.
5. Equilibration of Semen
Before storing the diluted semen at frozen state (-196°C), the practice is to
preserve it at much higher temperature (+5°C) for about 6 hours. This pre-freeze
storage of diluted semen is known as 'Equilibration of diluted semen. This is s6
done for the glycerol to bring about its beneficial action on the spermatozoa.
Glycerol permeates sperms and gives better resistance to withstand freezing stress.
During this equilibration period, filling of straws with diluted semen is
carried out inside a cold handling cabinet maintained at +5°C. Equilibration period
of 6 house is generally practised. At the end of equilibration period, the pre-freeze

97
motility is again recorded. Samples showing more than 60 per cent motility are
taken for "Test freeing". This is done by filling processed diluted semen rru2-ia 3
straws for each single which arc .initially kept either in big semen containers or in
mini-freezer firstly in liquid nitrogen vapour having a temperature between -120 to
-130°C and then plunged in liquid nitrogen. The entire treatment will take about
10-15 minutes. After this the revival rate is determined and satisfactory samples
are taken up for filling and further processings,
6. Different Packaging System of Frozen Semen
Packaging frozen semen in various single dose containers for storage and
.delivery systems started in U.S.A since 1954 where use of glass ampoules was
first developed. Japanese scientists developed a technique for freezing semen in
pellets. French, Danish and German were responsible for introduction of straw
technique. Other packing techniques such as pipettes, bulk sausages, gelatin
capsule etc. have been reported for frozen semen without any acceptance due to
high cost and lower fertility.
(I) The Ampoule Method
This method was mainly used in U.S.A. Semen is placed in glass ampoules,
sealed, frozen and preserved in liquid nitrogen. For insemination the ampoule is
thawed in warm water, ampoule.is : cut, semen drawn into glass catheter and used
as liquid semen.
Merits
(1) Since the semen is sealed inside ampoule, contamination during storage is
avoided.
(2) Identification of sample is possible as the bull no. etc. can be marked on the
ampoule.
Demerits
1. As the semen is frozen in a larger volume the freezability and fertility are
less.
2. It occupies more storage space in frozen semen containers.
3. About 8-10 per cent semen is lost while handling semen at the time of
thawing and insemination.
4. Use of glass catheters for Artificial Insemination has several drawbacks as
experienced with liquid semen. Hence this method is not popular.
(ii)The Pellet Method
Freezing is done by depositing 0.1 to 0.2 ml of semen on the depressions
created on the ice (solid CO2). The semen gets frozen and after minutes, the
pellets (like tablets) are collected' Tagoblet and stored, in liquid nitrogen at ^19&
C. At insemination a pellet is dissolved in 0.9 ml of warm diluents and used just
like liquid semen.
Merits:
(1) Economical method
(2) Occupies less storage space.

98
Demerits
1. Identification is difficult.
2. As the semen is stored uncovered they may get contaminated with organisms if
present in liquid nitrogen or other pellets.
3. When the pellets are handled by forceps they break and get attached to forceps
and results in loss of sperms.
4. The freezability is moderate.
5. Cumbersome procedure involving the necessity of seperate thawing solution.
glass rod for Artificial Insemination etc. Thus the potential for spreact of disease is
high and the pregnancy from wrong bull is not uncommon with this technique.
(iii) The Straw Technique '' Plastic straws were introduced in Denmark in 1940 for
packing liquid semen. The first technique for freezing of semen in straws using
liquid nitrogen vapour was developed by Adler in_l§60. These techniques were
later modified and refined by Cassou in 1965 and most of the procedures currently
employed with 'French straw' or French Straw' made of polyvinyl chloride was
introduced. In the year 1972 a plastic straw called 'mini tube' or German straws or
'Lanshut system' was developed in West Germany. A straw known as. U.S or
Coritinenial straws made of polypropylene was developed in United States.
Straw technique has got several advantages over pellet or ampoule
methods-and hence it is popular all over the world.
Merits:
1. Semen is processed in thin film, with its greater surface area to volume ratio,
there is rapid heat exchange and better revival rate.
2. Reduced volume of sernen is better tolerated by uterus. ,
3. The diameeter ATI7 catheter is small and hence passing cervix in heifers and
cows in late heat is easy.
4. Compjete delivery of semen into the uterus is possible.
5. identification of sample by colour of straw inning, colour of PVA sealing
powder or. coloured plastic" Beads is possible.
6. Use of steel gun is safer compared to glass catheter.
7. Use of disposable plastic sheath for each A.I. excludes spread of disease
during Artificial Insemination.
8. This occupies less storage space compared to ampoules.
9. With full automation for filling and sealing, it meets hygienic standards in
semen processing.
7. Printing of Straws
Before the diluted semen is filled into straws, the straws have to be lebelled
by printing the breed, name or no. of bull, date of collection, etc. by using an
automatic straw printing machine. The printing must become dry before the straws
are taken tip for filling.
8. Filling of Straws

99
Filling of straws can be done manually as well as by automatic machine.
Manual method is cheaper and easy, as such the method is described hereunder.
It requires vacuum pump, filling comb, rubber tube, straw clips and
polyvinyl alcohol (PVA) powder. The straws (15 medium or 20 mini) are clipped
together by using straw clips. The clipped straws are cooled to +5°C in the cold
handling cabinet. The straws in the clamps are fitted on the filling comb, which in
turn is fixed to vacuum pump through the rubber tube. Semen is taken in the bath
of the bubbler and filling is done by operating vacuum pump. Due to negative
pressure semen is drawn into the straws.
Appearance of two distinct bands in the factory seal indicates complete
filling of straws. An uniform air space is created in all the filled straws by pushing
the open ends of the straws on to the teeth of the bubbler.' The bubbler and the
bath are discarded after use is required in order to allow for expansion of the
semen during freezing.
In its absence the sealing will be pushed out by the frozen semen. Further
this air space also prevents contact of scissors with semen at the time of cutting of
straw before insemination, thus preventing possible contamination.
9. Sealing of Straws
The sealing powder, PVA is available in 10 different colours. The French
straws are also available in 16 different colours. The combination of both assures
160 positive identifications. The powder is spread on a clean glass dish to provide
a thickness of 4-5 mm. The open ends of filled straws are dipped into the powder
and when the powder penetrates 4-5 mm into the straw a satisfactory seal is made.
This is known as 'laboratory seal' which will appear as a single band. Immediately
after sealing, the straws are placed in water bath at +5°C for the equilibration
period to be completed. At the same time this allows the sealing to become firmer
and the excess powder on the ends falls to the bottom of water bath. The entire
operation of filling and sealing has to be done at +5°C within the cold handling
cabinet. At end the tubes are rolled and dried carefully with pre-cooled towels
since ice will form on damp straws and this will reduce freezability and storage
space.
10. Freezing the Straws in Liquid Nitrogen Vapour
Liquid Nitrogen vapour can be obtained from wide mouthed stainless steel
container (LR 320, LR 250) where the straws are also finally stored. For the sake
of economy and easy to work, the equilibrated straws after withdrawing from
water bath at +5°C (which were placed in a freezing rack) are now placed in a
mini freezer by holding in a horizontal position inside the straw racks at 4 to 5 cm
above the level of liquid nitrogen, there by exposing to the vapours of liquid
nitrogen. The temperature of semen reaches -130oC to - 150o by about 10-15
minutes. The main advantage of horizontal freezing is the efficiency and
simplicity of the method. The racks hold the straws at a constant level and the
straws are frozen at even rate all along the length of the straw.
11. Storage of Frozen Semen

100
Frozen semen may be stored at:
(i) -79°C by using solid CO2 (dry ice) and alconol.
(ii) -190°C by using liquid air
(iii) -196°C by using liquid nitrogen
(iv) -296°C by using liquid helium
Storage of frozen semen in liquid nitrogen is the most convenient/no1
accepted method all over the world. Soon after the temperature of straws reaches
about 140°C in Mini Freezer, the straws are collected by pre-cooled forceps and
finally transferred into pre-cooled goblets (a wide mouthed polythene tube like
container without handles to hold the straws filled-in with semen). Goblets
available in different capacities and sizes, e.g., bigger goblets hold 360 medium
straws, small goblets 100 straws and even smaller holds out 25 straws. These
goblets are then immersed in liquid nitrogen and kept in already identified
canisters, stainless steel container (with long handle) to hold goblets deep inside
liquid nitrogen of highly insulated double wall stainless steel wide mouthed ;
lemen containers (LR 320, LR 250).
12.Thawing of Frozen Semen (Melting)
Thawing means to melt or become liquid. Thawing of frozen semen should
be done immediately ' before use and as quick as possible to prevent
recrystallisation of the water into bigger crystals.
After thawing, frozen spermatozoa do not survive long as unfrozen semen
and also do not withstand freezing. Therefore, one must be certain that the semen
is going to be used soon once it has been thawed.
The time and temperature to be allowed for thawing depends upon size of the
packaging used. A universally adopted system does not exist. The most widely
practised temperature of 37-40o for 30 seconds is suitable to get optimum survival
of spermatozoa. Following steps should be taken shille thawing the semen.
1. The straw should be removed with the forceps from the liquid nitrogen.
2. The straw should be shaken vigorously once or twice to remove liquid
nitrogen fronxtfie cotton plug. .
3. Immediately after this procedure straw should be placed in warm water (37-
40oC) for only 30 seconds
4. Dry the straw with a tissue paper; also wipe the scissor.
5. Warm the chamber of the insemination gun by rubbing vigorously.
6. Hold the straw vertically with the cotton plug or ball downwards.
7. Cut the straw at right angles to remove the powder plug or sealed plug
through the air space.
8. Withdraw the piston of the syringe.
9. Place the straw in the warmed up chamber of syringe.
10. Take the sheath from the container and fix the sheath over the straw to
ensure the firm union between the straw and sheath.

101
13. Procedure for Loading the AJ. Gun
1. Identify the canister from which the desired semen is to be taken. Ascertain
the colour of the straw by reading of identification tag.
2. Remove the lid from the container, lift the proper canister upto the level of
the frost line. Never lift the canister above the neck level.
3. With a pair of tweezers grasp an individual straw and remove it, at the same
time lower the canister immediately back into the container.
4. With the wrist movement give one or two jerks to the straw to expel liquid
nitrogen trapped at end of factory seal.
5. Dip the straw into a clean waterbath at 37-40°C for 30 seconds. During this
period the entire straw must be completely submerged Twater bath. :
6. Remove the straw from bath, dry the straw with a clean tissue paper or
cotton. Inspect the straw carefully and discard straw with cracks or
defective seals. Semen must never come in contact with water.
7. Place the straw in the chamber of insemination gun. To obtain a perfect fit
with medium straw, it is essential that the laboratory seal is removed by
cutting at right angle through middle of the air space. The air space could
be brought to the top by gentle tapping of straw.
Make sure that the clipped end of straw has a straight clean cut with no
jagged edges. Straws cut at other angles or cut too short will result in back flow
and wastage of semen at the time of insemination.
8. Fit a sheath over the straw and the chamber of the insemination gun and
obtain a perfect fit between the extremity of the straw and the cone of the
sheath. Move the sheath about 1" up and check whether the straw follows
the sheath, if it is not correctly fixed it will not move upwards with the
sheath. Fix the sheath by locking the plastic "o" ring at the flange of the gun
with a rotating motion. Do not touch the sheath in the tip or middle portion,
handle only the base portion.
Sheaths are sterilized and packed in 50 or 40 numbers in a plastic packet.
The base of the sheath packet should be cut open by means of a small cut
made at an angle. The sheath packet should be kept inside a sheath
container made of aluminium and closed with rubber stopper on either side.
After removing each sheath, the container should be tightly closed to keep
it free from dust and contamination.
9. Extrude a tiny drop of semen to remove air bubble, to move the factory seal
and to ensure good fitting of the sheath
10. The post-thaw survival of spermatozoa is poor. For maximum reproductive
efficiency, thawed semen should be used immediately. So, do not thaw
more than one straw simultaneously.
Equipment Required for an Al programme
To simplify the list, all items of equipment are categorised under headings
appropriate to their specialised area of use. However, items of equipment which
are used in more than one area .are not necessarily duplicated in each section. It is

102
therefore important to refer to the whole list for checking equipment. The numbers
of items suggested only serve as a guide and should be modified according to
individual requirements.
A. Collection of Semen
1. AV outer casings (30 x 6 cm for young bulls, 40 x 6 cm for adult bulls, 20-
30 x 6 cm buffalo, 20 x 5.5 cm for ram and 15 x 5.5 cm for buck) at least 6
for each species.
2. AV liners and cones to suit above outer casings at least 12 for each species.
Genetic Markers Identified in the Bovine Species
Lactation
The Candidate Gene Approach
As reviewed by Bauman (1999) and Baldi (1999), administration of
recombinant growth hormone to dairy animals dramatically increases millk
production and demonstrates the critical role of the somatotropic axis in regulation
of lactation. Accordingly, the results presented in the next section focus on the
possible relationships between candidate genes, associated with the somatotropic
axis, and quantitative and qualitative milk production traits.

Somatocrinin (GHRH) and GHRH Receptor Polymorphisms. In the cascade of


events that control the pituitary GH secretion, the hypothalamic factor GHRH and
its receptor directly control the GH synthesis by the somatotropic cells. In 1995,
Moody et al. (1995a) described a restriction fragment length polymorphism
(RFLP) in the GHRH gene using the Haelll restriction enzyme. In a preliminary
study on 89 artificial insemination (AI) Holstein-Friesian bulls, we noted that the
rate genotype (AA, 7.7%) obtained after PCR-RFLP Haelll restriction enzyme
analysis is significantly favourable for fat percentage and fat yield. However, this
observation, this observation must be confirmed on a higher number of animals.
Recently, Connor et al. (1998) reported a polymorphism at the GHRH receptor
gene (located on chromosome 4) using Eco 571 enzyme that could modify GHRH
stimulation of the somatotropic cells (Connor et al., 1998).

Pituitary-Specific Transcription Factor (Pit-l) Polymorphism Biochemical and


ontogenetic studies have shown that Pit-l is the critical cell-specific transcription
factor for activating expression of the prolactin (PRL) and GH genes in the
anterior pituitary gland (Review in Tuggle and Trenkle, 1996). Pit-I has
subsequently been shown to be capable of activate other pituitary genes, including
the Pit-I gene itself, the GHRH receptor gene the gene coding for the β subunit
of thyroid - stimulating hormone (TSH) and the pituitary β 2 TSH receptor.
The regulatory regions recognized by PitI within the PRL, GH and Pit-I
gene promoters have been demonstrated to be required for appropriate pituitary-
specific expression of these genes both in cultured cell lines and transgenic
animals. In humans, different mutations of the Pit-I gent have been also reported

103
in patients with hypotituitarism or with sporadic combined pituitary hormone
deviancy .
Pit-I is a 201-amino-acid protein with a DND-binding POU domain which is
characteristic of a family of proteins, including Pit-I Oct-i, Oct-2, Unc-86 . The
POU domain is divided into two well-conserved regions. The C-terminal half,
called the POU domain is divided into two well-conserved regions. The C-
terminal half, called the POU-home domain, encodes a low-affinity DNA-binding
domain, whereas the N-terminal half, called the POU-specific domain, confers
high affinity to the POU domain and participates in protein-protein interactions .
In cattle, Woolard et al. (1994) reported a Hinft RELP in exon 6 of the Pit-I
gene which has been assigned to chromosome 1 (Moody et al., 1995b). Digestion
of the PCR products with the enzyme revealed two alleles: A (Hinfl-) and B (Hinfl
+). The frequencies of the three genotypes generated by the two alleles in a
sample of 89 Italian AI Holstein-Friesian sires were 2.2, 31.5 and 66.3% for AA,
AB and BB, respectively. In this study, fixed and mixed linear models fitted on
daughter yield deviations for milk and on directresses proofs for conformation
traits showed a superior effect of allele A for milk and protein yields, inferior for
fat percentage, and superior for body depth, angularity and rear leg set. Recently,
an additional study has been realized in our laboratory on 1100 Al Holstein bulls
commercialized by Semex Alliance (Canada) (unpublished data). In this sample,
the frequency of the AA genotype was higher then in the Italian bulls (9% vs 2.2).
The frequencies of the two other patterns were respectively 48% for AB and 42%
for BB patterns respectively. The statistical analysis revealed similar associations
as in our first study, except for fat yield, with a significant superiority of the A
allele on the B allete for milk yield, protein yield and for fat yield. These
preliminary investigations indicate that Pit-I is a promising new possibility to
select for increased protection and milk yield through selection for the A allele.
However, this hypothesis must be validated on a large number of animals and for
other breeds before definitive conclusion can be drawn.

