Ciência Animal, 22(1): 106-123, 2012 – Edição Especial CREATION OF FROZEN ARKS FOR MAMMALIAN SPECIES CONSERVATION (Criação de bancos biológicos criopreservados para conservação de mamíferos) Locatelli Yann 1,2 (PhD) 1MNHN, Réserve de la Haute Touche, 36290 Obterre, France. 2INRA, UMR 6175 Physiologie de la Reproduction et des Comportements, équipe Interactions Cellulaires et Fertilité, 37380 Nouzilly, France. ABSTRACT Preservation of endangered species represents a challenging perspective regarding quick decline of biodiversity. With benefit of human medicine and agronomic research developments, a wide range of reproductive biotechnologies are now available to facilitate conservation of mammals threatened with extinction. Cryopreservation of gametes, embryos and gonadal tissues are important to be performed for many endangered species on the basis of use of standardized methodologies. Examples of interspecific gestations have been reported for several mammalian species, indicating that some species can be restored using frozen embryos transfer even after extinction. This short review focuses on different illustrations of gametes and tissues cryopreservation among different mammalian species for creation of biological resource banks. INTRODUCTION Nowadays and whatever the species considered, rapid decline of populations is observed likely to confirm earths’s sixth mass extinction [9]. According to data from the International Union for Conservation of Nature (IUCN) more than 20% of mammals are threatened with extinction in their natural habitat. If conservation effort has shown to be efficient on Red List Index for mammals (indicator of the changing state of global biodiversity), it could not prevent its global decrease over the past three decades [33]. Ciência Animal, 22(1), 2012 Palestra apresentada no VI Congresso Norte Nordeste de Reprodução Animal, Fortaleza, CE, Brasil, 27 a 29 de junho de 2012. 106 Ciência Animal, 22(1): 106-123, 2012 – Edição Especial This phenomenon may underline apparition of new threatened species and the deterioration of status of some already threatened species as well. In some cases, population loss is fast and cannot be controlled despite protective measures developed in situ. Precisely designed in these cases, ex situ conservation programs are aimed in preservation of genetic variability of endangered species during restoration of their natural habitat. Ex situ programs for mammals consist of captive breeding and are generally performed by both governmental and non-governmental institutions, reserves or zoos. The objective of such breeding schemes is to preserve 90% of genetic diversity within species over 200 years, using non-parented founder animals. The number of founder animals and population to be kept in captivity depends on generation interval of the species but is generally estimated to be 50 and 250-500, respectively. The main difficulty in this aim is thus to prevent inbreeding and sustain heterozygosity to drive rational genitor exchange between institutions. Achievement of ex situ conservation is sometimes difficult especially because of genetic loss, inability to conduce genitor exchange or failure for the species to adapt to captive housing [7]. To prevent genetic loss and facilitate genetic exchange between conservation centers, assisted reproductive technologies developed for domestic species, such as semen recovery and cryopreservation coupled to artificial insemination (AI) have been proposed [18]. With development of second and third generations of animal biotechnologies (embryo transfer, in vitro fertilization, gametes micromanipulation, sperm sexing), new possibilities of both genetic management and conservation of endangered biodiversity is offered. To a greater extent, increasing knowledge in development and use of biotechnologies such as in vitro fertilization for livestock and domestic species, allow transposition to close related endangered species. With benefit of research efforts for human medicine and livestock production, cryopreservation methods for gametes, embryos or gonadal tissues based on slow freezing and vitrification turned from thought into possibility. Today, application of cryobiology for conservation of endangered biodiversity has appeared as an obligatory step for warranting achievement in conservation programs. Interspecific embryo transfer, demonstrated to be possible for some ovine [23], bovine [41], equine [68] and deer species [48], would offer possibilities to gave birth to extinct species using transfer to Ciência Animal, 22(1), 2012 Palestra apresentada no VI Congresso Norte Nordeste de Reprodução Animal, Fortaleza, CE, Brasil, 27 a 29 de junho de 2012. 