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ANIMAL CLONING AND GENOME EDITING IN BUFFALO, WITH
SPECIAL REFERENCE TO INDIA
Clonación de animales y edición del genoma del búfalo, con especial referencia a la India
Naresh Selokar*, Manoj Kumar Singh
Embryo Biotechnology Lab, Animal Biotechnology Division, ICAR-National Dairy Research Institute, Karnal, India-132001
*Correspondence email: naresh.selokar@icar.gov.in or selokarnareshlalaji@gmail.com
dujeron varios clones de búfalos y se están realizando intentos
para producir una población de más animales de élite. La clo-
nación de búfalos ha llegado desde los manuscritos cientícos
hasta las granjas de los agricultores. Recientemente, la edición
del genoma se ha convertido en una poderosa herramienta
para manipular los genomas de varias especies animales. Ya
se han producido varios modelos de ganado y se puede prever
que en el futuro se producirán más animales modelo utilizando
el sistema CRISPR-Cas. Estas historias de éxito han abierto
múltiples perspectivas para los investigadores indios. En este
artículo, ofrecemos una descripción general del progreso de
la clonación de búfalos en la India y nuevas iniciativas sobre
edición del genoma.
Palabras clave: Búfalo, Embrión, Clonación, edición del
genoma, CRISPR.
BUFFALO CLONING JOURNEY
Ten years ago, on February 6, 2009, a team of scientists
at the ICAR-National Dairy Research Institute (NDRI), Kar-
nal, created history in the eld of animal cloning research in
India. They produced the world’s rst cloned riverine bualo
calf, Samrupa, using an inexpensive, simple somatic cell nu-
clear transfer (SCNT) technique called handmade cloning [1].
This simple technique is less demanding regarding equipment,
skill, and time. It does not require sophisticated tools like mi-
cromanipulators and needle grinders to perform microscopic
manipulation of oocytes and somatic cells [2]. The world’s rst
cloned mammal, ‘Dolly’, the sheep, was born 13 years earli-
er at Scotland’s Roslin Institute using SCNT. SCNT is an ad-
vanced assisted reproduction technique in which a somatic cell
is transferred or fused with an enucleated oocyte. The recon-
structed embryo, thus produced, develops to the blastocyst
stage, at which it transplants into a surrogate mother, who car-
ries the pregnancy and delivers the ospring. Worldwide, more
than 20 animal species have been cloned using dierent SCNT
methods, including handmade cloning. Currently, China is at
ABSTRACT
India owns the best bualo breeds, particularly Murrah, which
is famous worldwide for high milk production. India’s white and
pink revolution cannot be imagined without the contribution of
bualo, and to achieve this, the best productive animals need to
be produced through scientic interventions. Animal cloning is
a technique used to produce multiple copies of the best animals
without normal reproduction. In India, bualo cloning has al-
ready happened, and India’s rst cloned bualo was produced
in 2009. Later, several bualo clones were produced, and at-
tempts are ongoing to produce a stock of more elite animals.
Bualo cloning has made its way from scientic manuscripts
to farmers’ farms. Recently, genome editing has emerged as
a powerful tool to manipulate the genomes of several animal
species. Various livestock models have already been pro-
duced, and it can be foreseen that more model animals will be
produced in the future using the CRISPR-Cas system. These
success stories have opened multiple prospects for Indian re-
searchers. In this article, we provide an overview of the prog-
ress of bualo cloning in India and new initiatives on genome
editing.
Keywords: Bualo, Embryo, Cloning, genome editing, CRIS-
PR.
RESUMEN
India posee las mejores razas de búfalos, en particular Murrah,
famosa en todo el mundo por su alta producción de leche. La
revolución blanca y rosa de la India no se puede imaginar sin
la contribución del búfalo y, para lograrlo, es necesario producir
los animales más productivos mediante intervenciones cientí-
cas. La clonación animal es una técnica utilizada para producir
múltiples copias de los mejores animales sin una reproducción
normal. En la India ya se ha clonado búfalos, y el primer búfalo
clonado del país se produjo en 2009. Posteriormente, se pro-
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the forefront of cattle and pig cloning, whereas India is heading
bualo cloning and has made signicant achievements.
In comparison to other farm animals, limited studies have
been done in the eld of bualo cloning. Researchers at NDRI
made the rst attempt to clone bualoes, Karnal, during 1994-
97, in which cells from fertilized or in vivo-produced embryos
were injected into enucleated oocytes to generate cloned em-
bryos [3]. This method of nuclear transfer using micromanipula-
tors was similar to a method that was used to produce the Dolly.
