Biotechnological Wildlife Management

Introduction to Biotechnology in Wildlife

Biotechnology refers to all the techniques that are associated with the improvement or altering of biological organisms. In wildlife management, biotechnology that is associated with the manipulation of reproduction may find a practical application, as many molecular techniques for studying genetic variation, species identification, scatology and searching for genes that are associated with genetic defects or other traits which may be of economic importance in the wildlife industry.

The adaptation of embryo biotechnologies for use in farmed cervids has been ongoing for several decades (Diagram by Veterian Key)

The reproductive techniques that are available for biotechnological wildlife management include artificial insemination, embryo transfer and multiple embryo transfers. A technique such as artificial insemination is routinely used in domesticated animals, but it has certain limitations in its application to wildlife.

The rapid movement of molecular techniques and studies of the genomes of a vast number of organisms have created new means for studying the genetic composition of an individual animal or a taxon. To date, the genomes of most domesticated animals have been mapped for a great number of different genetic markers. DNA technology has already shown an application in wildlife management in the identification of hybrids and the estimation of genetic variation for conservation purposes and genetic management decisions. Scatology is a relatively new non-invasive tool that can be used for obtaining genetic information from mammals. Scatology is based on extracting DNA from faecal material, which is then analysed with appropriate genetic markers, in a method that is similar to the use of blood or hair for this purpose. It is a useful technique when studying nocturnal or elusive wildlife because there is no need to capture or harm any animal.

Hair and tissue samples can be used in wildlife forensic sciences

For wildlife producers to make maximal use of the DNA technology that is currently available, it is recommended that biological samples such as hair, blood and/or tissue of animals be collected during the hunting season, or at any other opportunity, such as when animals have to be handled. Samples can also be obtained by using special biopsy darting and should be kept at a central facility where a DNA biological bank for the species or subspecies can be maintained and analysed as required. Wildlife ranchers need to follow the prescriptions for the correct collection and storage of the different samples. Hair samples should have a follicle that is intact to allow the extraction of sufficient DNA for analyses and should be stored in a paper, not a plastic container. All blood and tissue samples must be frozen at -4 °C as soon as possible after collection.

The wildlife biological Resource Centre and its partners in Biobank SA have created a biological resource bank that is dedicated to the acquisition, processing, banking, use and provision of biomaterials to the scientific and conservation communities. This bank is viable, diverse and representative of southern Africa’s wildlife populations. Banked biomaterials include tissue from muscles, kidneys, fat, livers, embryos, fibroblast cultures, blood, sperm, hair, eggshell, fluids, cells and more. Biomaterials are made available for research, biodiversity conservation and biotechnology development, and are used in many disciplines, including genetics, reproduction, nutrition, taxonomy and disease studies. Biomaterial from selected species or subspecies is also useful for the detection and monitoring of persistent organic pollutants and other potentially harmful substances that are found in the environment.

These biomaterials are made available to third parties with prior consent from the biomaterial’s ‘owner’, but only after the signing of a customised material transfer agreement. The training of staff from the national parks and provincial wildlife reserves, zoological gardens, animal breeders and laboratories to collect these biological samples is being done regularly, to secure good-quality biomaterials. Sampling kits are made available to persons who are tasked with the collection of wildlife biomaterials. The Biobank SA consortium acts as an integrated resource centre that links all the partner collections. The consortium’s operational arm, the Wildlife Biological Research Centre, is active in the development of relevant policies, regulations and legislation pertaining to biomaterials, including access and benefit-sharing systems. The main sponsor of the project is the Department of Science and Technology of the national government of South Africa.

 

Genetics as a Forensic Tool in Wildlife Management

Types of samples which can be used for wildlife forensic sciences

Genetics is a useful broad forensic tool in wildlife management using the white rhino as an example. DNA profiling method has been validated and optimised for rhino horns. This method is used routinely by the Veterinary Genetics Laboratory at Onderstepoort to identify rhino horns from stockpiles individually for security purposes to link recovered horns to individual poaching cases. This created the opportunity to link a poaching incident to a rhino horn trafficker, or where a poacher was caught, to linking the horn back to the carcass of an individual rhino. Each rhino poaching incident investigated has a collection of DNAs as part of the standard operating procedure.

