Alternative organism; Model organism

REVIEW
Alternatives to animal testing: A review
Sonali K. Doke, Shashikant C. Dhawale *
School of Pharmacy, SRTM University, Nanded 431 606, MS, India
Received 13 August 2013; accepted 10 November 2013
Available online 18 November 2013
KEYWORDS
Alternative organism;
Model organism;
3 Rs;
Laboratory animal;
Animal ethics
Abstract The number of animals used in research has increased with the advancement of research
and development in medical technology. Every year, millions of experimental animals are used all
over the world. The pain, distress and death experienced by the animals during scientific experiments have been a debating issue for a long time. Besides the major concern of ethics, there are
few more disadvantages of animal experimentation like requirement of skilled manpower, time consuming protocols and high cost. Various alternatives to animal testing were proposed to overcome
the drawbacks associated with animal experiments and avoid the unethical procedures. A strategy
of 3 Rs (i.e. reduction, refinement and replacement) is being applied for laboratory use of animals.
Different methods and alternative organisms are applied to implement this strategy. These methods
provide an alternative means for the drug and chemical testing, up to some levels. A brief account of
these alternatives and advantages associated is discussed in this review with examples. An integrated
application of these approaches would give an insight into minimum use of animals in scientific
experiments.
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Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
2. Three Rs: reduction, refinement and replacement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
2.1. Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
2.2. Refinement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
2.3. Replacement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
* Corresponding author. Tel.: +91 9970700030; fax: +91 2462229245.
E-mail addresses: [email protected] (S.K. Doke),
[email protected] (S.C. Dhawale).
Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
Saudi Pharmaceutical Journal (2015) 23, 223–229
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http://dx.doi.org/10.1016/j.jsps.2013.11.002
3. Alternative methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
3.1. Computer models. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
3.2. Cells and tissue cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
3.3. Alternative organisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
3.3.1. Lower vertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
3.3.2. Invertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226
3.3.3. Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
1. Introduction
Use of animals for various purposes like food, transportation,
pets, sports, recreation and companionship is as old as the human beings itself. Using animals for the purpose of research is
one of the extended uses. Various animals like mice, rats, hamsters, rabbits, fishes (examples – zebra fish, trout), birds
(mainly chicken), guinea pigs, amphibians (xenopus frogs),
primates, dogs, cats etc. are being used in research for a long
time (CULABBR, 1988). Drug testing and toxicological
screenings which are useful in the development of new treatments for infectious and non-infectious diseases is the main
purpose of such studies. Animals also serve as a tool to understand effects of medical procedures and surgical experiments.
Moreover, they are used to obtain products like vaccines, antibiotics etc. which are used in diagnostics as well as treatments
(Giacomotto and Segalat, 2010; Hendriksen, 2009, 2007). The
number of animals used in research has gone up with the
advancement in medical technology. Every year, millions of
experimental animals are used all over the world. For example,
in UK, 3.71 million animals were used for research in the year
2011 (www.rspca.org.uk). The total number of animals used in
the USA in the year 2009 was estimated to be 1,131,076, while
that in Germany reached up to 2.13 million in 2001 (Rusche,
2003). This huge population of experimental animals usually
comes from the breeding centers located in various universities
and national breeding centers. All of these are known as classA dealers, while the brokers who acquire the animals from miscellaneous sources (like auctions and animal shelters) are identified as class-B dealers. At few instances use of the wild
animals such as monkeys and birds is also followed (Baumans,
2005). In clinical testing laboratories, animals are isolated from
their groups and used as a tool irrespective of their natural instincts. For the experimental procedures, either a whole animal
or its organs and tissues are used. For this purpose animals are
euthanized (killed) by established methods. Many times, the
animals surviving the clinical testing are euthanized at the
end of an experiment to avoid the later pain and distress
(Rusche, 2003). In some cases (for example in LD 50 analysis)
animals die as a result of the experiment.
The pain, distress and death experienced by the animals
during scientific experiments have been a debating issue for a
long time. Argument is that being alive, animals have the
rights against pain and distress and hence, their use for experimentation is unethical and must be stopped (Rollin, 2003).
