230 in Vitro Maturation of Oocytes From Endangered Dorcas Gazelle (Gazella Dorcas Neglecta)

Abstract

In vitro maturation of oocytes recovered from dead animals provides an opportunity for rescuing genetic material for biodiversity conservation. The dorcas gazelle (Gazella dorcas) is regarded by the World Conservation Union (IUCN) as ‘vulnerable’ but the subspecies G. dorcas neglecta is thought to be endangered due to excessive hunting. A captive breeding program for dorcas gazelles has been developed at the Estacion Experimental de Zonas Aridas (CSIC) in the South of Spain where efforts have so far concentrated on natural breeding and on the development of sperm cryopreservation protocols. The aim of the present study was to explore the possibility of recovering and maturing in vitro healthy oocytes from animals that die suddenly for the establishment of a program to rescue female gametes. Ovaries of a dorcas female that died unexpectedly were collected about 7 h after death of the animal. Cumulus–oocyte complexes (COCs) were recovered by slicing the ovaries. Collection and washing of COCs were performed in warmed TCM-199-HEPES with antibiotics and polyvinyl alcohol. Degenerated oocytes or those with expanded cumulus cells were removed. A total of 15 COCs were cultured in TCM-199 with 10% heat-treated fetal calf serum, 10 μg/mL ovine FSH/LH, 1 µg/mL estradiol, and 0.1 mg/mL glutamine at 38.5°C under 5% CO2/air with high humidity. After 24 h of culture, matured oocytes, as revealed by the presence of a polar body, were activated with 7% ethanol for 10 min and further incubation for 3 h. Meiotic progression and activation were evaluated by staining with Hoechst 33342 and propidium iodide (1 μg/mL each) and visualization under a fluorescence microscope. Results at the end of incubations showed that 4/15 oocytes were degenerated, 4/15 were arrested at the MI stage, and 7/15 (46.7%) progressed to the MII stage. One oocyte was found to be at the 2-cell stage but it could not be established whether this was the result of the activation method used. These results demonstrate that it is possible to recover viable oocytes several hours after death and rescue them for subsequent in vitro maturation and fertilization. More studies are needed to characterize suitable conditions for oocyte maturation, fertilization, and culture in the dorcas gazelle. This would, in turn, help in the effort to rescue biomaterials from wildlife for generating offspring. This work was supported by the Spanish Ministry of Education and Science (REN 2003–01587) and Acciones Integradas (HI20030336).

In Vitro Maturation and Fertilization in the Nilgai (Boselaphus tragocamelus) using Oocytes and Spermatozoa Recovered Post‐mortem from Animals that had Died because of Foot and Mouth Disease Outbreak

“…In the present study, the average number of oocytes recovered per ovary was 36.9; of which, 22.9 were culture grade oocytes (fair and good quality). This number is higher than that observed in other antelope species such as kudu, impala, blesbok, blackbuck, springbok and black wildebeest (Loskutoff et al 1995;Winger et al 1997;Roldan et al 2005;Krisher et al 2006;Rao et al 2010). The high oocyte recovery in the present study may be because oocytes were recovered during the peak of breeding season (December) (Leslie 2008) when the ovarian follicular activity is at its highest level (Manjunatha et al 2009).…”

“…2004), blesbok (39%) (Brad et al. 2004), dorcas gazelle (47%) (Roldan et al. 2005) and black wildebeest (52%) (Brad et al.…”

“…Oocyte maturation in tissue culture medium 199 (TCM-199) supplemented with foetal calf serum and gonadotropins was used previously for IVM of oocytes of antelopes, such as the kudu and impala (Loskutoff et al 1995), black buck (Keller et al 1999;Rao et al 2010), blesbok (Winger et al 1997;Brad et al 2004), addax antelope (Hall-Woods et al 1999), black wildebeest (Brad et al 2004), dorcas gazelle (Roldan et al 2005), springbok (Brad et al 2004;Krisher et al 2006), eland and bongo (Wirtu et al 2009), and in cattle (Dominko and First 1997), approximately 80% of the immature oocytes reached the MII stage of the second meiotic division. In the present study, the proportion of oocytes (63%) that reached the MII stage was similar to that of the eland antelope (63%) (Wirtu et al 2009), higher than the proportions observed in other antelopes, such as the springbok (35%) (Brad et al 2004), blesbok (39%) (Brad et al 2004), dorcas gazelle (47%) (Roldan et al 2005) and black wildebeest (52%) (Brad et al 2004). However, it was lower than that observed in blackbuck (67%) (Rao et al 2010) and bongo antelope (81%) (Wirtu et al 2009).…”

“…One of the major problems in the implementation of ART in conservation programs is the lack of availability of biological material required for a better understanding of the fundamental biology of the male and female gametes (Andrabi and Maxwell 2007). It is in this context that post-mortem recovery of gametes from endangered animals following unexpected death could serve as a valuable source to understand the physiology of the species and to develop species-specific protocols for the production of competent oocytes (Loskutoff et al 1995;Hall-Woods et al 1999;Roldan et al 2005;Rao et al 2010), for in vitro fertilization (Raphael et al 1991;Loskutoff et al 1995;Keller et al 1999;Herrick et al 2004) and for somatic cell nuclear transfer technology (SCNT) to conserve endangered animals.…”

“…A few attempts have been made to explore the possibility of recovery, in vitro maturation (IVM) and IVF of oocytes collected from the ovaries of antelopes either from supervised culls or after their death as in kudu and impala (Loskutoff et al 1995), blesbok (Winger et al 1997;Brad et al 2004), addax antelope (Hall-Woods et al 1999), blackbuck (Keller et al 1999;Rao et al 2010), black wildebeest (Brad et al 2004), dorcas gazelle (Roldan et al 2005) and springbok (Brad et al 2004;Herrick et al 2004;Krisher et al 2006). In this study, oocyte recovery, IVM and IVF of oocytes collected post-mortem from the ovaries of nilgai were undertaken as a model for other endangered or threatened antelope species.…”