A. Conservation Genetics

Conservation genetics has great potential in saving and protecting the genetic integrity of specific species

This method can also indicate how closely related animals are. When small populations of wild animals become isolated due to habitat fragmentation, researchers use genetic techniques to determine whether those populations are already inbred or at risk of doing so.

Animal materials such as hair, bones, or carcasses, as well as blood samples and cheek swabs, if the animal is captured, are collected and used by the researchers to extract DNA. This DNA is then amplified to look for certain areas in the genomes. The arrangement of genes or base-pair sequences differs between individuals, communities, and species at various distinct sites on a genome. Two of these specific sites include the nuclear DNA which is the DNA contained in the cell’s nucleus and the other is the mitochondrial DNA which is the DNA located within the mitochondria of the cell.

After we amplify and produce numerous copies of these locations, DNA sequence analysis software is used to examine all of this data. We look at distinctive genome types, or DNA fingerprints, to examine diversity within individuals. When examining species variation, we look at the barcodes found in mitochondrial DNA.

Individual samples’ DNA barcodes and fingerprints are then compared to a genetic database of previously studied species and populations. If the population is found to be inbred, plans should be made on how to improve the genetic diversity of the population. This can be done by taking a few individuals from populations which have good genetic diversity and then introducing or trans-locating them into a population which is genetically inbred. This will assist with bringing the population back to higher genetic diversity.

This same technique can also be used in captive breeding when applied to endangered or critically endangered animals. Captive-bred animals with good genetic diversity could be reintroduced into the wild to help rescue the wild population genetically.

Genetics can also be utilised to identify species in areas where they previously were not known to exist.

B. Rhinoceros Horn DNA

DNA can be extracted from a living rhino’s horn to build a database and aid in the prosecution of illegal poachers

A DNA profiling method has been validated and implemented for rhinoceros horns by the Veterinary Genetic Laboratory (VGL) at the Faculty of Veterinary Sciences of the University of Pretoria. Since the start of the operation, the number of loci which can be used has almost doubled and the DNA extraction method uses some of the latest technology used for human forensic DNA extraction. This method is now standard practice when identifying rhino horns from stockpiles for security purposes. It is also used to link horns which have been recovered to individual poaching cases which assist in linking horn traffickers to a previous poaching incident or to a poacher who has been caught with horns in his possession. This technique has several advantages for the rhinoceros rancher which include:

• It identifies an individual animal which enables the rancher to claim ownership of the rhino.
• It can give the degree of genetic relation between individual rhinos which assists the owner in selecting breeding bulls which are minimally related to his cows from the database to prevent inbreeding.
• Identifies the sex of the animal.
• Differentiates between white and black rhinoceroses.

The use of forensic genetics in rhino horn poaching, therefore, has major benefits and can assist in preventing the illegal trade of these animals.

 

Reproductive Technology

An illustration of the process of reproductive technology. (Source: Trends in Biotechnology)

 

Although there is great potential in using reproductive technology to protect and conserve rare and endangered species, large amounts of basic research are still required to make it a success. Some of these concepts have been highly successful in domesticated farm animals, but wildlife species seem to be a lot more complex and individualised than domestic animals and have thus been less successful so far. The physical handling and limitations of wildlife may also be why there is still much less information and research done on these species.

Captive-bred and zoo animals play a vital role in the species survival of those wildlife species which are facing a drastic decline in numbers. They grant researchers the opportunity to study their reproductive physiology and make major advances in the assisted reproductive technology sector.

A. Artificial Insemination

Very first semen sample collected from a giraffe bull by Dr Luther-Binoir from GEOsperm South Africa. (Source: Dr. Francois Deacon)

Oocyte collection from southern white rhino females in Germany. (Source: Forschungsverbund Berlin e.V. (FVB))

Artificial Insemination, also known as AI, has major potential in the wildlife conservation sector and has already been used in multiple instances. It is the process of artificially introducing sperm into the reproductive tract of a female to fertilise her eggs. In wildlife conservation, many times captive donor animals will be used as it is easier to manage wildlife which is in a contained environment than it is to try and use wild roaming individuals. This is by no means an easy process. Captive wildlife on the other hand seems to be particularly difficult to breed in captivity. Although AI seems like a viable solution, some scientists are sceptical about the concept and whether interfering on such a level is acceptable.

