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Human reproductive techniques have been grabbing headlines over the
past few years and especially in recent weeks as developments take the public
by surprise. But elsewhere in the animal kingdom advances have been just
as dramatic, if relatively unsung, with scientists pioneering techniques
to improve artificial insemination and store frozen sperm, eggs and even
embryos. And it is a mark of how much progress there has been that scientists
are meeting next week to discuss setting up Europe’s first ‘frozen zoo’
– a bank of frozen genetic material from endangered species.

Their first meeting at Chester marks the start of a two-year programme
– funded by the European Commission – to thrash out the science, logistics
and implications of such a move. The bank would include frozen sperm, embryos
and, possibly, eggs. It would also be a resource for those involved in breeding
programmes and evolutionary research by storing genes which could be moved
easily between populations and by storing alternative forms of various genes
which might otherwise be lost in small captive groups of animals.

Bill Holt, a research fellow at the Institute of Zoology in London,
is the guiding spirit behind the project. He feels that although the technology
for such a bank is far from perfect, it has reached a stage where scientists
should start exploring the issues. ‘Although the techniques are not routine,
we feel they are coming to the point where we should start to think properly
about the way you would set up banks of frozen genetic material if you had
that opportunity and what questions you should be asking.’ The European
consortium will liaise with groups in the US and elsewhere who are also
examining the issues.

Much of the impetus for the project has stemmed from captive breeding
programmes and the need to ensure a genetic mix between populations of various
species in zoos and parks around the world. As Gordon Reid, curator in chief
at Chester Zoo, puts it: ‘Would you rather transfer three tonnes of elephant
or a flask of frozen sperm? Moving animals is a huge enterprise and very
stressful for the animals.’

In cattle, artificial insemination using frozen semen has been established
for over a decade, but in most other domestic species it is not the norm.
After years of research, farmers still do not routinely use frozen ram or
boar semen for breeding because the success rate is so low. Problems arise
from the quantity of viable semen required and the anatomy and physiology
of female sheep and pigs, as much as from the freezing itself. However,
this illustrates that reproductive technology is not automatically transferable
between species.

If the European project is to be successful, then developing the appropriate
freezing technology for a wide range of species is a prerequisite. Initially,
the bank would probably contain frozen semen and possibly some embryos.
Techniques to freeze eggs are not as well developed, so it may be some time
before they would be included in such a scheme.

Even with sperm, which are more easily preserved by freezing than the
other types of genetic material, a standard rate of treatment could damage
much of the cellular material. The rate of freezing and thawing is crucial
because it varies from species to species, let alone between taxonomic groups.
For example, sperm from different groups differ in their shape and size,
and they also have different protein and lipid compositions.

At the Institute of Zoology, researchers have devised a technique to
test tiny amounts of semen for their optimum freezing regime. This is important
because small animals, and even some large species such as cheetah, yield
very little semen. Using a so-called ‘cryomicroscope’ – one with a temperature
control system attached to its specimen chamber – they can test the sperm
for freezing and thawing at controlled rates. Results are recorded on video
and analysed to find out what works best for each species.

Eggs are fundamentally more difficult to freeze successfully than sperm.
There are several reasons. Different parts of their structure freeze and
thaw at different rates, and eggs with large yolk sacs tend to develop crystals
which damage the egg as it freezes. Also, the chromosomes in eggs are more
vulnerable to damage than those in sperm. Furthermore, eggs tend to become
harder to fertilise having undergone the freezing and thawing process. Eggs
from some species, most notably mice, have produced offspring after such
treatment, but much more research will be needed before frozen eggs are
common in any gene bank.

GLASS EMBRYOS

One technique that has been used with some success to preserve eggs
is a variant of the freezing process called vitrification. However, the
pioneering work on this process has been done on embryos, which are frozen
in a solution with a high glycerol and sucrose content. Under these conditions,
the embryos do not really freeze but become like glass. Using this technique,
damaging ice crystals cannot form. Human, cattle and sheep embryos have
been successfully preserved in this way. However, Holt feels that it needs
considerable refinement before it can be used for most exotic species.

Having successfully frozen genetic material, scientists need to test
that it is still viable once thawed. There are now several ways of testing
sperm. For example, putting sperm into cervical mucus, bovine serum albumin
or some artificial viscous polymer-based gels will show how vigorously the
sperm are moving – a good indication of their fertility.

The final step in the process, insemination, may also prove problematic,
even for domesticated animals. In pigs, for example, the uterus is proportionally
much bigger than in cows, so billions of sperm are needed if there is to
be a chance of fertilisation. Pig sperm are also more easily damaged through
freezing than bovine sperm. Cows are inseminated simply by inserting sperm
directly into the uterus using a catheter. This is impossible in sheep because
of their reproductive anatomy, and laparoscopy may be needed to ensure fertilisation.
Such problems are often compounded in exotic species by the small amounts
of semen that can be milked at any one time or by difficult-to-time oestrus.

A further problem in breeding programmes in semiwild populations is
how to identify individual animals, since the aim of most captive breeding
programmes is to minimise inbreeding to produce vigorous offspring. DNA
fingerprinting could be used to show how closely related individuals are.

