A review on reproductive biotechnologies for conservation of endangered mammalian species

This review describes the use of modern reproductive biotechnologies or assisted reproductive techniques (ART) including artificial insemination, embryo transfer/sexing, in vitro fertilization, gamete/embryo micromanipulation, semen sexing, genome resource banking, and somatic cell nuclear transfer (cloning) in conservation programs for endangered mammalian species. Such biotechnologies allow more offspring to be obtained from selected parents to ensure genetic diversity and may reduce the interval between generations. However, the application of reproductive biotechnologies for endangered free-living mammals is rarer than for endangered domestic breeds. Progress in ART for non-domestic species will continue at a slow pace due to limited resources, but also because the management and conservation of endangered species is biologically quite complex. In practice, current reproductive biotechnologies are species-specific or inefficient for many endangered animals because of insufficient knowledge on basic reproduction like estrous cycle, seasonality, structural anatomy, gamete physiology and site for semen deposition or embryo transfer of non-domestic species.     

Reproductive potential of domestic Ovis aries for preservation of threatened Ovis orientalis isphahanica: in vitro and in vivo studies

Although the potential use of reproductive biotechnology for safeguarding of endangered wildlife species is undoubted, initial evaluation of the genetic and reproductive relationship between the endangered mammals and those closely related species is indispensable. Isfahan mouflon Ovis orientalis isphahanica is now considered as a threatened species by International Union for the Conservation of Nature. Therefore, little is known about the biology of this species. This study was carried out to investigate the possible reproductive potential of domestic sheep for ex situ conservation of the Isfahan mouflon. Somatic cell cultures were taken from ear biopsies of the wild and domestic sheep and were used for karyotype analysis. Semen samples were collected by electroejaculator from the wild and domestic rams. The spermatological characteristics of the collected semen samples were determined and used for both cryopreservation and cross-insemination of the synchronized wild and domestic female sheep. To establish a cryobank for the threatened species biomaterials, freezed samples of the somatic cells and semen were transferred to a cryotank. The result suggested that Isfahan mouflon has conserved its chromosomal integrity as previously observed and contains the same chromosomal number as the domestic sheep (2n = 54). The semen samples of both species revealed similar cryoviability (>35% gross motility postthawing). Cross-insemination of both species resulted in successful pregnancy. It was suggested that domestic sheep possesses the required biological characteristics to be considered for safeguarding of the Isfahan mouflon.     

In vivo and in vitro fertilizing capacity of cryopreserved European mouflon [Ovis gmelini musimon] spermatozoa used to restore genetically rare and isolated populations

European mouflon sheep are an endangered species of ovidae residing primarily in the mountenous habitat of the islands of Sardinia and Corsica. The purpose of this study was to assess the fertilizing capacity of cryopreserved European mouflon spermatozoa after AI in synchronized mouflon and domestic ewes and after IVF in in vitro matured mouflon and domestic ewe oocytes collected by OPU technique. Domestic ram (Ovis aries) spermatozoa served as control. Semen was collected by artificial vagina from three mouflons and three domestic rams during the breeding season and was cryopreserved. At thawing, no significant differences in sperm viability were found between the wild and the domestic species (53.1 +/- 4.6% versus 56.0 +/- 4.7%) whereas the percentage of acrosome-intact sperm was lower in mouflon (55.5 +/- 4.6%) than in ram semen (62.7 +/- 3.1%; P < 0.05). Lambing rate did not differ between synchronized mouflon and domestic ewes (5/11 versus 8/12) after 150 and 156 days of pregnancy, respectively. After two OPU sessions, 87 and 132 oocytes were collected from three hyperstimulated mouflon and three domestic ewes. Cryopreserved/thawed semen was inseminated with an endoscope into the uterus of corresponding species during the non-breeding season. The oocytes were matured and fertilized in vitro; 61/73 mouflon and 81/101 domestic ewe oocytes were found to be fertilized. From these, we obtained 6/61 and 17/81 blastocysts. After vitrification and thawing, the hatching rate showed no significant difference between mouflon and sheep blastocysts (4/6 versus 14/17). In conclusion, our data showed that cryopreserved mouflon spermatozoa can be successfully used to carry out a genuine and complete program of genetic restoration in small and isolated groups of European mouflons.     

