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Ex-situ Germplasm Preservation Methodology for Farm
Animals
J.R. Dobrinsky
Germplasm & Gamete
Physiology Laboratory, Agricultural Research Service
U.S. Department of
Agriculture, Beltsville, Maryland 20705
USA
INTRODUCTION
Considerable
progress has been made in the improvement and simplification of preservation
procedures routinely used for sperm, oocytes and embryos, especially in
cattle. Conventional programmable
freezing and vitrification of germplasm and embryos have given veterinarians,
scientists and producers alternatives in their herd reproduction practices. While continuously being improved upon
for use in cattle, there are few methods available, tested or proven effective
with germplasm and embryos of other common domestic farm animals. This paper will discusses concepts and
breadth of germplasm preservation without elaborating on detailed technical
aspects. It will then discuss
novel development of germplasm preservation in a common farm species other than
cattle, illustrating how modern science can be applied to solve some of the
most difficult biological questions.
Finally, it will discuss conceptually how germplasm preservation may be
applied and implemented in propagating desirable genetics on a global basis.
EX-SITU GERMPLASM PRESERVATION
in situ (¹n sº“t›, s¶“-) adv. --in si·tu adj. In the original position. [Latin in sit¿ : in, in + sit¿, ablative of situs,
place.]
ex (µks) prep. 1. Not including; without [Latin.
See eghs .] eghs. Important derivatives are: ex-, exotic, external, extra-, strange, extreme. eghs. Out. 1.
Variant eks. a. EX, EX-from Latin ex, ex-,
out of, away from
preservation (prµz”…r-v³“sh…n) n. from preserve (pr¹-zûrv“) –tr. 1. To maintain in safety from
injury, peril, or harm; protect. 2. To keep in perfect or unaltered condition;
maintain unchanged. 3. To keep or maintain intact. 4. To prepare for future
use. 5. To prevent from decaying or spoiling.
derivatives:
•
ex situ = out
of original position
•
preservation
= maintain intact, unchanged, protected; prepared for future use
germplasm: sperm, egg or oocyte (gametes); germ cells that carry
haploid chromosomes
embryo: fertilized egg or oocyte possessing diploid chromosomes from
the sire and dam
inferred definition ex-situ germplasm
preservation: the maintenance and indefinite protection of sperm, eggs and
embryos outside the reproductive tract.
Preservation
potential Today, large numbers of animals are transported by
air freight from countries or regions in which nucleus herds are located to
countries or regions in which new breeding units are being established.
Transportation costs are extremely high, risk of disease transmission is a
threat, and health testing and import/export bureaucracy and requirements are
difficult and involved. These costs and associated constraints could be
considerably reduced by shipping gametes or embryos rather than live
animals. The use of embryos in
addition to sperm represents a major increase in the efficiency of transmitting
improved genetic potential. The ability to preserve maternal genetic
information indefinitely would enable the transmission of improved genetic
potential in a form other than the live animal. Desirable breeds possessing beneficial production
characteristics or valuable disease resistance traits are not in any one
country or region, yet they are desired globally to provide optimal production
and disease resistance. Disease transmission and other health concerns limit
live animal transport and subsequent propagation globally. Implementation of
methodologies for long-term embryo preservation and transfer would provide a
foundation for effective utilization of the world’s best genetic resources on a
global basis while modernizing production and enhancing genetic improvement
programs.
The
development of a repeatable method for the long-term preservation of gametes or
embryos would provide numerous applications, including: transport of paternal
and/or maternal germplasm, rapid regeneration or expansion of new and existing
lines, the ability to increase selection pressure in nucleus herds, extraction
or rescue of healthy stock from diseased herds, improve or eliminate quarantine
conditions and provide a method for the international export/import of
potential breeding stock. While maintaining genetic resources through embryo
banking, successful embryo preservation would enhance the further development
of other animal production technologies such as sperm sexing, artificial
insemination, in vitro fertilization and non-surgical embryo transfer. With the
increasing use of animals in human biomedical research, embryos from invaluable
genetic or transgenic lines used as organ donors for xenotransplantation could
be preserved for future use. Collectively, these technologies could be
instrumental in the continuous production of animals of high genetic merit
capable of having a significant impact on the improvement of livestock and
human medicine.
