The Conservation and genetic management of wildlife

Dr Ronnie de la Rey - Embryo Plus

Assisted Reproduction Technology (ART) For The Conservation And Genetic Management Of Wildlife.

Before the application of any assisted reproductive technique, it is vital to understand how natural reproduction is influenced by various factors, including those externally derived. Otherwise, efforts to develop artificial insemination and embryo transfer in unique taxa will undoubtedly fail or will not be reliable.

In this article, it will be evident how a variety of exogenous factors, including social and environmental conditions, can greatly influence the reproductive performance of captive animals. Such influences may be obvious (e.g., aggression in overcrowded conspecifics), but many times the influence may be more discrete (e.g., undetected pollutants).

Likewise, there is evidence to suggest that similar influences can affect free-ranging animals or those in situ. However, the presentations will lead many to conclude that in situ is probably more of a perception than a reality, since most species are confined within physical boundaries. For that reason, many managers of national parks and game reserves are increasingly aware of the need to manipulate control and manage reproduction so as to maintain a natural balance in delicate ecosystems.

Assisted reproduction should be interpreted in its broadest sense, and that would be any initiative taken, or stimulus introduced, that can enhance reproductive function (e.g., changing light cycles or other environmental manipulations; providing nutritional supplements that can overcome deficiencies, such as those often encountered with pregnancies in aged ruminants; or removing social stressors by either separating territorial rivals or uniting colony members).

Understanding species-specific factors that can influence fertility and either providing conditions that reflect those that are naturally found, or attempting to mimic them artificially can enhance natural reproduction. At the same time, assisted reproductive technology should also be viewed as a supplemental strategy that will inevitably become (based on the growing problems with governmental regulatory restrictions to control infectious disease outbreaks), one of the most important tools available for maintaining genetic diversity for populations ex situ as well as in situ.

Effects Of Environmental Pollutants On Reproduction.

Since the early 1990's, it has been established that a wide variety of natural and/or anthropogenic (man-made) chemicals in the environment are capable of modulating and/or adversely affecting or disrupting endocrine function in vertebrate organisms.

Environmental Pollutants.

It has been established that a wide variety of natural and man made chemicals in the environment are capable of modulating and/or adversely affecting or disrupting endocrine functions and reproductive failure in wildlife.

Illustrated by

1) the impact of pesticide on Honey Bee Queen Production.

2) Continued decline of African Penguins because of poor animal survival of young birds and the resulting low recruitment to the breeding population.

Current threats to the population include increased competition for a reduced food supply and marine pollution, particularly from oil spills. Social Suppression Of Reproduction.

Has been documented in approximately 120 mammalian and 200 bird species. Which has been written on the impact of environmental and social components of captivity on reproduction.

Management components include housing/exhibit facilities (area, configuration, and complexity), diet and presentation of diet, temperature, humidity, circadian cues, olfactory/auditory/tactile stimulation, social grouping, genetic compatibility for species/subspecies with little phenotypic variation, and interactions with human caretakers.

Any or all of these aspects of an animal’s environment, both during its early development and into adulthood, may impact reproduction at many levels: estrous cyclicity; courtship behavior; pair-bonding; copulation; pregnancy maintenance; parturition; and maternal care.

The captive breeding of endangered species has gained increased importance as a conservation tool since the rapid decline of wild populations.

Successful captive breeding is dependent on many factors. Often, the first step in the long sequence leading to a successful breeding outcome is pairing animals for mating. Determining which animals will be paired together is often done on the basis of the genetic compatibility of the individuals involved, especially in the case of critically endangered species.

However, in some cases, animals that may be genetically compatible are not behaviorally compatible, and so mating does not occur, despite the best efforts of zoo staff - often after many attempts, and months (or years) of housing the animals together. One way to assess behavioral compatibility is to try to mimic conditions that would occur in field
situations, where females of most species actively choose a mate.

There is overwhelming evidence to support the importance of female choice in mate selection in a wide variety of species. Males of many species advertise their "superior" quality through one or more signaling mechanisms (visual, vocal, tactile or chemical in nature) in order to attract and successfully mate with females. Females generally prefer traits that are difficult to maintain, which then provide valuable information about the quality of the potential mate.

Males that do not exhibit preferred traits are avoided by females. Understanding the basis of female choice can be a valuable tool to develop effective captive breeding programs for endangered species. In the cheetah (Acinonyx jubatus) such mating and breeding difficulties have been widely recorded.