Various acts and laws have been passed to bring the control
over unethical use of animals and minimize the pain to animals
during experimentation. For example, in 1824, the organization for animal rights was formed by the Royal Society for
the Prevention of Cruelty to Animals. In 1876, an act for prevention of cruelty to animal was formed in the UK (Balls,
1994). It came into existence in India, France and USA in
the year 1960, 1963 and 1966, respectively. At present, many
rules and acts are followed at the international level, to protect
the animals against the cruelty and misuse. The organizations
like ICH (International Conference on Harmonization of technical requirements for registration of pharmaceuticals for human use), CPCSEA (Committee for Purpose of Control and
Supervision on Experiments on Animal), NIH (National Institute of Health), and OECD (Organization for Economic Cooperation and Development) provide the guidelines for animal
house keeping, breeding, feeding, transportation, and mainly
for their use in scientific experiments (Rollin, 2003). Besides
the major concern of ethics, few more disadvantages of animal
experimentation are requirement of skilled/trained manpower
and time consuming protocols. Moreover, very high cost involved in breeding, housing and lengthy protocols of animal
experiments is another drawback (Balls, 1994).
2. Three Rs: reduction, refinement and replacement
Alternatives to animal testing were proposed to overcome
some of the drawbacks associated with animal experiments
and avoid the unethical procedures. A strategy of 3 Rs is being
applied which stands for reduction, refinement and replacement of laboratory use of animals (Ranganatha and Kuppast,
2012). Different methods and alternative organisms are applied to implement this strategy. The concept of replacement
of animals was first discussed in 1957 by Charles Hume and
William Russell at the Universities Federation for animal welfares (UFAW) (Balls, 1994). Russell and Burch (1959) suggested some ways to make the animal experiments more
humanly, which was later called as 3 Rs. This approach motivates the use of minimum number of animals i.e. ‘reduction’ in
the total number of animals used in an experiment. The use of
animals must be planned and ‘refined’ carefully in such a way
that pain and distress caused during the experiment should be
minimized. Moreover, if possible higher animals should be ‘replaced’ with alternative methodologies and lower organisms
(Ranganatha and Kuppast, 2012; Zurlo et al., 1996). Animal
replacement is defined as, ‘any scientific method employing
non-sentient material which may replace use of conscious living vertebrates in animal experimentation’. Two types of
replacements were distinguished as ‘relative’ and ‘absolute’
replacement. In relative replacement the animals are used but
not exposed to any distress during experiment. No use of
224 S.K. Doke, S.C. Dhawale
animals at any stage of experiment is identified as the absolute
replacement strategy (Balls, 1994).
2.1. Reduction
With the help of statistical support and careful selection of
study design one can produce meaningful scientific results of
an experiment. For example, in vitro cell culture is a good
way to screen the compounds at early stages. Use of the human
hepatocyte culture gives the information about how a drug
would be metabolized and eliminated from the body. Inclusion
of such method in study design helps to eliminate unsuitable
compounds in preliminary stages only and minimizes the use
of animals in further tastings (Kimber et al., 2001). Live animals and embryos are used to study effects of some compounds on embryo development. In vitro embryonic stem cell
culture test helps to reduce the number of live embryo used
and the compounds which are toxic toward developing embryo
(Gipson and Sugrue, 1994; De Silva et al., 1996). Also, sharing
or providing the discovered data (like characteristics of excipients for the test drug) avoids the necessity of animal studies.
2.2. Refinement
Enriching the cage environment by taking care of animals
reduces the stress on animals. Scientists should refine the animal facility so that pain, discomfort and distress during animal
life and scientific procedures are reduced. Moreover, under the
stress and discomfort there may be imbalance in hormonal
levels of animals leading to fluctuations in the results. Hence,
experiments need to be repeated which causes an increase in
the number of experimental animals. So refinement is necessary not only to improve the life of laboratory animals but also
to improve the quality of research (Hendriksen, 2009). For
example, it was observed that when mice genetically modified
to study Huntington’s disease were provided with a complex
cage environment with opportunity to nest, hide, gnaw and
forage, the disease progressed slowly than the mice in barren
cage. Also, such mice were found to mimic the progress of
the human disease more closely. Such a refinement provides
a very good model to treat the disease and also minimize stress
to the animals (De Silva et al., 1996).
2.3. Replacement
Various alternatives to the use of animals have been suggested,
such as in vitro models, cell cultures, computer models, and
new imaging/analyzing techniques (Balls, 2002). The in vitro
models provide the opportunity to study the cellular response
in a closed system, where the experimental conditions are
maintained. Such models provide preliminary information
for outcome of an experiment in vivo. For example, computer
models were used to study the working of the heart and to
select the potential drug candidates (Gipson and Sugrue,
1994). In many countries, in vitro cell cultures have replaced
the skin irritancy test and Draize eye irritancy test and use
of animals in those. Another example is, extraction of insulin
from the pancreas of pigs and cow, but now it is obtained from
the bacterial cultures which are lifeline drugs for diabetic
patients. This extracted insulin needs to be checked for its
purity, efficacy and dose. Use of animals was routine for such
checking, but now chromatography techniques are used for
checking the purity, efficacy and calculation of dosages of
drugs (Foreman et al., 1996). Overall, replacement substantially reduces the use of animals in various processes.