Growth Hormone Polymorphis, Considering its essential role in lactation


process, the GH gene (associated to chromosome region 19 q 26-qter in bovine
(Hediger et al. 1990) is a poetical target for studies of molecular variation in
association with genetic merit of dairy breeds. Using different restriction
enzymes, numerous polymorphisms have been reported in the bovine species.
Due to an insertion of a T at position +837 and a C-G transition at position
+838, a Mspl RFLP is present in the third intron of the bovine GH gene observed
a difference a difference in allele frequencies at the GH loci between two Danish
and Norwegian lines, the Mspl (-) haplotype being more frequent in the line
selected for milk aft. Lec et al. (1993, 1994) found also a significant association
of Mspl (-) with higher fat yield in Holstein cows. Ina recent study, Lagziel et al.
(1999) found a significant increasing effect of Mspl (-) allele on protein
percentage and kg of protein per year. For these authors, the observed effects are

104
due to a locus in linkage with GH but at some distance of it. In contrast, for Yao
et al. (1996), who have detected this polymorphic site by the SSCP methods, the
Mspl(+) allele has been found favourable for milk/ fat and protein yield. In this
study, the authors found an other polymorphism, due to an A-to-C transfusion
which changes the codon AGG t CGG in the fifth exon of the egene, in linkage
disequilibrium with the Mspl mutation. for the Mspl (+/-) pattern, located in the
third intron, is associated with a 0.9 kb insertion / deletion in the 3' flanking
region, potentially carrying transcription regulator sites. For these authors, this
polymorphism does not result in differences of indicators for genetic merit in 100
Holstein Al sires.
In the bovine GH gene, there are also other variation sites that could be
interesting for association studies with mill production traits. Hecht and
Geldermann (1996) have shown 7 mutations in the GH gene. Six of them have
been identified in the 5' flanking region and one in intron I. Some of these
variable sites are also potential binding sites for trans-acting factors
(CAAT/enhancer binding protein, polyoma virus enhancer A binding protein 3,
thyroid hormone response element) and therefore possibly involved in the
expression of the growth hormone gene. Recently, Rodrigues et al. (1998), have
identified a polymorphic site in the promoter region of the GH gene. This
polymorphism consists in the absence of an AAG trnucleotide localized 9
nucleotides upstream from the TATAAA sequence. Unfortunately, in these two
last studies, the association between production traits and the polymorphism was
not examined.
Therefore, it appears that selection for milk production traits based upon
conflicting GH genotype data is not a promising way, The major problem to
definitely conclude on the opportunity to selected an GH genotype is often due to
the limited number of genotyped animals in the published studies.

Growth hormone Receptor Polymorphism The CDNA of the bovine Tran


membrane GHR has been sequenced. It consists of 9 exons and is associated with
chromosome 20. As deduced from the human nucleotide sequence, the GHR is a
protein of 620 amino acids comprising an extracellular hormone binding domain
of 246 amino acids, a single 24 amino acid transmembrane region and a long
cytoplasmic domain. The extracellular domin contains seven cysteine residues and
five potential N-linked glycosylation sites and two boxes important for the signal
transduction after binding of the hormone to its receptor.
After hybridization with a homologous cDNA probe containing the
coding sequence for the intracellular C-terminal part of the receptor, we have
identified six restriction enzyme Taql bands (7.1/ 6.2, 5.7, 5.4, 4.2 and 3.3 kb).
The effect of this polymorphism on values for milk protein percentage was highly
significant (P<0.005) and favorable for the rare (6.6%) 5.7 and 5.4-kb patterns.
Recently, Moisio et al. (1998) detected three variants in the 3' flanking
region of the bGHR : GHR311, GHR235. The longer alleles GHR320 and GHR325 are

105
more frequent in the selected Finnish dairy breeds, whereas the shortest allele
GHR311 predominates in the native breeds.
IGF-I and IGF-I Receptor Polymorphisms. Mapped on chromosome , the
IGF-1 gene showed a SnaBl polymorphism in the 5' flanking region (Ge et al.,
1997). However, in 152 Holstein bulls from select sires and A1 organizations,
PTAs for fat, Protein, and net merit did not tend to be significantly related to a
IGFI/SnaBl polymorphic Moreover, in the same study, it was reported that no
SnaBI polymorphism at IGF-I gene and Alul polymorphism at GH codon 127
interaction effects were detected. Moreover using the Single Strand
Conformational Polymorphism (SSCP) method, noted a significant difference in
allete frequencies in seventy-six genotypes Angus cattle from lines selected for
high or low blood serum IGF-I concentration that could influence the lactation
process. In 1996, have located the IGF-1 receptor on bovine chromosome 21.
Digestion of IGF-i receptor PCR products with Taql restriction enzyme revealed a
polymorphism with two alleles (A and B). However, these authors concluded that
usefulness of this IGF- receptor polymorphism in studies designed to identify
economically important quantitative trait loci in cattle may be limited because of
the low B allele frequency and because of the low B allele frequency and because
this allele is only present in Bos indices cattle. To our knowledge, no new
information have been published to validate or not this hypothesis.
THE POSITIONAL CLONING APPROACH
In order to generate linkage disequilibrium between the alleles of the QTL
and the genetic marker, most studies have crossed inbred lines to construct F1
individuals heterozygous for both loci. When this method is not a viable option
(in dairy cattle for example), alternative options exist. The progeny of a single
sire, heterozygous for a or more genetic markers may be analyzed. If a specific
sire is also heterozygous for a linked QTL, then the progeny of this sire that inherit
different paternal alleles will have different means for the quantitative trait. This
method, the 'daughter design', has been applied to dairy cattle data and extensively
analyzed. In 1990, Weller et al proposed an alternative 'granddaughter design', in
which sons of a sire heterozygous for the genetic marker are genotyped, while the
quantitative traits are analyzed using the granddaughter records. Although the
magnitude of the QTL effect measured in the granddaughter design will be only
half of the effect measured in the daughter design, the number of progeny
analyzed for quantitative traits can be much greater. For many population
structures, it is possible to genotype less than half as may individuals and still
obtain greater power with the granddaughter design than with the daughter design.
Using the granddaughter design, Andersson-Eklund and reported a linkage
between the amylase-1 locus and a quantitative trait locus influencing fat content
in milk. In an other report, the granddaughter designs was used to study
chromosome substitution effects associated with k-casein and β -lactoglobulin in
Holstein cattle. The most severe limitation on an analyses of this type has been the
lack of suitable genetic markers. Most previous studies on dairy cattle have used

106
blood groups, blood proteins and milk proteins. The number of such markers is
limited. Presently, two published linkage maps exist for cattle. That cover most
of the genome and contain a large selection of micro satellite markers. With the
availability of highly informative marker maps, it has recently become possible to
map quantitative trait loci underlying the genetic variation of multifactor trait.
Although micro satellite linkage maps with intervals of 5-10 cM are sufficient for
identification of loci with large effects on phenotype, characterization of loci with
smaller effects, such as QTL, require higher resolution.
IN the case of meat production, the GH gene was also considered as a
possible candidte gene. reported an association between a Taql RFLP
polymorphism and birth weight and shoulders width of calves. For the Taql RFLP
was associated with growth performance at the 7th and 13th moths of age for
Belgian Blue White bulls. In a recent study, presented an approach to evaluate
the support for GH1 gene as QTL for growth and carcase composition within the
context of genome-wide-map-based cloning strategies. To establish candidacy, a
bacteria artificial chromosome (BAC) clone containing a putative candidate gene
is physically assigned to an anchored linkage maps in order to localize the gene
relative to an identified QTL effect. Micro satellite loci derived from BAC clones
containing an established candidate gene are integrated into linkage map
facilitating the evaluation by interval analysis of the statistical support for QTL
identify. In their results, the author conclude that, wide the exception of ether
extractable fat and adjusted fat, there is no compelling evidence for association
between breed-specific Augus and Brahman GHI alleles and any of the growth,
carcse composition and quality characters. Nevertheless, this strategy defines an
efficient and cost effective approach for the positional candidate cloning of QTL
in domesticated livestock species. An other important finding in the area of meat
production is the positional cloning of the muscular hypertrophy (mh) gene
responsible for the double-muscling nature of the Belgian Blue White (BBW).
The doubled-muscling animals have leaner carcasses then those not doubled-
muscling and exhibit greater muscle (about 20%) mass with less fat. their
hyperplasia is due to an increase in the number of muscle fibers rather than the
individual diameter., In the case of BBW, the author reposted an 1-bp deletion
coding sequence for the bioactive carboxy-terminal domain of the myostatin gene
that inactivate the protein.

THE POSITIONAL CLONING APPROACH


An economically important use of markers in pigs was for the prediction of
the halothane (Hal) genotype. The halothane locus is the best studied region of the
porcine genime. This locus controls the so-called Porcine Stress Syndrome which
includes susceptibility to malignant hyperthermia and poor meat quality. In 1991,
Fujii et al. identified a C to T mutation at nucleotide position 1843 in the porcine
RYR gene. To day, an accurate DNA-based test is used. A successful MAS was
applied in Swede to reduce the incidence of the Halothane gene prior to the

107
identification of the causative mutation. A second major gene of economical
importance in pigs is the Rendement Napole (RN) gene that influences meat
quality. This gene has been mapped using a set of markers, represented by micro
satellites and blood protein. Recently, the RN loci is more accurately assigned to
the 15q24-q25 region by and confirmed. While geneticists are seeking the causal
mutation, the linked DNA markers identified so far, might be already, used in a
Marker Assisted Selection , reproduction traits meat procudtion. Finally, using
markers flunking the IGF-1 and GH locus, found a potential association of a QTL,
located in the interval between the markers and IGF-1, with average daily gain in
one family. No association was found for the GH locus.
Genetic Markers Identified in Chicken
The Candidate Gene approach
I chicken, the GH gene was also studied. Selection for feed conversion, eg
production or growth have been shown to affect GH and GH-receptor levels. The
GH gene is highly polymorphic disease resistance and egg production. the GH
allele co-selected with resistance was associated with a delayed onset of ovulation
but a higher persistency of ovulation a age progressed, resulting in an overall
increase of egg production by 15%. The resistance associated GH was dominant
for the onset of ovulation and recessive for the persistency of egg production.
The positional cloning approach
In F 1992, 100 RFLP loci were mapped in the chicken. As yet, 700 chicken
micro satellite markers are available (NAGRP, 1998). In their results, found a G
to A substitution in the NRAMPI (natural resistance associated macrophage
protein) gene that is specific to a S.typhimurium. On the contrary, presented a
possible associated between resistance to salmonellosis trait with a linkage group
on chromosome 5 using backcross populations form a cross between susceptible
and resistant inbred lines. To refine the location of the resistance gene, additional
markers to lie in this region were selected. To day, the gene on chromosome 5
affecting resistance to salmonellosis in chicken is still not found.

108
GENETIC ENGINEERING OF ANIMALS

INTRODUCTION
Genetic engineering of animals is becoming a reality as the later chapters in
this Volume will illustrate. Genetic engineering is the field of manipulating the
DNA of a cell or of an animal in order to alter the genetic information contained
within the organism's genome. The standard techniques of recombinant DNA are
used. A number of excellent textbooks are now available that explain in clear
fashion how to work with DNA to clone genes, to alter known genes, and so on.
One convenient aspect of DNA is that the same techniques can be used whether
the source of material is from humans, bacteria, yeast, plants, cattle, or chickens.
Essentially any DNA can be recombined with any other DNA.
My major charge is to examine the techniques for gene transfer into cells
and to look in detail at those techniques, useful for inserting genes into whole
animals (1). There are 4 well-established groups of techniques—viral (e.g., simian
virus 40), chemical (e.g., calcium phosphate), physical (e.g., microinjection), and
fusion (e.g., liposomes or protoplasts)—and one new procedure (i.e., retroviruses).
VIRAL TECHNIQUES
DNA viruses, such as simian virus 40 (SV40) with DNA as the nucleic acid
in their core, have been employed for several years as gene transfer vectors. An
adenovirus vector has recently been developed that will efficiently infect animal
and human cells (including hematopoietic cells) with the result that one or a few
copies of the recombinant virus are integrated into the host cell's genome. One
subcategory of DNA viruses should be mentioned: bovine papilloma virus. This
viral DNA replicates extrachromo-somally so that bovine papilloma virus-based
vectors may prove to be useful for maintaining genes in cells in a nonintegrated
manner. The advantage of DNA viruses is that they are effective and well-studied
vectors for gene transfer in plants, animals, and bacteria. The disadvantages are
that many of the most widely studied viruses can replicate within cells and some
can kill the host cell. In addition, they are much less efficient at transformation
than retroviruses (see below). Thus, because they are a potential hazard and they
have low efficiency, their wide-spread use in animals appears unlikely at the
present.
Another procedure actively used for insertion of genes into tissue culture
cells is calcium phosphate-mediated DNA uptake. The original procedure of
Graham and van der Eb (6) was modified by Wigler et al. (12) in order to insert
into the genome of; mammalian cells growing in culture a fragment of DNA
carrying one or more genes. A large number of different genes have been
transferred into tissue culture cells using this technique.
Transfection is carried out by pipetting a suspension of DNA, com-plexed
into small precipitates with calcium phosphate, onto a monolayer of cells growing
in a tissue culture dish. A number of protocols are used to increase the efficiency
of transfection in different cell types: for example, diethylaminoethyl dextran can

109
be employed instead of calcium phosphate or the cells can be shocked with
glycerol after 2 hours of incubation. The efficiency of the process varies with the
cell line. Under optimal conditions and very receptive cells (i.e., mouse L cells),
one cell in 10 to 10 can be obtained that has integrated and- expressed the
exogenous DMA. Because the-usual efficiency is 10 - to 10 , a procedure is
required or detect the occasional transfected cell. In other words, a gene must be
present that can protect the cell from a lethal selective agent that is added to the
incubation medium-ox. that complements a genetic defect [i.e., hypoxanthine-
guanine phosphoribosyl transferase (HPRT) or thymidine kinase]. The transfected
cell will survive while all others are killed. Attempts to obtain transfected cells
without selective pressure have generally been unsuccessful.
Transfection appears to work poorly in suspension cells, as, for example,
bone marrow cells: Efficiencies can only be estimated, but the value is probably
one cell in 10 or 10 . Using the powerful selection system offered by the mutant
dihydrofolate reductase gene (isolated from 3T6-R400 cells) that provides
exceptional resistance to methotrexate, Carr et al. (3) reported that the calcium
phosphate transfer technique can be successfully employed to obtain mouse bone
marrow cells that contain a functional exogenous DHFR, gene. The permanently
transfected cells can partially re-populate a lethally irradiated mouse. These results
support the studies of Cline et al. (2,4) who reported successful transfer of a
functional dihydrofolate reductase gene into the bone marrow of mice. However,
the presence of the dihydrofolate reductase gene has not been confirmed with
DNA hybridization studies and, until such experiments are reported, the efficiency
of the calcium phosphate procedure is uncertain.
The advantages of chemical techniques are that they are easy and inex-
pensive to perform, require no special equipment, and they do not require an
infectious agent, so they are very safe. The disadvantages are that uptake and
expression of exogenous DNA is low and they insert multiple copies of the
transferred gene linked head-to-tail. Thus, the low efficiency makes their use in
animals unlikely unless the procedure is coupled with a strong selective advantage.
PHYSICAL TECHNIQUES
Microinjection and electroporation are the 2 principal classes of physical
techniques. Electroporation, a relatively new procedure, is the transport of DNA
directly across a cell membrane by means of an electric current (9). It has been
used to transfer a variety of genes into a number of different cells including the
immunoglobulin K gene into B cells (10). Its potential for gene transfer into
animals is uncertain.
Microinjection has been used for a number or years and has advantage of
high efficiency (up to one cell in 5 injected can be permanently transfected).
However, the distinct disadvantage is that only one cell at a time can be injected.
Transfection of a large number of hematopoietic stem cells, for example, is not
feasible.