Genetic
Repository
Determining what kind of genetic resources are necessary for genetic
repopulation and maintenance of genetic diversity are dependent on a number of
factors which include: 1) species involved; 2) population status of the specie;
3) gamete availability; 4) laboratory expertise; 5) available biotechnologies
for the specie; 6) germplasm research and repository systems; 7) contingency
plans for germplasm maintenance, deposition and strategic use; 8) strategic
planning for germplasm use in repopulation. For example, in cattle, most of the technology for
cryopreserving bull sperm and cow
embryos are highly commercialized and satisfactory for use in germplasm
conservation. However, the swine
industry is much different. Do to
physiological and cellular differences between pigs and most other mammals,
their germplasm and embryos are more difficult to handle in vitro as well as
for long term preservation.
Laboratory expertise is crucial for developing new or adapting current
technologies to work with each and every species, and no two species are much
alike. The more rare an animal
becomes, the more difficult
it becomes to obtain the
germplasm or embryos
needed, as well
as adapting existing technologies to their unique embryos. A strong and well funded research
program with state-of-the-art equipment and well trained personnel are
required.
Gamete
Preservation For over 40years,
bull studs around the world have been peddling frozen bull semen for use in
artificial insemination (AI). In
the US, over 90% of the dairy cattle are bred by AI. Within the last few years, boar studs have been established
where most semen is extended and maintained at 18oC for up to 7
days. Over 30% of pigs bred in the
US today are by bred AI, and its use has been increasing dramatically over just
the last 2 years. The producers
telephone or email in orders then
rely on over-the-ground delivery of semen to be used with AI. The network is quite vast, and fully
commercialized businesses.
However, most other farm animal species do not have this type of commercialized
support, and thus, for these species, most of the reproductive biotechnologies
remain at the research level.
In
cattle and swine, the use of sperm for germplasm preservation has many
advantages: 1) ease of collection; 2) reliable protocols for semen maintenance
and preservation; 3) renewable resource over time; 4) reliable insemination
protocols; 5) high pregnancy rates with AI; 6) minimal disease transmission
with AI. However, there are some
disadvantages to using preserved semen coupled with AI: 1) frozen sperm must be
monitored and maintained on the stud or the farm; 2) timing of AI to the estrus
cycle of the female becomes crucial for timing of insemination; 3) training is
necessary for handling preserved semen and performing AI; 4) germplasm itself
contains only a haploid component of chromosomes, i.e. there is no maternal DNA
present; 5) reliable protocols are available for cattle and swine, while
protocols for other species are experimental or confined to research
laboratories; 6) protocols for rare or endangered animals require a model
system as genetic resources from them are in low to no availability.
Eggs,
or as we in science refer to them as oocytes, are the least understood and
least researched area of gamete preservation. Because of their size (such a large individual cell), their volume
to surface area ratio and high water content make them extremely sensitive to
cooling and/or cryopreservation.
Although successful methodology is available in laboratories, that is
where it is confined at the present day.
Oocyte preservation would be useful for maintaining maternal
genetics. However, there is a much
simpler way to preserve maternal genetics... embryo preservation.
Embryo
Preservation At the
time of fertilization, the joining of a single sperm cell from the male with a mature
oocyte or egg from the female results in the formation of an embryo. The embryo contains haploid chromosomes
from both the sire and dam, that join together to make a diploid eucaryote. Thus, by utilizing embryo transfer,
both maternal and paternal genetics can be conserved over time and germplasm
can be propagated from a diploid chromosome source. During early embryonic development, embryos reside in the
uterine horns (in vivo), where they develop rapidly and change morphologically
(appearance, structure) over time prior to implantation and the establishment
of pregnancy. While in the uterus,
the morula and blastocyst stage embryos can be surgically and/or transferred
back into surrogate embryo recipients.
After transfer, these embryos can develop into normal, live
offspring. Non-surgical embryo
transfer in cattle is commercialized globally and completed on farm. Surgical embryo collection and transfer
methods are routine procedures in many pig reproduction laboratories. Other species are confined to the
laboratory, and reliable procedures
are not fully developed.
The important message is, the use of embryos in addition to sperm
represents a major increase in the efficiency of transmitting improved genetic
potential.