3. Alternative methods
Various methods have been suggested to avoid the animal use
in experimentation. These methods provide an alternative
means for the drug and chemical testing, up to some levels.
Advantages associated with these methods are, time efficiency,
requires less man power, and cost effectiveness. These methods
are described in detail as follows3.1. Computer models
Computers can help to understand the various basic principles
of biology. Specialized computer models and software programs
help to design new medicines. Computer generated simulations
are used to predict the various possible biological and toxic
effects of a chemical or potential drug candidate without animal
dissection. Only the most promising molecules obtained from
primary screening are used for in vivo experimentation. For
example, to know the receptor binding site of a drug, in vivo
experimentation is necessary. Software known as Computer
Aided Drug Design (CADD) is used to predict the receptor
binding site for a potential drug molecule. CADD works to identify probable binding site and hence avoids testing of unwanted
chemicals having no biological activity. Also, with the help of
such software programs we can tailor make a new drug for the
specific binding site and then in final stage animal testing is done
to obtain confirmatory results (Vedani, 1991). Hence, the total
number of experimental animals is lowered and the objectives
of Russel and Burche’s 3 Rs are achieved.
Another popular tool is the Structure Activity Relationship
(SARs) computer programs. It predicts biological activity of a
drug candidate based on the presence of chemical moieties attached to the parent compound. Quantitative Structure Activity Relationship (QSAR) is the mathematical description of the
relationship between physicochemical properties of a drug
molecule and its biological activity (Knight et al., 2006). The
activities like carcinogenicity and mutagenicity of a potential
drug candidate are well predicted by the computer database.
The recent QSAR software shows more appropriate results
while predicting the carcinogenicity of any molecule. The
advantages of computer models over conventional animal
models are the speed and relatively inexpensive procedures
(Matthews and Contrera, 1998). A very good example is a
study by Dewhurst et al. (1994) which assessed the effectiveness of computer models versus the traditional laboratory
practices. In this comparative study, two groups of undergraduate students performed an experiment with the traditional wet
lab approach and computer assisted learning (CAL), respectively. CAL is an interactive computer assisted learning
(CAL) program without involvement of real experimental
tools. At the end of the study both the groups were assessed
for the knowledge gain (through test questionnaires, calculations, and interpretation). It was found that the students
performing CAL had a better problem solving attitude.
Moreover, the cost of new techniques was much less than
the traditional laboratory practices (Dewhurst et al., 1994).
Alternatives to animal testing: A review 225
3.2. Cells and tissue cultures
Use of in vitro cell and tissue cultures which involves growth of
cells outside the body in laboratory environment can be an
important alternative for animal experiments. The cells and tissues from the liver, kidney, brain, skin etc. are removed from
an animal and can be kept outside the body, in suitable growth
medium, for few days to several months or even for few years.
In vitro culture of animal/human cells includes their isolation
from each other and growing as a monolayer over the surface
of culture plates/flasks. Cellular components like membrane
fragments, cellular enzymes can also be used. Various types
of cultures like cell culture, callus culture, tissue culture and organ culture are used for various purposes. Benefits associated
with techniques are, easy to follow, less time consuming and
are less expensive. These methodologies are routinely used
for preliminary screening of potential drug molecules/chemicals to check their toxicity and efficacy (Shay and Wright,
2000; Steinhoff et al., 2000). Almost all cosmetics, drugs and
chemicals are tested for their toxicity and efficacy, using these
tests. For example, eye irritancy test. To check the irritancy of
chemicals previously Draize test was used, which requires animals (mainly rabbit). It is very painful and every time a new
animal is used. Ke Ping Xu and coworkers suggested an alternative which uses bovine corneal organ culture. The bovine
cornea is cultured up to three weeks in laboratory and various
analytical methods are used to evaluate the toxicological effect
of test chemical irritancy in vitro (Xu et al., 2000).
3.3. Alternative organisms
The ethical issues have posed many restrictions over the experimental use of higher model vertebrates like guinea pig, rats,
dogs, monkeys etc. Therefore, use of alternative organisms
has been proposed. Different model organisms are used to replace experimental animals (Table 1).
3.3.1. Lower vertebrates
Lower vertebrates are an attractive option because of the
genetic relatedness to the higher vertebrates including mammals. Moreover, there are less ethical problems involved in
the experimental use of lower vertebrates.