110
An area where microinjection has had spectacular success is in transferring
genes into fertilized mouse eggs (see Fig. 1). Gordon et al. (5) first demonstrated
that if plasmid DNA--is microinjected into one of the 2 pronuclei of a recently
fertilized mouse egg, and the ovum is then placed into the oviduct of , a
pseudopregnant female, -£he egg could develop into a normal mouse carrying..the
plasmid DNA.-in revery- fiell of its body. Furthermore, the injected DNA can be
transmitted to offspring in a normal Mendel-ian manner. Mice carrying an
exogenous gene in eheir genome are called "transgenic."
Hammer et al. (7) used this technique to partially correct a mouse with a
defect in its growth hormone production. By attaching a rat growth hormone gene
to an active regulatory sequence (specifically, the promoter that normally directs
the synthesis of metallothionein messenger RNA in mice), they obtained a
recombinant DNA construct that actively produces growth hormone in the
genetically defective mouse. Although the level of growth hormone production is
inappropriately controlled—that is, influenced by signals that normally regulate
metallothionein synthesis—these experiments do show that microinj ection can be
used as a delivery system that can put a gene into every cell of an animal's body.
The major advantage of microinjection of fertilized eggs is that transgenic animals
can be obtained. The disadvantages are that the procedure still results in many
eggs being damaged during the injection process, and that regulation of the
inserted gene is still not controlled well.
FUSION TECHNIQUES
These include procedures where a "container" of some sort is first loaded
with DNA and then the carrier is fused with the target cell. Well-studied systems
are liposomes (which are lipid vesicles), red blood cell ghosts (lysed and resealed
erythrocytes), and protoplasts (bacteria with their cell wall removed). The
advantages of fusion techniques are that they are somewhat more efficient than
chemical techniques, and they do not require an infectious agent so they are safe.
The disadvantage is that they are more complicated than chemical techniques. At
present it appears unlikely that they will be used in preference to other techniques
unless further work establishes that they are in some way superior.
RNA VIRUSES (RETROVIRUSES)
There are a number of advantages of vectors derived from retroviruses as a
gene delivery system. First, up to 100% of cells can be infected and can express
the integrated viral (and exogenous) genes ; this is in contrast to chemical methods
where, although most cells take,in the-DNA, as shown by positive assays after 48
hours, only one cell in 10 to 10 stably expresses the exogenous gene. (Second, as
many cells as desired can be infected simultaneously;") 10 to 10 is a convenient
number for a simple protocol. (Third, under appropriate conditions the DNA can
integrate as a single copy at a single, albeit random, site, whereas the chemical and
physical techniques often result in the insertion of multiple copies of the
transferred geneV all linked head-to-tail in tandem repeats. Fourth, although
integration is random with respect to the host genome, it is precise wi.th respect to

111
the viral genome—that is, the structure of the integrated DNA is known. Fifth, the
infection and long-term harboring of the retroviral vector usually does not harm
cells, finally, a wide and controllable host range is available. A number of
retroviral vector systems have been developed, the most common one being the
use of vectors based on Moloney murine leukemia virus.)
Life Cycle and Structure
The details of the life cycle of retroviruses have been reviewed recently
(11). In brief, the retrovirus, composed of an RNA-protein core and a glycoprotein
envelope, enters a cell where the RNA acts as a template for the reverse
transcription of the genetic information into a double strand of DNA. This DNA
can precisely integrate as a single copy, called a pro-virus, at a random location in
the genome of the host.
Although much has been learned about the regulatory features of retro-
viruses, uncertainties remain. Those features of the proviral structure that are
thought to be necessary for transcription and transmission of the viral genome are
(see Fig. 2): a long terminal repeat (LTR) Sequence on each end, containing
regulatory signals for initiating and terminating transcription; sequences required
for reverse transcription and others for proviral integration; short sequences (called
here, for short, r~ and r ) immediately adjacent to each LTR and necessary for
reverse transcription; the packaging sequence, called i|i in Moloney murine
leukemia virus, necessary for the viral RNA to be packaged into an infectious viral
particle; and the donor (D) and acceptor (A) splice sites.
Retroviral RNA is synthesized from the proviral DNA by the host cell's own RNA
polymerase. A portion of this RNA is used in the cell's transla-tional machinery to
synthesize the viral proteins that go into the final viral particles along with the
genomic RNA. These viral particles bud off from the cell and can then infect other
cells.
From experimental studies as well as from the existence of a number of
naturally occurring defective viruses, it is known that almost all of the regions
coding for viral proteins can be deleted and some or all of these sequences
replaced with other DNA. Once the viral genes are deleted, the retroviral vector
becomes defective. In order to obtain infectious viral particles, a cell harboring a
defective provirus must be infected with a "helper" virus, which carries all the
viral functions needed—that is, the genes for gag, pol, and env.
Use as Gene Delivery System
The proviral DNA for the desired retrovirus is isolated and inserted into a
convenient plasmid. The viral genes can then be replaced with the exogenous
genes of choice by standard recombinant DNA techniques. This construct is used
to transfect tissue culture cells (i.e., NIH 3T3 cells) by a convenient gene transfer
procedure (i.e., calcium phosphate). After infecting the cells with a helper virus
(such as intact Moloney murine leukemia virus), infectious viral particles,
possessing both the retroviral vector and the helper virus, bud off from the cells
into the surrounding medium. This particle-containing supernatant is collected and

112
used to infect bone marrow cells in culture or, more simply, freshly extracted bone
marrow is incubated directly with the cells budding the viral particles. The marrow
cells are removed and injected intravenously into a mouse whose bone marrow has
been killed by X-rays (lethally irradiated). The animal is then studied to determine
if the transferred marrow cells express the desired gene from the vector.
An improvement of this procedure is to treat bone marrow cells with a
retroviral particle that can deliver the vector but which will not itself produce a
spreading infection. Mann et al. (8) have developed a technique for accomplishing
this goal. The regulatory signal 1(1 (Fig. 2) contains a sequence, the exact .size
and structure of which are not yet known, that must be present in the viral RNA
.for it to be packaged into a viral particle. A helper virus was constructed with this
sequence deleted (4>~) by making use of conveniajjt Restriction endonuclease
sites (Ball and Psjtl) flanking the i|i sequenee._in Moloney murine .leukemia virus.
The 1(1 helper is able to produce all the viral proteins required to make a particle,
but the particle does not package its own RNA. Since the retroviral vector has a
sequence, it is packaged. Consequently, the particle can just infect once; it is only
a delivery system for the vector, not an infectious agent.
In order to use the i)Tahelper - virus conveniently, a line of N1H 3T3
cells._was established with the helper proviral DNA permanently integrated; i|i
helper viral RNA is produced constitutively. The transfection of this cell line
(called i|i-2) with the retroviral vector DNA results 48 hours later in a supernatant
that contains viral particles with only the vector. I will discuss in. a.subsequent
chapter (W.F. Anderson et al., this Volume) the use of this system to transfer
functional genes into intact animals.
Properties still needed for an optimal delivery system can be visualized. An
ideal delivery system not only would be stable but also would be tissue-specific.
When transferring a gene into the hematopoietic system, the isolated bone marrow
can be treated. But no other tissue, except skin cells, can be removed, treated, and
replaced at present. Since many viruses are known to infect city specific tissues
(that is, to bind to receptors that are present only on certain cell types), a retroviral
particle containing a coat glycoprotein that recognizes only hematopoieLli: sLem
cells would permit the retroviral vector to be given intravenously with little danger
that cells other than those in the marrow would be infected. Such specificity could
permit the transfer of genes into an animal's liver or pancreas, for example. One
problem, however, is that cell replication appears to be necessary for integration. It
would not be possible to infect nondividing brain cells, for example, as far as we
sow know.
The optimal system not only would deliver the vector specifically into the
cell type of choice but would also direct the vector to a predetermined
chromosomal site. Specific insertion into a selected site of a chromosome by
means of homologous recombination can be readily achieved in lower organisms
but appears to be a formidable task in mammals, whether retroviral vectors or

113
plasmid-based vectors are used. Present evidence suggests that homologous, site-
specific integration occurs at a very low level, when it occurs at all, in mammals.
CONCLUSION
All 5 types of gene transfer procedures discussed above are of use for
various types of tissue culture experiments. However, only 2 procedures,
microinjection and retroviruses, have proven to be effective in inserting functional
genes into intact animals. Microinjection is valuable for producing germline gene
transfer while retroviruses are at present only useful for somatic cell gene transfer
into bone marrow cells. Progress is rapid, however, and Improved techniques for
genetic engineering of animals will undoubtedly arise over the next several years.

114
MANIPULATION OF GENES IN VITRO AND IN VIVO
INTRODUCTION
The genetic engineering of animals Is by no means a new concept. Since
the beginnings of history, man has attempted to improve his livestock and
domestic animals through selective breeding. Such classical breeding schemes,
though successful in many aspects, are limited by at least 2 major constraints.
First, genetic information can be exchanged only between members of the same or
very closely related species. Second, these schemes depend on naturally occurring
mutations or variants that may arise at very-low frequencies.
Recombinant DNA technology holds the promise of overcoming both of
these limitations. Because recombination is performed in vitro rather than in vivo,
genetic information can be exchanged between completely divergent species.
Also, the ability to manipulate genes in vitro makes it possible to generate mutants
in days or weeks rather than in millions of years of evolutionary history. In this
brief chapter, I describe 2 types of gene manipulations that may in the future be of
special use in agricultural genetic engineering.
GENE REPLACEMENT
This method Involves exchanges between chromosomal and cloned DNA
sequences. The endogenous chromosomal sequences can either be completely
removed (a deletion) or they can be replaced by closely related sequences (e.g., a
point mutant) or completely unrelated sequences. A major purpose of such
replacement experiments is to determine gene function. Suppose that we think that
a given gene plays a critical role in some physiological function, e.g., growth.
Then obviously animals lacking this gene should show abnormal growth
characteristics. If instead they grow normally, then the original hypothesis is
disproven.
An example of a typical gene replacement experiment in the lower eu-
karyote Saccharomyces cerevisiae is shown in Fig. 1. In this experiment, we were
interested in determining the function of the gene CUP1 which encodes a small,
cysteine-rich, copper-binding protein called copperthionein. First, the CUP1 gene,
together with 5' and 3' flanking sequences, was cloned into a bacterial plasmid.
Second, all of the CUP I coding sequences (but not the flanking sequences) were
removed from the plasmid and replaced by the heterolbgous URA3 gene. Third,
the plasmid was digested with a restriction enzyme that cleaves in the flanking
sequences and thereby generates recombinogenic termini. Finally, the plasmid
DNA fragment was transformed into a ura3 recipient strain carrying a single copy
of CUPl. We anticipated that a double recombination event would result in
eviction of the resident CUPl sequences and their replacement by URA3
sequences. Gel transfer hybridization analysis of the DNA from URA3+
transformants confirmed this prediction. As expected, the resulting strains were
incapable of copperUiionein synthesis as judged by polyacrylamide gel electro-
phoresis of [ S]-cysteine-labeled proteins (1).

115
Because there has been considerable debate about the possible detoxifying
or homeostatic functions of metallothionein, it was of great interest to study the
physiology of these strains. Three major findings were obtained. First,
copperthionein protects cells against poisoning by excess copper in the medium.
Second, copperthionein feedback regulates the transcription of its own structural
gene; in the absence of copperthionein, there is a high, constitutive level of
transcription from an episomal CUPl promoter. We currently believe that this is
simply due to the ability of thionein to chelate intracellular copper that would
otherwise be available to activate transcription factors. Third, copperthionein Is
not required for normal growth, mating, sporulation, or germination. Surprisingly,
strains lacking copperthionein also accumulated normal levels of total cellular
copper and of the copper-dependent form of superoxide dismutase. From these
results, we conclude that the major function of copperthionein is to act as a
scavenger that maintains low levels of free intracellular copper. At high copper
concentrations this protects the cells against the CUP1 gene. Previous speculations
as to the role of thionein^in copper transport or enzyme activation are ruled out by
these results (1).
At present, gene replacement experiments have 'been conducted success-
fully only in yeast and bacteria, but the recent demonstration of low-level,
homologous recombination in cultured mammalian cells miy soon make it
possible to conduct similar experiments in animals. If so, a number of Important
applications can be envisioned. For example, it might be possible to manipulate
complex, feedback-regulated physiological systems, such as fat production, by
eliminating one hormone gene while amplifying another. It might also be possible
to improve the nutritional value of milk by replacing the endogenous gene with
one containing a more-desirable amino acid compositionIn this method,-?
transcriptional_.c6iitrol sequences from one gene are joined to structural
sequences from a second gene that encodes a desirable gene product. The fusion
gene is then Introduced" into a host tell that is capable of carrying out any post-
translational modifications required to render the gene product biologically active.
Ideally, the host cell should also be capable of prolonged, high-density growth.
Vectors containing the bovine papilloma virus replicon and metallothionein
promoter sequences have proven especially useful for the overproduction of
mammalian gene products. In a typical experiment, coding sequences for human
growth hormone were joined to mouse metallothionein-I gene upstream
sequences. The fusion was made so as to retain all of the metallothionein
transcriptional control sequences and the transcription initiation site, and all of the
growth hormone translated sequences, translation initiation codon, and 3'
translated sequences. When the fusion gene was introduced into mouse C127 cells
on a bovine papilloma virus vector, essentially all of the transformed foci
produced detectable levels of growth hormone. Moreover, the hormone was
appropriately modified in that the amino-terminal leader sequence was precisely
removed and the processed polypeptide was secreted into the culture medium. The

116
highest producers secreted as much as 100-200 mg/1 per day of mature hormone
and were capable of continued growth and production for up to 40 days in culture
(2).
The practical applications of this technology are obvious since essentially
any protein, including specifically altered polypeptides derived from mutant
genes, can be overproduced at will. Special interest will focus on products such as
animal growth hormones and fertility hormones.
PROSPECTS
Molecular biologists have developed a large arsenal of techniques to
isolate, analyze, recombine, mutate, and transfer genes. Nevertheless, we are still a
long way from realizing all the possible benefits from this technology in practical
areas such as agriculture. One important step will be to move from simple model
systems, such as yeast and cultured mammalian cells, to livestock animals. A
second will be to start studying the complicated questions of the effects of gene-
gene and gene-environment interactions on phenotypes. Clearly, these endeavors
will greatly benefit from interactions between molecular biologists and the more
traditional practitioners of animal engineering.