Unfortunately, no blastocyst-stage embryo could be produced
during that time. The possible reasons could be 1) lack of tech-
nical advances at that time, since highly skilled micromanipu-
lation methods were used; 2) improper culture conditions for
SCNT embryos; 3) not much research had been done in SCNT
during the late nineties; therefore, the researchers did not have
much information on SCNT. Later, in 2009, a simplied method
of SCNT, named handmade cloning, was used for reprogram-
ming dierentiated somatic cells of adult animals. This method
was used successfully at NDRI, Karnal, for producing blasto-
cyst-stage embryos, and India’s rst cloned farm animal was
born in 2009 [4]. Subsequently, many cloned bualoes were
produced [1]. Later, in 2017, several years after the birth of In-
dia’s rst cloned bualo, another research institute, ICAR- Cen-
tral Institute for Research on Bualo (CIRB), Hisar, transferred
the bualo cloning technology to the eld, away from the clon-
ing laboratory [5]. CIRB also produced seven cloned copies
from a single breeding bull.
METHOD OF BUFFALO CLONING
The method of producing cloned embryos appears to
be quite simple and straightforward. In principle, the donor ge-
nome is fused with the cytoplasm of an enucleated ooplasm,
followed by activation of the recombined ooplasm to stimulate
embryonic development to the blastocyst stage, at which the
embryos can be either transferred to suitably synchronized
recipients or cryopreserved for future use. Two dierent ver-
sions of SCNT have been used to produce cloned embryos in
bualo [1]. The rst is the traditional micromanipulation-based
method of SCNT, which was used to produce the rst cloned
mammal, ‘Dolly.’ This method is well-established and has been
extensively used in most cloning laboratories. To date, 23 ani-
mal species, including bualo, have been successfully cloned
using this method. An alternative method called Hand-made
cloning (HMC), which was developed as a simplied alternative
to micromanipulation-based SCNT [2], has been successfully
used for the production of cloned ospring in many farm animal
species such as cattle, pig, sheep, and goat, and bualo. A sig-
nicant advantage of HMC over the micromanipulation-based
method is that there is no requirement for micromanipulators
and their tools-making instruments for enucleation and fusion
and for a highly skilled workforce to operate those instruments
[2]. This signicantly reduces the cost of establishing a cloning
laboratory, so such laboratories can be set up in developing
countries where the availability of funds and technical expertise
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are major constraints. In addition to the multiple advantages of
HMC, one practical problem is that two oocytes are used for the
reconstruction of a single embryo, which increases the require-
ments of oocytes to generate cloned embryos, and the use of
two recipient oocytes to generate a single cloned embryo may
be responsible for higher mitochondrial heteroplasmy. We ad-
opted this simplied method of SCNT developed by Vajta et
al. (2001) and incorporated several modications in the basic
procedures, resulting in ecient enucleation, fusion, and acti-
vation, leading to a high blastocyst development rate [1]. Pub-
lished reports suggest that with a higher blastocyst production
rate, HMC can be used as an alternative method of SCNT.
APPLICATION OF BUFFALO CLONING IN INDIA
Potential applications of bualo cloning in India are
1) make multiple copies of elite bualoes such as high milk-pro-
ducing females or proven breeding bulls; 2) creation of trans-
genic bualoes that harbor human genes in their genome and
can serve as bioreactors to produce therapeutic proteins like
insulin and blood clotting factors, preferably in milk and 3) cre-
ation of disease model bualoes that are designed to express
certain human diseases. Despite multiple applications, bualo
cloning needs improvement since, on average, less than 6-8
% of the transferred cloned embryos produce healthy ospring
[6]. Faulty or improper reprogramming of dierentiated somatic
cells is considered a major problem behind the low success of
cloning technology. Joint research eorts are going on at two
laboratories in India, namely CIRB, Hisar, and NDRI, Karnal,
to unravel the exact cause of faulty reprogramming and to im-
prove bualo cloning eciency.
CURRENT STATUS OF BUFFALO CLONING
RESEARCH
Since Dolly died at the age of 6 years, the fear has been
spread that clones could not survive as long as their original
donors due to premature aging and genetic abnormalities. Re-
searchers who produced Dolly spent more than 15 years un-
raveling the issue of aging in cloned animals. They recently
published a paper in the Nature Journal that proved premature
aging in clones was a wrong perception and that four genomic
copies of Dolly have normal growth, health, and aging process-
es [7]. This study tells us that premature aging in cloned ani-
mals has been exaggerated. The same aging principle can be
applied to bualo since the basic reprogramming mechanisms
are similar across the species. In addition to the wrong per-
ception of the premature aging of cloned animals, people are
also worried about the safety of products such as milk, meat,
and semen obtained from cloned animals. In 2006, the U.S.