Genetic rescue of an endangered mammal by cross-species nuclear transfer using post-mortem somatic cells

Since the advent of procedures for cloning animals, conservation biologists have proposed using this technology to preserve endangered mammals. Here we report the successful cloning of a wild endangered animal, Ovis orientalis musimon, using oocytes collected from a closely related, domesticated species, Ovis aries. We injected enucleated sheep oocytes with granulosa cells collected from two female mouflons found dead in the pasture. Blastocyst-stage cloned embryos transferred into sheep foster mothers established two pregnancies, one of which produced an apparently normal mouflon. Our findings support the use of cloning for the expansion of critically endangered populations.     

Surgical transfer of in vivo produced farmed European polecat (Mustela putorius) embryos

Surgical embryo transfer of farmed European polecat (Mustela putorius) was investigated as part of an ex situ preservation project. The long-term objective of the project is to develop effective technology for ex situ conservation of the European mink (Mustela lutreola), which is a highly endangered aboriginal European species. Twenty European polecat females, which served as a model species for the European mink, were humanely killed 4-9 days after first mating and embryos were recovered from oviducts and uteri. Donor-recipient pairs (n = 16) were generated by mating the donors (n = 20) once a day for 2 consecutive days with fertile males and by mating the corresponding recipients (n = 16) on the same days with vasectomized males. An embryo recovery rate of 70% (200 recovered embryos/284 corpora lutea) was achieved from 20 donors. Morulae and blastocysts were recovered between Days 5 and 9 after first mating and were regarded as the best developmental stages for uterine embryo transfer. A total of 172 embryos were transferred surgically under general anaesthesia into the ovarian third of the left uterine horn of 16 recipients with a thin glass capillary. Eleven recipients (69%) produced 72 pups equivalent to an average success rate of 42% (72 pups/172 transferred embryos). The average litter size was 4.5 (range 0-9). These results with this model species, farmed European polecat, demonstrate the potential of embryo transfer as an effective method for the preservation of the endangered European mink (M. lutreola). These species are closely related and have a similar reproductive physiology. However, success of applying embryo transfer in conserving European mink is still dependent on further studies both into its reproductive physiology and developing of improved flushing techniques for anaesthetized donors and the successful transfer of frozen-thawed embryos.     

Membrane integrity and fertilizing potential of cryopreserved spermatozoa in European mouflon

There is a pressing need to develop and use assisted reproductive techniques in wildlife species living in small and captive groups. We evaluated the effect of freezing on membrane integrity and fertilizing capacity of European mouflon (Ovis gmelini musimon) spermatozoa collected during the breeding season. After thawing, the percentage of live spermatozoa, stained with fluorescein isothiocynate labeled Pisum Sativum agglutinin and propidium iodide, was 47% of which 19% showed intact acrosomal membrane. After culture in TCM 199+10% FCS, the number of live spermatozoa was significantly (P<0.01) lower than in a medium with oviductal epithelial cells. The absence of oviductal cells decreased significantly the fertilization rates (P<0.05), 24.0 vs. 63.1 with oviductal epithelial cells and 59.1 in vivo of in vitro matured ovine oocytes. Polyspermic fertilization rate of oocytes was lower (P<0.05) with oviductal epithelial cells (1.6) than in absence of cells (12.8). However, the percentage of embryos that reached blastocyst stage was significantly higher in vivo than in vitro. These results provide interesting preliminary data for the development of genetic resource banks for European mouflon.     