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STEM CELLS
Stem cells are unspecialized cells that renew themselves for long periods by
repeated cell division. These stem cell cab be induced under specialized
physiological and experimental conditions to become cells with specialized
functions. The two main types of stem cells that are used by scientists in research
are:

• Embryonic stem cells


• Adult stem cells
• Embryonic Stem Cells

As the name suggests, these stem cells are obtained from embryos. The
embryonic stem cells are basically derived from the embryos that develop from
eggs that have been fertilized in vitro ( in an artificial environment). Naturally
developing embryos are not used to derive the same. The embryos from which
human stem cells are obtained are typically 4-5 days old and bear resemblance to
hollow microscopic ball of cell called blastocyst. The blastocyst has three main
layers:

• Trophoblast that surrounds the blastocyst


• Blastocoel a hollow cavity
• Inner cell mass, a group of 30 cells concentrated at a pole.

Embryonic cells are grown in vitro in a process called cell culture. In the
process, the inner cell mass of bastocyst is transferred into a culture dish
containing a nutrient broth celled culture media. The inner surface of the culture
dish is coated with an adherent material (chemically treated cells from mouse in
most cases). This is to provide support for the growing embryonic cells and act as
substratum during advanced growth. This layer is called the feeder layer. Feeder
cells not only act as a substratum but also release nutrients into the media. This is
the reason why feeder-less cultures are being preferred owning to risk of
contamination and transmission of viruses from the feeder layer in case of mouse
cells.
When the embryonic cells reach a certain stage of growth, they start
crowding the plate and are transferred to a separate plate with fresh culture media.
This process is repeated and is called subculture. The subsequent cycles of
subculture is called a passage. After 6 months, the 30 odd cell of the inner cell
mass divide into millions of embryonic cells. The cells that continue dividing
without differentiating are called pleuripotent cells and form an embryonic cell
line. These cells are then frozen in liquid nitrogen and stored.
The process of characterization, which is a series of tests performed on
growing cells to check for signs of differentiation, is an important step. Some of
the prominent tests include physical observation of cells to check for

118
morphological changes and the use of surface markers found only on
undifferentiated cells. Loss of the markers indicates differentiation of cells. As
long as the cells are separated, they do not differentiate, but once allowed to clump
or aggregate, they form embryoid bodies and differentiate further to form various
other cells (muscle, nerve, etc.). The process of controlling the differentiation
process by means of altering media, or introducing genes into cells to force
embryonic cells to differentiate into specific organs is called directed
differentiation. This is one of the processed used in stem cell therapy procedures.
ADULT STEM CELLS
These stem cells are undifferentiated cells found among differentiated
cells, which yield specialized cells that produce various individual organs.
Adult stem cells maintain and repair tissues and organs in which they are found.
One of the striking aspects of these cells is that their origins are still unknown.
Adult stem cells are also called somatic stem cells.
In the past decade, studies have revealed the presence of adult stem cells
in many organs. There is also evidence about the ability of these cells to
differentiate into various type of cells. Interest in adult stem cells began with the
discovery that the bone marrow mainly contains two types of cells, the
hematopoietic cells that from all the blood cells of the body and the bone marrow
stromal cells. The stromal cells are responsible for production of bone, cartilage,
fat and fibrous connective tissue.
Adult stem cells, distributed sparingly in the body and along with stem
cells, remain quiescent for a number of years before being activated by disease or
injuries. The major adult organs that contain stem cells are the brain, bone
marrow, peripheral blood, blood vessels, skeletal muscle, skin and liver. Much of
the research in the areas is directed ways of culturing cells for therapeutic
purposes. A very important property of the adult stem cells is trans differentiation
of platicity. It is the ability of the adult stem cell from one tissue to differentiate
into another. Such kind of plasticity has been observed in case of bone marrow
and brain cells where cells have trans differentiated into muscle, bone and cardiac
cells.
In spite of accumulating data on these cells, many questions regarding them
are unanswered, such as: What is the source of adult stem cells? How do they
transdifferentiate? What are the signaling events that are responsible for the
process and can these cells be stimulated to grow in grow in large enough numbers
to produce fresh for transplantations?
APPLICATIONS OF STEM CELLS
This section details the different uses of stem cells in practical fields of
application.
ACADEMIC INTEREST
There are a number of areas that can benefit from the use of stem cells.
These cells offer vital clues to the entire process of differentiation and lead us
toward events that trigger the process. While signals that switch on and turn off

119
the genes are being probed, not much is known about the actual mechanisms of
these signals. Studies using stem cells are currently trying to unravel the mystery.
THERAPEUTICS
Another important area that benefits from the use of stem cells is that of drug
designing and testing. Potential drugs can be tested and screened fro using
differentiated stem cells. Cancer cell lines are already being used to test various
anti-cancer drugs. The challenge scientists face in this regard is to maintain
conditions and alter them sufficiently to stimulate the differentiation of cells.
The most important areas of application of stem cells are in the production
of vital organs and tissues. Currently, transplantations from donors are the only
means of therapy in certain cases of organ failure or tissue damage. This is,
however, fraught with difficulties of transplant rejection and lack of sufficient
number of donors. The ability to differentiate cells into particular organs or
tissues might offer hope to many such patients suffering from diseases including
Parkinson’s and Alzheimer’s diseases, spinal cord injury, stroke, burns, heart
disease, diabetes, osteoarthritis and rheumatoid arthritis. Scientists have
successfully transplanted bone marrow cell in the heart of mice and observed the
differentiation f cells into heart muscle cells while in some other quarters,
scientists claim to have successfully differentiated adult stem cells into insulin-
producing cells offering a treatment for diabetes.
SKIN TRANSPLANTATION
Burn victims can look forward to new hope since techniques involving stem
cells are currently being used in skin grafts as transplantation experiments. The
skin keratinocyte stem cells from hair follicles are being used for this purpose.
The patients’ own epidermal layer can be rejuvenated using this approach without
the risk of rejections.

BRAIN CELL TRANSPLANTATIONS


Recent studies negated the earlier concept that brain cells do not divide.
Growing evidences have isolated neural stem cells from embryos and adults and
have convinced scientists about the use of these in curing various ailments such
as, stroke, spinal cord injury and neurodegenerative diseases, such as Parkinson’s
disease, by neural stem cell transplantation.
CLINICAL APPLICATIONS OF HEMATOPOIETIC STEM CELLS
(HSCS)
HSCs are used in treatment of caners of the blood, such as leukemia an
lymphoma that result from the uncontrolled proliferation of white blood cells. In
these kinds of applications, the patient’s own cancerous HSCs are destroyed by
various therapies and the HSCs collected form a snatching donor are introduced
in the body. The curcial element in this procedure is the compatibility of the
donor, which in most cases are blood relatives. HSCs are also used in the
treatment of hereditary blood disorders, such as different types of anaemia and
inborn errors of metabolism.

120
A host of primary applications of HSCs are also used for the treatment of
autoimmune diseases such as, diabetes and rheumatoid arthritis. However, the
major hurdle that scientists face with such therapies are the life-threatening
instances of graft rejection by the host’s body. Research is currently on to
outfox the host’s immune system and in exploiting the property of plasticity to
differentiate HSCs into specific organs and tissues.
STEM CELLS AND GENE THERAPY
Non-embryonic stem cells have used in cell-based gene therapy studies. In
this approach, stem cells are removed from the body and genetically modified
cells are introduced instead. Modified stem cells act as carriers for the desired
gene that is to be replaced. The major advantage of such as approach is that stem
cells are self renewing population of cells and thus reduce or eliminate the need
for repeated administrations of the gene therapies. The only types of stem cells
that have been used in gene therapy are the HSCs.
ETHICAL ISSUES IN STEM CELL THERAPY
Stem cell therapy has raised some vary important question in ethics and
legal practice. Some, of these questions are discussed in this section.
It is permissible to create life with an objective to destroy it ?
This is one of the questions that have been raised by the academic and
ethical communities across the world. Even as the potentials of stem cell based
therapies emerge, there have been growing debates about the ethics on this issue.
While religious heads across the world have deplored the used of embryos for
stem cell culture branding it as “,murder”, scientist are divided on the issue of
whether an embryo uis animate or not.
Is it justified to seek human eggs for scientific research?
The biggest source of stem cells is human eggs that are raised in vitro.
Since the average number of egg cells that a normal women can produce is less,
various chemical stimulants are used to stimulate the production of a large number
of eggs. This is fraught with side effects and the morality behind such unnatural
procedures is also being debates. In many cases, patients undergoing in vitro
fertilization treatment are unsuspecting victims of such unnatural procedures is
also being debated. In many cases, patients undergoing in vitro fertilization
treatment are unsuspecting victims of such procedures, and the eggs that are
produced are harvested without their consent. This trend is prevalent in Third
World countries where the awareness levels are low and legislations are practically
non-existent.
PROTECTING DONOR IDENTITY
The issue of protecting the donors’ identity is an extremely sensitive one
considering the fact that these donors could be targets for the media and antistem
cell groups. Stem cell therapy depends heavily on the availability and willingness
of healthy donors. Failure to safeguard their interests would only result in
reticence on the part of the donors, which would ultimately stifle the entire field.

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APPLICATIONS OF BOVINE CLONING
POTENTIAL USE IN BREEDING SCHEMES
In cattle, most of the genetic improvement is achievement is achieved
through Artificial Insemination (A1) from selected bulls and embryo transfer
from selected bull dams to generated the next generation of A1 bulls. However
the diffusion potential from highly valuable bulls can be impaired by accidental
death of the progenitor. Somatic cloning allows to secure this risk by generating a
genocopy of the bull once it has been proved to be superior. Recent work in Italy
has shown that a live offspring could be obtained from an aged Al bull using
lymphoid blood cells as a source of nuclei.
Female cloning could also be a very useful tool to improve annual genetic
gain in conventional dairy breeding schemes that are already using intensive
embryo transfer. Cloning of female embryos recovered form elite cows mated to a
donors for the genetic programme could be chosen. If we consider milk
production trait (heritability H2=0.25), calculations indicate that the precision for
genetic evaluation of a clone of 5 females is an high as that of a bull evaluated
from the lactation of at least 25 daughters. Furthermore, the precision of
evaluation is increased by the fact that the performances of one given genotype are
evaluated through several copies of this genotype that are placed in different
environmental conditions. Calculations by indicate that the use of somatic cloning
instead of embryo cloning from elite cows could improve by 20% the annual
genetic gain provide that set of 3 to 5 cloned heifers could be obtained from each
selected donor. In this situation, adult somatic cloning would result in a higher
annual genetic progress because the first lactation of the bull dam donor of
somatic cells is already .
Cloningh could also be used for diffusion of some elite genotypes that are
well characterized and have interesting genes such as those involved in longevity
or disease resistance. A good example of such application is provided by the
work. They established a primary cell line from mural granulose cells collected by
aspirating the ovarian follicles from a Frieasan dairy cow of high genetic merit,
using ultrasound-guided Ovum Pick Upvaginal probe (OPU) and demonstrated
that it was possible to obtain at least 10 cloned genetically identical to the adult
elite donor cow.
Table 4. Possible impact of cloning on annual genetic gain in dairy cattle.
Origin of donor cells Age of female Nb of calves per Annual genetic
donors clone gain*
Embryos 4 years 3 +5%
5 +10%
Fetal fibroblasts 4 years 3 -3%
5 +1%

Adult fibroblasts 2 years 3 +21%


5 27%

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comparision with standard breeding scheme involving embryo from 2 years old
females.
In beef breeds, potential applications for cloning do exist, breeding
schemes have to consider criteria of meat quality. Such measurements could be
made directly on the carcass of clones of a selected bill and not on his offspring.
This would also save time in the selection programme by allowing to multiply
very valuable phenotypes. That is the case in Japan where there is a shortage of
top quality meat with specific infiltration of fat in the muscle. As it is known that
in the same breed, the carcass quality is a highly variable trait, adult cloning from
the best phenotypes of the breed could contribute to standardize top quality meat.
Therefore, different groups are activity developing nuclear transfer in cattle in
order to fit this objective.
CLONING FOR ENDANGERED BREEDS
If used on a very large scale, somatic cloning could lead to a potential risk
of decreasing genetic diversity. This could happen if thousands of the same
animal were diffused in livestock which is far form being is not the case. On the
opposite, somatic cloning could be a valuable tool for maintaining and developing
endangered breeds of cows or other species. Provided seman from different bulls
is stored, a very limited number of females cab be multiplied by cloning and then
further inseminated by the stored seman in order to re-create different families and
re-establish a minimum of genetic diversity. In New Zealand, somatic cloning
was used to preserve the last surviving cow of the Enderby island cattle breed and
at least 4 calves were generated from this single surviving female. This
application f cloning has to be considered right possible to store the genetic
material from endangered breeds after a very small skin boilpsy of the live animal.
multiplication of the cells in vitro and freezing of the cells in liquid nitrogen for a
further use when nuclear transfer efficiency has improved.
BOVINE CLONES AS ANIMAL MODELS
The generation of genetically identical offspring from the same parent
animal can provide a valuable tool for agronomical research. In cattle, sets of
cloned calves cab be sed a animal models for experiments on pathology studies,
nutrition behavior or reproduction evolution. In France, at INRA, we have
developed such an approach to minimize the number of cattle that are required for
an experimental project can be minimized. For example, feeding behavior as
assessed by quantity of food intake, number of daily meals or rumination time,
indicate that the coefficient of variation for the criteria is significantly reduced
between cloned animals compared to control non cloned animals. Fore
reproduction studies, the developmental ocyte competence can be studied through
repeated owum pick up and IVF on a limited number of cloned heifers.
At the laboratory leval, in vitro development of nuclear transfer embryos
derived from differentiated cells fused to enucleated oocytes provides a valuable
tool to investigate nuclear reprogramming. The interactions between the oocyte
cytoplasm components and the nucleus can be studied in relation to their

123
respective cell cycle stage, in order to define the optimal conditions for
development up to the blastocyst stage. The most important point is to avoid any
DNA damage and to maintain a correct oocytes as recipients for a variety of donor
nuclei from other species opens the way to better understand the species-
specificities in early development. Trans-species nuclear transfer would have
potential clinical application for human medicine by allowing the generation of
human embryonic stem of the ethical issues associated with the use of human IVF
embryos to generate human therapeutic cell line.
ARE CLONES IDENTICAL?
Genetically identical animals obtained by nuclear transfer have the same nuclear
genome but may present some phonotypical differences due to epigenetic
phenomena. The most visible of these differences concern the cost color. For
example a clone of Hholstein breed calves may show black and white spotting
differences. The black color is due to the presence of a pigment (melanin)
contained in melanocytes. These melanocytes are derived from stem cells,
melanoblasts located in the neural crest. The migration of melanocytes towards
hair follicles is determined genetically but also depends on fetal growth (Seidel,
1985). In cattle, cloned embryos derived from the same cell source are usually
transferred to the uterus of different recipient females which provide different
uterine environment for fetal growth that can partly explain different migration of
melanocytes.
Furthermore, during the nuclear transfer procedure itself, the recipient
cytoplasms are prepared from oocytes of different genetic origin. These recipient
cytoplasm contain mitrochondrial DNA and maternal transcripts. Meanwhile the
mitochondrial DNA sequence is very limited compared to the bovine nuclear
genome, it is more susceptible to mutation and its role is important for the energy
metabolism of the cell. During nuclear transfer, differential mitochondrial
genotype segregation can occur within the same clone but in cattle, the factors
affected by this cytopolasmic inherence remain to be determined.

