Int. J. Morphol., 24(2):285-292, 2006. Obtaiment of Pudu (Pudu pudu) deer Embryos by the Somatic Nuclear Transfer Technique Obtención de Embriones de Pudú (Pudu pudu) Mediante Técnicas de Transferencia Nuclear Somática *Felipe Venegas; **Michel Guillomot; **Xavier Vignon; **Jean-Luc Servely; **Chistophe Audouard; *Enrique Montiel; **Daniel Le Bourhis; **Sandrine Perón; ***Pilar Soto & *Mariana Rojas VENEGAS, F.; GUILLOMOT, M.; VIGNON, X.; SERVELY, J-L-; AUDOUARD, C.; MONTIEL, E.; LE BOURHIS, D.; PERÓN, S.; SOTO, P. & ROJAS, M. Obtaiment of Pudu pudu deer embryos by the somatic nuclear transfer technique. Int. J. Morphol., 24(2):285-292, 2006. SUMMARY: The objective of this project is to produce pudu embryos, obtained by means of fibroblast transfer taken from the ear of one animal, to the oocytes of a domestic ruminant such as cattle. In further work, the pudu embryos will be transferred to the uterus of synchronized recipient females of another species. Biopsies 1mm in size were obtained from the outer rim of the ear of two male pudu deer belonging to the Buin Zoological park, Santiago, Chile. The cell lines have been established and kept according to protocols used in cattle. The oocytes are obtained through a puncture in the oocyte-cumulus complex (OCC) from cow ovaries recovered from the butcher. Each oocyte is enucleated and fused with an isolated fibroblast which is inserted beneath the pellucid zone. The fusion of cell membranes is achieved by means of electric shocks. In respect to the timetable, we have observed that a stage of two blastomers is reached on the second day, and morulae of 8 to 16 cells on the third day. On the fourth day it has differenciated as a blastocyst and on the seventh day it finally hacht from the pellucid zone. The obtainment of embryonic blastocytes indicates that it is possible to obtain pudu embryos through heterospecific cloning, even though the percentage of success is relatively low. The viability of the embryos obtained in this manner after in uterus transfer remains still to be verified. KEY WORDS: Pudu deer; Cervidae; Ruminant; Cloning. INTRODUCTION The pudu (Pudu pudu) is the smallest of South rate caused by genetic illnesses (Codeff, 1991 and Mella et American deer species; it reaches no more than 40 cm at the al., 2002). withers and weights less than 15 kg (Blumberg, 2002). It is a deer native to Chile and Argentine and can be found from The Pudu is a ruminant belonging to the Order sea level up to 1700 meters of altitude. In Chile it is distributed Artiodactyle, Family Odocoileinae, which incluyes the from 35º10'S (VII Region) to 46º45'S (XI Region). This genders Hippocamelus and Pudu. In turn, two species belong species has been considered endangered in accordance with to the gender Pudu: Pudu mephistophiles, which inhabits Perú the Red Book from the World Union for Nature (U.I.C.N.) and Colombia, and the southern pudu or Pudu pudu native of and the appendix 1 of the CITES (Glade, 1993). Chile and Argentine. The pudu has 70 chromosomes (Spotorno & Fernández-Donoso, 1975 and Spotorno & Walker, 2000). This species has suffered from the gradual destruction of its habitat, poaching and chasing by feral dogs. At present Activity patterns of the pudu in its natural habitat have the population can be found forming small isolated groups been studied (Wetterberg, 1972; Eldridge et al., 1987), as well that cannot reproduce between them. As a consequence, the as its behavior in captivity (Macnamara & Eldridge, 1987 increased in-breeding has resulted in an increase in the death and Cortes et al., 1988). Studies have also been carried out in * Laboratory of Compared Embryology; ICBM; Department of Anatomy and Developmental Biology; Medical School, University of Chile. ** Institut National de la Recherche Agronomique; Jouy en Josas; Francia. *** Buin-Zoological Park, Santiago - Chile. Grant ECOS-CONICYT C03 B02 285 VENEGAS, F.; GUILLOMOT, M.; VIGNON, X.; SERVELY, J-L-; AUDOUARD, C.; MONTIEL, E.; LE BOURHIS, D.; PERÓN, S.; SOTO, P. & ROJAS, M. order to establish the relationship between LH and testosterone species at risk of extinction. These techniques include Artifi- levels to the development of the antlers in the male deer (Re- cial Insemination, Embryonic Transplant, In Vitro Fertilization yes et al., 1993). and the intracytoplasmic injection of sperm cells (Comizzoli et al., 2000). At present, assisted reproduction technique is The seasonal changes of the pudu testicles are also being fine-tunned in deer species. The “Proyecto Arca” in well known, as well as the variations of cortisol, IGF-1 and Argentina is an example. Their objective is to return the “father triodothyronine levels in males (Reyes et al., 1997). Other David’s deer” (Elaphurus davidianus) to it’s country of origin, endocrine studies in adult males pudu, concerning the levels not only because this species has become extinct in China, of FSH, LH, testosterone and prolactine in adult males have but because of the necessity of the development of a been published (Bubenik et al., 2000). Bartos