It's a question of breeding

Saving an endangered species is never easy, and if there is only a small population in-breeding is a problem. But last year a group of gazelles were rescued from extinction using artificial insemination. Could animal sperm banks be the answer?

The average male pig produces an impressive half a litre of seminal fluid containing approximately 85 billion sperm. It takes about 10 minutes to deliver the goods. Professor Bill Holt and Dr Amanda Pickard, from the Institute of Zoology in London, are interested in pig sperm; they're using it as a model for how gazelle sperm might behave. The team is setting up a sperm bank to help save a species of gazelle that is extinct in the wild. Their research, which encompasses conservation as much as the dynamics of sperm, is already having far-reaching implications for other sperm banks.

The average male pig produces an impressive half a litre of seminal fluid containing approximately 85 billion sperm. It takes about 10 minutes to deliver the goods. Professor Bill Holt and Dr Amanda Pickard, from the Institute of Zoology in London, are interested in pig sperm; they're using it as a model for how gazelle sperm might behave. The team is setting up a sperm bank to help save a species of gazelle that is extinct in the wild. Their research, which encompasses conservation as much as the dynamics of sperm, is already having far-reaching implications for other sperm banks.

The Mohor gazelle went extinct in the wild in 1968; fortunately a Spanish officer, Commandante Estalayo, kept a small group alive in what was formerly the Spanish Sahara. Seventeen animals were brought to Almería in south east Spain three years later. It is from this group that all the Mohor gazelles in the world are descended. The problem with any small population is in-breeding. The situation looked even worse for Cuvier's gazelle which was believed to have gone extinct in 1975 leaving only three individuals in captivity. Bill Holt and his team offered to collaborate with Drs Teresa Abaigar and Mar Cano from the Spanish institute, Estaçión Experimental de Zonas Aridas in Almería, to create a sperm bank in an attempt to prevent the gazelle population from becoming too in-bred.

Collecting gazelle sperm isn't straightforward. For a start, since the team was using the pig as its model, it was a little optimistic about the amount of sperm needed. Female pigs have an unusually long vaginal tract: the sperm have to swim the equivalent of a 50km marathon to reach their goal. Gazelles don't need quite as much sperm. What they do need, like any other female who wants to get pregnant, is potent sperm. Not every sperm is capable of swimming the distances required or even fulfilling its function at the end of the journey, so the Institute of Zoology, in conjunction with Sheffield's Sense and Vision Systems Ltd, developed a program to determine how mobile sperm are. The greater the motility of the sperm, the more likely they are to be fertile.

Samples of the gazelle sperm are mixed with cryoprotectants, chemicals which are supposed to protect their delicate membranes during the freezing process, and egg yolk. The solution is then frozen at 196Å¡C in liquid nitrogen. The samples are injected into the tip of the gazelles' uterus within two minutes of defrosting.

Unfortunately, sperm do not survive freezing and thawing particularly well. The problem is twofold: the ice crystals themselves disrupt the sperm's membranes, and secondly, as water in the solution crystallises into ice, the other chemicals become more concentrated. "The poor sperm find themselves in a toxic medium," says Dr Lisa Thurston, who is studying pig sperm at the Institute of Zoology. "For the past 20 years people have been looking at the cryoprotectant and instead the variation in ability to survive freezing could be to do with genetics," she adds. Thurston is attempting to find the combination of genes which are responsible for creating sperm membranes capable of withstanding freezing. She theorises that an animal with these genes will produce sperm that have channels in their membrane which allow water to flow easily in and out. This animal will thus have sperm that can withstand being frozen. She also believes that shape matters: seminal fluid contains different populations of sperm which, though functionally normal, differ in shape. Semen that has large amounts of one sort of sperm can survive freezing better, perhaps because the shape of this kind of sperm is structurally well able to cope with being frozen. So far Thurston has found a marker - a fragment of DNA from the genes she is looking for - and this is good enough for pig farmers. They can now carry out blood tests on boars to determine whether they have the marker and will thus supply copious amounts of freezable sperm. "Once we locate the genes, we'll be able to screen endangered species in the same way," says Thurston.

Not wanting to put all its eggs in one basket, the Institute of Zoology had adopted another approach. It's known that sperm can survive inside the reproductive tract of females, particularly within insects and reptiles, and also some mammals like bats. Researchers have started to cultivate surface cells from the female reproductive tract and use them as a medium for storing sperm. So far sperm kept in this way seems to be viable. In this case, the research is principally aimed at conserving marsupials because no one has successfully managed to freeze kangaroo sperm.

Of course, there is no point injecting the carefully defrosted sperm into females at an inappropriate time. Normally blood or urine samples are used to determine when animals are in season, but as this would be stressful for semi-wild animals, Holt and his colleagues use gazelle dung instead. They found that they could tell when the females were ready to release their eggs by charting the amount of progesterone, a female hormone, that is present in the dung. They correlated the hormone levels with the times when males naturally mated with the females.

Until this research was carried out, this basic information on the length of the gazelles' reproductive cycle was not known. This method also works for black rhino and could be used to manage wild populations. Holt also analysed elephant dung. Initially he discovered that there seemed to be no correlation between female elephants' cycles and the progesterone level in their droppings. Fortunately another hormone, pregnaetriol, can be used instead. Holt has developed another reproductive technique. He inserted a small device into the gazelles' reproductive tract which releases progesterone so that all the females would become fertile simultaneously. This worked for about half the group: the other half did not become pregnant straight away.

So far 20 gazelles have been successfully reintroduced to Morocco between 1993 and 1999. Seventeen Cuvier's gazelles were released into Tunisia for the first time last year. With careful breeding, and by building up sperm banks, in-breeding in endangered species can be reduced. However, it's far better to begin sperm banks before numbers are at a seriously low level. With this aim in mind, the Institute of Zoology has been collaborating on a number of projects. Andy Hartley, a lab technician based at the institute, has been to Russia to collect tiger semen from zoos in Moscow; Holt has started to work with Dr Gordon McGregor-Reid, the director of Chester Zoo, on a cichlid fish sperm bank. Cichlids are indigenous to Lake Victoria in East Africa but their numbers have been decimated due to the introduction of the predatory Nile perch.

"These threatened cichlids are particularly interesting in that there were previously several hundred species existing in a 'species' flock," says Reid, "As an evolutionary phenomenon worthy of study, this ranks with the famous Galapagos finches of Darwin." Hold and Reid are trying to create a gene bank in case reintroductions to the wild prove to be necessary. They've enlisted the help of amateur fish enthusiasts.

But how on earth do you manage to extract fish sperm? "Not always easy," says Reid, "Tiny quantities are gently milked off using a very fine pipette. Urine contamination of the sample can be a problem." A further problem is that each different species of fish needs different cryoprotectants in which to freeze the sperm. "We're some way off our goal," admits Reid.

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