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Embryo transfer in large domestic animals
The term 'embryo transfer', taken literally, refers solely to the collection Of
an embryo from a donor animal and its placement into the uterine tube or uterus of
a recipient. However, by common usage, it has become accepted to cover a whole
range of allied techniques, including super ovulation of the donor, and storage and
manipulation of embryos in vitro.
The first successful embryo transfer was carried out over 100 years ago in
rabbits but it was some time before the technique was successfully applied to farm
animals produced the first lamb by embryo transfer, but despite intense research
effort, the first calf was not born until 1951 Even then, it was not until much later
that the technique had advanced sufficiently .to be of practical use in cattle
breeding . Since then, embryo transfer has been used successfully to increase the
reproductive rate of cattle, horses, sheep, goats and pigs.
Embryo transfer has been applied most extensively in the cow;
consequently the technology has advanced most rapidly in this species. In the
early 1970s, general anaesthesia and laparotomy were necessary for both recovery
and transfer of bovine embryos, and embryo transfer was used in the UK and
North America mainly for the rapid multiplication of imported exotic beef breeds.
With the advent of efficient non-surgical techniques for recovery of embryos, and
effective methods for preserving embryos in liquid nitrogen in the latter part of the
decade, demand for embryo transfer services increased dramatically in both the
beef and dairy industries.
According to figures collected by the European Embryo, Transfer
Association, the total number of bovine embryos transferred in the EU in 1997
topped 118 000, and the trend continues upward. In excess of 23 000 of those
transfers were carried out in the UK, although the current depression in UK
agriculture is likely to result in a significant downturn in these figures over
subsequent years. The majority of embryos are collected and transferred on the
same farm, but national and international trade in frozen embryos contributes
significantly to these figures. :
The commercial application of embryo transfer has been much more
restricted in the domestic species. A reluctance on the part of the breed
associations to register progeny produced by embryo transfer has partly been
responsible for this in the horse, and economic factors, together with the need for
surgery, have militated against widespread use in pigs, sheep and goats.
Applications of embryo transfer;
Over the years embryo transfer has been, and continues to be, a valuable
research tool. It has been used exclusive]}: in studies of uterine capacity, on the
uterine environment, the maternal recognition of pregnancy, embryo-uterine
relationships and endocrinology of pregnancy. Embryo transfer has also been μM
in disease transmission studies and to investigate the genetics of reproduction: for
instance, litter size, gestation length, birth weight and postnatal production. The

125
rapid development of new technologies is now expanding the scope of embryo
transfer in research.
The production of identical twins and clones will accelerate progress in
many fields of research, and the ability' to manipulate fertilisation and modify the
genome of the early embryo will advance the frontiers of knowledge. However,
the most practical application of embryo transfer today depends on its capacity to
increase the reproductive rate of female animals. The rapid uptake of embryo
transfer by cattle breeders, in particular, has depended on the ability to increase the
number of progeny from valuable brood cows, either as a means of rapid herd
improvement or to produce surplus embryos, pregnancies or stock for sale.
Genetic improvement
The rate of genetic improvement within a breed depends on four variables:
the amount of genetic variation for the traits under question, the accuracy with
which the parents of the next generation can be selected, the selection intensity
and the generation interval. Embryo transfer can be used to influence all four
variables and improve rates of progress.
Genetic variation
This can be increased by introduction of a breed genetically superior for the
desired traits. Embryo transfer can be used both to introduce the breed in question
through the medium of frozen embryos, and to increase the reproductive rate of
resulting females to facilitate its rapid distribution.

Selection of dams
The breeding value of females can be calculated from their own
performance and conformation data, together with that of close relatives. The
accuracy of the calculation depends on the numbers of relatives available for
recording, and embryo transfer can be used to increase numbers in species with a
low reproductive rate, allowing, for instance, sibling or progeny testing of cows.
Selection intensity
In dairy cattle, the majority of female offspring in a herd are needed as
replacements to produce the next generation. Using embryo transfer to increase the
reproductive rate of the best cows, selection can be restricted to the top 5-10% of
females. Similarly, in a national bull selection programme the use of embryo
transfer would allow the proportion of bull dams selected to be reduced from, say,
2 to 1 %, by ensuring that a bull calf was produced from virtually every mating.
Generation interval
A method for dairy cattle improvement using embryo transfer intensively
on selected individuals within one nucleus herd has been proposed by Nicholas
and Smith (1983). Sets of full and half siblings, within this type of scheme, can be
recorded for the traits in question in a uniform environment, and the selection of
males and females to produce the next generation can then be made on the basis of
sibling testing rather than progeny testing as in conventional improvement
schemes. Breeding programmes based on this system have become known as

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MOET (multiple ovulation and embryo transfer) schemes, and have been applied
in practice in dairy and beef cattle and sheep. The practical application of MOET
in dairy cattle has been described.
This approach allows a dramatic reduction in the generation interval, and
consequently allows the opportunity for more rapid genetic improvement than can
be achieved with the application of a traditional progeny-testing system.
Genetic screening
Embryo transfer has also been used to expedite the screening of both dams
and sires for genetic defects such as syndactyly in cattle.
Disease control
There is increasing evidence to suggest that embryos are unlikely to spread
viral and bacterial diseases when transferred into recipients. The zona pellucida
would appear to be an effective barrier to infection of the embryonic cells from the
uterine environment, and washing of embryos 'or treating with trypsin has been
shown to remove viral contamination from the zona pellucida in vitro cites data
from several authors. For example, 407 embryos transferred from enzootic bovine
leucosis-seropositive donors have resulted in no seropositive recipients or calves.
Similarly, 67 embryos transferred from blue tongue virus-infected donors and 62
embryos (trypsin-treated) transferred from infectious bovine rhinotracheitis virus-
infected donors resulted in seronegative recipients and calves. Embryo transfer has
also been effectively used for the introduction of new blood lines into specific
pathogen-free pig herds. Sufficient transfers have been conducted with embryos
from bovine leucosis, infectious bovine rhinotracheitis, blue tongue (cattle),-
Brucella abortus (cattle), foot-and-mouth disease (cattle) and pseudorabies (pigs)-
infected donors to determine that these microorganisms will not be transmitted via
embryos, provided they are washed properly (Stringfellow and Seidel, 1998). As
the results of current and future experimental work become available, it is likely
that similar conclusions will be drawn for other pathogens. However, more field
trials will be necessary before the risk of disease transmission by embryos can be
fully assessed. For instance demonstrated the presence of bovine viral diarrhoea
(BVD) virus antigen within follicles and oocytes of persistently infected cattle,
thereby throwing some doubt on the effectiveness of embryo washing for
prevention of transmission of BVD via embryo transfer.
It is also likely that the risk of disease transmission through the transfer of
in 'vitro-produced embryos is greater than in those produced in vivo. The use of
abattoir-derived cells for co-culture in some production systems increases the risk.
There may also be differences between in vitro and in vivo-produced embryos that
affect risk. For instance, demonstrated differences in the zona pellu-cida between
embryos derived from the two sources. It seems, therefore, that conclusions drawn
from research involving in vivo-derived embryos cannot necessarily be
extrapolated to those produced in .vitro.

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Import and export
The development of efficient methods for the cryopreservation of embryos
of the cow, sheep and goat have stimulated a growing international trade in genetic
material. Economy and convenience have been major considerations, but many
governments have now made import regulations for embryos less stringent than
those for live animals or semen, in recognition of the relatively lower risk of
introduction of disease by embryo transfer. An additional advantage of embryo
transfer in this situation lies in the fact that a calf resulting from an imported
embryo transferred into an indigenous recipient acquires colostral immunity to
local diseases, and consequently may thrive better than an animal imported on the
hoof.
Circumvention of infertility
Embryo transfer techniques have proved valuable in the diagnosis,
treatment and circumvention of certain types of infertility in cows Careful
screening of donors is necessary to ensure that the infertility is due to injury,
disease or senility and is not of genetic origin; otherwise reproductive problems
could be propagated.

Twinning in cattle
Studies have shown that the efficiency of beef production from suckler
herds could be increased by twinning in intensively managed units.
Genetic selection for twinning has largely been unsuccessful and
gonadotrophin treatments to increase ovulation rates are not reliable. Twinning by
embryo transfer, by transfer of either two embryos or one embryo to a previously
inseminated recipient, is a practical alternative. The relatively high cost of embryo
transfer has precluded practical application in the past. However, the technology
of in vitro fertilisation applied to oocytes aspirated from abattoir ovaries
dramatically reduced unit costs per embryo, opening up possibilities for
commercial application of twinning to improve beef production. However,
pregnancy rates achieved in the field have not been good enough for the technique
to be economically viable.
Conservation
Embryo transfer can be used to increase the population of rare or
endangered breeds or species, provided there are recipients of a more plentiful
breed or species that will accept the embryo.
Superovulation
Single embryos can be recovered and transferred to other cows 6-8 days
after service at natural oestrus, but because of the high costs involved, this is not
usually an economic procedure in the practical situation. Consequently, a critical
aspect of embryo transfer technology is the use of gonadotrophins to induce
multiple ovulations in the ovaries of the donor cow (superovulation). For optimum
response, gonadotrophin treatment is initiated on days 9-14 (oestrus = day 0) of a
normal oestrous cycle, coinciding with the emergence of the second follicular

128
wave. Prostaglandin is administered 48-72 hours later to cause regression of the
mid-cycle corpus luteum and induce oestrus, which usually occurs 40-56 hours
later. Behavioural manifestations of oestrus are usually normal, and it is common
practice to inseminate donors on at least two occasions 12-18 hours apart when
using frozen semen as ovulations may occur over a prolonged period of time. The
superovulated donor would appear to be a sensitive indicator of the fertility of
semen (Newcomb et al., 1978a), and only bulls of high fertility should be used.
EMPRYOTRANSFER IN URGE DOMESTIC ANIMALS
Several different gonadotrophins have been used to superovulate cattle and
these include equine chorionic gonadotrophin (eCG), pituitary follicle-stimulating
hormone (FSH) of porcine (Elsden et al., 1978), equine or ovine origin, and
human menopausal gonadotrophin (hMG).
eCG has a longer biological half-life in the cow than either FSH or hMG;
consequently, a single injection of 2000-3000 IU will induce superovulation. FSH
and hMG require a multiple injection treatment regimen for optimum effect; for
instance, porcine FSH is usually administered twice daily for 4-5 days. The long
half-life of eCG can be a disadvantage, as its effect persists even after the induced
oestrus, and in some cows embryo transport is adversely affected. This is manifest,
over large numbers, by a poorer recovery rate of embryos after superovulation
with eCG, compared with other gonadotrophins. There is evidence that an eCG
anti-serum administered at oestrus will improve results . The presence of sub-
stantial luteinising hormone (LH) activity in eCG, and in crude FSH preparations'
used for superovulation, can adversely affect the viability of some ovulated
oocytes by causing premature maturation . Fertilisation failure has also been
attributed to abnormalities of oocyte maturation , and to asynchrony between
maturation of the oocyte and the follicle. The problems are compounded by
deficiencies in sperm transport in superovulated animals, resulting in reduced
numbers of sperm in the uterine tube at the time of fertilisation. There is some
evidence that the use of the more purified FSH preparations now available for
superovulation will improve fertilisation rates and embryo quality.
Donor cows can be superovulated repeatedly at approximately 6-8-week
intervals with no adverse effect on subsequent fertility, but ovarian response to
superovulation treatment is very variable, both between animals and between
treatments of the same animal. With experience, variability can be reduced by
adjusting dose rates, but this still remains one of the problem areas of embryo
transfer, with some donors yielding no embryos and occasionally 30 or more being
recovered. One of the sources of variation would appear to be the presence or
absence of a dominant follicle, as the former has been shown to depress response
to superovulation. Removal of the dominant follicle prior to the start of FSH
treatment - for instance, by aspiration - can improve response in some cases.
Further progress in the understanding of ovarian follicular dynamics in cattle may
be the best option for improving superovulation treatments in the future.

129
Collection of embryos
In the cow, the egg usually enters the uterus on day 4 after oestrus, at which
time non-surgical embryo recovery becomes feasible by flushing the uterus
through the cervix. Collection attempts are usually made on day 6, 7 or 8 after
oestrus, but recovery and successful transfer are possible up to day 16.
There are several methods of non-surgical embryo recovery in use, but the
commonest fall broadly into the types depicted in Figures 35.2 and 35.3. The
earliest reported method was the variable-distance three-way but the fixed-distance
three-way is the most common technique in use in the UK. This method has the
advantage of a continuous flow of medium within the distal third of the uterine
horn, and a consequent efficient flush of the region of the horn where the majority
of early-stage embryos are situated. The ebb and flow two-way technique is
simpler, but requires larger volumes of flushing medium and is more time-
consuming. In non-superovulated cattle, a skilled operator using these techniques
can recover an egg in six or seven out of 10 attempts. The results that can be
expected from superovulated donors are summarised.
Embryos are located under a stereoscopic microscope after settling and
siphoning or aspiration of the flushing medium, or more commonly, after filtering
through a commercially available embryo filter to concentrate the embryos in a
small volume of medium. A modified phosphate-buffered saline (PBS) is
commonly used both for flushing the uterus and for storage. Embryos can be kept
in PBS on the bench for at least 8 hours with no loss of viability, and can be
cultured for up to 48 hours with acceptable results on transfer. It is also possible to
cool embryos to +4°C and maintain them in a state of suspended development for
up to 3 days, or store them long-term by deep-freezing Day 7 bovine embryos are
about 150-190 |im in diameter, and are still within the zona pellucida and at the
late morula or blastocyst stage of development.They can be handled easily using a
micropipette, and are evaluated under the microscope at 50-1 OOx magnification.
An assessment of viability can then be made by taking into account the stage of
development relative to age, and the appearance of the cells. Embryos are usually
classified as good, moderate or poor in quality, and this can be related to
pregnancy rate on transfer.
Transfer
Many factors will affect the suitability of a recipient for embryo transfer.
The animals used should be healthy, fertile heifers or young cows that are in good
body condition, and can be reasonably expected to calve normally at term.
Nutritional status should be good, and ideally the recipient should be on a rising
plane for at least 6 weeks before and after transfer to achieve optimum results.
It has been shown conclusively that the oestrous cycle of the donor and
recipient should be closely synchronised if transferred embryos are to survive. An
asynchrony of more than 24 hours results in a marked reduction in pregnancy rate;
it is more economic to freeze and transfer later, when suitable recipients are avail-
able, than to step outside these limits. Of great importance too is the side of the