Preservation
vs. Cryopreservation Whether the
source of germplasm propagation be gametes or embryos, they must be preserved
for short or long term for prospective use over time. There are a few options available. Short term storage includes: 1) culture; 2) hypothermic
storage; long term storage = cryopreservation. However, these techniques are typically used with freshly
recovered gametes and embryos, or previously cultured embryos. The problem is this: fresh or cultured
gametes and embryos are perishable entities, and can only survive in vitro
(outside the reproductive tract) for only a short period of time in
culture.
Depending
on the species, sperm can remain viable post ejaculation for up to 24-48 h at
room temperature, although viability is reduced over time. If extended in a
nutritious medium, sperm can prolong their fertilizing capacity and viability
up to 7 days. However, extended
semen does survive over these lengths of time, but their viability is
continuously decreasing.
Refrigerating extended bull sperm can help prolong its fertilizing
capacity, however, boar semen may not be cooled below 15oC, as boar
sperm possesses a high lipid content that is extremely sensitive to cooling,
and death will occur. Bovine sperm
can easily be frozen and maintained indefinitely. It is the basis for the AI industry and is responsible for
the practical commercialization of the cattle AI. To a limited extent, boar semen can be cryopreserved. However, 2x109 sperm are
needed to AI a female. Boar sperm
are extremely sensitive to cooling below 15oC, and although
protected with present day extenders, many sperm perish after freezing, and too
few are often available for use in AI.
Much research needs to be continued in boar semen preservation to make
it as easy and reliable as it is in cattle.
Oocytes
can be maintained in culture for a short period of time. Depending on the species, freshly
ovulated mature oocytes can be cultured up to 10-15 h before they lose there
developmental competence. Immature
oocytes collected from ovarian follicles can be cultured up to 24-48 h and
matured in vitro, depending on the species. However, oocyte culture is terminal if the oocytes are not
fertilized within their maturation window. Oocyte cryopreservation is very dismal, as in most species
maybe 10-20% survive any form of cold storage, and very few retain normal
developmental competence.
Embryos
can be cultured up to seven days, however, the longer the culture period in
vitro, the less likelihood of individual embryo development in vivo after
transfer. Whether it be sperm, eggs or embryos, the problem is directly related
to mother nature. We as scientists
have not created the ideal culture
medium which will support in vitro embryo development equivalent to the in vivo
environment mother nature has provided in the reproductive tract. So, how do we
get around this and still be able to handle embryos in vitro? We could figure
out mother nature’s recipe for success, of which science has yet to crack. Or
rather, if there was methodology available for the indefinite storage of
embryos, then the factor of culturing embryos in vitro would be minimized, and
they could be transferred shortly after recovery from long term preservation.
PROBLEMS WITH PRESERVATION
Patience and persistence needed...
This
part of the manuscript is an illustration of how preservation methodology was
established for a species whose gametes and embryos are extremely sensitive to
cooling. Explained is the
following: 1) why the gamete and embryonic cells from this species are
sensitive to chilling injury; 2) what research was committed to determine what
parts of the embryos are permanently disrupted during cooling or
cryopreservation; 3) what inhibitory methods were employed to deter chilling
injury; 4) application of the new methodology for the production of live
offspring after transfer of cryopreserved embryos; 5) Practical situations
where this new technology will be an invaluable tool for animal production.
Methods
exist to adequately, but not optimally, preserve germplasm and embryos from
genetically superior animals of most of our livestock species except the pig.
While methods exist to preserve boar sperm, little success until recently has
been realized in preserving pig oocytes and embryos. As stated previously, the
use of embryos in addition to sperm represents a major increase in the
efficiency of transmitting improved genetic potential.
In
1989, the first live offspring were reported after transfer of frozen pig
embryos. For many years, isolated reports
in the scientific literature stated production of live offspring after transfer
of frozen embryos. Unfortunately,
many of these reports were never or could never be repeated in controlled
scientific studies. Why are cow
embryos so easy or routine to cryopreserve, yet this technology has never
worked for pig embryos? Evolution
is one contributing factor to consider. During evolutionary development of
mammals, the pig must have developed a little differently than other domestic
farm animals, or even humans. One
pronounced evolutionary difference was in early embryonic development. Unlike
cattle and human embryos, pig embryos suffer from severe sensitivity to
hypothermic (below normal body temperature) conditions, which limit their
ability to withstand many conventional methods of cryopreservation. This sensitivity has been attributed to
a high lipid content of pig embryos.
