Alpaca World Magazine Spring 2005 - Classical MileEnd Alpacas

it does for a smaller embryo. In
fact, it takes 5–10 times as long to
equilibrate (bring the concentration
of cryoprotectant high enough) in a
7 day llama or alpaca embryo as it
does to equilibrate a bovine embryo
still in the zona
✱ all known penetrating cryoprotectants
are toxic to living cells if the exposure
time is too great.
A hatched blastocyst, the kind of
embryo we get from the llamas and
alpacas, looks like a little translucent
soccer ball about half a millimeter in
diameter. The living part is the spherical
envelope of cells that surrounds a
central volume of aqueous solution.
This envelope is quite fl imsy if you push
against it, but it is very resistant to
puncture. It’s almost as if it were made
of a fi ne nylon mesh.
It was two years ago that I fi nally
accepted the fact that it would never
be possible to freeze these hatched
embryos unless I could reduce the
total volume of aqueous solution inside
the envelope of cells and control the
concentration of cryoprotectant in that
volume. Understanding that and actually
doing something about it were two
very different things. I wanted to inject
cryoprotectant into this central volume
so it didn’t have to migrate through the
cells I was trying to protect in order to
get inside, but all my efforts to puncture
the envelope, even with a very sharp
glass micropipette, were in vain.
The standard approach used for
microinjection through the zona, the
technique used for injection of a single
sperm or for cloning purposes, involves
the use of a holding pipette to suck
the surface of the zona and hold it
tight against the opening at the tip.
This holding pipette is presented from
one side of the zona while an injection
pipette is poked at it from the opposite
side. This just didn’t work in the case of
a hatched blastocyst. The envelope just
invaginated ahead of the sharp tip of the
injection pipette.
It became obvious that it would
take a new approach, creation of a
new device, for injection into and
aspiration from the central volume of a
hatched embryo. I tried several different
approaches to this problem before
fi nally settling on a combination pipette,
a holding pipette with an injection
pipette inside it. This device, which I
was able to cobble together from items
I had around the lab and could buy
at the local hobby shop, I called the
co-axial micro injection system (CMIS)
for purposes of scientifi c writing about
it. In conversations about it with other
researchers, however, it soon got the
nickname ‘the Dracula Pipette’ because
of its suck-and-puncture function.
Before we could inject into these
embryos, hardly any of them survived
any of our freeze and thaw experiments.
As soon as we got the Dracula Pipette
Above, from top to bottom:
A 16 cell llama embryo
Llama embryo hatching
A seven day llama hatched blastocyst
A cross section of seven day llama embryo
Far left:
Llama embryo
up and running it became obvious
that almost all survived, regardless of
the specifi c protocol used. No matter
what type of cryoprotectant or its
concentration (within a wide range)
almost all the frozen/thawed embryos
survived. In fact, only the third embryo
processed using the Dracula system
resulted in our fi rst-ever pregnancy from
a frozen embryo!
This breakthrough device allowed us
to inject cryoprotectant directly into the
central volume of a hatched blastocyst
and then to remove almost all of the
resulting fl uid from inside the envelope
of living cells. This shortened the total
time of exposure to cryoprotectant
for these cells and resulted in a
higher intracellular concentration of
cryoprotectant at the moment of the
freeze. Then, immediately after the
thaw, we could again grasp the defl ated
envelope and reinfl ate it with a culture
solution to allow the cryoprotectant
inside the cells to migrate out in both
directions, toward the central volume as
well as toward the outside.
All that remained was to fi nd the
very best combination of solutions
and timing to freeze these embryos
for commercial uses. We and other
camelid researchers around the world
are working on that fi ne-tuning right now.
Because almost every embryo survives
the freeze to tell us its story, we are
fi nally closing in on a protocol that will
give us virtually the same survival and
pregnancy rate for frozen embryos as for
fresh embryos.
At the same time, llama and alpaca
breeders in almost every country where
these camelids exist are applying for
government permits to allow importation
of frozen embryos. In the US, UK, EU,
Chile, Australia and New Zealand,
regulators are fi nding formal requests
for importation of frozen llama and
alpaca embryos on their desks. Within
a year or two, it should be possible
to move frozen hatched blastocysts
of llamas and alpacas to just about
anywhere from just about anywhere.
As one longtime alpaca breeder in the
US put it, ‘This will really change the
landscape.’
Because we have had experience with
moving live llamas from South America
to North America, we understand
two other, less obvious, advantages
of moving embryos rather than live
animals. The fi rst is animal welfare.
No matter how good the intentions or
what precautions are taken, pre and
post embarkation quarantines, testing
and transport are very stressful for the
animals involved. Almost always some
die, and the others often suffer negative
physical and psychological effects for
years afterward. Embryo collection and
movement will makes these stresses
just a bad memory, a relic of the past.
Also, the movement of live animals
results in the loss of their genetic
potential for the exporting country.
In Peru, for example, buyers from
Australia and the US skimmed the
cream of the alpaca breeding stock
and shipped them out of the country,
forever damaging the ability of Peruvian
breeders to produce the fi ne fi bre that
was a national heritage. By contrast, if
only frozen embryos had been exported
from Peru, all of the pre-existing genetic
potential could have been preserved.
In the future, there can be several
centres in each country where fi ne
alpacas or llamas exist or are wanted,
to fl ush their embryos for export or to
thaw and transfer high-quality imported
embryos. International trade in camelid
genetics will become routine, and a
robust world market will open up for
even the smallest producers of quality
genetics, no matter where they live.
I believe that alpaca and llama
breeders everywhere will give new
consideration to the possibility of
doing embryo transfer work themselves
rather than depending on high-priced
professionals. As one famous vet who
had told us that ET in llamas was
impossible said when I told him we
had our fi rst ET pregnancy ten years
ago, ‘Well, it’s not rocket science, is
it?’ In fact, all it takes is a small hand,
some sensitivity of the fi ngertips and
some sensitivity to the animals. The
supplies necessary are cheap and
easy to get, thanks to the popularity of
bovine ET work. A few years back I did a
demonstration programme of practical
llama ET in Argentina, in the open under
rough conditions. This resulted in a
70% pregnancy rate of the transferred
embryos.
ET requires rectal palpation, and at
fi rst Sally and I were petrifi ed that we
eventually would rupture the rectum of
one of our llamas. This hasn’t happened,
and we now have over 10,000
palpations behind us. The danger is still
there and we are still very careful, but
the record proves that this work can be
done with very little risk to the donor
and recipient females.
Serious breeders of alpacas and
llamas can put this valuable tool,
embryo transfer, which holds the
promise of a tenfold increase in the
production of select females, to work for
themselves. Thanks to the breakthrough
in freezing of embryos they can look
forward to being able to buy great new
genetics from anywhere on earth and
to being able to sell their best genetic
production to other serious breeders no
matter where they are.
Alpaca World Magazine Spring 2005 | 31