130
transfer. Pregnancy rates are greatly reduced unless the embryo is placed in the
lumen of the uterine horn on the same side as the corpus luteum.
Embryos can be transferred either surgically or non-surgically. For surgical
transfer, the uterus is exposed through a flank incision under local anaesthesia. A
puncture is made with a blunt needle and the embryo transferred to the lumen
using a fine pipette or catheter . Under controlled conditions, a pregnancy rate of
approximately 70% can be consistently achieved with embryos transferred on the
same day as recovery.
Similar equipment to that used for artificial insemination can be used for
non-surgical transfer, but stricter asepsis must be observed and the embryo is
usually placed some distance into the appropriate uterine horn. Success appears to
be skill-related, suggesting that trauma to the endo-metrium may be a limiting
factor with this technique. However, experienced and dextrous individuals can
achieve a pregnancy rate approaching that of surgical transfer, and as a
consequence welfare considerations must mitigate against continued use of the
surgical technique. In practice, non-surgical transfer has almost totally superseded
the surgical method.
TRANSFER IN SHEEP AND
Embryo transfer techniques are well established in sheep, mainly because
the ewe has been used extensively in research as a low-cost model for the cow.
Commercial use of the technique in sheep has not been widespread, with rapid
multiplication of recently imported breeds and MOET schemes for breed
improvement being the commonest applications. The use of embryo transfer in the
goat rapidly expanded in the late 1980s, in line with the increased demand for
valuable purebred Angora and Cashmere stock in the UK and Australasia, but has
subsequently decreased to a low level.
Techniques for superovulation, embryo recovery and transfer are very
similar for both species. The gonadotrophin preparations used are the same as
those used in the cow, and they are administered in similar treatment regimens
Gonadotrophin treatment is usually initiated mid- to late cycle and prostaglandin
F2 or analogues is administered 24-72 hours later, inducing oestrus within 24-36
hours. Oestrus and ovulation can also be controlled by progesterone or
progestogen administration in the form of injections, implants or vaginal sponges.
Sheep are treated for 12-14 days and goats for 14-18 days; using this method,
superovulation can be induced outside the breeding season.
Insemination is commonly achieved by natural service or artificially, using
freshly collected semen. However, fertilisation failure can occur commonly in the
ewe, particularly when the ovarian response to goriadotrophin is high. This can be
overcome by surgical insemination directly into the uterus, either by laparotomy or
by a laparoscopic technique.
Surgical techniques for the recovery of embryos from the ewe and the doe
have changed very little since the first reports and involve general anaesthesia,
midline laparotomy and flushing of the catheterised uterus and uterine tube. Non-

131
surgical recovery in the ewe has been reported, although the tortuous nature of the
cervix makes catheter passage very difficult. Laparoscopy has been shown to be as
effective as laparotomy and is now widely used. The tran-scervical passage of
catheters is much easier in the doe, and non-surgical techniques could be more
successful in this species.
Collections are normally carried out 3-7 days after oestrus, and embryos
can be evaluated and handled in the laboratory in a similar manner to the cow.
Most transfers are performed using general anaesthesia and midline
laparotomy or laparoscopy. Embryos earlier than the eight-cell stage of devel-
opment are best transferred to the uterine tube, and later-stage embryos to the
uterus. Uterine transfer of day 6 and 7 embryos by laparoscope is as effective as
laparotomy in the ewe, and has the advantage of not requiring exter-iorisation of
the tract. Recipients are synchronised using prostaglandin F2ct treatments or
intravaginal progestogens, and oestrus is detected by the use of a harnessed,
vasectomised ram or buck.
The requirements for synchrony of oestrus between donor and recipient
are similar to the cow.
EMBRYO TRANSFER IN THE MALE
Embryo transfer in the mare is a relatively new procedure compared to the
cow and many breed societies will not register the progeny . This, coupled with
the difficulty in inducing superovulation, has limited the commercial application.
The major uses, apart from the production of multiple offspring, are for the
production of foals from subfertile mares, for the removal of the risks of gestation
and parturition from older valuable brood mares, and for the production of foals
from mares while they are in competition.
Limited success has been achieved with superovulation in mares using
large doses of equine pituitary extract injected daily during dioestrus. In this study,
two embryos were collected per mare compared with 0.65 embryos per untreated
control. Porcine FSH was even less effective and eCG has been shown to have no
effect at all on fojlicular development in the mare .Consequently, it is routine for
most groups involved in equine embryo transfer to collect single embryos.
Embryos are recovered non-surgically 6-8 days post-ovulation using a
Foley-type catheter and an ebb and flow flush of the uterus with modified PBS.
Day 9 embryos have been found to be less viable on transfer, possibly due to their
relatively large size and consequent predisposition to handling damage. The use of
prostaglandin on the day of recovery allows repeat collections to be made at
approximately 17-18-day intervals without compromising embryo recovery.
The early equine embryo grows very rapidly and can usually be seen with
the naked eye in flushing media, ranging from 0.1 to 4.5 mm in diameter from 6-9
days post-ovulation.
The degree of synchrony between donor and recipient is not so critical in
the mare as in other large domestic species. found no difference in pregnancy rate
between recipients ovulating 1 day before or up to 3 days after the donor, although

132
those ovulating after tended to be best. Ovulation can be synchronised using
prostaglandin F2 or progesterone and human chorionic gonadotrophin (hCG)
treatments. Transfer can be performed either non-surgically through the cervix or
surgically through a flank incision. As with the cow, the results with non-surgical
transfer are more variable than with surgical transfer, and are dependent on
operator skill. Pregnancy rates of 50-70% can, however, be achieved by an
experienced technician.
Embryo transfer has not been widely used in the pig, except as a research
procedure. Potentially the major applications in the pig are international
movement of genetic material and disease control, either to establish disease-free
herds from infected donors or for the introduction of new bloodlines into specific
pathogen-free herds.
The basic procedures in pigs are well established. When superovulation is
required, gonadotrophins such as eCG are best administered during the early
follicular phase of the cycle, 15 or 16 days after the onset of oestrus. Oestrus then
occurs 3-f-4 days later, and an average of 25-30 ovulations' may be expected
following a dose of 1000-1500 IU eCG. Synchronisation of oestrus of donors and
recipients can be easily achieved by use of the oral progesto-gen altrenogest.
Embryo recovery in the pig is generally very successful and involves
general anaesthesia and mid-ventral laparotomy 3-7 days after oestrus, although
endoscopic procedures have been used successfully to recover porcine embryos.
Ovulation in pigs occurs 36-40 hours after the onset of oestrus, and embryos
remain in the uterine tubes for less than 48 hours after ovulation. Consequently,
embryo recovery from the uterine horns is the general practice. Modified PBS is
flushed into the uterus from the fimbrial end of the uterine tube and is collected
through a cannula in the uterine horn. Donors can be used for collection two or
three times if care is taken with the surgery.
Average recovery rates of over 90% can be achieved, and embryos can be
stored for short periods in modified PBS before transfer. Embryos must be
maintained at a temperature above 15°C in the laboratory, as they are extremely
sensitive to cooling. Embryo survival after culture periods of more than 24 hours
is low.
Transfers are also performed using midline surgery, the usual method being
to use a fine pipette that is passed through a puncture in the isthmus of the uterine
rube and into the uterus. Embryos need only be transferred to one uterine horn,
from which they will migrate throughout the uterus. About 14 embryos are
routinely transferred to each recipient, but a minimum of four are required to
establish pregnancy . Optimum pregnancy rates of 70% and embryo survival rates
of 60-65% are achieved when day 3-7 embryos are transferred to recipients that
were in oestrus on the same day or 1-2 days after the donor. Embryos can also be
transferred endoscopically, and recently successful non-surgical transfer has been
reported .

133
CRYOPRESERVATION OF EMBRYOS
The earliest report of successful cryopreservation of mammalian embryos
was This group used the mouse as an experimental animal and demonstrated the
importance of cooling rate, thawing rate and cryoprotectant on embryo survival.
Initial attempts to apply the best method for the mouse to the cow resulted in the
birth of a calf, but the success rate was very low. The sheep was subsequently used
experimentally as a model for the cow, and soon practical methods for both
species were developed and later extended to the goat and the horse. There are
variations between species, however, in the stages of embryonic development that
tolerate exposure to low temperatures.
The early experiments with mouse embryos had demonstrated that embryos
from one cell to the blastocyst stage could survive deep-freezing. However,
showed that early bovine embryos were sensitive to cooling, but an increased
tolerance developed once they had reached the compacted morula or blastocyst
stage. Consequently, interest centred on day 6, 7 or 8 embryos in this species,
particularly in view of the fact that these stages are readily recovered non-
surgically, are easily handled and stored on the bench, and can be successfully
transferred surgically or non-surgically into recipients.
In the mare, embryos do not enter the uterus from the uterine tube until day
6, at which stage they can be successfully recovered non-surgically. Day 6
embryos have been successfully frozen in the mare, but later-stage embryos do not
withstand freezing so well using conventional protocols. However, day 7 and 8
equine embryos have been successfully frozen recently by equilibrating with a
high concentration (4 molar) of glycerol, but stepping down to 2 M prior to
freezing, suggesting that poor permeability to the cryoprotectant may be a problem
with later-stage equine embryos.
In contrast, pig embryos at any stage of development appear to be
extremely intolerant of cooling There has, however, been a report of the birth of
piglets after transfer of frozen/thawed expanded blastocysts (Hayashi et al., 1989),
although with a low rate of success. More recently Dobrinsky et al. (1998)
reported live births after transfer of vitrified hatched blastocysts, and development
of this technology may be the way forward.
The principles of cryopreservation in the larger domestic animals are best
discussed by referring to the cow, as techniques are well documented in this
species. The important features of successful bovine programmes are applicable to
sheep, goats and mares, including the use of a modified PBS (Whittingham, 1971)
as a freezing medium and glycerol as a cryoprotectant.
The cryoprotective effect of compounds such as glycerol depends on their
presence intracellularly consequently a period of equilibration is necessary.
Embryos can be placed directly into 1.5 M glycerol in PBS at room temperature
and equilibration will occur in 10-15 minutes without deleterious effect. In
contrast, it is important that glycerol is removed slowly from the embryo after
thawing, in order to avoid osmotic lysis of cells.

134
This can be achieved by serial dilution in four to six steps of 10 minutes
each, and gradually decreasing concentrations of glycerol in PBS. Alternatively, a
sucrose gradient can be used. Sucrose does not permeate the embryonic cell
membrane and when added to the medium during cryoprotectant removal, the
resulting high extracellular osmotic pressure prevents the intermittent swelling of
blastomeres that would otherwise occur during stepwise cryoprotectant removal.
Several authors have reported an improvement in embryo survival after thawing in
a sucrose gradient compared with the stepwise method.
Glycerol can be removed in one step by transferring the thawed embryo
directly into 0.25-l.OM sucrose in PBS for 10-20 minutes before placing in PBS.
The latter is the basis of a 'one-step' procedure for direct transfer of embryos after
thawing. This technique requires that the embryo is placed in a plastic straw in a
column of medium containing glycerol. The remainder of the straw is then filled
with a column of 0.25-l.OM sucrose in a medium separated from the embryo by
air bubbles. The fluid columns are mixed after thawing by shaking the straw and
the embryo is transferred non-surgically after an equilibration period. More
recently, ethylene glycol has been shown to be an effective cryoprotectant for
bovine embryos. Ethylene glycol diffuses across the cell membrane much more
rapidly than glycerol, allowing direct transfer of frozen-thawed embryos without
cryoprotectant removal. Ethylene glycol is now rapidly superseding glycerol as the
cryoprotectant of choice for field use where the practical benefits of 'one-step'
thaw and direct transfer are appreciated.
The 6-8-day bovine embryo does not appear to be adversely affected by
rapid temperature change above -7°C, and embryos suspended in freezing medium
and sealed in plastic straws can be placed directly into the freezing machine at this
temperature and left to equilibrate rapidly. Induction of ice formation, or 'seeding',
in the freezing medium is necessary once the temperature has reached the true
freezing point of the medium; otherwise supercooling, spontaneous freezing and
intracellular ice formation will occur, with a consequent adverse effect on embryo
viability . Seeding is usually accomplished by pinching the straw gently with a
pair of forceps cooled in liquid nitrogen, but some modern freezing machines
include automatic seeding in the programme. Most laboratories are using
programmable freezing machines to obtain the precise cooling rates necessary for
optimal embryo survival but it is possible to use a relatively simple device with
good success.
The damage incurred by cells during freezing and thawing is thought to be
mainly caused by the formation of intracellular ice and the dehydration of the
embryo. The cells dehydrate during cooling, as the water in the medium
crystallises to form extracellular ice and the solute portion becomes increasingly
hypertonic. The cryoprotectant helps protect the cells from the damaging effects of
hypertonicity, and intracellular ice formation is minimised if the cells are allowed
sufficient time to dehydrate before they reach the temperature at which they would

135
freeze internally. It is evident, therefore, that the cooling rate, plunge temperature
and thawing rate will be critical in balancing these effects for optimal survival.
Slow cooling from the seeding temperature (0.3-0.5° C/min, plunging
between -30 and. -40°C) and a rapid thaw (approximately 360°C/ minute) are
favoured by most laboratories. Using this type of technique, very acceptable
results can be achieved in commercial embryo transfer programmes.
A further cause of damage to the embryo during freezing is the formation of
random fracture planes in the extracellular ice during rapid cooling to the storage
temperature, and possibly also during rapid thaw. Fracture planes involving the
embryo itself will cause varying degrees of cell damage and consequently affect
viability. Damage restricted to the zona, in the form of cracks or holes, does not
appear to be of any significance, although the presence of a zona, intact or
otherwise, may well be beneficial in that it acts as a physical barrier to the growth
of extracellular ice.
The optimum thawing rate depends on the method used for freezing. When
embryos are frozen slowly and plunged into liquid nitrogen between -30 and
-40°C, rapid thawing is essential to prevent residual water in the cells crystallising
during warming. Current common practice is to thaw the straw for 10 seconds in
air at ambient temperature and then to plunge into a water bath at 30°C when the
cryo-protectant is glycerol, or in the case of ethylene glycol, to plunge direct into a
20°C water bath.
Although there are probably as many different techniques for freezing
embryos as there are groups involved in bovine embryo transfer, the majority
differ only in minor detail. Good results are more dependent on fastidious
attention to detail in the laboratory and on good management and critical selection
of suitable recipients, than to minor changes in equilibration times or cooling rates.
Future research is needed to simplify techniques without prejudicing embryo
survival. Field transfer of frozen/thawed embryos can now be carried out without
the use of a laboratory set-up but the protocols for freezing embryos are time-
consuming and still require the use of sophisticated, programmable freezing
machines.
Rail and Fahy (1985), however, showed that mouse embryos could be
successfully cryopre-served by a simple, rapid vitrification technique requiring
minimal equipment. This is a new approach dependent on the fact that
concentrated solutions of cryoprotectants in PBS do not crystallise when cooled to
low temperatures but become increasingly viscous and form a glass-like solid. Rail
and Fahy used a mixture of four cryoprotectants in the mouse, but Massip et al.
(1986) have reported successful vitrification of bovine embryos using a simplified
technique that resulted in seven pregnancies from 13 transfers (53.8%). The
embryos were equilibrated in two steps with 25% glycerol and 25% 1,2-
propanediol, and then immediately plunged into liquid nitrogen. A rapid thaw
technique and one-step dilution of cryoprotectant in 1 M sucrose in PBS was used,
and this suggests that a one-step transfer procedure could be applied successfully,

136
making available an effective field technique, both for freezing and thawing,
which requires minimal laboratory equipment.Have improved the vitrification
technique by using an open pulled straw as an embryo container. This allows a
much faster cooling and warming rate (over 20 000°C/min), and very encouraging
pregnancy rates have been produced from in vitro-derived embryos vitrified at
both the oocyte and blastocyst stage of development.
There is no doubt that simple field methods for freezing and thawing
bovine embryos are becoming more widely used. Whether they are generally
applied will depend on the comparative results. Bovine embryos are still expensive
to recover, and most embryos collected commercially are potentially valuable.
Consequently, even a few per cent advantage in pregnancy rate would mean
continuous use of programmable freezing machines and laboratory thawing for a
few years yet.
MANIPULATION OF EMBRYOS
The technologies associated with manipulation of oocytes and embryos are
advancing at a very rapid rate. Successful cloning from somatic cells has been
reported in the ewe and the cow, and foreign genes have been injected into the
nucleus and incorporated into the genome of single-cell, fertilised eggs of pigs
sheep and cows. Many other manipulations of the fertilisation process, such as
androgenesis, gynogenesis and parthenogenesis, may also be possible in future
.Some of the simpler manipulations such as embryo-splitting, sexing and in vitro
embryo production are currently being applied commercially in cattle breeding,
but others may soon follow.
In vitro production of embryos
Preovulatory oocytes collected from ovaries and fertilised in vitro have
resulted in live calves, lambs , goat kids and piglets.The techniques for in vitro
production of embryos have developed particularly rapidly in cattle, since
demonstrated that maturation and fertilisation proceeded most efficiently at 39°C
and described a method for capacitating sperm in vitro using heparin.
For experimental purposes the vast number of oocytes normally wasted in
the abattoir can be recovered by harvesting the ovaries and releasing the oocytes
from 2-5 mm follicles by aspiration. Maturation is achieved by culturing the
oocytes for 20-24 hours in medium containing bovine serum and hormones. Most
oocytes will reach the second metaphase stage of meiosis during culture, but not
all have the full potential for development.
Matured oocytes are then cultured with sperm capacitated in vitro and up to
90% fertilisation can be achieved with some bulls.
Embryos must then be cultured for 6-9 days to ensure that they are at the morula
or blastocyst stage of development before they can be frozen of transferred to the
uterus of a recipient. By applying these techniques to abattoir ovaries, banks of
cheap embryos have been made available for commercial transfer on a limited
scale in the UK .

