The Programme for the Study, Conservation and Recovery of the Far Eastern Leopard in the Russian Far East is an independent project being carried out as part of the ongoing research at the Russian Academy of Sciences on the study of animals that have been included on the Russian Federation’s Red List of Endangered Species, as well as other particularly species of animals of particular importance living in Russia. The research programme has been established as part of the work of the Severtsov Institute of Ecology and Evolution at the Russian Academy of Sciences (IEE RAS) in compliance with directive No. 12300-128 of the Presidium of the Russian Academy of Sciences dated February 29, 2008.

The research supervisor is Academician Dmitry Pavlov, Director of the IEE RAS; and Head of the research is Doctor of Biology Vyacheslav Rozhnov, Deputy Director of the IEE RAS.

The programme was drafted by the Severtsov Institute of Ecology and Evolution of Animals at the Russian Academy of Sciences, the Biology and Soil Science Institute at the Russian Academy of Sciences’ Far Eastern branch, the Pacific Institute of Geography at the Russian Academy of Sciences’ Far Eastern branch and the Komarov Ussuri National Nature Reserve, which is overseen by the Russian Academy of Sciences’ Far Eastern branch.

The Head of the programme is Doctor of Biology Vyacheslav Rozhnov.

The programme is intended to develop a scientific basis for leopard conservation and the leopard’s reintroduction to its former natural habitat in the Russian Far East.

The Programme’s Objectives and Structure

1.   Studying and monitoring the status of the existing population of Far Eastern leopards

The study and monitoring of the Far Eastern leopard population is focused on the following four main areas:

• Studying the animals’ migration using GPS Argos collars

This involves capturing leopards with foot snares and then tranquilising the animals so as to fit them with GPS Argos collars, which supply information on their migration routes and activities via the Argos satellite system.

• Creating a GIS-linked database of the results of the individual identification of Far Eastern leopards, using camera traps and non-invasive molecular genetic methods

This entails installing camera traps in leopard habitats, gathering samples of faeces for molecular and genetic analysis and the individual identification of an animal. This data will then be summarised using geo-information technologies.

• Monitoring the animals’ reproductive status using non-invasive methods

The work here involves collecting samples of leopards’ faeces for immune-enzyme analysis of hormone levels and also to determine the animal’s sex, the activity of males and females’ reproductive system and stress levels

• Carrying out zoological and veterinary examinations on the Far Eastern leopard population and other predators with whom leopards have biotic relationships

Modern medical equipment will be used to conduct veterinary examination on leopards and samples of faeces will be collected to study parasites carried by leopards and other predators.

Reasons for choosing the areas of the study

Assessing and monitoring the Far Eastern leopard population and the density of the species are amongst the study’s the priority objectives and these require the use of modern methods. At present, no accurate information on leopard numbers has yet been collected, with figures ranging from 22 to 31 (Pikunov et al., 1997; Pikunov et al., 2000) and 48 to 50 (Aramilev, Fomenko, 2000). Most research has relied on studying the size, number and location of predators’ tracks in the winter (Matyushkin et al., 1996). However, this method is not accurate and there are no hard statistics to support it (Karanth, Nichols, 1998). This has resulted in subjective estimations and discrepancies in the analysis and interpretation of the same source material (Miquelle, 2000). It is important to optimise the existing counting methods and develop new counting methods based on the signal biological field elements and the identification of individual animals using camera traps.

Although we know a significant amount about the biology of the Far Eastern leopard, more research needs to be done into this animal’s ability to adapt to the ever-changing conditions of the modern environment around it. In order to do this, scientists need to research their habitat structure, analyse the long-term developments in the forest ecosystems of the Russian Far East and make projections of leopard habitats by using GIS technologies to predict the leopard’s range.

Another important aspect of the programme is the research into the structural and functional organisation of the populations of the leopard’s main prey, including ungulates (roe deer and sika deer) and other animals (badgers, raccoon dogs and Manchurian hares), and of the main rival predators (Amur tigers, brown and Asiatic black bears and wolves). Of particular importance is the research into the specifics and implications of the inter-population relationship between the two species of big cats, namely, the Amur tiger and the Far Eastern leopard.

A critical factor for the survival of many species of big cats is the abundance and distribution of food (Kitchings, Story, 1984; Jakson, Alborn, 1988; Lukarevsky, 1993; and S. Naidenko, K. Hupe, 2002) and the animals’ exposure to human activity (Breitenmoser et al., 1993; and A. Gavashelishvili and V. Lukarevsky, 2008). The research will focus on establishing the relationship between the size of the leopard’s home range and its gender and age, as well as abiotic (snow depth), biotic and man-made factors. Determining the density of the populations of the main species of ungulates and comparing them with the leopards’ home range could help understand the role of ungulates in the spatial distribution of leopards. In addition, the non-invasive hormone monitoring of the animals will be used for studying the effects of all these factors on the leopard’s physiology (their stress levels and the activity of their reproductive system).