Cattle and human embryos have less lipid. Simply, pig embryos cooled below 15oC do not
implant after surgical transfer to recipients. This sensitivity of pig embryos to chilling is not apparent
in embryos at the morula to blastocyst stage from other domestic and laboratory
species, such as cattle, sheep, rodents and rabbits. Thus, conventional methods of cryopreservation (such as
programmable rate freezing) that rely on slow, controlled cooling during early
ice formation are not suitable nor viable for whole pig embryo
cryopreservation.
Our
laboratory has been involved in developing vitrification procedures for
cryopreservation of pig embryos.
Vitrification is the rapid cooling of liquid medium in the absence of
ice crystal formation. The
solution forms an amorphous glass as a result of rapid cooling by direct
submersion of the embryo in a plastic straw into liquid nitrogen. The glass retains the normal
molecular/ionic distributions of a liquid but remains in an extremely viscous,
supercooled form. The glass is
devoid of all ice crystals, and embryos are not subjected to the physical
damage that is associated with ice crystal formation during freezing.
Vitrification is simple,
inexpensive, and has been developed as a viable alternative to conventional
freezing of domestic and laboratory animal embryos. Cattle embryos have been
successfully cryopreserved with vitrification and under field conditions
without any reduction in pregnancy rate.
For
pig embryos, we hypothesized that vitrification might reduce the possibility of
both intracellular and extracellular ice formation while rapid cooling of
embryos could bypass or out-race
detrimental chilling-induced cellular changes that take place during slow
cooling. Vitrification worked as we obtained 30-40% survival and subsequent
development of vitrified hatched blastocysts in vitro. Good, yes, but not good
enough. Finding any survival and development after vitrification was great, but
at the going rate, you would need about 100 embryos to even come close to a
normal litter size after cryopreservation and embryo transfer. The question
became: What is happening in the other 60-70% of the embryos being destroyed by
cryopreservation?
We
observed major cellular disruption following vitrification. Cryopreservation of
any kind can be extremely disruptive to the cellular organization of embryos.
Ice crystal formation can lyse plasma membranes, storage in liquid nitrogen can
denature critical intracellular functions and organelles, and the central
cytoarchitecture (cell skeleton or cytoskeleton) of a cell can be destroyed.
The cytoskeleton is complex network of protein constituents, microfilaments and
microtubules, distributed throughout the a cell. They give 3-dimensionality and
mechanical strength to the surface of a cell, including the membrane, and
provides a system of fibers which impart polarity while regulating cell shape,
cell movement, and the plane of cell division. Documenting cellular damage
during or after cryopreservation provides useful information for understanding
cellular sensitivities to cryopreservation and can lead to improved protocols
for embryo cryopreservation. Recently, research has characterized critical
cellular players in the hypothermic sensitivity of pig embryos.
Utilizing
advanced flourescent, laser scanning confocal microscopy, we observed that
vitrified embryos which were damaged or destroyed after cryopreservation had
disrupted outer membranes and cytoskeleton. During cryopreservation, solutes
used to protect cellular structures wreak havoc on the entire embryonic
cytoskeleton, cause its disruptions, then leave the embryo partially or wholly
destroyed. At this point, we knew the following: 1) pig embryos at the
appropriate stage could survive cryopreservation; 2) 60-70% of cryopreserved
embryos did not survive after recovery from cryopreservation; 3) the plasma
membrane and its linked cytoskeleton were disrupted in many pig embryos
following cryopreservation.
We
hypothesized that cytoskeletal disruption from cryopreservation might be
prevented if we could stabilize the cytoskeleton prior to cryopreservation.
Hatched blastocyst stage pig embryos were vitrified under the influence of
cytoskeletal stabilization with a microfilament inhibitor. Embryonic survival
and subsequent development in vitro improved 2.5-fold (80-90%). We then wanted
to check the in vivo developmental competence of cytoskeletal stabilized and
vitrified pig embryos. In our first trials, 2 of 5 recipients of
stabilized/vitrified embryos farrowed 10 live and normal offspring, being the
first report of live offspring produced after transfer of pig embryos
cryopreserved by vitrification. Of these mature offspring, 2 boars and 2 gilts
successfully proved their reproductive capacity, as the boars each inseminated
3 gilts, and the 2 gilt offspring were mated and farrowed normal litters. In
our second trial, utilizing trans-oviductal uterine catheterization (TUC) for
surgical embryo transfer, 4 of
7 recipients farrowed 26 live offspring. Stabilized
vitrification is a viable method for the long term preservation of pig embryos,
a first for maternal genetics in swine.