137
Co-culture of early embryos with bovine uterine tube epithelial cells (Lu et
al., 1988), or buffalo rat liver cells in medium supplemented with serum, has
produced very acceptable rates of development in practice. Some groups,
however, have reported an increase in abortion, dystocia, perinatal loss and
anomolies in in vitro produced calves, and that their birth weight averaged
significantly higher than in vivo-produced controls. The use of serum and/or co-
culture may be implicated and further research is necessary to reduce the incidence
of these problems. It is not surprising, therefore, that culture of early embryos in
chemically defined (serum-free) media without co-culture has now become the
method of choice.
More recently, a technique for aspiration of oocytes from the ovaries of live
cows has been described.This has opened up the prospect of a dramatic increase in
embryo production from valuable pedigree cattle where slaughter is not an option.
Van der were able to collect an average of 9.4 oocytes per aspiration from cows
aspirated twice weekly over a 3-month period. Follicle aspiration was achieved
using a transvaginal, ultrasound-guided puncture technique, and there appeared to
be no significant detrimental effect of repeat sampling on the ovary or genital
tract.
Follicle aspiration and in vitro fertilisation in practice are particularly
applicable to repeat breeders that are unsuitable for conventional embryo transfer,
or normal cows that do not respond to superovulation treatments.
Micromanipulation
Division of embryos using microsurgical instruments is a practical method
for creating identical siblings. Identical quadruplet sheep, triplet calves , and twin
horses and pigs have been produced by separation of blastomeres of two-, four-
and eight-cell embryos. Success rates are high when embryos are divided into two,
but decline considerably when they are quartered. Identical quintuplets have been
produced in sheep by mixing cells of four-and eight-cell embryos, when the more
advanced cells apparently developed into the fetus and the less advanced
contributed to the placenta.
A simpler and more practical method of producing identical twins from
morulae and early blastocysts involves microsurgical division of the embryo into
two groups of cells, and immediate transfer into recipients .The half-embryos can
be replaced in surrogate zonae pellucidae before transfer or transferred naked.
Pregnancy rates of 50% or more per half-embryo have been reported, resulting in a
net pregnancy rate of over 100% per original embryo
Embryo division is being used commercially to increase the number of
progeny produced in bovine embryo transfer programmes, and could also be a
valuable method for producing identical twins for research.
Sex determination
The efficiency of livestock breeding enterprises would be considerably
increased if it were possible to routinely predetermine the sex of offspring. In the
past the vast majority of claims to alter the sex ratio significantly by the separation

138
of X and Y chromosome-bearing spermatozoa have not been substantiated in
practice. Recently, however, a method has been described for separation of sper-
matozoa on the basis of their DNA content, by flourescent labelling and cell
sorting . Field trials in the USA have demostrated that acceptable pregnancy rates
can be achieved after insemination of heifers with sexed, unfrozen sperm. Also, it
was notable that in the majority of the most recent trials cited, the pregnancy rate
with sexed frozen sperm was within 90% of unsexed frozen controls. Frozen sexed
bull semen is now commercially available in the UK, but significant production
limitations due to cost and the speed of sorting means that only a limited number
of bulls will be available to breeders for a year or two.
Embryos have been sexed by cytological methods.These involve chromo-
some analysis of cells in metaphase that have been sampled from the embryo
using an embryo division technique. However, biopsy and karyotyping procedures
are tedious, time-consuming and relatively inaccurate, making them impractical
for routine commercial use.
An alternative approach is to use an antibody to the HY antigen, a protein
present on the surface of male mammalian cells. The HY antibody binds to male
embryos and can be detected by adding a fluorescently labelled antibody directed
against the first, such that male embryos fluoresce in appropriate light. This
procedure sexes mouse embryos with 80% accuracy but has not been successfully
applied to large domestic species.
A more accurate method for determination of sex has become available
with the development of DNA technology and polymerase chain reaction (PCR),
utilising primers that target DNA sequences specific to the Y chromosome. Only a
few cells need to be sampled for testing, with a consequent minimal effect on
embryo viability. Several versions have been developed for commercial use in
combination with PCR technology, but the relatively high cost of the test has
prevented widespread application until recently.
The manipulation of eggs and embryos of the large domestic species will
have a major impact on the efficiency of animal production in the future. Although
much of the research involved is in its infancy, it is growing fast and there are
exciting prospects ahead for the geneticist and the animal breeder. It should be
remembered, however, that it is through embryo transfer that new developments
may be exploited and therefore the' use of this breeding technique is likely to
continue to expand.
Cryopreservation
Longer-term storage of semen is achieved through cryopreservation.
Cryopreservation maintains the fertile life of semen virtually indefinitely, although
a large proportion of individual spermatozoa fail to survive the considerable
stresses of freezing and thawing. For sperm to survive freezing, they need to be
extended in a diluent that contains not only substances that protect them against
cold shock, but also cryoprotectants, such as glycerol, which protect them from the
deleterious consequences of freezing.

139
The general responses of cells to freezing were not understood until long
after empirical methods of cryopreservation had become widely adopted. Initially,
as the temperature of the external medium falls below its freezing point, crystals of
pure water start to form. The concentration of solutes in the unfrozen part of the
medium therefore rises as, in consequence, does its osmotic pressure. Ice crystals
do not extend into the cell at this stage, as they are excluded by the cell membrane.
Thus, the intracel-lular contents undergo a period of supercooling, during which
the cell loses water to the unfrozen part of the extracellular medium by osmosis. A
variable degree of cell dehydration follows, which is terminated by the formation
of intracellu-lar ice crystals. Thus, damage can occur to cells in one of two ways.
Where a substantial degree of cellular dehydration occurs, the high concentrations
of solutes in the residual intracellular water can be damaging, whereas, if only
slight dehydration occurs, large ice crystals can form within the cell, which cause
physical damage to its internal and bounding membranes. The degree to which
each affects the cell is determined by the rate of cooling - the slower the rate, the
more dehydration, the faster the rate, the greater the damage by ice formation - and
the size of the cell, such that the larger the cell, the slower its inherent rate of
dehydration.
Cryoprotective agents may either penetrate or remain outside the "cell, but
both act by binding water and therefore alter the availability of water either for
dehydrative loss or for ice crystal formation. Penetrating cryoprotectants, such as
gly- cerol or dimethyl sulfoxide (DMSO), appear not only to reduce the loss of
water from the cell, thereby reducing solute damage, but also to bind it in a form
that renders it unavailable for crystal formation, thereby reducing the effects of
intracellular ice formation. Non-penetrating cryoprotectants, such as disaccharides
or proteins, may hasten dehydration during very rapid cooling, thereby minimising
intracellular ice formation. Notwithstanding the aforegoing, precise understanding
of the mode of action of cryoprotectants still remains elusive, and much
information relating to their practical use remains empirical.
Glycerol is the main primary cryoprotectant used in preparing mammalian
semen for freezing, despite the fact that it has some directly toxic effects upon
sperm .Concentrations of glycerol depend upon the species and the other compo-
nents of the diluent. For example, diluents for bovine semen that contain
disaccharides can utilise lower percentages (3-4%) of glycerol than diluents that
lack such disaccharides, which have a final glycerol concentration of at least 7% .
Whether the toxic effects of glycerol are exacerbated at high temperatures
has been a matter of debate. Certainly considered that the addition of glycerol at
28°C was more dam- aging to bovine sperm than its addition at 4°C, although
reviewing the (by then) copious literature, concluded that the effects of
temperature of glycerolisation were equivocal, Nevertheless, normal practice in
commercial bovine AI centres is that where the final concentration of glycerol is
high (> 7%), a primary dilution of the semen is made with a diluent containing
little or no glycerol, with glycerolisation being carried out after reducing the

140
temperature to 4°C; whereas diluents that utilise lower final concentrations (<5%)
are added in one step, at 30°C. With boar semen, by contrast, the toxicity of
glycerol at high temperatures is much less equivocal, and low-temperature gly-
cerolisation is desirable.
Originally, diluted semen was placed in glass ampoules for freezing in a
mixture of alcohol and solid carbon dioxide at -79°C, or drops of diluted semen
were placed directly on to the surface of a ' block of solid carbon dioxide where
they froze in pellet form . Long-term storage at -79°C was not satisfactory,
however, as deterioration occurred at that temperature. Storage in liquid nitrogen
at -196°C has subsequently become established as the standard medium for long-
term preservation of semen and, over the 40 years for which it has been practised,
has maintained sperm fertility unscathed. At the present time, semen is frozen in
one of two main ways. Diluted semen is packed into thin, plastic tubes of 0.25 or
0.5 ml capacity, then, in the simpler techniques, these tubes ('straws' or 'paillettes')
are suspended in the vapour of liquid nitrogen, which is at about -120°C, for about
10 minutes .The straws are then plunged into the liquid nitrogen . More recently, a
greater degree of control of freezing rate has been exercised by the use of
microprocessor-controlled freezers, with improved sperm survival justifying the
increased cost of the processing.
Thawing of the semen needs to be rapid; slow thawing allows
recrystallisation of ice within the cells, causing membrane damage. In practice, the
rate of thawing is rarely critical, the surface area-to-volume ratio of the 0.25 ml
tubes being so great that any temperature of the thawing water between 0 and
40°C will thaw adequately. Of greater importance is the temperature control of the
thawed semen. This should not be allowed to cool below the final temperature
achieved during thawing; otherwise substantial sperm losses can occur. Rethawed
spermatozoa are as sensitive to fluctuations in temperature as are their unfrozen
counterparts.

Collection, handling and storage of semen


Semen is usually collected by an artificial vagina, although
electroejaculation is occasionally used. After assessment for motility, density and
morphology, the semen is diluted into insemination doses.
The degree of dilution and the diluents with which the semen is diluted
depend upon its subsequent use. Most semen is cryopreserved, although some is
used after simple extension and chilling to 4°C. The practice of using an ambient
temperature diluent is virtually unique to New Zealand, where the practice is made
possible by the low incidence of infectious disease and is required for the highly
seasonal pattern of reproduction in its dairy cows.
For cryopreservation, or for use at 4°C, the semen is first extended with a
diluent based upon either egg yolk or skimmed milk which contains antibiotics for
the control of contaminating bacteria. The semen is then cooled to 4°C. If it is

141
destined for use in this form, the motility of the sperm will be reassessed, then the
semen released for use. If the semen is destined for cryopreservation, glycerol is
also added, then the semen packed into 0.25 or 0.5 ml paillettes, or 0.5 or 1.0 ml
glass ampoules. The semen is then equilibrated for 1-4 hours. It was originally
considered that this was the period over which glycerol penetrated the sperm,
although more recent observations indicate that the penetration of glycerol is very
rapid and that most of the equilibration period is concerned with membrane
stabilisation during exposure to low temperatures. The semen is then frozen in the
vapour of liquid nitrogen or in a microprocessor-controlled freezer. The semen
thereafter remains in liquid nitrogen until thawed for use. Freezing in alcohol and
solid carbon dioxide or in pellets on blocks of solid carbon dioxide, although
formerly used widely, has now virtually ceased.
The ability to perform an intrauterine insemination in cattle means that a
relatively low dose of sperm is required to achieve acceptable pregnancy rates.
Typically, of the 20-30 million sperm that are required in each insemination dose,
6-7 million survive freezing, a figure that is generally regarded as the minimum
dose compatible with acceptable fertility. Lower numbers of sperm can be used
where unfrozen semen is used.
The use of ambient temperature diluents is precluded in most continental
countries by the risk of contamination of the semen with foot-and-mouth disease
virus. However, it has many advantages over other methods of extension, for,
whereas cryopre-served semen requires about 20 million spermatozoa per
insemination, semen extended in ambient temperature diluents can achieve
acceptable fertility with less than 2.5 million sperm per insemination. Initial work
on ambient temperature diluents for bovine semen was based upon the use of the
FVT diluent but the development of ambient temperature dilution in New Zealand
has subsequently been based upon the use of the Caprogen diluent . With the
subsequent modifications that have taken place, the Caprogen diluent is now
capable of maintaining sperm viability and acceptable conception rates for up to 5
days, with an insemination dose of between 0.5 and 2.5 million sperm per
insemination.
Insemination
Cows ovulate at about 12 hours after the end of the oestrus period. The
ideal time for insemination is therefore 6-24 hours prior to ovulation . Where the
technician service provided by an AI centre is used, the optimum insemination
times achieved in practice are on the same day (morning or afternoon) where
oestrus is first observed in the morning, or on the morning of the next day, where
oestrus is first observed in the afternoon. However, it is claimed that it is possible
to achieve a better timing of insemination in relation to the most fertile period of
oestrus by farmers inseminating their own cattle at appropriate intervals after the
first observation of oestrus. For this reason, and because of the cost of using AI
centre technicians, technician services have fallen somewhat into disfavour,
compared to farmers' own ('do-it-yourself') inseminations.