The use of collars with satellite-linked GPS transmitters, which is a new practice in the study of the Far Eastern leopard, and of non-invasive methods (molecular, genetic and hormone-based) will allow scientists to monitor individual leopards, thereby taking their research to a new level.

A decline of genetic diversity in the population leads to certain morphological and physiological pathologies that are detrimental to the survival of individual animals and the population as a whole, as they affect the constitution of the heart, kidneys and genitals and weaken the immune system, making animals prone to catching various diseases. Low leopard numbers over a period of over 50 years have put the species at risk of low genetic diversity.

Therefore, it is necessary to hold zoological, veterinary and parasitologic examinations of both Far Eastern leopards and those predators with which leopards come into contact.

Methodology for the study of the existing Far Eastern leopard population

The following methods will be used to study the Far Eastern leopard.

• Tracking individual leopards using satellite-linked GPS Argos collars.

Modern technical devices will be used to track individual predators, such as GPS Argos collars manufactured by New Zealand’s Sirtrack, the US firm Telonics or Canada’s Lotek, as well as ES-PAS transmitters made in Russia. In addition, a molecular and genetic databank of information collected from faeces samples will be created. These methods need further development.

Far Eastern leopards will be caught and fitted with satellite-linked collars in the Kedrovaya Pad and Borisovskoye Plato nature reserves in the spring, from April to June, and in the autumn, from September to November. In the autumn, the work will be suspended if temperatures drop below minus 10 Celsius or if over 5 centimetres of snow falls, to make sure there is no risk of an animal freezing its paw.

The Aldrich foot snares, manufactured by Canada’s Margo Supplies Ltd., have already been tested during the capture of Amur tigers in the Far East and will be used to catch leopards so that Argos collars can be fitted to them. The snares will be placed along the leopards’ likely routes. Radio beacons which issue a different signal when the trap is activated will be used to lower the risk of causing harm to the animals; these radio beacons were developed during the Amur tiger research programme.

Air rifles manufactured by Dan­inject, which were tested on the Amur tiger, will be used to tranquilise the leopards. Zoletil and Domitor will be the drugs used, which are currently used for tranquilising all large predators, including tigers and leopards.

• Identifying individual leopards.

 There are two main approaches to identifying individual Far Eastern leopards:

- photo identification (using camera traps) and

- identification based on molecular and genetic methods.

Normally, individual leopards are identified visually by the pattern on their coats. Camera traps will be set up in such a way as to take photographs of the leopard from both sides simultaneously so as to obtain images of both sides of the animal’s body.

Instead of classic ecological approaches, which can be quite problematic, molecular and genetic approaches will be used to assess big cat numbers, their gender composition and range. Non-invasive methods of collecting biological material, such as faeces, urine and hair samples, have proved their worth in the molecular and genetic study of wild animals (Ernest et аl., 2000; Fеrnаndо et al., 2003; Perez et al., 2006; Hausknech et аl., 2007; Murphy et al., 2007; and V. Rozhnov et al, 2009). However, scientists have yet to conduct extensive comprehensive research on the Far Eastern leopard population based on molecular and genetic methods.

These non-invasive molecular and genetic methods used to identify individual animals are based on nuclear DNA microsatellite analysis. The structure of these DNA sequences is unique for each animal. This kind of technology has long been used to hold environmental and zoological examinations of various groups of mammals, including the Amur tiger (V. Rozhnov et al., 2009), the Bengal tiger (Bhagvatula, Singh, 2006), the puma (Ernest et al., 2000) and the Arab leopard (Pe1ez et аl., 2006). Statistical approaches and computer software, including GIMLET (Valiere, 2002), will be used in addition to non-invasive methods, which carry a high risk of error.

A cynological method of identifying animals will also be used for the first time in the study of the Far Eastern leopard. The method was developed by scientists at the Russian Academy of Sciences (Sokolov et al., 1990) and is based on the uniqueness of the odours produced by an animal performing its vital functions. During field studies, samples of leopards’ faeces, urine and paw prints will be collected in locations where an animal has left its odour marks. In laboratories, specially trained dogs then identify them by sniffing individual samples. The samples will be kept in the odour bank for at least 10 years.

• Monitoring an animal’s individual reproductive status.