This
validated technology will provide a foundation for effective utilization and
transport of the world’s best genetic resources on a global basis while
modernizing pork production and enhancing genetic improvement programs. Although an important breakthrough,
much work still needs to be continued in order to gain the ability to
cryopreserve earlier stage pig embryos that are essential for the pathogen-free
propagation of desirable genetics.
Thus, more intensive research needs to be conducted to characterize
cellular and molecular disruptions during and after cryopreservation, so that
we can further improve methodology for preserving earlier stage,
developmentally competent swine embryos.
As
stated earlier, until recently, the swine industry has had no successful method
for preserving pig embryos. The ability to preserve maternal genetic
information indefinitely would enable the transmission of improved genetic
potential in a form other than the live animal, a first for maternal genetics
in swine. Desirable breeds possessing beneficial production characteristics or
valuable disease resistance traits are not in any one country or region, yet
need to be globally available to ensure optimal production and disease
resistance. Disease transmission and other health concerns limit live animal transport
and subsequent propagation globally. Implementation of methodologies for
long-term embryo preservation and transfer in swine would provide a foundation
for effective utilization of the world’s best genetic resources on a global
basis while modernizing pork production and enhancing genetic improvement
programs.
Practical
Applications The
development of a repeatable method for the long-term preservation of pig
embryos would provide numerous applications, including: transport of maternal
germplasm, rapid regeneration or expansion of new and existing lines, the
ability to increase selection pressure in nucleus herds, extraction or rescue
of healthy stock from diseased herds, improve or eliminate quarantine
conditions and provide a method for the international export/import of
potential breeding stock. While maintaining genetic resources through embryo
banking, successful embryo preservation would enhance the further development
of other animal production technologies such as sperm sexing, artificial insemination,
in vitro fertilization and non-surgical embryo transfer. With the increasing
use of swine in human biomedical research, embryos from invaluable genetic or
transgenic lines of pigs used as organ donors for xenotransplantation could be
preserved for future use. Collectively, these technologies could be
instrumental in the continuous production of animals of high genetic merit
capable of having a significant impact on the improvement of the world swine
population and human medicine.
For
example, genetic seedstock companies have nucleus herds throughout the world,
or want desirable genetics outside their region or country. However, the
desired genetics must come from a non-disease validated herd, or a herd that is
positive for any such disease, or from a region or country that is positive for
such diseases. The bottom line is, the pigs must come from possible infected
sources, which is a terrible risk to take for genetics advancement in their
lines. International policy dictates that the animals cannot come into the
country with the possibility of bringing in potential devastating disease risks
with them. How do you get around this dilemma? Embryos. Embryos can be
recovered from the reproductive tracts of donor females from the herd
maintaining the desirable genetics. After recovery, embryos are washed to
remove any possible pathogens that may be on the surface of the embryos that
were acquired from the mother while residing in vivo. This is an example for
genetic rescue from a diseased herd. That is great, but, as said before,
embryos are perishable, and must be preserved if remaining in vitro for any
extended amount of time. If we could preserve embryos indefinitely, embryos
from the desired genetics herd could be collected, washed, preserved, and then
shipped to the location where the genetic repopulation is to occur. The
existing lines could now be regenerated in a disease-free environment, and
eventually integrated into breeding programs.
In
another example, a researcher or genetic supplier has over 20 years in
development of specific nucleus herd or invaluable lines of pigs. All these
pigs are produced and propagated in one huge confinement facility. The 20 year
old wiring in the barn fails, and there is a fire; or some disease or sickness
comes over the entire herd. Bottom line... the entire herd is devastated or
wiped out. The only pigs of this type in the entire world were in this barn,
and now everything is gone. All those years of work, toil and trouble... gone!
Not a pig left! What do you do? If you had embryos stored indefinitely, by
regeneration through embryo transfer, you could feasiblely have your line of
pigs back up as a reproducing herd within a couple of years.
