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Regular Article |
b Dipartimento di Strutture, Funzioni e Patologie degli Animali e Biotecnologie, Università di Teramo, 64100 Teramo, Italy
c Department of Animal Reproduction, University of Agriculture, 30150 Krakòw, Poland
d Department of Gene Expression and Development, Roslin Institute, Edinburgh, United Kingdom
e Centro Recupero Fauna Selvatica, Azienda Foreste Demaniali, Sassari, Italy
f Istituto Zootecnico e Caseario per la Sardegna, 07040 Olmedo, Sassari, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IVF/ART, ovum pick-up, pregnancy, stress
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One species classified as vulnerable (i.e., facing a high risk of extinction in the wild in the medium-term future) is the autochthonous population of European mouflon, which inhabit only the islands of Sardinia and Corsica [2]. In Sardinia, the expansion of sheep farming has eroded the available habitat of the European mouflon, resulting in a reduction in their numbers and their dispersal into small, isolated groups. Such developments threaten the survival of the European mouflon as a population's probability of extinction is directly correlated with its variability and inversely correlated with density and immigration rate [3]. It is generally accepted that without conservation efforts, it is very probable that this wild sheep population will continue to decline and may very soon be lost [2]. Whereas the outright protection of certain areas of occupancy of Ovis orientalis musimon may still be the best option, in most areas this is not feasible due to the presence of pasture land and human settlements. In such instances of substantially reduced populations, restoring biodiversity can be achieved either by traditional approaches such as translocation, captive breeding, or both; or by the modern alternative of applying an integrated package of reproductive biotechnologies. The traditional approaches suffer from a number of limitations; animals may die during transport, the survivors may not integrate into the breeding population, and introduced animals may disrupt current inhabitants or even threaten the population's health [4, 5]. In reality, few traditional breeding programs have met the ultimate goal of safeguarding targeted species [6]. On the other hand, a number of advantages of modern reproductive biotechnologies have already been demonstrated in domestic animals [7]. These include extending the reproductive potential of individuals (by hormonally stimulating multiple follicle development and by semen collection), preserving genetic diversity by cryopreservation of gametes and embryos, and unlimited outcrossing by the use of in vitro fertilization. Despite these advantages, no complete program of genetic management by reproductive biotechnologies has yet been undertaken for any endangered mammalian species [8].
We present for the first time the successful employment of an integrated package of reproductive biotechnologies to preserve an endangered species, the wild European mouflon. As the donor resources available for different species will vary in practice, our goal is to devise systems for generating high embryo and, ultimately, offspring yields using oocytes derived from different types of donors. Thus, oocytes were collected from animals of various ages, including both prepubertal and recently deceased females. These techniques, coupled with storage of germ plasm and embryos, represent important conservation measures for the maintenance of genetic diversity.
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MATERIALS AND METHODS |
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All animal experiments were performed in accordance with DPR 27/1/1992 (the Animal Protection Regulations of Italy) which conform to European Community regulation 86/609, and which adhere to guidelines established in the Guide for Care and Use of Laboratory Animals as adopted and promulgated by the Society for the Study of Reproduction. In order to establish permissive circumstances for oocyte recovery, a number of variables such as age, hormonal status, and sedation were examined. Oocytes were aspirated from female mouflons maintained either permanently or on a temporary basis at the Wildlife Rescue Centre in Sardinia. In certain females, recovery of immature oocytes was performed directly, whereas others were subjected to hormonal priming during the nonbreeding season (April–June 2000) in order to increase the number and size of ovarian follicles. Mouflon females were synchronized by insertion of Norgestomet s.c. implants (Crestar, Intervet, Boxmeer, Holland) for 12 days. Follicular growth was primed with 4.8 mg of ovine FSH (Ovagen, ICP, Auckland, New Zealand) given in 6 equal doses every 12 h on the 10th, 11th, and 12th days of implant insertion [9]. Animals were anesthetized using a combination of acepromazine maleate and pentothal sodium on the day following final hormone injection, and ovaries were exposed by midventral laparotomy. For oocyte recovery, all visible follicles were aspirated using a 5-ml syringe fitted with a 20-gauge needle. Tissue culture medium (TCM)-199 supplemented with 5% calf serum, 0.05 mg/ml heparin, and 0.05 mg/ml gentamycin sulfate was used for oocyte collection. Oocytes from individual donors were maintained separately during all in vitro procedures. Particular care was taken to avoid postoperative adhesions by carefully washing reproductive organs with saline and subsequently coating ovaries with hydrocortisone acetate ointment (Cortison Chemicetina, Pharmacia & Upjohn, Milan, Italy). To reduce handling stress, 19 mg flufenacine decanoate (Moditen Depot, Squibb, Hertfordshire, U.K.) was administered before treatment.