et al. (1998) methodological work applicable to native deer. studied the hormonal levels of two males housed together with females, in order to assess dominance characteristics. By other Some zoos have successfully managed to keep pudus hand, Hershkovitz (1982), reported that pudu have a in captivity, as opposed to other south american endangered gestational period of 7 months (between 202 to 223 days). cervids such as the Taruca (Hippocamelus antisense) and the southern Huemul (Hippocamelus bisulcus), both of which The publication by Schurer & Sliwa (2002) provides species do not survive in captivity (Rojas et al., 2004, 2005). much more information: the average length of gestation Hick (1969) reported the unsuccessful raising of a pudu at observed in this study was 210 days; the average weight at Cologne Zoo. Pudus are currently kept at Doué-la-Fontaine birth was 890 g (55 newborn animals were assessed), the zoo in France and in the Wild Animal Park of the Zoological female usually being as heavy as the males. Offspring below Society of San Diego, USA, where reproduction of the 600 g did not survive, nor did those weighing more than 1000 shouther huemul has been achieved. (Hershkovitz). g Guzmán et al. (1986), described some characteristics of birth: the female gives birth to a single foal of reddish tabby In South American zoos, banks with of pudu sperm color, with whitish stripes along the back and thighs. These are kept in order to aid artificial insemination of this species stripes disappear at the age of three months. (Santiago Metropolitan Zoo, in Chile and also in Buenos Ai- res Zoo, Argentina). Diverse activities have been carried out The pudu placenta, like that of other deers is in parallel to generate an increase in the reproduction rate in oligocotyledonary, villous, and epithelio-chorial. The placenta other vulnerable and endangered deer all over the world. had only one intact cotyledon and a second atrophying Nevertheless, classic techniques of embryo production have cotyledon. The larger cotyledon observed measured 8 x 1 cm. proved to be difficult. From this point of view, Locatelly & The placenta weighed 110 g. There is intensive interdigitation Mermillod (2005) stated that reproductive biotechnology and of villi with the caruncular epithelial folds. Thus, an intimate assisted reproduction methods in endangered cervids could contact between trophoblast and endometrial epithelium is help conservation programs. established. The caruncles are composed of stroma and blood vessels, covered by endometrial epithelium. There are no Restoration of species by transfer of somatic nuclei into glands (Harrison & Hamilton, 1952; Hamilton et al., 1960; enucleated recipient oocytes has already been considered Sadlier, 1987 and Drion et al., 2003) (Moratelli et al., 2002). Calves of an endangered breed of cattle (Enderby Island) adapted to extreme climatic conditions were Although natural reproduction should be the preferred born after nuclear transfer of granulose cells into enucleated method in order to increase the population of a certain species, oocytes from domestic cows and the ulterior transfer of the when wild populations are decreasing in number this is not resulting embryos into domestic cow recipients (Wells et al., longer possible. The pudu reproduction program developed 1998, 2000). Additionally, a pregnancy was observed after the by Reyes (1991) has been a contribution to the country but transfer of embryos reconstructed from cells of argali (Ovis has not been able to improve the situation. Reproduction ammon) and enucleated oocytes of domestic sheep (White et technology could be an important support by establishing al., 1999). In the giant panda, blastocysts have also been obtained genetic reserve banks of endangered animals in risk of after nuclear transfer of panda cells into rabbit enucleated extinction (Holt et al., 1996; Holt & Pickard, 1999; Watson oocytes (Chen, 1999). Six day embryos of the Saola deer & Holt, 2001 and Holt et al., 2002) and for the conservation (Pseudoryx Nghetinhensis) have also been obtained by means of wild animals. of the cloning method and are currently kept frozen, waiting to be implanted on to a surrogate mother (Service Presse INRA). It is thought that reproduction of wild species could be aided by the use of stored genetic material (Wildt et al., Furthermore, it has been demonstrated that bovine 1997). from zoological parks and universities, specially those oocyte cytoplasm could also be used as recipient for somatic 286 Obtaiment of Pudu pudu deer embryos by the somatic nuclear transfer technique. Int. J. Morphol., 24(2):285-292, 2006. cells from different mammalian species (Dominko et al., Once in the laboratory, the sample was shaved and rinsed to 1999). Cloning might even serve a useful purpose with species remove the loosen hairs. The sample was then placed on a that have never bred in captivity, such as the saola (Comizzoli culture dish were micro fragments of tissue were cut and et al., 2000) y el huemul (Rojas et al., 2005).