3.3.1.1. Example – Danio rerio. Danio rerio, commonly called
as zebra fish, is a small freshwater fish with an approximate
length of 2–4 cm. It has a nearly transparent body during early
development, which helps easy visual access to the internal
anatomy. The optical clarity allows direct observation of
developmental stages, identification of phenotypic traits during mutagenesis, easy screening, assessment of endpoint of toxicity testing and direct observation of gene expression through
light microscopy. Small size, short life cycle and high fecundity
favor its laboratory use.
The working space, cost of laboratory solutions, test chemicals and the manpower involved are reduced by opting D. rerio as an alternative to animals (Hill et al., 2005). Its embryos
and larvae can be developed and used for testing in cell culture
plates and Petri dishes. Whole genome sequence availability
makes Zebra fish an attractive option for molecular and genetic research. From infancy to the adult stage it is used in a variety of applications, mainly for the detection of various
toxicological studies of chemicals and pharmaceuticals. It is
also having wide applications in the investigation of cancer,
heart diseases, neurological malfunctions, behavioral diseases
and to observe the mutations and problems in organ development due to exposure to test molecules. Modeling of certain
human diseases in zebra fish could be used to ameliorate the
disease phenotype and malfunctions in organ development
(Peterson et al., 2008).
3.3.2. Invertebrates
Invertebrate organisms are widely used as an alternative for
laboratory use of animals. They have been used to study
Table 1 Selected examples of organisms as alternatives for laboratory use of animals.
Alternative organism Remarks
Prokaryotes
Escherichia coli Model for molecular and genetic studies
Bacillus subtilis Model for cellular differentiation
Caulobacter crescentus
Protists
Dictyostelium discoideum Model for molecular and genetic studies
Fungi
Neurospora crassa Model for genetic study, circardian rhythm and metabolic regulation studies
Saccharomyces cerevisiae
Schizosaccharomyces pombe Model for molecular and genetic studies
Aspergillus nidulans
Lower vertebrate
Danio rerio/zebrafish
Invertebrates
Amphimedon queenslandica Studies on evolution, developmental biology and comparative genomics
Aplysia sp./sea slug Neurobiology
Caenorhabditis elegans Genetic development studies
Drosophila melanogaster Genetics and neurology research
Hydra/Cnidaria To understand the process of regeneration and morphogenesis
226 S.K. Doke, S.C. Dhawale
various diseases like Parkinson’s disease, endocrine and memory dysfunction, muscle dystrophy, wound healing, cell aging,
programmed cell death, retrovirus biology, diabetes and toxicological testing (Lagadic and Caquet, 1998). Invertebrates
have an undeveloped organ system and do not have the adaptive immune system, which poses some limitations for their use
in human diseases. However, they hold numerous benefits,
such as a brief life cycle, small size and simple anatomy, so that
a large number of invertebrates can be studied in a single
experiment within a short period with less ethical problems.
Their cost of housing is less compared to the animals. For
example, thousands of flies could be accommodated in a shelter where only few mice can be kept (Wilson-Sanders, 2011).
3.3.2.1. Example – Drosophila melanogaster. Drosophila melanogaster, also known as fruit fly is one of the most widely studied invertebrates in research (Gilbert, 2008). It has a well
studied genome which enables study of molecular mechanisms
underlying the human diseases. Its complete genome has been
sequenced and annotated, which encodes more than 14,000
genes on four chromosomes. Only three genes carry the bulk
of genome of D. melanogaster. Nearly 75% of the genes involved in human diseases are believed to have a functional
homolog in the fly (Reiter et al., 2001; Wilson-Sanders,
2011). D. melanogaster requires extremely low cost of maintenance, propagation and screening as compared to the other
mammal based models. It also produces the results very rapidly due to a short life cycle. Fruit fly possesses four stages
in life cycle – the embryo, the larva, the pupa and the adult.
Each stage of fly has its own advantage, hence considered as
a multiple model organism to study the various concepts (Pandey and Nichols, 2011). The Embryo is frequently used to
study the cell fate determination, neuronal development, axon
path finding, organogenesis, fundamental developments and to
examine pattern formation. The larva is used to study the
physiological and developmental processes and behaviors like
foraging. The adult fly is a very complex organism. The functions of various structures like the heart, lungs, gut, kidney and
reproductive tract are equivalent as that of mammals (Rothenfluh and Heberlein, 2002).