Many cheetah pairings never result in breeding and low libido and lack of estrus is a common
problem with this species in captivity. Genetic and physiological studies alone have failed to detect differences between breeders and non-breeders. Research with captive cheetahs at the Toronto Zoo and Mountain View Farms suggests that visual signals exhibited by male cheetahs are critically important to female mate choice.

They have presented female cheetahs with life-sized realistic male cheetah models, and analyzed their behavioral responses. Results showed that female cheetahs display strong individual preferences for particular traits, including size, coat pattern and age-related signals. These preferences can be used to predict which females, will accept which males, and they hope will aid in successful pairings.

In captive breeding situations, one of the most important measures of reproductive status is when an animal is in estrus and ready to breed. Using behavioral measures can be extremely useful here, as illustrated by a study that have recently completed on Indian rhinoceros. All species of rhinoceros have proven to be very difficult to breed in captivity (Czekala and Callison, 1996; Dinerstein,Wemmer and Mishra, 1988; Foose and van Strien, 1997; Fouraker and Wagener, 1996; Hindle, Mostl and Hodges,1992).

There are a number of reasons for low reproductive rates in this species, both in the wild and in captivity. These include 1) long gestation periods, 2) long inter-birth intervals and 3) late reproductive maturity. Another factor that limits reproduction rates in captivity is that Indian rhinos are solitary in the wild, with adult males and females associating only for breeding (Fouraker and Wagener, 1996; Laurie, 1982; Nowak, 1999).

Placing a pair together when either animal is not sexually receptive can result in serious aggression, especially as neither captive animal can run away. Unfortunately, it is not always easy to recognize the state of sexual readiness in a rhino, and to make matters worse, males and females often appear to come into breeding condition at different times (Gowda, 1967; Tong, 1961; Vahala, Spala & Spitalsky,1993).

Animal Temperament.

Behavioral differences are relevant for the captive management of endangered species, because how individual animals respond to challenges and changes in their environment may have an impact on reproduction and well being. How animals cope with stress is strongly influenced by temperament and environmental changes.

The Okapi, solitary, forest-dealing animals in their natural habitat are virtually impossible to mimic in captivity, because of the size of their territories, density and variety of the rainforest in Zaire. I appear to be stress sensitive because of poor survivability during transportation from the wild to captivity and sub optimal reproduction.

Nutrition.

Proper nutrition and dietary husbandry is fundamental for reproduction success.
Genetic Manipulation

The need to replenish and improve bloodlines in population of cattle
and other farm animals was initially met by importing of postnatal
animals.

Artificial insemination developed as the first assisted reproduction technology.

The first report in the world of the application of AI came from an Italian who in 1780 had successfully inseminated dogs. Several other reports came and the Russians built an AI centre for horses in 1919 and AI for cattle began in 1930. Freezing of semen had commenced on a limited scale in 1960. Development of extenders of semen took place and is still ongoing today.

Semen: A Dynamic Tool In The Farm Animal Breeding System Is Also Developing To An Important Tool In Our Wildlife Situation.

The use of ART eliminates the problems of distance and time as major obstacles in captive breeding programmes. In addition, it offers the opportunity to incorporate new resources of gametes or embryos harvested from:

• free-living individuals from the wild,
• animals carrying dangerous transmissible diseases such as tuberculosis, foot and mouth disease, salmonellosis etc. after germ-eradication-protocols,

• from animals with permanent behavioural disorders (not linked to genetic defects),

• from post-reproductive or dead individuals.

There are several gamete recovery techniques, which can be applied to zoo and free-living animals.

These include for sperm cells:


• electro-ejaculation,

• manual semen collection and,

• use of artificial vagina (both exclusively conditioned captive individuals),

• post coital vaginal flushing,

• testicular or epididymidal sperm aspiration (biopsy technique),

• sperm preparation from the Cauda epididymidis (after castration or post mortem)

These include for oocytes:

• endoscopically or ultrasonographically-guided ovum-pick-up (OPU),

• oviductal flushing after induced ovulation (requires normally oviductal extirpation),

• follicle aspiration and/or ovarian slicing in isolated gonads (after castration or post mortem),

• ovarian graft technique (after castration or post mortem).

In addition to gamete collection for artificial insemination (AI), in vitro fertilization (IVF) or synchronic gamete transfer, so called gamete-intrafallopian-tube-transfer (GIFT), pre-implanted embryos can also be collected successfully from non-domestic species during a defined species-specific time window (Dresser, 1986; Pitra, et al., 1991; Summer, 1986). In principle, the different embryo flushing techniques are relative similar with the exception of the approach of obtaining access to the lumen of the uterus.