The cryopreservation of ovarian tissue: uses and indications in veterinary medicine

Animal experiments have shown that cryopreservation of the ovarian cortex, containing primordial follicles, could be used to preserve gametes thereby restoring fertility in humans and animals. During the last 100 years, many hundreds of species have been lost, and a third of the breeding animals are threatened with extinction. To preserve genetic diversity, notably for the conservation of endangered species, it is essential to conserve female and male gametes. Today, biotechnologies such as artificial insemination and embryo transfer are used in breeding programs and are well developed. However, even using these advanced techniques, there are problems due to the limited number of individuals used as the source of gametes, so that the risk of inbreeding is high, even in large populations. To preserve genetic diversity, it is necessary to create gene banks of male and female gametes and embryos, using a very large number of individual donors. Cryopreservation of ovarian tissue could present a means for enlarging the gene pool. Cryopreserved ovarian tissue could be used in auto- or xenografts, or for in vitro maturation (IVM) of primordial follicles. In this review, we describe the processes for cryopreservation of ovarian tissue and the various possibilities for using it.     

Assisted reproductive technologies in the reproductive management of small ruminants

In modern agriculture, assisted reproductive technologies are being used for out of season oestrus induction, enhancement of reproductive performance and genetic improvement. In addition, they can have substantial contribution in preservation of endangered species or breeds, as well as in eradication programs of various diseases. While their applications are widespread in cattle, in small ruminants it is almost restricted to artificial insemination. The main limitations of a wider application in small ruminants are the naturally occurring anoestrus period, the variability of response to superovulatory treatments, the fertilisation failure and the need of surgery for collection and transfer of gametes and embryos. Nonetheless, during the last 30 years, considerable progress has been made in sheep and goat embryo technologies, especially in the fields of oestrus synchronisation, superovulation and in vitro embryo production. This paper reviews the status of assisted reproductive technologies in sheep, analysing the prospects offered by recent advances in in vivo and in vitro embryo production from mature and juvenile lambs.     

Current progress on assisted reproduction in dogs and cats: in vitro embryo production

The objective of the development of assisted reproduction techniques in dogs and cats is their application to non-domestic canine and feline species, most of which are considered threatened or endangered. Among these techniques, an entirely in vitro system for embryo production is effectively an important tool for conservation of wildlife. In the last decade, progress has been made in embryo production in carnivores. It has been shown that canine oocytes can resume meiosis in vitro and that these oocytes can be fertilized and developed in vitro, although at a much lower rate than most other domestic animal oocytes. The reason lies in the dissimilarities of reproductive physiology of the dog compared to other species and the lack of precise information concerning the oviductal environment, in which oocyte maturation, fertilization and early embryonic development take place. Successful in vitro embryo production in the domestic cat has been attained with oocytes matured in vitro, and kittens were born after transfer of IVM/IVF derived embryos. On the basis of these results the in vitro fertilization of oocytes has also been applied in several non-domestic feline species. The effectiveness of such protocols in the preservation of genetic material of rare species can be improved by developing better techniques for long-term storage of gametes. In dogs and cats sperm cells have been successfully frozen and the cryopreservation of oocytes would greatly increase their availability for a range of reproductive technologies. Cryopreserved cat oocytes can be fertilized successfully and their development in vitro after fertilization is enhanced when mature oocytes are frozen. Thus refined techniques of oocyte maturation and fertilization in vitro coupled with oocyte cryopreservation could allow for an easy establishment of genetic combinations when male and female gametes in the desired combination are not simultaneously available, and the propagation of endangered carnivores would be facilitated.     

Chromosome variation in the embryos of domestic animals

Chromosome abnormalities in the embryos of domestic animals are mostly eliminated during development. De novo chromosome abnormalities in the embryos of domestic animals have been detected in a larger proportion of embryos produced by in vitro fertilization and somatic cell nuclear transfer than in those produced by natural mating or artificial insemination. The increased incidence of abnormalities in embryos produced in vitro provides evidence for an influence of the embryo production procedures on chromosome stability. Research strategies involving cytogenetics, molecular biology and reproductive biotechnologies hold the promise of yielding insight into the mechanisms underlying chromosome instability in embryos and the impact of the in vitro environment on the chromosome make-up of embryos.     