107 Ciência Animal, 22(1): 106-123, 2012 – Edição Especial surrogate females. An illustration of possible samples for creation of frozen ark and their potential use is represented in Figure 1. Figure 1: possible samples to cryopreserve and their potential use for constitution of mammalian frozen arks. Sperm and testicular tissue cryobanking Since the first success of semen cryopreservation and discovery of the cryoprotecting properties of glycerol by Polge et al. in 1949 [60], success in sperm freezing was achieved in many mammalian species that allowed the creation of sperm banks for human and livestock species. It’s interesting to note that methods of cryopreservation of sperm based on glycerol supplementation and controlled slow freezing have not changed much from the principle described by Polge or Lovelock et Polge [50,60]. Sperm resource banks would allow to both conserve rare genetics for long periods and to facilitate realization of ex situ programs [36]. Based on ongoing ex situ program data, Harnal et al. [31] showed in silico that sperm resource banks may Ciência Animal, 22(1), 2012 Palestra apresentada no VI Congresso Norte Nordeste de Reprodução Animal, Fortaleza, CE, Brasil, 27 a 29 de junho de 2012. 108 Ciência Animal, 22(1): 106-123, 2012 – Edição Especial represent efficient tools for prevention of inbreeding and enhancing genetic diversity for different mammalian species (tiger, Przewalski’s horse and eld’s deer). In wild mammals, the first difficulty consists of recovery of good quality ejaculates. In most cases sperm recovery from non-domesticated animals involves in electro-ejaculation under general anaesthesia, which can be associated with over- stimulation of accessory sex glands and affect seminal plasma composition as demonstrated in sheep [12]. Interestingly semen may be recovered from valuable males post mortem via flushing of cauda epididymis, allowing recovery of highly concentrate of semen. Depending on high variability among mammalian species for the role that cauda epididymis plays in maturation and storage of sperm [42], this methodology may allow to recover between 1 to 30 ejaculates equivalent. This method may be particularly advantageous in preventing genetic loss of valuable adult males. Seasonal component is also important to consider and semen recovery must be of course recovered during the breeding season of the species [47]. In our opinion, successful semen cryopreservation could be obtained with slow freezing for all mammals using empiric approaches for assessment of extenders, cryoprotectant (CPA) toxicities, efficiencies and evaluation of the intrinsic permeability of sperm membrane. However, efficiency of cryopreservation varies highly among species, individuals within species, and even between ejaculates from individuals, which is largely attributed to the differences the biophysical characteristics among cell types or seminal plasma composition. The primary objective in sperm cryoconservation is preservation of fertility which may rely on both integrity of DNA, cellular machinery and organites including the acrosome, and preservation of motility. Good quality semen (ejaculated or epididymal) from exotic species was successfully cryopreserved using different extenders and methodologies mainly based on buffered egg yolk or milk media and using glycerol as cryoprotective agent [34-35]. Table 1 summarizes the different protocols generally employed for cryopreservation of semen from exotic or endangered species amongst different families. Interestingly, despite existing differences in membrane permeability or CPA sensitivity according to species, few variations are observed in methodology employed, thus resulting sometimes in poor transposition to target species. Ciência Animal, 22(1), 2012 Palestra apresentada no VI Congresso Norte Nordeste de Reprodução Animal, Fortaleza, CE, Brasil, 27 a 29 de junho de 2012. 109 Ciência Animal, 22(1): 106-123, 2012 – Edição Especial Table 1. Protocols employed for semen cryopreservation among different mammalian species. Family Extender 1 Extender 2 References species cervidae Cervus elaphus Citrate- fructose-egg Citrate - fructose - egg [44]. yolk (20%) yolk (20%) glycerol (4% final) Tris – glucose – citrate – / [6] egg yolk – glycerol 4% final Cervus ssp. Lactose - egg yolk Hyper-osmotic skim [47] (20%) milk -glycerol (4% final) Bovidae Capra Falconeri Necessity of washing of Skim milk - glycerol Locatelli, ejaculate (7% final) unpublished Skim Milk Oryx damah Lactose – TEA - glucose Lactose – TEA – [62] - egg yolk (20%) glucose - egg yolk (20%)-glycerol 5% final Camelidae Lama glama Tris – egg yolk (10%) Tris – egg yolk (10%) [11] – glycerol 7% Rhinocerotidae Rhinoceros unicornis Lactose - egg yolk - / [32,67] glycerol TEST-egg yolk extender, 6.25% Me2SO Felidae Acinonyx jubatus Tes - tris – glucose - egg Tes