In
a final example, a company has produced an invaluable line of pigs developed
for human organ transplantation. If a devastating incidence would occur to this
herd, the whole company could perish. They could preserve embryos for an
insurance policy on their unique line, insuring indefinite recovery possibility
for their line anytime in the future, for any purpose.
The
cattle industry has had this technology available since the early 1980s, and it
is primarily used for the propagation of genetically or economically superior
animals. Embryo preservation and transfer revolutionized cattle breeding on a
global basis. These technologies are in their relative infancy for the swine
industry, and the possibilities are limitless.
PROPAGATION
OF GLOBAL GENETICS
Considerations, Cooperation and Vision
A
future scenario
Desirable genetics for a specific disease resistant line of pigs has
been located in a country. The herd happens to be partially positive with an
infective pathogen, inhibiting the transport of breeder pigs out of the herd. A
research team is sent in to isolate, cryopreserve and recover 500 pathogen-free
embryos from this line. The aseptic embryos are transported out of the country
to be transferred into recipients and propagated in a pathogen-free
environment. After ample propagation while maintaining genetic diversity,
founder-line pigs from this line will be integrated into hybrid production
systems.
A
future solution A
future solution will be developed following common scientific method.
Objective: Can we develop a
pathogen-free system for the global propagation of swine genetics?
Rationale: The development of a
repeatable method for the long-term preservation of pathogen-free pig embryos
would provide numerous practical applications, including: transport of maternal
germplasm, rapid regeneration or expansion of new and existing lines, the
ability to increase selection pressure in nucleus herds, extraction of healthy
stock from diseased herds, improve or eliminate quarantine conditions and
provide a method for the international export/import of potential breeding
stock. While maintaining genetic resources through embryo banking, successful
embryo cryopreservation would enhance the further development of other animal
production technologies such as sperm sexing, artificial insemination, in vitro
fertilization and non-surgical embryo transfer. With the increasing use of
swine in human biomedical research, embryos from valuable genetic or transgenic
lines of pigs used as organ donors in xenotransplantation could be preserved
for future use. Collectively, these technologies could be instrumental in the
continuous production of animals of high genetic merit capable of having a
significant impact on the improvement of the world swine population and human
medicine.
Experimental Design:
1) Establishment
of a global network of swine genetics, based on gene mapping for production,
reproduction and disease resistance traits. This will allow researchers, industry and producers access
to information on desirable global genetics. 2) Implementation of disease control guidelines, to minimize
any possibility for accidental or unwanted disease contamination; including
facility, animal, embryo and human contamination. These guidelines will establish methodology for the access
to, and embryo collection procedures from a possible infected herd, embryo
handling procedures as defined by international transport guidelines set forth
and governed by the International Embryo Transfer Society. 3) Locate herds containing lines of
desirable genetics regionally, nationally or internationally. 4) Propagation of embryos from line(s)
of animals of desired genetics by established laboratory methodologies. 5) Aseptic collection and preservation
of embryos. 6) Long-term
(indefinite) storage of embryos in a Germplasm Repository. Regional Repositories will be set up
internationally in association with proposed bio-secure zones. 7) Establishment of global
pathogen-free bio-secure zones for the propagation of rescued genetics. Internationally procure zones with
unique bio-secure features, such as islands, where contaminated traffic can be
fully controlled. Cryopreserved
embryos will be aseptically transferred into pathogen-free recipient animals.
Farrowing and development of F-1 founder lines will occur in the bio-secure
zones. Propagation will continue
to fulfill minimal requirements of founder line animals needed, as well as
founder F-1 and F-2 line embryos for long-term preservation. 8) Release of germplasm and/or live
animals for integration into hybrid production systems. 9) Continual location, propagation and
inventory maintenance for desired lines of animals in a genetic conservation
program.
Expected Results:
1) Preservation
and conservation of valuable genetic resources in reasonable and economical manner.
2) Development of
pathogen-free system for the rescue of valuable swine genetics.
3) Establishment
of global bio-secure zones for propagation of valuable lines of pigs.
In conclusion, it
is quite evident from the cattle industry what an impact germplasm or embryo
preservation can have on the global production herds. Although the swine industry is far behind cattle, research
has uncovered developmental differences between species and allowed solutions
to be found. If we can do it with
pigs, we can do it with other domestic farm animal species too. All it takes is
asking the right questions and having good experimental method, while being
patient observant and persistent.
Good luck.
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