For the purpose of monitoring the reproductive status of individual Far Eastern leopards, non-invasive methods will be used to identify an animal’s sex, assess the activity of its reproductive system and its stress levels.

To identify a leopard’s sex by the level of steroid hormones in its faeces, researchers will have to determine the proportion of the metabolites of the male and female gonadal steroid hormones (testosterone, progesterone and estradiol), which is considered a reliable way to determine the sex of other big cats, such as tigers, lynxes and leopard cats (Naidenko, Rozhnov, 2009). The above non-invasive methods, which need to be further validated and improved, will be used to obtain the physiological profiles of Far Eastern leopards living in the wild.

Assessing the quality of the sperm of male leopards will allow researchers to analyse the periods of their sexual activity and compare the quality of the sperm of leopards living in the wild and those bred in captivity.

The duration of the active period of the leopard’s reproductive system is still unknown. Tracking changes in an animal’s reproductive system related to aging will help find the correct conclusion.

Methods of diagnosing pregnancy in a leopard by using non-invasive methods have not been developed yet, although similar research has been conducted for other species of big cats (Brown et al., 2001; and Graham et al., 2006). It is extremely important to assess female leopards’ reproductive status for the effective management of the leopard population and the recovery of the Far Eastern leopard population in the north of the region.

The success of a particular non-invasive approach to assessing an animal’s hormonal status depends, to a large degree, on the type of antibodies used for immunoenzymometric analysis and radioimmunoassay. The Laboratory of Behaviour and Behaviourial Ecology of Mammals at the A.N. Severtsov Institute of Ecology and Evolution is the only laboratory in Russia which conducts non-invasive studies of leopards’ hormonal status, both on its own (Pavlova et al., 2007; and Pavlova, Naidenko, 2008) and in collaboration with foreign researchers (Jewgenow et al., 2006а,b; and Dehnhard et al., 2008). The laboratory is currently conducting a study of eight species of big cats (Eurasian lynxes, bobcats, European wildcats, leopard cats, Pallas’s cats, Amur tigers, leopards and snow leopards).

• The zoological and veterinary examination of the Far Eastern leopard.

After being caught, all leopards will be examined for morphological defects (cryptorchidism, curled tails and curled fur). This will allow scientists to determine changes occurring in the population due to genetic drift, inbreeding or any other reasons.

As part of the programme, veterinaries will perform ultrasounds on all captured animals, examine their genitals and kidneys and compare the results with data on leopards held in captivity. Samples of blood and faeces taken from each captured animal will help assess the state of their immune system (by determining the proportion of cell elements and the concentration of immunoglobulin) and their clinical state, including the presence of various parasites.

2. The study of the taxonomical status of leopards living in the Russian Far East and the adjacent territories.

If Far Eastern leopards are to be reintroduced to their habitat, these should be animals with the same taxonomical status as those who inhabited the area previously. Zoo leopards either belong to the Far Eastern subspecies or are related to leopards living in Northern China, whose taxonomic status is yet to be determined.

Researchers in the Russian Far East and the adjacent areas described several distinct types of leopard: Felis orientalis Schlegel, 1857 (Korean Peninsula); Leopardus japonensis Gray, 1862 (Northern China); Felis fontanieri Milne­Edwards, 1867 (not far from Beijing); Felis chinensis Gray, 1867 (hills to the west of Beijing); and Felis villosa Bonhote, 1903 (Amur Bay). Their taxonomical status is still not clear, although some pilot works have been published (Uphyrkina et al., 2001, 2002; and Sorokin et al., 2010) that could help clarify the issue.

The programme includes an independent section aimed at the recovery of the Far Eastern leopard, which involves gathering tissue samples (skull bone tissue, skin and claws) in museum collections for the molecular and genetic study of DNA, as well as the molecular and genetic analysis of the founder group of zoo animals which will be used for the reintroduction of the northern population of the Far Eastern leopard.

3. Preparing a new founder group of animals for the reintroduction of the northern population of the Far Eastern leopard to the Ussuri Nature Reserve

The experiment to select and prepare a new founder group of animals for the reintroduction of the northern population of the Far Eastern leopard will be carried out in the Komarov Ussuri National Nature Reserve, which is overseen by the Far Eastern branch of the Russian Academy of Sciences. The Far Eastern leopard used to inhabit this area (Abramov et al., 2003) and was considered an ordinary species normally to be found in the reserve and the adjacent areas. In the 1930s and the 1940s, the Ussuri Nature Reserve, as well as other nature reserves, were instructed to kill all predators, leopards included. In 1956, leopard hunting was officially prohibited but the increasing human activities in leopard habitats, particularly park deer breeding, had a negative impact on the population’s genetic viability. These factors, combined with no-holds-barred poaching, led to a significant reduction in leopard numbers and the loss of a significant part of its habitat. The last time leopard tracks were seen in the Ussuri reserve was in 1975.