These methods include:

• non-surgical Trans cervical technique (e.g. wild cattle, antelopes, zebras, bongos, bears, monkeys, great apes),

• surgical technique, which requires a laparotomy (e.g. monkeys, small and big cats, sheep and deer species, mustilids).

The minimal invasive laparoscopic flushing techniques are not as yet well established for non-domestic species, however, endoscopically-guided procedures are widely used for embryo transfer [ET] into the recipients (Howard, 1998).

Most new and advanced develops in ART are generated by the life sciences either directly or indirectly linked to research on human or livestock reproduction (Trounson, et al., 1998). Until now, conservation biology has not made a substantial contribution to this field, mainly because of limited resources available for independent developments*. The direct application of established ART developed for domestic species or humans in captive breeding programs has only limited success. Frequently, there are major modifications or new developments necessary to accommodate the specific reproductive anatomy and physiology of the target species.

The necessary changes of the established technologies affect the procedures themselves e.g. ovulation induction, super ovulation, gamete cryo-preservation (timing, dosage and composition of ingredients) as well as the design of the specific ART equipment applied.

The following are required for the development of novel ART tools in non-domestic species. There are several operative problems if customized equipment originally developed for humans or livestock is applied to exotic species. Non-domestic animals are normally chemical or mechanical restrained for reproductive intervention, however some species (elephants, great apes, rhinoceroses) have been trained to accept a certain level of manipulations (Hildebrandt, et al., 1998). The specific ART tools should be designed to minimize the total manipulation time (reduction of stress and/or anaesthetic risk). Mechanical and/or electrical equipment should always meet safety standards to minimize the risk of severe injury of the animal in case of unsuspected defence behaviour or due to improper use by the operator. In this context is it important that ART procedures always correspond with the general guidelines of animal welfare.

The priority for the development of novel ART instruments should focus mainly on non-invasive or minimal-invasive procedures. The golden standard for the design of ART instrumentation in non-domestic species is reached if the ART equipment can be applied during the daily routine in captivity or integrated in necessary field operations in the wild e.g. translocation, dehorning, collaring etc. The portability of the equipment is a highly desirable feature to avoid long transport for the patient to a special facility.

• However, undiscovered evolutionary mechanism to control for example delayed implantation (present in many species), superfetation (European brown hare, Lepus europaeus), sperm storage (e.g. bates, sloth, birds), induced ovulation (e.g. cats) etc. have the potential of generating new pioneering reproductive techniques.

Several reports appeared where AI (semen) was used in wildlife conservation to mention some of them:

1)AI was used to enhance propagation of giant pandas at the Wolong Breeding Centre.

The China Research and Conservation Center for the Giant Panda in the Wolong Nature Reserve (Wolong Breeding Center) was established in 1982, and currently is the largest such breeding facility in the world. Wolong also has the highest number of breeding males (two wild-born and two captive-born) in the ex situ population. Propagation continues to improve and illustrate the important role of ex situ breeding programs. To enhance pregnancy success, Wolong Breeding Center uses the combined practice of natural mating and AI using semen from non-breeding males.

Although the number of offspring increases, some individuals still do not breed naturally. The efficient use of AI would facilitate genetic management of the population. Therefore, the goal of a recent analysis of breeding records was to:

1) assess reproductive success in giant pandas at the Wolong Breeding Center using both natural mating and AI; and 2) determine the efficiency of AI only without natural breeding in giant pandas.

2)One of the most successful examples of artificial insemination as a conservation management tool has been the production and genetic management of over 87 black-footed ferrets and selected re-introduction into their previous home range (Howard et al., 2001). While AI has the capability to supplement inbred populations, prevent disease transmission associated with the movement of whole animals, and overcome mate incompatibility, little effort has been devoted to this area in Australia.

AI into the urogenital sinus has been successful in the koala (Phascolarctos cinereus) with 6 live births from 11 inseminations reported (Johnson et al., 1999 abst.). Super-ovulated brush-tailed possums (Trichosurus vulpecula) and tammar wallabies (Macropus eugenii) inseminated into the uterus resulted in 1-4 cell embryos (Molinia et al., 1998a).

Embryo transfer – the ability to collect pre-implantation embryos, freeze them, store them for extended periods of time, thaw them when needed or when foster dams are available, and finally transfer them into the reproductive tracts of foster dams offers a very visible alternative.