Cloning advances and challenges for conservation

Although controversy surrounds cloning efforts, the cloning of animals to assist efforts to preserve genetic variation in support of endangered species conservation efforts has attracted serious interest. A recent report by Loi et al. describing the cloning of a mouflon (a species of wild sheep) in a domestic sheep surrogate points to potential conservation opportunities and additional challenges in the evaluation of appropriate technologies for present and future efforts to conserve gene pools of endangered species.     

Does asymmetric gene flow among matrilines maintain the evolutionary potential of the European eel?

Using evolutionary theory to predict the dynamics of populations is one of the aims of evolutionary conservation. In endangered species, with geographic range extending over continuous areas, the predictive capacity of evolutionary‐based conservation measures greatly depends on the accurate identification of reproductive units. The endangered European eel (Anguilla anguilla) is a highly migratory fish species with declining population due to a steep recruitment collapse in the beginning of the 1980s. Despite punctual observations of genetic structure, the population is viewed as a single panmictic reproductive unit. To understand the possible origin of the detected structure in this species, we used a combination of mitochondrial and nuclear loci to indirectly evaluate the possible existence of cryptic demes. For that, 403 glass eels from three successive cohorts arriving at a single location were screened for phenotypic and genetic diversity, while controlling for possible geographic variation. Over the 3 years of sampling, we consistently identified three major matrilines which we hypothesized to represent demes. Interestingly, not only we found that population genetic models support the existence of those matriline‐driven demes over a completely panmictic mode of reproduction, but also we found evidence for asymmetric gene flow amongst those demes. We uphold the suggestion that the detection of demes related to those matrilines reflect a fragmented spawning ground, a conceptually plausible consequence of the low abundance that the European eel has been experiencing for three decades. Furthermore, we suggest that this cryptic organization may contribute to the maintenance of the adaptive potential of the species.     

Molecular DNA identity of the mouflon of Cyprus (Ovis orientalis ophion,Bovidae): Near Eastern origin and divergence from Western Mediterranean conspecific populations

The mouflon population of Cyprus (Ovis orientalis ophion) comprises historically preserved feral descendants of sheep domesticated during the Neolithic. We determined genetic identity of this taxon in order to elucidate its systematic placement and enforce its protection. We used 12 loci of microsatellite DNA to infer genetic relationships between the Cypriot mouflon and either long-time isolated (Corsica, Sardinia) or recently introduced (central Italy) European mouflons (O. o. musimon). We also sequenced the mitochondrial DNA (mtDNA) Cytochrome-b gene to infer the origin of the Cypriot mouflon including many National Centre for Biotechnology Information (NCBI) entries of European and Near Eastern conspecifics. Microsatellites disclosed net divergence between Western Mediterranean and Cypriot mouflon. The latter was included in the highly heterogeneous Near Eastern O. orientalis mtDNA group, Iran representing the most credited region as the source for its ancient introduction to Cyprus. Both international and national legislation protect the mouflon of Cyprus as a wild taxon (O. o. ophion). However, the IUCN Red List of Threatened Species and NCBI include the Cypriot mouflon as subspecies of its respective domestic species, the sheep (O. aries). Unfortunately, people charged with crime against protected mouflon may benefit from such taxonomic inconsistency between legislation and databases, as the latter can frustrate molecular DNA forensic outcomes. Until a definitive light can be shed on Near Eastern O. orientalis systematics, we suggest that the Cypriot mouflon should be unvaryingly referred to as O. o. ophion in order not to impair conservation in the country where it resides.     