In Vitro Maturation, Fertilization, and Culture
Methods of in vitro embryo production were adapted from those previously described for domestic sheep [9]. Maturation medium was bicarbonate-buffered TCM-199 with an osmolarity of 275 mOsm and a glutamine concentration of 2 mM. This medium also contained 10% fetal bovine serum (FBS), 5 µg/ml FSH (Ovagen) 5 µg/ml LH, 1 µg/ml estradiol, 0.3 mM sodium pyruvate, and 100 µM cysteamine. Oocytes were incubated in 0.4 ml of medium in 4-well dishes (Nunc, Nunclon, Denmark) under mineral oil. In vitro maturation was carried out at 39°C for 24 h in a humidified atmosphere with 5% CO2. Following maturation, oocytes were partially denuded by a short incubation in Hepes-TCM-199 containing 300 IU/ml hyaluronidase and by gentle pipetting. Pools of cryopreserved semen from 4 mouflon rams were used for all in vitro fertilization (IVF) procedures. Motile spermatozoa were selected by a 1-h swim-up procedure and incubated (1 x 106 spermatozoa/ml) with oocytes in bicarbonate-buffered synthetic oviduct fluid (SOF) containing 20% (v/v) heat-inactivated estrous sheep serum, 2.9 mM calcium lactate, and 16 µM isoproterenol at 39°C with 5% CO2 in humidified air for 20–22 h. Presumptive zygotes were incubated in 20-µl drops of SOF supplemented with 2% (v/v) basal medium Eagle-essential amino acids, 1% (v/v) modified Eagle medium-nonessential amino acids, 1 mM glutamine, and 8 mg/ml fatty acid-free BSA at 39°C in an atmosphere of 5% CO2, 7% O2, 88% N2, and maximum humidity. On the 3rd and 5th days of culture (Day 0 equals the day of fertilization), 5% charcoal stripped-FBS was added to the medium. Culture was continued for 7 or 8 days following fertilization, and embryos that had developed to the blastocyst stage were either cryopreserved or immediately transferred to synchronized recipients. All chemicals, unless other indicated, were obtained from Sigma Chemical Company (St. Louis, MO).
Cryopreservation Protocols
All semen used in this study was collected during the breeding season (August–October). Ejaculates were diluted in medium containing Tris, citric acid, and fructose at 30°C, cooled over 2 h to 4°C, and then diluted to a final concentration of 2 x 108 spermatozoa/ml with Tris-egg yolk and glycerol (3.2% v/v). Treated semen was transferred to 0.25-ml straws (IMV, L'Aigle, France), cooled at -70°C for 5 min, and immersed in liquid nitrogen.
Methods for cryoconservation of embryos were adapted from previously described procedures [10]. Dulbecco PBS supplemented with 0.3 mM sodium pyruvate and 20% FBS was used as the basic vitrification solution. Blastocysts were processed for vitrification at room temperature under the following regime: 10% glycerol for 5 min followed by 10% glycerol + 20% ethylene glycol for 5 min. Embryos were then transferred to 25% glycerol + 25% ethylene glycol and loaded into the center of 0.25-ml straws separated by air bubbles from 2 columns of 0.5 M sucrose solution, and immediately immersed in liquid nitrogen.
Granulosa cells were collected into TCM-199 plus 20% FBS at 4°C and then transferred into medium containing 7% dimethyl sulfoxide. Cells were aliquoted into 200-µl lots and stored overnight at -20°C before immersion in liquid nitrogen.
Transfer to Recipients
Blastocysts were surgically transferred to recipient ewes 7 days after the onset of natural estrus. Pregnancies were confirmed by ultrasonography (Aloka, 7.5 MHz high resolution linear probe) and allowed to continue to term.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although initial studies using 6 hormonally primed animals revealed that the majority of females produced very limited (1–4) numbers of follicles, one animal in particular showed an excellent follicular response in both ovum pick-up sessions (14 and 12 oocytes recovered at first and second sessions, respectively). This animal was the only one of the group tested to have been raised at the Wildlife Rescue Centre and, as a result, had adjusted to regular handling. The remaining animals arrived at the rescue center as wild adults, and we assumed that the repeated capturing, which was necessary in order to administer hormones and collect oocytes, induced stresses that affected their reproductive performance. To reduce the inevitable stress that results from handling wild animals, we elected to administer a long-lasting tranquilizer. The effective flufenacine decanoate dosage for mouflon was established by using domestic sheep of an autochthonous breed (Sarda) with a similar body weight. Following a second gonadotropin stimulation administered under tranquilizer, follicle numbers increased by an average of 2 per animal compared with the initial studies performed without sedatives.
