By F.J. de Haas van Dorsser MA VetMB MRCVS

Breeding Centre for Endangered Arabian WildlifeSharjah, UAE

Over the last hundred years Arabian species such as the ostrich, cheetah and Caspian turtle have become extinct and many more Arabian species are destined for a similar fate if loss of natural habitat and poaching continues. As a result, breeding programmes with very small founder populations have been created. This, however, leads to complications of inevitable reduction in genetic material (see Inbreeding and Genetics), inbreeding and loss of adaptability. Such populations are rarely self-sustainable and eventually die out.

There are examples of species with tiny founder populations that have bred successfully in captivity such as the cheetah, the Golden lion tamarin and the snow leopard, but inbreeding has reduced their reproductive ability. The reproductive rate of these species has occasionally been good enough to allow the stocking of zoos, but has generally been too poor to allow the species to survive in the wild. This raises the controversial, and much debated, issue of how best to conserve endangered species.

Breeding programmes that work solely to furnish zoos with endangered species have only a minor role in conservation. If zoos are to retain their function in conservation beside education, they must be aiming to breed only those species which can be re-introduced into the wild. The Arabian oryx project in Saudi Arabia, Jordan and Qatar was a successful effort at large scale breeding using wild-caught stock as well as captive bred animals. Unfortunately the re-introduction into their natural habitat failed because the poaching problem had not been solved and the Arabian oryx is once again endangered. There is a need to conserve the genetic material of wild-caught animals before the only founders of such programmes are ones that have been captive bred.

What is a 'Frozen Zoo'

The 'frozen zoo' can be defined as a collection of animal genes in the form of frozen semen and embryos. In practical terms this is a collection of sperm-holding straws stored in liquid nitrogen tanks.

Modern technology allows the indefinite preservation of genetic material through the freezing of semen and embryos (unfortunately, ova do not freeze very well). The implication of being able to freeze semen and embryos is that the species can be kept forever, even when they are extinct, as their genes would be preserved. When the threat to the preserved species has been controlled, implantation and development of an embryo can then be carried out. On the research front it allows much to be learned about species-specific reproductive physiology and will help develop the technology required for related species not yet endangered. Thus when the time comes to make conservation efforts for one of these species, the technology would already be available.

The Uses of the 'Frozen Zoo'

The genes from wild-caught endangered animals are very valuable as they will be needed to increase the founder population of breeding groups. Domestic cattle are constantly being improved genetically by means of artificial breeding. The advantage of speeding up the genetic progress in a breeding programme can similarly be used for exotic species. The frozen zoo could then provide the valuable material to produce genetically superior stock to that which natural breeding in one herd or colony could have produced. Furthermore, the production of genetically healthier animals will automatically reduce the inbreeding problems caused by loss of heterozygosity.

There are several ways in which this type of assisted breeding can be done. Semen from a sperm bank can be used for Artificial Insemination (AI) (see Assisted Breeding Techniques: Artificial Insemination) of captive females. Further techniques include Embryo Transfer (ET) (see Assisted Breeding Techniques: Embryo Transfer) and In-Vitro Fertilisation (IVF) (see Assisted Breeding Techniques: In-Vitro Fertilisation), both of which are skillful methods that are very species-specific.

The success rate of assisted breeding is low compared to natural breeding, but it is very useful for animals which are anatomically or physiologically unable to breed naturally. It bypasses the hurdles of 'infertile'animals that fail to produce oocytes or ovulate, have blocked fallopian tubes or are unable to sustain a pregnancy. Also, it allows a female to donate oocytes and carry a natural pregnancy, potentially doubling her breeding rate.

Creating a Frozen Zoo

Males hugely overproduce sperm which facilitates the establishment of a sperm bank. There are various methods of collecting semen including training to mount as done in horses, camels (here in the UAE), primates and certain wild animals, manual stimulation as is done in dogs, but far most common method in wild animals is electroejaculation. This method uses an electrical stimulus over the accessory sex glands to elicit ejaculation and is performed under heavy sedation or general anaesthetic. Semen can also be collected post mortem if it is retrieved soon enough.

Each species has different semen composition and this changes the freezing technique and quality post-thawing. Current research is geared to establish semen cryopreservation protocols for many endangered species. The semen quality is fully evaluated for its normal parameters and any morphological abnormalities are noted (these are particularly important if genetic). The movement of the sperm is assessed as to its capability to reach the ova, and the viability of the sperm and the acrosome integrity are assessed using special stains. Only good sperm samples are banked and these are handled carefully in order to retain their competence. Washed and concentrated semen are drawn up in straws and are then frozen in liquid nitrogen and stored in tanks.

Females produce very few oocytes each cycle, greatly limiting the use of female genetic material. However, using a specific hormone protocol it is possible to stimulate the ovary to produce more, called superovulation, enabling 10-20 oocytes to be collected at one time. Each species does vary though in its ability to be stimulated in this way.
The oocytes are then inseminated in an incubated petri-dish to produce embryos by IVF. Embryos are more resistant to being frozen than oocytes and will develop into normal offspring after implantation into a female. If there are too few females the embryos can be used for interspecies transfer into a related species. Zebras have been born from ponies using this method.

Currently in the Arabian peninsula, work has been carried out on several endangered species. An Arabian leopard, sandgazelle, Asian cheetah and Gordon's wildcat sperm bank has been set up in the Breeding Centre for Endangered Arabian Wildlife (BCEAW) in Sharjah, UAE. Already, IVF for the Arabian leopard and Gordon's wildcat has resulted in cryopreservation of embyros. These will be kept frozen until suitable recipient mothers become available. This is a fast growing area of research, however there is still a lot of work to be done on endangered species to enable a significant amount of material to be cryopreserved before it is too late.

Human assisted reproduction research has developed a great deal in the last decade, with improved preservation of genetic material, improved sperm injection techniques, embryo sexing, sperm sexing and cloning. Human research has the advantage of greater funding but is, fortunately and unfortunately, curtailed by ethics. Nonetheless, such techniques are carried through and are being applied to animal breeding, though many, as yet, are too expensive. The number of institutions actively involved in assisted reproduction of exotic animals is still small.

Modern technology allows assisted breeding in wild animals without the need to keep them in captivity, but requires close collaboration between wildlife managers and scientists. Much research is called for, so it remains imperative that zoos, wildlife centres and field stations co-operate on assisted reproduction projects to harvest opportunistic data on large-scale wild animal manipulations to facilitate the growth of developing frozen zoos.