Live Births from Domestic Dog (Canis familiaris) Embryos Produced by In Vitro Fertilization

Development of assisted reproductive technologies (ART) in the dog has resisted progress for decades, due to their unique reproductive physiology. This lack of progress is remarkable given the critical role ART could play in conserving endangered canid species or eradicating heritable disease through gene-editing technologies—an approach that would also advance the dog as a biomedical model. Over 350 heritable disorders/traits in dogs are homologous with human conditions, almost twice the number of any other species. Here we report the first live births from in vitro fertilized embryos in the dog. Adding to the practical significance, these embryos had also been cryopreserved. Changes in handling of both gametes enabled this progress. The medium previously used to capacitate sperm excluded magnesium because it delayed spontaneous acrosome exocytosis. We found that magnesium significantly enhanced sperm hyperactivation and ability to undergo physiologically-induced acrosome exocytosis, two functions essential to fertilize an egg. Unlike other mammals, dogs ovulate a primary oocyte, which reaches metaphase II on Days 4–5 after the luteinizing hormone (LH) surge. We found that only on Day 6 are oocytes consistently able to be fertilized. In vitro fertilization of Day 6 oocytes with sperm capacitated in medium supplemented with magnesium resulted in high rates of embryo development (78.8%, n = 146). Intra-oviductal transfer of nineteen cryopreserved, in vitro fertilization (IVF)-derived embryos resulted in seven live, healthy puppies. Development of IVF enables modern genetic approaches to be applied more efficiently in dogs, and for gamete rescue to conserve endangered canid species.     

Factors affecting the efficiency of embryo transfer in the domestic ferret (Mustela putorius furo)

Embryo transfer (ET) to recipient females is a foundational strategy for a number of assisted reproductive technologies, including cloning by somatic cell nuclear transfer. In an attempt to develop efficient ET in domestic ferrets, factors affecting development of transferred embryo were investigated. Unilateral and bilateral transfer of zygotes or blastocysts in the oviduct or uterus was evaluated in recipient nulliparous or primiparous females. Developing fetuses were collected from recipient animals 21 days post-copulation and examined. The percentage of fetal formation was different (P<0.05) for unilateral and bilateral transfer of zygotes (71%) in nulliparous females with bilateral transfer (56%) in primiparous recipients. The percentage (90%) of fetal formation in nulliparous recipients following unilateral transfer of blastocysts was higher (P<0.05) than that observed in primiparous recipients with bilateral ET (73%). Notably, the percentage of fetal formation was higher (P<0.05) when blastocyts were transferred as compared to zygotes (90% versus 71%). Transuterine migration of embryos occurred following all unilateral transfers and also in approximately 50% of bilateral transfers with different number of embryos in each uterine horn. These data will help to facilitate the development of assisted reproductive strategies in the ferret and could lead to the use of this species for modeling human disease and for conservation of the endangered Mustelidae species such as black-footed ferret and European mink.     

Status, habitat use, and vulnerability of the European ggrayling in Austrian waters

The European grayling Thymallus thymallus is widely distributed in Austria, occurring in all eight rural provinces. However, in recent years, an increasing number of studies report severe declines in population sizes. Since 1997, the grayling has had the status of an endangered species in Austria. In 1997 the multidisciplinary research programme 'Local Adaptation, Threat, and Conservation of European Grayling' has carried out three projects in Upper Austria, Salzburg, and Carinthia. Research has included repeated electro-fishing in selected stretches of eight rivers and the collection of data on habitat characteristics, fish species composition, population abundance and size distribution, growth and body condition, as well reproductive timing, and migratory activities of grayling. Furthermore, genetic and morphological variation among grayling populations has been studied as well as the number, habitat use and feeding activity of cormorants. Evidence is provided for the existence of distinct negative effects caused by single factors or combinations of factors on grayling stocks in each of the river stretches studied.     