Generally, follicles from stimulated females reached a size approximately 2–3 mm larger than those of nonstimulated females, although considerable variation in follicle numbers was observed between donors (range of variability, 1–8 and 1–15 follicles per nonstimulated and stimulated donor, respectively). The majority of animals used were of fertile age, although one female was at the end of her reproductive life and, as a result, only a single oocyte was recovered. We have previously demonstrated [11] that some oocytes collected from animals as young as a 4-wk-old prepubertal domestic sheep are fully capable of reproducing, and thus, here we stimulated a 7-mo-old prepubertal mouflon. After an initial gonadotropin stimulation, 12 oocytes were recovered, whereas a second stimulation yielded a further 11 oocytes. This number of oocytes recovered represents more than twice the average recovery (5.7 oocytes per session) from an adult stimulated donor.
During oocyte recovery, particular care was taken to avoid damage to reproductive organs (see Materials and Methods), both to allow future natural reproduction, and also to allow repeat oocyte collections. The treatment as applied here had previously been demonstrated to be effective for repeat oocyte recovery in domestic sheep (Ovis aries) [12]. In our study, from 5 nonstimulated mouflons and from 8 twice-stimulated mouflons, respectively, 4 and 6 resulted in pregnancy, or they gave birth following natural mating. Three animals were not pregnant; 1 died due to chronic pneumonia, whereas the probable cause of fertility disorders of 2 other females was their advanced age.
Genome Resource Bank
All semen samples used in this study were obtained from 4 mouflons, 2 of which were only months old, and 2 which were captured as adults (approximately 8 yr and 5 yr of age). After a short period of exposure to domestic ewes in natural estrus, it was possible to obtain ejaculates using an artificial vagina. Semen from 3 of these mouflons was stored frozen for up to 10 yr, and for 1 yr from the fourth animal. In order to determine the developmental capacity of stored semen, in vitro-matured oocytes from domestic sheep were fertilized in vitro by frozen-thawed mouflon sperm. The proportion of sheep-mouflon embryos that developed in vitro was similar for all males and was also similar to the proportion of domestic sheep embryos routinely produced in vitro in our laboratory (34% mouflon-sheep developed to the blastocyst stage vs. 38% of control sheep). A number of these embryos were transferred to synchronized domestic sheep recipients, and healthy cross mouflon-sheep offspring were delivered.
Twenty-three of the in vitro-produced mouflon embryos were cryopreserved using routine procedures developed for domestic sheep embryos.
During in vitro maturation procedures, granulosa cells representing 17 mouflon genotypes were collected and cryopreserved for eventual future cloning using domestic sheep ooplasts.
In Vitro Embryo Production
The success of preliminary experiments with in vitro development of cross mouflon-sheep embryos prompted attempts at the in vitro production of pure mouflon embryos. With the exception of a small number of oocytes, all mouflon oocytes were cultured without further selection (Fig. 1, A and B). Semen from 4 previously tested mouflon rams was used for all in vitro fertilizations of mouflon oocytes (Fig. 1C), and more than 70% of embryos had cleaved by 48 h postinsemination (Table 1). There were no significant differences between nonstimulated, stimulated, and death donors in cleavage (67% vs. 71% vs. 82%) nor blastocyst rate (33% vs. 37% vs. 36%). Optimal embryo development (37% to blastocysts) was obtained with oocytes derived from groups of hormonally primed females, and there were no significant differences between the proportion of blastocysts derived from stimulated donors on 2 separate occasions (13 of 39 and 21 of 53; 33% and 40%). The most successful donor (Fig. 2A) produced 7 and 5 blastocysts from the first and second experiments, respectively (mean, 46% blastocyst; Fig. 1D). Also, a significant proportion of the oocytes derived from prepubertal mouflon donors developed to the blastocyst stage (mean, 8 of 23, 35%).