Food and Drug Administration (FDA) examined the results of
extensive studies that were conducted in dierent countries
such as the U.S., U.K., Japan, China, and New Zeeland, and
recommended that products obtained from cloned animals are
similar in terms of constituents and nutrients to the products
of non-cloned animals [8]. Therefore, consumers are expected
to benet from consistent milk, meat, and semen from cloned
animals, including bualo.
During the last ten years, research and improvement
in bualo cloning techniques have produced several bualo
clones that are normal, healthy, and fertile [1,6]. These advanc-
es were made possible by signicant improvements in blasto-
cyst production rate and reduced health risks to born clones.
Bualo cloning can allow breeders and farmers to produce
identical copies of their best animals, particularly proven breed-
ing bulls, to exploit maximum production potential from them.
By considering the potential utility of bualo cloning in India, the
Indian Council of Agriculture Research has been working on a
mega research project to improve bualo’s production potential
using semen of clones of elite breeding bulls, and our team is
heading towards this goal.
METHODS USED TO MODIFY GENOME OF
DOMESTIC ANIMALS
Conventionally, genetic changes in the livestock genome
can be achieved by selective breeding, in which continual mat-
ing of favorable elite animals with unfavorable animals over
many generations leads to the up-gradation of the specic
alleles in the genome of the targeted animal population [9].
In dairy husbandry, semen from bulls of high milk-producing
breeds (e.g., Holstein Friesian bulls) have been used to insem-
inate the low milk-producing breeds (e.g., Tharparkar cows)
with the aim of producing upgraded female calves with im-
FIGURE 1. The cloned bualo, named Garima, born at
ICAR-National Dairy Research Institute, Karnal on 6th June,
2009 with the birth weight of 43 kg, has produced seven
healthy and normal progeny. This cloned female has been
growing well and does not have any physiological abnor-
malities
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proved milk production trait. A well-known ‘Karan Fries’ breed
of cattle was developed by the crossing of Holstein Friesian
with Tharparkar at the National Dairy Research Institute, Kar-
nal, using the principle of conventional breeding (http://www.
ndri.res.in/ndri/Design/livestock_farm.html, accessed on Sep-
tember 10, 2021). With the selective breeding, milk production
in India has increased from 55 million tonnes in 1991-92 to 187
million tonnes in 2018-19, almost triple (http://www.nddb.org/
information/stats/milkprodindia, accessed on September 10,
2021), titled India becomes world’s largest producer of milk.
It is perceived that selective breeding continues to be a vital
approach to producing desirable animals. Moreover, breeding
allows the genome modication of those genetic traits which
are naturally available in the population.
The employment of engineered nucleases would save the
eight to ten generations of back-crossing required in conven-
tional breeding to modify animals and allow the addition of new
genes or the deletion of unfavorable genes. In FIG. 2, we have
schematically shown the comparison between traditional breed-
ing and current genetic engineering methods; both can be used
to produce desirable animals. Before the birth of the famed ‘Dol-
ly’ (born in 1996), the world’s rst genetically modied bovine,
Herman the Bull, was produced by manipulation of the early
embryo stage with the human lactoferrin gene [10]. Eighty-three
calves were produced from this modied bull, and all calves in-
herited the lactoferrin production gene [11]. This work has been
opening unlimited promises to livestock research, such as ma-
nipulating genes that aect milk and meat production, the pro-
duction of essential proteins in milk, and producing model ani-
mals for biomedical or veterinary applications. Multiple research
success stories have already demonstrated that genetic engi-
neering with reproductive technologies (e.g., SCNT and IVF)
can be used to manipulate the livestock genome [12]. However,
wide-scale applications have always been a challenge due to
the extremely poor eciency of these techniques.
CRISPR EDITING
Engineered nucleases, including zinc nger nucleas-
es (ZFNs), transcriptional activator-like eector nucleases
(TALENs), and the clustered regularly interspaced short pal-
indromic repeats (CRISPR)–CRISPR-associated protein (Cas)
system have been used for targeted modications of the ge-
nomes of many organisms. Before engineered nucleases were
developed, ecient genome manipulations, particularly knock-
ing-out of genes, could only be achieved by using the inecient
homologous recombination (HR) in pluripotent embryonic stem
(ES) cells. Thousands of mice lines were produced using the
FIGURE 2. Conventional breeding and new genome editing as tools to manipulate the genome of domestic animals. In con-
ventional breeding, unfavorable animal population (lower milk producers) inseminated with the semen of favorable animals
(Bulls of high milk producers) for many generations (8-10), which resulting in the up-gradation of the specic trait (milk
production). In genome editing, new genes can add to genome (e.g. lactoferrin in milk) or existing genes can delete (e.g.