Successful transfer of cryopreserved embryos in Camel- (Camelius dromedarius) reported – (Central Veterinary Research Laboratory, Dubai).

Ovum pick up and In - vitrofertilization

Intra vaginal pick up of oocytes from the ovaries by means of a probe and needle under ultrasound observation.

Oocytes can be harvested from the ovaries collected from animals that died shortly, matured and fertilized in the laboratory.

This procedure is becoming very popular and already used in a variety of work in the wildlife programmes.

Ovarian Tissue Banking

Potential in animal conservation: The development of ovarian tissue banking may have significant implications for use in animal conservation in years to come. Several specialised reproductive tissue storage centres have been established that cryobank ovarian tissue from valuable, rare, and endangered species using technologies that were established in laboratory species.

Many Zoos worldwide are well aware of this technology and cryobanking of reproductive tissues from deceased animals is becoming increasingly common practice, thereby preserving the genetic diversity and storing tissues for assisted reproductive technologies in the future. Even tissue of sexually immature females can be harvested because the primordial follicles are already present at a very young age. Following ovarian grafting into the right hormonal environment, the immature tissue will become morphologically similar to adult tissue.

Cloning And Gene Transfer

Potential applications of cloning go well beyond the population envisioned replication of valuable animals or endangered species. This is because targeted genetic modification can be made in donor cells before nuclear transfer.

Before embarking on a consideration of the technical issues involved in cloning, it is worthwhile considering the conservation-related goals that this technology would, or could, serve. At its simplest level, cloning is just one of several ways to increase the numbers of individuals within a population. Clearly, natural breeding is the method of choice with thriving populations; they do not present any conservation issues.

When the species in question lives freely in the wild, but the population is in decline, conservation biologists begin to seek methods of stopping or reversing the threatening processes. Many such threats exist, including habitat loss through the activities of humans, hunting, effects of pollution on fertility and fecundity, and destruction by introduced predators or, indeed, poor diet through loss of prey species.

In a few cases, these threats can be alleviated, but this may require the developmental of national, and even international, policies in support of conservation goals. Assisted reproductive technologies may then come into their own as supporting measures; here, their purpose is usually to assist with genetic management, where an important common aim is the avoidance of inbreeding depression with its resultant exposure of rare, and often deleterious, alleles.

Even without any input from assisted reproduction, the management of small populations is highly focused upon the avoidance of inbreeding, because if there is to be a realistic prospect of reintroducing the animals to their original habitat they should be given the best possible chance of survival. The Mauritius kestrel provides a good example of successful reintroduction without assisted breeding; the population declined to about 9 individuals in the early 1970s, four were reintroduced to the island of Mauritius, and the population is now estimated at 700-800 (Groombridge et al., 2000).

Genetic analyses have revealed that, in comparison with the pre-crash individuals, the population has now become extremely homogeneous and therefore poorly equipped to adapt to changes in environmental conditions. Nevertheless this has so far not proved to be a problem. A second example, where assisted reproduction played a major role is provided by the Black-footed ferret. This species declined almost to the point of extinction in the 1970s - 1980s, but a group of 18 animals were captured by the Wyoming Game and Fish Department in co-operation with US Fish & Wildlife service (Thorne and Oakleaf, 1991).

A species recovery strategy was developed, in which assisted reproduction within a captive-breeding group played a key role. The captive-breeding programme proved such a success that re-introductions have now been successful in several states of the USA (for review, see Howard et al., 2003). How could cloning have helped with these two examples? Populations that have declined to such low numbers of individuals possess minimal genetic variation and it would therefore seem a good idea to avoid any further losses of diversity. A subsequent generation resulting from natural breeding and AI would contain some, but not all, of the genetic variability possessed by its parents.

Some loss would occur if any of the individuals failed to breed, a strong possibility with such small populations. If cloning could be guaranteed to be 100% successful, one could argue that a good strategy would be to clone every individual (not too onerous if the population size is only 9-18), then allow the offspring to mature and breed naturally.