The Ussuri Nature Reserve is one of the best places for the reintroduction of the Far Eastern leopard. It occupies an area beyond the Razdolnaya River, a natural boundary separating the current habitat from the sites that are likely to be selected for the reintroduction of the northern population of leopards.

The experiment in the Ussuri Nature Reserve is based on the programme developed for the recovery of the Persian leopard in the Caucasus (Rozhnov, Lukarevsky, 2008), which is being implemented in Sochi National Park, where the Centre for the Breeding and Rehabilitation of the Persian Leopard has already been built.

The initial stage of the project, the creation of a new founder group of animals for the recovery of the northern population of the Far Eastern leopard, involves the following activities:

• Checking the area for the presence of parasites.

 All predators which might come into contact with leopards in the area designated as a reintroduction site will be subject to a parasitologic and epizootic examination lest they transmit diseases. Preliminary studies (Yesaulova et.al., 2010) have shown that helminths found in the Ussuri Nature Reserve are typical of the predators inhabiting the area and do not pose a particular threat to the Far Eastern leopard, therefore, the reserve can be used to reintroduce the northern population of the leopard.

• Creating reintroduction sites for the adaptation, breeding and rehabilitation of leopards near Kordon Peishula. 
  
  

 The reintroduction sites will be organised according to a system similar to the one used at the Centre for the Breeding and Rehabilitation of the Persian Leopard in the Caucasus (Lukarevsky, Rozhnov, 2007; and Rozhnov, Lukarevsky, 2008), though on a smaller scale.

To ensure the best conditions for the animals’ adaptation to the environment, the reintroduction sites will be built in the area where leopards will be released into the wild. The physical geography of Kordon Peishula is similar to that of the Khasansky Region, which is known as a Far Eastern leopard habitat. The reintroduction sites will have conditions similar to their natural habitat with natural environs and interiors.

Four reintroduction sites, each 40 square metres in size, will be built for breeding purposes. They will be large enough and will have everything that is needed to observe and manage the animals.

A reintroduction site for the rehabilitation of captive-bred animals will be built separately and will occupy an area of 1 hectare. The enclosure will have a six-metre high fence of Rabitz-type double twist steel-wire mesh, with the wire diameter being 0.4 centimetres. The mesh will be held up by seven-metre high posts (100 to 150 millimetres in diameter), that will be dug into the ground at a distance of 3 metres from one another and covered with concrete at the base. To prevent animals from climbing up and down the fence, a multitier electrical fence will be installed inside and outside the enclosure. The enclosure will be divided into two parts with a four-and-a-half metre high partition panel with two passages, each having remotely-operated drop-down gates. It will also have a small section for a feedbox (3 metres by 4 metres in size, with a two-metre high wall and a mesh roof) that can also be used to manage the animals.

• Creating a new founder group of animals to produce leopard offspring and later release them into the reserve.

 For breeding purposes, a founder group of animals, consisting of two male leopards and two or three female leopards to be taken from zoos, will be created at Kordon Peishula in the Ussuri Nature Reserve over the course of two or three years. Molecular and genetic methods will be used to examine all the animals involved in the project to confirm that they belong to the Far Eastern subspecies.

Henceforth, orphaned cubs captured in the Khasansky and Nadezhdinsky regions of the Primorye Territory could also be part of the programme.

• Taking care of the food supply for the Far Eastern leopard in the areas bordering on the nature reserve.

According to V. Korkishko’s studies (1983, 1986), the main species of prey that are key to the survival of the Far Eastern leopard are roe deer and sika deer.

The creation of a food supply for the leopards (mainly roe deer in order to reduce the rivalry between leopards and tigers over food resources) using biotechnical methods is scheduled to begin on the adjacent territory of the Orlinoye National Experimental Game Preserve.

4. Drafting a new version of the Strategy for the Conservation of the Far Eastern Leopard and preparing proposals for state environmental agencies regarding measures to conserve the Far Eastern leopard

The current strategy for the conservation of the Far Eastern leopard in Russia was developed in 1999 for a ten-year period and now it requires revision and an analysis of the current situation.

Based on the results of their studies, scientists will submit proposals for the conservation of the Far Eastern leopard to the state environmental agencies.

Participants in the Programme

Scientists from research institutes, including the Far Eastern branch of the Russian Academy of Sciences, as well as staff at nature reserves and other institutions will be involved in the programme. In addition, university students and postgraduate students will be invited to work on the project.