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Supporting conservation efforts

EDUARDO ROLDáN

Museo Nacional de Ciencias Naturales, CSIC

MONTSERRAT GOMENDIO

Museo Nacional de Ciencias Naturales, CSIC

The role of a germplasm and tissue bank in the conservation of endangered species

The various biological materials that are preserved in a germplasm and tissue bank can ensure the genetic diversity of a species almost indefinitely. Stored gametes, embryos and somatic tissues represent a means of overcoming barriers of space and time, allowing the transfer of genetic material between populations and its use many years after the death of the donor animal.

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The shrinking and fragmentation of animal populations is an increasingly common phenomenon. As a result, the number of species that are threatened with extinction is growing all the time. In general, the main cause of decline in population size is habitat HIGHLIGHTSProfile: Eduardo Roldán and Montserrat Gomendio
loss resulting from the pressure of the exponential growth of the human population and intensive use of resources that this entails. However, in other species the most important threat is over-exploitation.


What can we do to reverse species loss?
There is broad consensus that the best strategy for the conservation of biodiversity is the preservation of the natural environment (in situ conservation). However, since it is not always possible to preserve natural habitats, this strategy needs to be backed up with other conservation approaches focusing actions more directly on the animal species concerned The conservation of endangered species implies maintaining gene banks with genetic resources from natural populationsor obtaining, storing and using biological materials taken from them. This second case (known as ex-situ conservation) involves the design and implementation of measures such as captive breeding programmes and/or banks of genetic resources to conserve biomaterials. It should be noted that the two types of initiative, namely, captive breeding and genetic resource banks, do not necessarily have to go together. Some conservation efforts aimed at endangered species involve maintaining a bank of genetic resources from wild populations, but do not rely on captive breeding. Setting up a repository of biological samples in a gene bank so as to conserve as much biodiversity as possible is nevertheless a crucial initiative that should be used whenever possible and it needs to start early, before the decline of biological diversity of the species concerned is too far advanced.

Both types of efforts, in-situ and ex-situ conservation, should not be seen as mutually exclusive alternatives. On the contrary, they should be considered complementary and interacting approaches, as they enrich each other and bolster conservation efforts, making them more likely to succeed. A number of examples currently exist, such as the Iberian lynx conservation programme, demonstrating the advantages of advancing on these two fronts simultaneously.


Consequences of shrinking animal populations
Natural populations may decrease in size because individuals fail to reproduce, because mortality increases, or for a combination of these two reasons. The smaller number of individuals increases the probability that related animals will mate with one another, One of the primary objectives of the conservation efforts using gene banks is to try to preserve as much genetic diversity as possiblehence leads to increased inbreeding and reduced genetic variability. This is already known to have negative effects on female reproduction and the survival of offspring and juveniles, but there is as yet less information about the effects on male reproduction. Our research group has conducted a number of studies on these issues, and found that males with high levels of inbreeding suffered a reduction in the quality of their sperm, in terms of both motility and morphology. Given that some of the sperm parameters affected by inbreeding are crucial for male fertility, it is likely that related males will suffer reduced fertility. This is particularly obvious in extreme cases such as that of the Florida panther, in which males showed a very high proportion of abnormal sperm (about 90%). It also seems to be true of the Iberian lynx, in which we also found a high proportion of abnormal sperm in the semen.

To date, most studies have examined basic sperm characteristics. A study of the impact on sperm function (e.g., mechanisms that prepare sperm to play its part in fertilisation) has yet to be undertaken.

It is also of particular interest to determine the effect of inbreeding on sperm DNA, and how this might affect embryo viability. Our research has shown for the first time that DNA damage to the sperm increases in parallel with inbreeding, and that it can reach surprisingly high levels. It has also been found that the level of male DNA damage is related to sperm quality and offspring’s survival chances.


  Creating a repository of biological samples in a gene bank is crucial. / Photo: Eduardo Roldán.



Why preserve biological material from endangered species?
Given that the reduction in the size of animal populations causes a decrease in genetic variability, one of the primary objectives of conservation efforts involving a gene bank is to try to preserve as much genetic diversity as possible. It was for this purpose that the Banco de Germoplasma y Tejidos de Especies Amenazadas gene bank was set up, made possible by an agreement between the CSIC and the Ministry of the Environment.

Since its inception, one of the Bank’s objectives has been to support conservation initiatives focusing on the Iberian lynx and the European mink. Over time, other animals of special interest have been included, such as various other species of lynx and South American cats. In collaboration with other institutions, we are also running conservation initiatives on emblematic species such as North African gazelles and the giant panda. Currently the Bank operates as a research facility of the CSIC’s Museo Nacional de Ciencias Naturales (National Museum of Natural Science) and is a part of the centre’s structure.