The probability of losing genetic diversity would then be reduced, especially if each parent had given rise to more than 2 identical copies of itself. This suggests the emergence of an interesting and novel principle in animal conservation, where the individuals are effectively induced to reproduce asexually, somewhat like plants, thus improving their long-term fitness. The important question, however, is how could this be achieved, if at all? Current success rates with nuclear transfer in mammals are currently very low (less than 0.1 - 5% of reconstructed embryos result in a live birth; Di Berardino, 2001; Wakayama and Yanagimachi, 2001). Assuming that 2 - 3 oocytes could have been recovered from each of two females, the realistic chance of obtaining a single offspring would be somewhere between 0.0006% and 0.3%, in other words vanishingly small. To date the cloning of birds has not been accomplished, therefore the Mauritius kestrel example could not have been addressed at all with this technology. Such arguments suggest that attempting to apply cloning technology to highly endangered species is hopelessly optimistic given current technology. However, should this rule out the idea of cloning altogether? Increased success rates could be expected when dealing with larger populations; however, even assuming an optimistic 5% success rate it would be necessary to produce 20 reconstructed embryos for every successful live offspring.

The best way to maximise success might therefore be to concentrate upon litter-bearing species with multiple ovulations. This would immediately rule out many of the larger mammals, especially the giant panda that only ovulates 1-2 oocytes per year (Hodges et al., 1984; Kleiman, 1983; Knight et al., 1985). Nevertheless, this is such a popular choice of candidate species that a special research programme has been initiated in China.

Paradoxically, this argument leads towards the application of cloning technologies to endangered rodents, in which other and more traditional assisted reproductive technologies have been largely overlooked. Although there are 330 rodent species listed as Endangered (IUCN Red list, 2000), techniques such as AI, semen freezing and embryo transfer have not been applied successfully to any.

It would be feasible to collect and cryopreserve ovarian slices from many individuals of such species, prepare fibroblast cell lines from muscle or skin, with the expectation that this approach might be ultimately successful. Having collected samples from many individuals, it might be feasible to regenerate offspring that represent the genetic variability of the founder populations, and use methods allied to those currently being developed for the laboratory mouse.

The message here is that any attempt to clone such species should be
approached on a grand scale, where sufficient numbers of offspring
could be generated to maintain a genetically diverse population.
As mentioned earlier, there is some merit in considering whether
cloning technologies might be applicable to non-mammalian vertebrate.
This possibility seems to have gone unnoticed for many years by
conservation biologists, despite the fact that amphibians (Rana pipiens
and Xenopus laevis) were first cloned in pioneering experimental work
carried out in the1960s (Briggs and King, 1960; Gurdon, 1962).

Amphibian populations all over the world are currently experiencing significant declines, possibly due to emerging diseases such as Chytridiomycosis and ranaviruses (Daszak et al., 1999). This phenomenon, popularly known as the .amphibian extinction crisis. signals the urgent need for the implementation of protective measures, and it is surprising that there is a dearth of studies on methods of assisted reproduction in these species.

Some research on the collection and freezing of spermatozoa has recently been published (Beesley et al., 1998; Browne et al., 1998; Mugnano et al., 1998), but most of this work has commenced within the last 5 years. Nuclear transfer in fishes has been studied for about 40 years in China by T. C. Tung (quoted by Yan, 2000), and more recently by Yan (1998), Niwa et al. (1999) and Wakamatsu et al. (2001).

This research has revealed an apparently very plastic system in which viable offspring and sometimes fertile can be produced from nucleocytoplasmic hybrids made between species, genera and even subfamilies. It would seem logical that cloning might be successfully applied to threatened species of fish, and that it might even be relatively straightforward to mass-produce the offspring.

The eggs are often produced in large quantities, sometimes thousands or millions at a time, and they are also physically large in comparison to mammalian oocytes. It is possible to culture fish cells in vitro, so making an extensive collection of cell lines from endangered fishes is perhaps a current conservation priority.

Knowing how to obtain the eggs may be the most challenging aspect of such a programme. The extreme diversity of fish species is reflected in a corresponding diversity of reproductive systems. In the context of cloning biology, an interesting example of a population that reproduces entirely by parthenogenesis (equivalent to cloning) is provided by a species of molly Poecilia formosa, which occurs naturally as all female populations.

They breed by gynogenesis, females mating with another sympatric Poecilia species, but spermatozoa play no role in fertilisation. Mating simply stimulates egg development, with the male not contributing to the genome of the offspring, and all-female clones are produced (Paxton and Eschmeyer, 1998). This example, albeit extreme, underlines an important and relevant principle, namely that while the detrimental effects of inbreeding are widely recognised as something to be avoided, the specific consequences of inbreeding differ between species. The reasons are unclear, and could be a matter of chance, but they could also reflect the environmental conditions within which species have evolved.