DNA samples from high-value animals or animals facing extinction can be collected and stored for future usage. Saving DNA, sperm, and embryos from highly valuable living or recently deceased animals can be done for later use. These samples can be frozen and used later to produce high-value or good-quality genetic offspring. One genetically high value can for instance ‘father’ a whole bunch of offspring without having to move anywhere. The downside of this is that inbreeding is a potential risk if accurate record-keeping is not done. It is therefore important to have a variety of different samples from different donors to prevent one or two high-quality animals from being the only source of genetics. Breeding programmes use DNA profiling to avoid inbreeding or to maximise diversity.

The two main struggles in AI with wildlife are the lack of knowledge and understanding of the reproductive tract of these diversely different species and the difficulty which arises when handling them. Scientists are still researching the crucial factors for the success of such procedures, such as what types of hormones to give the females to accept the sperm as well as when exactly the females are fertile. Up until now, artificial insemination in wild animals has had limited results.

In 1973, the wolf underwent the first successful artificial insemination of a wild species using semen that had been previously frozen.

Successful nonsurgical artificial insemination of a Persian leopard. (Source: Asghar Khamseh)

The majority of attempts to artificially inseminate wild-caught Felidae (cats) have failed, despite a significant amount of time and effort being put towards it. In 1980, the London Zoo was able to surgically inseminate a puma with a fresh semen sample and in 1981, successful nonsurgical insemination was done on a Persian Leopard in the Cincinnati Zoo with fresh semen. Since 2003, scientists at the Smithsonian Conservation Biology Institute have attempted artificial insemination of cheetahs without success, largely because female cheetahs respond inconsistently to hormone treatments.

In wildlife, for both semen collection from the male and insemination of the female anaesthesia is required which always poses a great risk. Unfortunately, it seems like the fertility of semen collected through AI compared to natural ejaculation is questionable and semen starts to die as soon as they are collected. This is the case even when using a fresh semen sample.

B. Embryo Transfer

Pregnancy monitoring of recipient female cheetah who has received two embryos. On the left is an ultrasonograph 32 days post transfer where the arrow points to the embryonic vesicle filled with fluid; on the right is a radiography 62 days post transfer indicating mineralised skeletons of two cubs

The 2 cheetah cubs two days after birth

 

Embryo transfer is a procedure whereby fertilised ova and early embryos are removed from a female donor, also known as the genetic mother, and placed into the reproductive system of a female receiver, often known as the foster mother. In the recipient female, the embryos grow into full-term foetuses and live young. The future advancement of interspecies embryo transfers to the point at which embryos can be taken from threatened species and given to surrogates of more common species, significantly boosting the donor species’ reproductive capacity will be greatly beneficial to wildlife conservation.

An ovary from a Barbary sheep showing the superovulatory response to FSH in a Zoological Park in Mexico

 

Through precisely timed injections of prostaglandins, such as Lutalyse and Estrumate, it is possible to synchronise the donor and recipient animals during embryo transfer. The ovaries are stimulated by these hormone analogues to start a new cycle. Fertility hormone injections, such as follicle-stimulating hormone (FSH), are used to induce superovulation in the donor. However, in wildlife, optimal drugs and dosages are still to be refined. With ungulates, superovulation has been fairly successful and an FSH-stimulated Eland cow was able to produce 31 embryos which could be collected. Other species such as the zebra have been highly unresponsive to fertility drugs.

Felines are mostly induced or reflex ovulators and require copulation to ovulate and are therefore rather difficult to synchronise donors and recipients through superovulation. In future, it is hoped that domestic cats will be able to serve as surrogates for incubating embryos from small endangered wild cats such as the black-footed cat. A lot more research is required before successful embryo transfers of felines will be possible.

Much more research is required in this particular field as it appears that the hormone regimen that results in the best superovulation response within a single species is rather individualised.