Sperm cryopreservation in wild animals

Extinction of a species represents the loss of a resource evolved through eons of mutations and natural selection. Reproductive technologies, including artificial insemination, embryo transfer, in vitro fertilization, gamete/embryo micromanipulation, semen sexing, and genome resource banking (GRB) have all been developed with the aim of solving existing problems and preserving genetic material for conservation purposes. Although protocols from domestic or non-threatened related species have been extrapolated to nondomestic and endangered species, usually these reproductive technologies are species-specific and inefficient in many nondomestic species because of insufficient knowledge on their basic reproduction biology and the need for species-specific customization. Since spermatozoa are usually more accessible and come in large numbers compared to oocytes and embryos, they are considered the primary cell type preserved in most emerging GRBs. For this purpose, semen from endangered species is currently cryopreserved to avail long-term storage. Due to the intractability of most exotic species, semen collection without chemical restraint is limited to only a handful of species and individuals. Viable epididymal spermatozoa can be obtained from dead or castrated animals, but this resource is limited. Electroejaculation, artificial vagina, abdominal massage, and/or transrectal, ultrasound-guided, massage of the accessory sex glands of living animals are viable alternative methods of semen collection. The ultimate goal is to adapt and optimize collection and cryopreservation protocols for each species, making it feasible, among other things, to collect gametes in the wild and introduce them into captive or isolated populations to increase genetic diversity. Recent advances in these fields have allowed the establishment of GRBs for many threatened species.     

Erodium maritimum (Geraniaceae), a species with an uneven and fragmented distribution along the Western Mediterranean and European Atlantic coasts,has a weak genetic structure

Erodium maritimum L. is an annual species presenting heterogeneous, sometimes very small, and distant populations, distributed along a discontinuous coastal strip of the European Atlantic and the central and western Mediterranean basin. The aim of this study is to investigate genetic variation and geographic structure changes across its large distribution. Fourteen populations of E. maritimum were studied using AFLP fingerprints, together with their population sizes, reproductive systems and flower visitors. AFLP markers revealed the genetic structure of the species to be weak. Many individuals from one population clustered together with those of other populations, showing a high degree of genetic admixture. Despite having a self‐compatible reproductive system, populations (especially the largest ones) showed high levels of genetic polymorphism, and the majority of genetic variation was contained within populations. The low genetic structure suggests high levels of gene flow, which might be explained through the dispersability of the species’ fruits. Finally, recommendations are provided for management strategies to facilitate the conservation of this endangered species.     

濒危植物居群恢复的遗传学考量

本文针对濒危植物居群的遗传多样性、生殖适合度、基因流、近交和远交衰退等遗传学问题在居群恢复过程中的应用进行了探讨。濒危植物居群的回归重建,既面临遗传多样性的迅速丧失、近交衰退等遗传风险,还因回归引种地存在较多近缘种而带来远交衰退的风险,最终导致遗传适应性降低,生境适应性变窄,繁殖和竞争能力减弱。为提高濒危物种保护的质量和效率,在构建回归居群时,应分批次从同一来源居群的不同母株采集材料,确保种源的遗传纯正性和遗传组成的多样性,还应使回归居群尽可能远离近缘广布种。另外,还需要对回归种群进行持续的监测和管理才能保证回归引种的成功。     

The analysis of chromatin remodeling and the staining for DNA methylation and histone acetylation do not provide definitive indicators of the developmental ability of inter-species cloned embryos

The restricted supply of oocytes in the domestic dog limits the development of reproductive biotechnologies in this species. Inter-species somatic cell nuclear transfer could be an alternative for cloning animals whose oocytes are difficult to obtain. In this study, the possibility of cloning dog embryos using pig oocytes was investigated by evaluating nuclear remodeling. Chromatin remodeling, assessed by premature chromosome condensation, pseudo-pronuclei formation, DNA methylation and histone acetylation, along with the developmental ability was compared between intra- and inter-species cloned embryos. The incidence of premature chromosome condensation was significantly higher in intra-species cloned embryos relative to inter-species cloned embryos (87.2% vs. 61.7%; P<0.05), but comparable pseudo-pronuclei formation was observed in both (85.3% vs. 75.8%). None of the inter-species cloned embryos developed beyond the 8-cell stage while 18.3% of intra-species cloned embryos developed to the blastocyst stage. The relative level of both DNA methylation and histone acetylation was similar between intra- and inter-species cloned embryos at all times examined. These results suggest that although partial chromatin remodeling occurs, further investigation is needed to be able to use pig oocytes as recipient oocytes in dog cloning.