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Interspecies Pregnancy Progression and Development of Newborn Mouflons
All foster mothers used in this study were domestic sheep (Ovis aries). Our initial studies led to the successful delivery of a number of mouflon-sheep crosses and, as a result, attempts were made to transfer pure mouflon blastocysts to domestic sheep recipients. In total, 10 pairs of embryos were transferred 7 days after the onset of natural estrus. The proportion of interspecies pregnancies as determined by ultrasonography was 70% at 40 days and 50% at 80 days following transfer, and 3 of the recipients delivered a total of 4 normal healthy mouflons (Fig. 2B). The birth weight of all newborn animals was within the normal range for this species (the singleton male weighed 3.8 kg, the female weighed 3.5 kg, and the twins weighed 2.4 kg [male] and 2.5 kg [female]). In a further 2 ongoing pregnancies the recipients are carrying embryos derived from oocytes collected postmortem.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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With cryopreservation of the appropriate genetic resources (gametes, embryos, somatic cells), the potential exists to maintain the genetic diversity of small populations at risk of inbreeding, such as those found in zoos [20]. Small populations can rapidly lose genetic diversity and cryopreservation allows individuals to contribute even after death, effectively extending generation length [21]. By preserving gametes, embryos, or somatic cells in liquid nitrogen, the individuals contributing the germ plasm or entire DNA can continue to provide genetic material.
The pregnancy rate reported here is similar to that seen with domestic sheep monospecies pregnancies with in vitro-produced embryos [9]. This pregnancy rate is also similar to that reported for other interspecies pregnancies in the Bovidae family, such as when gaur embryos are transferred into the domestic cow [16], although in the latter study, all offspring died within 1 wk of birth. A separate study [22] reported the transfer of in vitro-produced red sheep embryos into mouflon and domestic sheep recipients. No pregnancies resulted in the mouflon recipients, whereas a pregnancy rate of 38.5% was reported in the domestic sheep. The relevance of this study in terms of interspecies transfer is unclear because the red sheep donors are considered to be hybrids of Armenian mouflon and urial sheep (Ovis orientalis gmelini x Ovis vignei arkal) [3], and the mouflon recipients were not detailed taxonomically.
The success of interspecies embryo transfer depends upon selection of the appropriate recipient, generally on the basis on similarities in reproductive characteristics. If it has previously been demonstrated that the 2 species selected for interspecies embryo transfer are capable of generating hybrids, then the chances of a successful gestation are increased [6]. When unsuccessful, reasons cited for gestation failure include immunological rejection by the recipient female, the formation of inadequate placentae, or both (for review see [6, 8, 23]).
Recent attempts to reproduce North American bison (Bison bison) confirm the considerable degree of stress induced by the restraint and handling of wild animals [24], and stress-induced disruption of gonadotropin secretion has been widely documented in a related species, the domestic sheep [25, 26]. The effects of repeated capture have also been studied in mouflon [27]. Following tranquilizer administration, animals in our study improved their follicular response; furthermore, the handling of mouflons became more efficient and safe.
The aim of conservation, according to a recent symposium on the Conservation of Wild Living Resources [28], should be to maintain biological diversity at the ecosystem, population, species, and genetic levels. Although the ultimate goal is to establish self-sustaining wild populations within the appropriate ecosystems, in many instances the depth of resources available will be insufficient, and a primary aim should be the preservation of genetic variability in small, threatened populations. For this purpose, modern reproductive biotechnologies theoretically confer many advantages over more traditional approaches, although until now they have not proven sufficiently robust for routine use. Many attempts to conserve species will be carried out under less than ideal circumstances, and here we demonstrated that reproductive biotechnologies can be used to preserve endangered species not only outside the breeding season, but also in extreme circumstances in which only prepubertal or postmortem material is available. Other aspects of this approach, collection of both haploid and diploid genotypes and cryopreservation, contribute to the maintenance of genetic biodiversity even after the death of the germinal and somatic cell donors [29].
Here we have used the wild European mouflon to demonstrate for the first time the potential of using an integrated package of modern reproductive procedures to rescue a threatened species. We have shown that substantial yields of oocytes, embryos, and pregnancies can be achieved independent of age, breeding season, and donor treatment, and suggest that these procedures can be applied to preserve other species, or at the very least, the 30 species and subspecies of Ovis included in the Red List of Threatened Animals [1].
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Correspondence: Grazyna Ptak, Dipartimento di Strutture, Funzioni e Patologie degli Animali e Biotecnologie, Università di Teramo, Piazza Aldo Moro no. 45, 64100 Teramo, Italy. FAX: 39 0861 411285; gptak{at}tiscalinet.it 
Accepted: October 30, 2001.
Received: July 2, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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