Thus a species that lives in small isolated groups may be less affected by inbreeding depression than one that is normally found in large groups, where it would be unusual to have restricted choice of mate. These considerations are relevant when contemplating the desirability of a cloning programme; survival and fitness of offspring may differ between species for the same reasons.
The arguments presented above have ignored the shortcomings of the technology in its current state of development. Since the first sheep were produced by nuclear transplantation (Campbell et al., 1996; Wells et al., 1997; Wilmut et al., 1997) a multitude of studies have revealed that most cloned mammals suffer from various developmental abnormalities.

Abnormalities have included extended gestation, inadequate placental formation and histological defects in most organs, including kidney, brain, the cardiovascular system and muscle. (Barnes, 2000; Chavatte-Palmer et al., 2000; De Sousa et al., 2001; Hammer et al., 2001; Hill et al., 1999, 2001; Renard et al., 2002). These effects have been attributed to inefficient reprogramming and imprinting of the donor genome (Betts et al., 2001; De Sousa et al., 1999; Fairburn et al., 2002; Heyman et al., 2002; Humpherys et al., 2001; Kang et al., 2002; Oback and Wells, 2001; Renard et al., 2002; Wakayama and Yanagimachi, 1999, 2001; Young and Fairburn, 2000; Young et al., 2001), a process that occurs naturally during gametogenesis and early development, and governs whether certain genes are expressed from the maternal or paternal chromosomes.

This problem alone strongly indicates the avoidance of cloning as a method of assisted reproduction in mammals until these difficulties are resolved (Solter, 2000). However, there is no harm in collecting cells and tissues from wildlife species so that they could be used in the long-term, a strategy currently being used in connection with attempts to prevent extinction of the Northern hairy-nosed wombat in Australia (Wolvekamp et al., 2001).

A further complicating factor to bear in mind when considering the application of cloning technology to wildlife using stored somatic cells is that of nucleocytoplasmic compatibility. If the only available oocytes were from a different species, as has frequently been proposed, then the resultant offspring would not be true members of either species.

Such a strategy has been proposed for the Giant panda (Chen et al., 1999), where in the absence of panda oocytes consideration has been given to the use of bear, and even rabbit, oocytes. Extensive studies of nucleocytoplasmic compatibility have been undertaken, and some researchers have proposed schemes for replacing the mitochondria of one species with those of the somatic cell donor (Meirelles et al., 2001).

The biological problems associated with the cloning of mammals have stimulated considerable debate about the ethical aspects of the procedure, both among scientists and the general public. Serious developmental biologists in the forefront of cloning research are emphasising the need for caution, while encouraging continued research. Unfortunately, those few that abandon caution tend to be both highly vociferous and good at capturing media attention.

Hence, a highly publicised plan to clone a Tasmanian tiger using DNA recovered from a single alcohol-fixed museum specimen has generated considerable publicity worldwide, attracted funding for the project and raised public expectations of success (for commentaries in Science and a serious UK newspaper, see: Anon, 2002; eek, 2002 respectively).

Examples of this sort do manage, however, to create the impression among the conservation community that reproductive and developmental biologists are unthinking zealots who only want to carry out the latest hi-tech enterprise. Unfortunately, the TV and newspapers are not usually interested in protestations of caution, so this impression is very difficult to rectify.

It is therefore very important that reproductive biologists working in conservation biology try to dampen the, often, well meaning, excessive claims that arise from time to time. Fortunately, some journalists are interested in presenting a more balanced and considered view and solicit opinion from a wide range of experts before putting pen to paper.

Despite the various arguments that can be adduced in favour of cloning programmes for various groups of species, both extant and extinct (for reviews and proposals, see Lanza et al., 2000; Ryder and Benirschke, 1997; Stone, 1997; Wells, 2000), success will ultimately depend upon the extent to which detailed background knowledge is available.

Currently this is a major limiting factor as, almost by definition, it is difficult to amass background knowledge of species that is in such decline as to merit this type of last-ditch strategy. Acquisition of scholarly knowledge about a species reproductive systems, together with a detailed understanding of cellular and developmental processes peculiar to that species, can only be undertaken within small and focused research programmes.

From a purely practical point of view this consideration alone rules out the widespread application of cloning technology in conservation; there are far too many species and far too few suitably funded and skilled researchers. However, it highlights the need for fundamental research that addresses the species of interest; in a sense, funding such research may prove to be the greatest challenge of all.the greatest challenge of all.