beta-lactoglobulin from milk) from genome, possibly in one generation
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13th World Bualo Congress ~ 13er Congreso Mundial de Búfalos / Lectures / Biotechnology & Omics Technologies ___________________
HR approaches; however, the translational to other species,
including livestock, was limited by the absence of genuine ES
cells in these species. In recent years, genome manipulation
has gained tremendous momentum due to the discoveries of
engineered nucleases, popularly named genome nucleases
or scissors, namely ZFNs, TALENs, and CRISPR. In principle,
these designer nucleases create double-strand breaks (DSBs)
at specic desired sites in the genomic DNA. DSBs are repaired
either by the non-homologous end joining (NHEJ) or the homol-
ogous directed repair (HDR) procedures, which have been ex-
ploited to manipulate the genome [13]. NHEJ is an error-prone
procedure involving interrupted joining of the broken ends of
DNA, creating insertions and deletions (indels) of nucleotides
at cleavage sites, leading to disruption of gene function. HDR
is a more accurate procedure, which requires homologous DNA
sequences as templates for repair; therefore, a designed nucle-
otide sequence can be integrated into the genome by supplying
it as an exogenous DNA template for HR events. FIG. 3 rep-
resents how designer nucleases work.
POSSIBLE OPPORTUNITIES TO INDIA
India possesses a large number of domestic animals:
about 192.49 million cattle, 109.85 million bualoes, 74.26 mil-
lion sheep, and 148.88 million goats. Milk and milk products,
meat and meat products, eggs and eggs products are integral
to the Indian diet. To satisfy the demand for food for the ev-
er-growing population, the country needs the continuous and
sustainable production of milk, meat, and other animal prod-
ucts. Climate change, emergence and re-emergence of dis-
ease pathogens, and non-availability of fodders are new chal-
lenges that might be overcome through scientic intervention
for sustainable animal production. We believe that the CRIS-
PR-Cas system and other sophisticated genome engineering
techniques could be able to manage some of these concerns.
A few futuristic examples are: 1) the production of bulls carrying
disease resistance (e.g., FMD, rinderpest, and black quarter)
or stress-tolerant (e.g., extreme heat or extreme cold) genes.
The semen of such bulls would be valuable to produce toler-
ance osprings. 2) The generation of bulls, which produce only
spermatozoa with the X chromosome, produced sorted semen,
producing female ospring. Male ospring are unprotable to
farmers due to the ban on cattle slaughter and mechanization
of agriculture. 3) The production of veterinary vaccines (e.g.,
against FMD and rinderpest) or human therapeutic proteins
(e.g., insulin) in the mammary glands of dairy animals for clin-
ical uses.
According to existing knowledge and technologies avail-
able in India, the bualo will be a preferred choice among do-
mestic animals for exploring CRISPR-Cas applications. Ex-
FIGURE 3. How molecular scissors/designer nucleases work in embryos: Molecular scissors are delivered in a one cell
stage embryos, they specically cleavage the target DNA causing a double-strand break (DSB). The DSB is subsequently
repaired either by the error-prone non-homologous end joining pathway (NHEJ), or the homology-directed repair (HDR)
processes. NHEJ leads to gene disruption due to insertions or deletions (indels) of nucleotides, whereas HDR leads to gene
correction or insertion if provided with donor template
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perimental working with bualos has several advantageous
aspects: 1) ample availability of oocytes for manipulations, 2)
most assisted reproductive techniques, such as IVM, IVF, IVC,
ET, and MOET, have been demonstrated, 3) SCNT has suc-
cessfully been proved and is currently being used in the cloning
of breeding bulls and elite females, and 4) scientic expertise
and technical personnel are available, including sta for e-
cient manipulation of oocytes and embryo transfer. Our lab re-
cently initiated a proof of principle study aiming to produce ma-
nipulated bualoes expressing improved production traits and
health. Two major limitations need to be addressed to reach
this aim: 1) poor eciency of bualo cloning success rate, and
2) the complete annotation of the bualo genome, which still
needs to be fully accomplished. Basic and applied aspects of
research are required to achieve successful gene editing in the
bualo species.
CONFLICT-OF-INTEREST STATEMENT
The authors have no conicts of interest to declare.
ACKNOWLEDGMENT
Financial support has been received from the Indian
Council of Agricultural Research (ICAR), New Delhi and the
Department of Biotechnology (DBT) to continue bualo cloning
work and to initiate the genome editing studies in bualo.
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