Types of biological material that are worth preserving
The primary focus is on preserving “germplasm”, i.e. semen (sperm), eggs or embryos, using the appropriate cryopreservation protocols. This germplasm can subsequently be used for a variety of assisted reproduction techniques. It is also worth preserving tissues and somatic cells as they can potentially be used in nuclear transfer techniques (cloning) or to produce pluripotent stem cells.


Assisted reproduction techniques suitable for use with threatened species
Assisted reproduction can offer a solution to the problems caused by inbreeding. Reproductive technologies can be a useful complement to conservation efforts because they facilitate genetic management and exchange between populations. However, it is not easy to implement assisted reproduction technologies in wild animals. It takes a considerable effort to put together teams of people who have the necessary backgrounds and experience, all of whom are working on different programmes, and to arrange the conditions for the capture, collection and processing of samples (in many cases in the field). It is also necessary to conduct preliminary research (usually using model species) so as to explore appropriate conditions for processing, evaluating and storing biological material and, last but not least, sources of funding need to be secured to initiate the work and, above all, to ensure it has continuity.

When organising endangered species breeding programmes, opportunities arise to collect and store gametes under more controlled conditions and from individuals that enjoy better health and nutrition. A captive breeding programme can be integrated within a broader strategy of ex-situ conservation and, where possible, should serve to support in-situ conservation measures. Thus, the experience on reproductive health issues deriving from the Iberian lynx ex-situ conservation programme has been used to collect, handle and store cells from the male germ line of individuals in the wild.

In some species, including the Iberian lynx, some animals also unfortunately killed while crossing roads. The existence of a germbank means that it is possible to collect and rescue germ cells and somatic tissues from these animals that would otherwise not be able to reproduce.

The various biological material that are preserved in the germplasm and tissue bank ensure genetic diversity of the species almost indefinitely. Gametes, embryos and somatic tissues preserved in this way may be used without limitations of time and space, as they can allow genetic material to be transferred between populations and be used many years after the death of the donor animal.


  Dama gazelle spermatozoa. / Photo: Eduardo Roldán.



Collection and cryopreservation of semen. Artificial insemination
The storage of spermatozoa by cryopreservation is an essential tool in the conservation of gametes and in genetic management that is suitable for both captive populations and for those in the wild. As part of a project to develop assisted reproduction techniques for three species of North African gazelles (Cuvier’s gazelle, dama gazelle and dorcas gazelle) we have examined factors affecting the success of semen cryopreservation. As a result of this series of studies, we obtained the world’s first gazelle to be born using artificial insemination with cryopreserved semen.

In turn, in our work on freezing semen from the Iberian lynx, we have frozen sperm from this species, using protocols previously evaluated with the domestic cat and the bobcat. These preliminary steps have allowed us to initiate systematic conservation of semen from both males in the captive breeding programme and those living in the wild in Doñana. Furthermore, using these techniques it has been possible to recover and cryopreserve sperm from epididymis of dead animals killed on the road or which have died from disease.


Cloning by nuclear transfer
This technique has attracted a lot of attention recently. There has been much debate about the potential application of cloning in the conservation of endangered species, as its possible use as a means of ‘rescuing’ or ‘resurrecting’ extinct species like the Tasmanian tiger, the mammoth or the Pyrenean Ibex, has been suggested. However, Preserving “germplasm”, i.e. semen (sperm), eggs or embryos, following the appropriate cryopreservation protocols, is of considerable value for conservationapart from the technical difficulties of this, the conservation value is highly questionable. These isolated cases aside, there are nevertheless clear benefits to be gained from the use of cloning in the conservation of endangered species.

Contrary to some of the arguments made in the past, cloning could be of immense use in conservation efforts helping to preserve and even increase the genetic variation in populations. Cloning could be used to avoid the loss of unique and valuable genotypes and enable animals to reproduce without taking them out of their natural surroundings.

For these reasons, we have begun to collect and store tissues and somatic cells from endangered species. In particular, we have devoted a lot of effort to cooperation with in-situ and ex-situ Iberian lynx conservation programmes to perfect techniques for tissue culture and cell and tissue cryopreservation, both derived from autopsies of animals dying as a result of accident or disease, such as biopsies of the animals examined during follow-up of populations in nature or during health checks and reproductive examinations. As a result of this work, our endangered species gene and tissue bank currently stores tissue and viable cells from over 250 individuals of Iberian lynx, making it an extremely valuable repository for the conservation of the species.