142
Cows are inseminated just into the short uterine body. Insemination into the
cervix produces a lower fertilisation rate, while insemination deeper into the uterus
runs the risks of either inseminating into the uterine horn contralateral to the
ovulation site, or scoring the endometrium with the tip of the insemination
catheter. Reduced fertility is the consequence of both of the latter two errors. The
standard technique of insemination is to grasp the cervix through the rectum with
the left hand. A catheter, into the tip of which a paillette of semen has been
inserted is then passed into the vagina and manipulated into and through the cervix
by the right hand. This technique, the rectovaginal method of insemination,
requires considerable practice for success. The vulval lips are opened by
downwards pressure from the arm in the rectum, while the circular folds of vaginal
mucosa are obliterated by pushing the cervix forward. The catheter is initially
inserted pointing upwards at an angle of about 30° to avoid entering the urethral
meatus or fossa, and is then moved horizontally until it engages in the external os
of the cervix. The left hand squeezes the anterior vagina on to the caudally
projecting external os of the cervix, thereby obliterating the fornix of the vagina
and facilitating entry of the catheter into the cervix. Entry into the external os is
accompanied by a characteristic 'gritty' sensation. The catheter is then introduced
through the convoluted cervical canal by manipulation of the cervix through the
rectal wall. One finger is placed over the internal os of the cervix, so that the tip of
the catheter can be palpated as it emerges from the cervical canal .
As soon as the catheter has emerged, deposition of semen into the uterus begins;
the catheter is advanced no deeper into the uterus. In this way, semen should be
equally distributed between the two uterine horns .
No forward pressure should be exerted on the catheter with the right hand,
for the uterine wall is friable and easily penetrated if the catheter moves suddenly.
The most common fault of insemination is twisting die cervix in the left hand, so
that one uterine horn is partly occluded. Alternatively, the catheter may be partly
withdrawn during the deposition of semen, resulting in a partially intracervical
insemination. Penetration of the cervical canal of maiden cattle is difficult at
oestrus, and virtually impossible at other stages of the oestrous cycle; such animals
are therefore often beyond the capabilities of inexperienced inseminators.
However, the cervix of parous cattle can, with greater or lesser difficulty, be
traversed at most stages of the oestrous cycle and early pregnancy. It is therefore
imperative that it is known whether an animal is likely to be pregnant before
insemination is attempted, for abortion can be induced if an insemination catheter
penetrates the fetal membranes or if infection is introduced into a pregnant uterus
by poor insemination hygiene.
Management of insemination
Insemination can be performed at an observed or induced oestrus. The
former is more common in dairy cattle, for considerable opportunity exists for the
observation of oestrus, but in beef cattle and dairy or beef heifers the time required
for observation makes the use of induced oestrus relatively more attractive. Oest

143
us can be induced and synchronised by the use of prostaglandin F2o (PGF2ct) or its
analogues, progesterone-like hormones, or combinations of progesterone and a
luteolytic agent. Most such regimens require the use of fixed-time insemination,
although, particularly in the case of PGF2a, the accuracy with which the timing of
oestrus can be predicted is sufficiently imprecise for some observation to be
advisable, and reinsemination performed if animals exhibit signs of oestrus after
fixed-time insemination has occurred.
Fertility to AI is generally very similar to that achieved at natural service,
with a calving rate to a single insemination of around 50% . The true fertilisation
rate is much higher than this, at around 90%, but subsequent embryonic losses
bring the apparent figure to the lower value . In practice, AI companies are
generally unable to obtain complete data on the calving rates, so they estimate
fertility from the proportion of cows which are represented for insemination by
either 49 or 60-90 days after the initial service. The proportion of cows that are not
re-presented is closely related to the proportion which actually have become and
remained pregnant. The figure thereby obtained, the non-return rate (NRR), is an
overestimate of calving rate, but is generally in fixed ratio to the calving rate, so is
usable for monitoring fertility. AI centres therefore use the 'non-return rate' to
monitor both the fertility of their bulls and the results obtained by their
technicians. Bulls or technicians that produce consistently low figures are
generally slaughtered or dismissed, respectively. No such control exists over the
technical proficiency of farmers who inseminate their own cattle, nor, necessarily,
over the fertility of the bulls they use.
Regulation of cattle AI varies from country to country, but is generally
under some form of state control. In the UK, AI centres are licensed by the
Ministry of Agriculture, with the right to distribute semen, or to employ
technicians to inseminate cattle, also being granted by ministry licence. Further
licences grant individual farmers the right to inseminate their own cattle and to
store frozen semen on their farms.
Artificial insemination of sheep has recently been comprehensively
reviewed. The sheep is less amenable to artificial insemination than is the cow,
since oestrus cannot readily be detected without the presence of rams,
insemination is less straightforward and ovine semen is less easy to freeze than
bovine semen. In Eastern Europe, South America and Australasia, AI is widely
used in sheep-breeding programmes, but its use is much less widespread in
Western Europe and North America, mainly due to the high costs of handling and
inseminating sheep compared with the costs of natural service.
Ewes normally display oestrous behaviour only in the presence of a ram. In
order to determine the time at which AI should be performed, it is therefore
necessary either to control the timing of oestrus or to detect it with male animals.
In the former situation, pharmacological methods are used to induce and
synchronise oestrus, so that the time of the fertile period is defined. In the latter
situation, either raddled, vasectomised rams or intact rams with an abdominal

144
apron to prevent intromission are used to detect oestrus. The cost:benefit ratio for
the use of AI in sheep has therefore to be considered carefully. Where there are
substantial costs associated with AI, either of maintaining rams, of drugs (plus
their administration) for oestrus synchronisation, or of the procedures associated
with insemination itself, these have to be set against the financial benefits gained
from the superior carcass or wool characteristics of the progeny born to AI.
However, the most important limitation of the use of AI in sheep is in the
method of insemination, since it is difficult to achieve an intrauterine insemination
because the cervical canal of the ewe is so tortuous. Since intracervical AI results
in both a lower conception rate and a lower number of lambs born per ewe than
with natural service, a number of methods of insemination have been devised that
try to circumvent the cervix. All of these attempt, with varying success, to achieve
an economically viable compromise between fertility, technical difficulty and
numbers of sperm needed
for insemination. The methods in widespread use are:
• intravaginal
• intracervical
• transcervical intrauterine
• laparoscopic intrauterine.
These methods will be considered below.
Collection, handling and storage of semen
Most inseminations of ewes are performed using semen that was collected
on the day of insemination. Such semen is normally extended by the addition of
simple diluents, although direct insemination of raw semen is still practised in
some regions. The semen is collected by an artificial vagina or electroejaculation
and subjected to routine examination for motility and density. It is then diluted
with a simple diluent, to a final volume and sperm content that depend upon the
route by which it is to be inseminated. The number of sperm and the volume of
diluted semen used for insemination depend upon the route of insemination,
whether the insemination is undertaken during the natural breeding season or after
induction of out-of-season breeding, and whether the semen is cryo-preserved.
Recommendations for sperm numbers and insemination volume Diluents that are
in routine use for unfrozen semen include buffers that contain glucose, egg yolk-
citrate or egg yolk-phosphate solutions, or heat-treated cows' milk. After dilution,
the semen is cooled and stored at either +15°C or +4°C until used. The semen has
to be used within 8 hours of collection, as fertility declines substantially after this
time.
However, although such simple diluents are capable of supporting sperm
viability for the relatively brief periods demanded for direct insemination,
cryopreservation is required for long-term storage of semen. Unfortunately,
cryopreservation of ovine semen is not particularly straightforward, since the
process of cryopreservation causes a significant level of damage to ovine
spermatozoa. Thus, they are less able to survive in the female genital tract than are

145
unfrozen cells. In particular, the passage of the cervix appears to be very much
more difficult for cryopreserved than fresh sperm, so, as a generalisation,
insemination routes that require sperm to traverse a significant length of the
cervical canal are not well suited to cryopreserved semen. Hence, frozen semen is
not really appropriate for use in intravaginal insemination, unless large numbers of
spermatozoa are used and a lower than normal conception rate is accepted.
There are also a number of difficulties encountered when semen is frozen
for intracervical insemination. These arise as a relatively large number of sperm
have to be contained within the limited volume of inseminate that can be placed
within the ovine cervix. Since the anatomy of the cervical canal limits the
insemination volume to below about 0.25 ml the dilution rates are limited to
between 1:1 and 1:4. In consequence, insufficient protection can be afforded to the
sperm by the diluent against cold shock and freezing damage generally resulting in
mediocre post-freezing survival of functional sperm.
For intrauterine insemination, in which lower numbers of spermatozoa are
required, far more satisfactory dilution rates of semen can be achieved, so
cryopreservation is more successful. Furthermore, since the insemination site does
not require sperm to traverse the cervix, the sperm do not have to survive in such a
robust state as is needed for intracervical insemination. Hence, the conception
rates that are achieved with the use of cryopreserved semen for intrauterine
insemination are commensurate with those of natural service after oestrus
synchronisation.
As with most other species, it has been noted that there is much variation in
the ability of the semen of individual rams to survive cryopreservation. However,
the semen of most rams has been relatively successfully frozen in pellet form on
the surface of blocks of solid carbon dioxide or in paillettes in the vapour of liquid
nitrogen, using diluents based upon egg yolk, skimmed milk, citrate and/or
lactose. The problem of low dilution rates for semen that is to be used for
intracervical insemination was addressed by Salamon, by preparing a range of
different diluents for freezing semen at dilution ratios of 1:1 to .1:4, in which the
constituents were present in higher concentrations at the diluents to be used at
lower dilution rates. In this way, compensation is achieved for the effects of the
ratio between diluent and seminal plasma. Dealt with the same problem by using
two different diluents (an egg yolk-lactose diluent and a glycerolised skimmed
milk diluent) in varying proportions depending upon the final dilution rate to be
achieved.
Nevertheless, despite the use of higher numbers of cryopreserved sperm for
intracervical insemination than when using fresh semen, conception rates are
below those of natural service or fresh semen insemination. Conception rates of
65-80% are typical when this method is used in the ewes' breeding season, with a
somewhat lower figure when for out-of-season breeding regimes. Embryonic
mortality is generally considered to be similar with frozen and fresh semen,

146
although some reports exist of higher embryonic losses after the use of frozen
semen.
Insemination
Vaginal insemination deposits semen into the cranial part of the vagina,
without attempting to locate the cervix. The requirements of this method, in terms
of both technical proficiency and handling facilities for the sheep, are minimal.
However, as previously described, this method requires large numbers of
spermatozoa per insemination and is not really amenable for use with stored
semen. Moreover, conception rates are also poor after pharmacological oestrus
synchronisation, so intravaginal insemination is best suited to use after oestrus
detection during the natural breeding season. The ideal timing of insemination is
before ovulation, i.e. 12-18 hours after the onset of oestrus. Highest conception
rates are therefore achieved when the timing of insemination is optimised by
drafting ewes for insemination twice per day.
Intracervical insemination is best achieved with the hindquarters of the ewe
elevated. After cleaning of the perineum, the vagina :.s opened with a duck-billed
speculum and the cervix located. The insemination catheter is then inserted as far
as possible into the cervix. Penetration of the cervix is typically 0-2 cm. The
conception rate is highly correlated with the depth of penetration and, hence, the
technical proficiency of the inseminator. Conception rates achieved with the use of
unfrozen semen by this method are adequate after pharmacological methods of
oestrus synchronisation. The ideal time for insemination is 55 ± 1 hour after
removal of progesterone sponges, or 15—17 hours after the onset of detected
oestrus.
The method of direct intrauterine, laparoscopic insemination was developed
to overcome many of the difficulties of intravaginal and intracervical
insemination. In this method, ewes are restrained in a cradle and laparoscopy is
per-formed close to the udder, whereupon the uterus is located and semen injected
into the uterine lumen. The semen can be introduced to the uterus via a simple
pipette or by the use of specialised insemination equipment. With oestrus-
synchronised ewes, the ideal timing of insemination is between 68 and 72 hours
after withdrawal of progesterone sponges. Conception rates to frozen semen
inseminated by this method are higher than for intracervical insemination, because
of better cryopreservation of sperm and a site of insemination that avoids sperm
having to traverse the cervix. Conversely, the laparo-scopy is technically
demanding and there are far greater implications for the welfare of the ewe's than
with intracervical insemination.
Use of laparoscopic Al is- already widespread throughout Australasia, but
the method has had only a limited uptake in Western Europe, mainly due to
concerns for the welfare of its subjects and, as with sheep Al in general, the lack of
a clear cost benefit. However, laparoscopic intrauterine insemination of ewes is
undoubtedly the most significant development in sheep Al, for it circumvents
many of the problems of the traditional methods.The numbers of sperm required

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for insemination are lower and the volume of inseminate is proportionally greater,
allowing more appropriate dilution rates and, therefore, better preservation of
sperm. Hence, conception rates are closer than those of natural service, and
embryonic mortality is reduced to an acceptable level. The method also allows for
the possibility of genuine progeny testing of rams, as semen from an individual
sire can be used in many flocks, over prolonged periods of time.
Laparoscopic Al is therefore already the basis of several sire referencing
schemes world-wide.
Finally, a method that attempts to achieve an intrauterine insemination via
the cervical route has also been developed. In this method, transcer-vical
intrauterine insemination, the cervix is fixed by being grasped with forceps, and an
insemination needle is introduced as far as possible through its lumen. Although
better conception rates can be achieved by this method than by conventional
intracervical insemination, both the retraction of the cervix and its penetration of
the cervical lumen are associated with significant levels of damage. It is probable,
with the development of expertise in the method, that its use will become more
widespread but, at present, it seems to have little to commend it over laparoscopic
insemination.
Artificial insemination of goats is generally very similar to that of sheep.
However, it is much easier to achieve an intrauterine insemination via the cervix
of the goat than in the ewe. Moreover, conception rates to frozen semen tend to be
much higher in the goat than in the sheep, since, in addition to the better site of
insemination, caprine semen appears to survive cryopreservation better than ovine
semen. As a consequence, in the substantial flocks of goats that are maintained for
milk production and in the small flocks of goats maintained by many amateur
farmers, Al has been making an increasing contribution to breeding. Thus, in
France, for example, the numbers of goat inseminations increased 4-fold during
the 1980s In the Western world, AI of goats is based predominantly upon frozen
semen, inseminated by an intracervical route .
Cryopreservation of goat semen differs from that of the ram in one
important aspect: namely, that the seminal plasma must be removed before
dilution in media that contain egg yolk. A phos-pholipase that is secreted by the
bulbourethral gland coagulates egg yolk media and hydrolyses lecithin to fatty
acids and spermicidal lysolecithins . Therefore, diluents for goat semen have either
been based upon skimmed milk, in which the sperm survive adequately, or the
seminal plasma has been removed before using egg yolk-based diluents .Where
yolk-based diluents are to be used, washing the spermatozoa is achieved by
dilution in Krebs-Ringer phosphate solution and centrifugation.
For direct insemination, caprine semen can be extended in a skimmed milk-
glucose diluent or, after removal of seminal plasma, in an egg yolk-citrate • or egg
yolk-tris-fructose diluent. For cryopreservation, egg-yolk-based diluents are more
widely used than skimmed milk-based ones, although acceptable results can be
achieved w;th skimmed milk. Glycerolisation can be in one or two steps; for

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example, recommend a primary dilution in glycerol-free egg yolk-citrate diluent,
followed by addition of diluent containing 14% glycerol once the semen has
reached 4°C. However, when final concentrations of egg yolk are kept below 2.0%
of the diluent, egg-yolk-based diluents can be used without removal of seminal
plasma.
For intracervical insemination, there are similar problems of sperm
numbers in relation to the volume of inseminate to those that apply in the sheep.
Hence, Salamon also suggested variable composition extenders (which also
contain a final concentration of egg yolk of less than 2.0%) for one-step dilution of
goat semen for use in intracervical insemination at dilution rates of between 1:1
and 1:4. Semen can be frozen either by the pellet method (i.e. on the surface of
solid carbon dioxide) or in paillettes suspended over the vapour of liquid nitrogen.
Since highly acceptable post-thaw recovery rates are achieved with the latter
method, the convenience of handling semen means that it is usually the preferred
method of cryopreservation. Although much variation exists between individual
animals, overall results with doses of 50 million motile frozen-thawed sperm,
inseminated into the uterus, are comparable with those of natural service. Yet the
relationship between numbers of spermatozoa upon fertility is not well known,
since few data from large-scale surveys exist on the subject. Nevertheless, sug-
gests that between 100 and 125 million spermatozoa are used per insemination,
while Evans recommend similar numbers to those used in sheep.
Similar routes of insemination can be used in the doe to those employed in
the ewe. Intravaginal insemination is not widely used, so intracervical
insemination is the most widely practised method. In a significant number of does,
an intrauterine insemination can be achieved via the intracervical route, since the
caprine cervix is relatively easier tcr traverse than the ovine cervix. Interestingly,
although intrauterine insemination can be achieved simply by direct pressure of
the insemination catheter upon the cervix, many technicians prefer to deposit at
least some of the semen into the cervical canal, in case the intrauterine
insemination had been entirely into a single uterine horn. Because the intracervical
route is relatively successful, the impetus to the use of laparoscopic intrauterine
insemination has been far less compelling in the doe than in the ewe.

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