The response of flies to many drugs which are acting on
CNS is similar to that observed in mammals. The brain of
the adult fly is quite extraordinary because more than
100,000 neurons form the discreet circuits, which mediate various complex behaviors like circadian rhythms, learning and
memory, feeding, sleep, courtship, aggression, grooming and
flight navigation (Pandey and Nichols, 2011; Rothenfluh and
Heberlein, 2002; Wolf and Rockman, 2008). Number of
molecular and genetic tools has been made available to study
Drosophila. Due to many similarities in development and
behavioral activities, fruit fly served as a unique and sensitive
model for the study of human genetics and diseases (Beckingham et al., 2005). It is also used to express the protein products
found in human diseases and to compare the resulting pathologic conditions. Fruit fly serves as an important tool to investigate neurodegenerative diseases like Alzheimers, Parkinson’s,
disease and Huntington’s disease (Bonini and Fortini, 2003;
Iijima and Iijima-Ando, 2008; Iijima et al., 2004). It is used
in primary small molecule discovery validation as well as in
the target discovery processes by taking advantage of the
sophisticated genetics available in it. In 1994 the Nobel Prize
for physiology and medicine was awarded to Ed Lewis for
his pioneering research defining gene structure in flies, as well
as to Eric Weischaus and Christiane Nusslein-Volhard for
their studies investigating embryogenesis (Iijima et al., 2004).
3.3.2.2. Example – Caenorhabditis elegans. Caenorhabditis elegans is a eukaryotic nematode. This multi cellular organism is
approximately 1 mm in length and has a very short generation
time. Complete life cycle of this hermaphrodite is about 2–
3 weeks. Embryogenesis occurs in 12 h and an adult form is
developed in 2.5 days. It is transparent, genetically amenable
and has simple cellular complexity. Hence, was selected as a
model organism by Nobel laureate Brenner (Barr, 2003;
Strange, 2007). Life cycle of C. elegans proceeds through various complex developmental stages like embryogenesis, morphogenesis and growth to an adult. This is one of the most
commonly used model organisms for research purposes. Information obtained can be applicable to more complex organisms
like humans. As a model, C. elegans have been used to study
various neurological disorders like Huntington’s disease, Parkinson’s disease, Alzheimer’s diseases; various immune disorders as well as cancer, diabetes. It has served development
and testing of the therapeutic agents for treatment of these diseases (Artal-Sanz et al., 2006; Faber et al., 1999; Link et al.,
2001; Nass et al., 2008; Pujol et al., 2008).
3.3.3. Microorganisms
3.3.3.1. Example – Saccharomyces cerevisiae. Brewing yeast,
Saccharomyces cerevisiae is the most popular and important
model organism due to its rapid growth, ease of replica plating
and mutant isolation, dispersed cells, well defined genetic system and highly versatile DNA transformation system. Yeasts
can be grown in solid or liquid culture and isolated as colonies
derived from a single cell on solid media. The generation time
is very short i.e. about 90 min, hence it is very easy to grow a
large population and analyze it (Mell and Burgess, 2002).
Whole genome of this unicellular fungus has been sequenced
in 1996. The nuclear genome contains about 16 chromosomes
with more than 13 million base pairs. It also contains an extra
nuclear genome in the mitochondria. The budding yeast carries
its genetic information in the form of 6000 genes. The number
and size of genes are relatively small and the density of genes is
very high. Best characterized and studied genome makes S.
cerveisiae one of the most ideal eukaryotic microorganisms
for the biological studies. Presence of similar cellular architecture and rudimentary life cycle like multi cellular eukaryotes is
another advantage. The numerous membrane-bound organelles like nucleus, peroxisome, mitochondria and the organelles
of secretary pathway also mimic the functions of mammalian
cells (Mell and Burgess, 2002). This brewing yeast is used to
understand programmed cell death, cell death regulators in humans and is very useful in cancer research (Madeo et al., 2002).
S. cerevisiae helps to understand the fundamental aspects of
cellular biology in neurodegenerative diseases like Alzheimer’s,
Parkinson’s and Huntington’s diseases by studying the endogenous or heterologous proteins that lay at the root of these diseases (Pereira et al., 2012; Siggers and Lesser, 2008).
4. Conclusion
Animal ethics is an issue as important as the human welfare.
More efforts need to be undertaken for effective implementation
Alternatives to animal testing: A review 227
of 3 Rs during laboratory use of animals. Various alternatives to
animal use have been suggested, which need to be implemented
in an effective manner. For this integration of various computer
models, bioinformatics tools, in vitro cell cultures, enzymatic
screens and model organisms are necessary. Use of modern analytical techniques, data acquisition and statistical procedures to
analyze the results of alternative protocols can provide dependable outcomes. These integrated approaches would result in
minimum involvement of animals in scientific procedures.
Acknowledgements
Authors are thankful to Prof. S. Mohan Karuppayil, former
Director, School of Life Sciences, SRTM University for his
kind support.
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