Production of gametes from embryonic stem cells and somatic cells
Embryonic stem cells are pluripotent cells produced from early embryos before the point at which germ tissue layers form. These cells exhibit indefinite proliferative capacity. Several research groups have shown that mouse embryonic stem cells can differentiate into primordial germ cells (PGCs), and subsequently, into early-stage gametes (oocytes and sperm). The immature mouse sperm cells produced in culture from embryonic stem cells are able to produce live offspring.

These possibilities depend on the availability of embryonic stem cells. However, it is not easy to obtain embryonic stem cells from embryos of wild species produced in vitro due to the limited availability of material and technical difficulties. It is therefore worth considering using the nuclear transfer of somatic cells with heterologous oocytes in order to obtain stem cells of these species. In this context, nuclear transfer could be much more efficient than when used for reproductive purposes.

Besides embryonic stem cells it has been possible to develop induced pluripotent cells (iPS) from somatic cells of mice and humans. This method, which does not involve the use of cells from embryos, uses a minimum combination of genes to transform a somatic cell, for example, a fibroblast, into a stem cell with characteristics similar to those of embryonic stem cells.

These results, which have revolutionised the field of regenerative medicine, also have important implications in the use of these cells for the conservation of endangered species because the fact that it has been possible to obtain haploid gametes from embryonic stem cells suggests that it might also be possible to generate gametes from these induced pluripotent cells.

The use of embryonic stem cells or induced pluripotent cells has outstanding implications because, again thinking of possible applications to endangered species, it may allow “synthetic” gametes to be produced. It is conceivable that in the future sperm generated in this way –with a virtually unlimited supply– may be used in assisted reproduction of endangered species.

In short, the biodiversity of our planet is facing a crisis and we need to devise strategies to stop this decline and, as far as possible, reverse it. We humans are largely responsible for this situation and, therefore, have an obligation to act. Scientists working in a variety of fields can all make a significant contribution.

The development and implementation of assisted reproductive technologies for threatened species based on biological material preserved in gene and tissue banks can offer management options to promote gene flow. This task, which must involve many players, requires infrastructure and ongoing funding.  

Profile: Eduardo Roldán and Montserrat Gomendio

Eduardo Roldán
Has an honours degree in Veterinary Science, and PhD in Biological Sciences and is a Research Professor at the CSIC’s Museo Nacional de Ciencias Naturales (National Museum of Natural Sciences) in Madrid, where he co-directs the Ecology and Reproduction Biology Group (GEBIR). He has made postdoctoral study visits at the University of Hawaii (United States) and the AFRC Institute of Animal Physiology (Cambridge, UK) with support from the Rockefeller Foundation and the Lalor Foundation. He was a Senior Research Scientist at the Babraham Institute (Cambridge) and Full Professor and Director of Animal Reproduction and Developmental Biology Programme at the University of London. He is also a member of the Real Academia de Ciencias Veterinarias del Instituto de España (Royal Academy of Veterinary Sciences of the Institute of Spain).

He specialises in the reproductive biology of mammals, with a particular focus on gametes, fertilisation and embryonic development and the application of reproductive biotechnologies to domestic animals and wildlife, including endangered species. In 2005, jointly with fellow CSIC researcher Montserrat Gomendio, his work led to the birth of a baby Mohor gazelle, a critically endangered species, using frozen semen for assisted reproduction techniques.


Montserrat Gomendio
Has a PhD in zoology from the University of Cambridge (United Kingdom). After completing her doctorate she became a Research Fellow at Trinity Hall and associate professor in the Department of Zoology, University of Cambridge. She joined the CSIC’s Museo Nacional de Ciencias Naturales (National Museum of Natural Sciences) in 1991, where jointly with Eduardo Roldán, she set up the Ecology and Reproduction Biology Group (GEBIR). She is a corresponding member of the Academia de Ciencias Exactas, Físicas y Naturales (Academy of Exact, Physical and Natural Sciences).

Her lines of work include reproductive ecology, evolution of reproductive strategies, the evolution of parental care, the role of sexual selection in speciation, reproduction of endangered species, the deleterious effects of inbreeding, and biodiversity conservation.

She was the Director of the Museo Nacional de Ciencias Naturales from 1997 to 2002 and Vice President for Organisation and Institutional Relations of the CSIC from 2003 to 2004. She is currently a CSIC Research Professor at the Museo Nacional de Ciencias Naturales in Madrid.

Published in No. 03


  • ® Fundación General CSIC.
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  • Lychnos. ISSN: 2171-6463 (Spanish print edition),
    2172-0207 (English print edition), 2174-5102 (online edition)
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