However, the technological feasibility of creating a frozen genome bank
will not be the only problem to tax the scientists meeting in Chester next
week. They must also address basic logistical issues such as where the bank
should be sited, whether it needs to be duplicated in case of accidental
damage, how it should be managed, and how best to arrange the material in
storage. Storage is crucial if disease is not to be transmitted between
samples. Already, researchers at the Royal Veterinary College in London
are trying to discover whether viruses can be transferred between frozen
samples. Their results will dictate whether, for example, genetic material
from ungulates and birds can be safely stored together, or whether they
will need separate flasks. Animals might also be screened for diseases or
dosed with antibiotics before they become donors to the bank.

And any bank which offers genetic material for use worldwide will have
to tackle the practical questions: how should we control the movement of
animal material between countries, which species should be preserved, how
much genetic material should be stored and in what form. These sorts of
decisions depend on the role envisaged for the bank. If, for example, it
is to provide a resource for evolutionary studies or gene splicing, then
it might be more convenient to have DNA stored in various forms including
nonreproductive tissue.

The choice of database is not an inconsiderable question either. Peter
Bennett, another research fellow at the Institute of Zoology, has the tough
task of developing guidelines for a database. It will need not only to keep
track of the genetic material in stock, but to integrate with other such
databases worldwide, providing data on suitable breeding matches and the
best time to use material.

Obviously, the database would include everyday information on species,
sex, pedigree and identifying characteristics. But its structure could get
a lot more complex depending on the remit of the frozen zoo. If, for example,
it included supporting small, endangered popu-lations, the database would
need to hold information that would allow users to maximise genetic variety.
In this case, the zoo would contain genetic material from a wide selection
of individuals from a population and the database would need to record their
degree of relatedness. ‘I’ve got to insure that the right information is
recorded initially,’ says Bennett. ‘For example, if a pedigree of an animal
is unknown we have to ask whether the whole thing is very useful, as we
can’t then minimise inbreeding.’

Once the technical and logistical issues are in hand, Holt sees the
biggest problem as finding a backer for a genome bank. Currently there are
no estimates of cost, but any commitment would have to be open-ended – there
is little point in initiating such a bank and then pulling the plugs a few
years later. Any backer must be convinced of its value, either scientifically
or commercially.

OUTSIDE INTEREST

Fortunately, the signs are promising. It is already apparent that interest
in a frozen zoo comes not just from people concerned with conserving rare
species. Last year, the UN’s Food and Agricultural Organization proposed
the establishment of an international genetic resource bank. The Directorate
of Fisheries within the European Commission has also discussed the idea.
And there is interest from biomedical scientists who want to preserve genetic
material from as many species as possible in the hope that it will be medically
useful in the future.

But could a frozen zoo ever replace conventional conservation methods?
Sceptics might say that it is simpler to keep wildlife frozen in a flask
than to go to the trouble of protecting their natural habitats. ‘Anything
that gives companies or whoever an excuse to damage environments is a concern,’
says Bennett. He believes that conserving species is about conserving habitats,
but adds: ‘This (gene bank) is not something that can be used without keeping
live populations.’

Reid believes that the technology provides its own safeguards. ‘I don’t
think that the technology is reliable enough – the longer you hold samples
the more their fertility will decrease. You must have an actively managed
population to ensure that a species will survive in perpetuity.’ He also
argues that people want to see live animals, so there are commercial and
public interest reasons why conventional zoos will survive. ‘It’s unlikely
we will end up with a nightmarish world of frozen populations.’

Over the next two years the European consortium should be in a position
to answer this and other imponderables. But next week they will concentrate
on ensuring that they start out with the right questions.

Jane Seymour is a freelance journalist

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WHO’S IN?

If A frozen genome bank for endangered animals is set up in Europe,
then the first species to be included will be those of particular interest
to the scientists planning the venture.

Bill Holt at the Institute of Zoology in London, wants to see the clouded
leopard (Neofelis nebulosa) included. Destruction of its forest habitat
in Southeast Asia means that the leopards are endangered in the wild, and
there are fewer than 250 in captivity worldwide. The British population
of 18 animals is particularly important as it includes the sole descendants
of some genetic lines.

In captivity, clouded leopards are difficult to breed. Females and males
are very aggressive towards one another unless they have known each other
from a very young age – something that is not easy to arrange in a small
population close to the end of its reproductive life. Holding frozen semen
from this group would help artificial insemination and allow breeding between
animals in Britain and the US, thus improving the genetic viability of the
world population.

Another species that researchers at the Institute of Zoology would like
included in a European frozen zoo is the scimitar horned oryx (Oryx dammah).
Its natural habitat has now shrunk to the south Sahara where hunting is
driving it to the brink of extinction. Groups are held in captivity in Britain,
New Zealand, Australia and the US, with a total of around 2000 worldwide.
Again, it would improve the genetic viability of this species if it was
managed on a worldwide basis, something which would be easier to accomplish
with a bank of frozen genetic material.

Gordon Reid, curator in chief at Chester Zoo, has a particular interest
in cichlid fish from Lake Victoria. Around 80 per cent of the indigenous
cichlid population has been wiped out through being eaten by Nile perch
which were introduced into the lake around 30 years ago. Chester Zoo coordinates
the British part of a successful breeding programme for the cichlids but
they cannot be reintroduced to the wild until the number of perch has fallen.

At present, captive populations in Europe and the US contain around
30 of the 300-odd species originally found in the lake. There are several
thousand individuals at each site. Freezing cichlid sperm and eggs would
allow the time gap between generations to be increased, making the population
size more manageable while it awaits its return to the wild. And if material
is stored for several generations new individuals could be invigorated by
introducing genes from a previous generation.