Cloning for the Twenty-First Century and Its Place in Endangered Species Conservation.

Cloning as it relates to the animal kingdom generally refers to the production of genetically identical individuals. Because cloning is increasingly the subject of renewed attention as a tool for rescuing endangered or extinct species, it seems timely to dissect the role of the numerous reproductive techniques encompassed by this term in animal species conservation. Although cloning is typically associated with somatic cell nuclear transfer, the recent advent of additional techniques that allow genome replication without genetic recombination demands that the use of induced pluripotent stem cells to generate gametes or embryos, as well as older methods such as embryo splitting, all be included in this discussion. Additionally, the phenomenon of natural cloning (e.g., a subset of fish, birds, invertebrates, and reptilian species that reproduce via parthenogenesis) must also be pointed out. Beyond the biology of these techniques are practical considerations and the ethics of using cloning and associated procedures in endangered or extinct species. All of these must be examined in concert to determine whether cloning has a place in species conservation. Therefore, we synthesize progress in cloning and associated techniques and dissect the practical and ethical aspects of these methods as they pertain to endangered species conservation.

Information Flow between Parents and Offspring Is Essential for Successful Development.

Over the last several decades, the sciences of developmental biology and physiology have expanded and intertwined their scope enormously [...].

Cryo-scanning electron microscopy demonstrates that ice morphology is not associated with the post-thaw survival of domestic boar (Sus domesticus) spermatozoa: A comparison of directional and conventional freezing methods.

Directional freezing (in 2 or 10 ml hollow glass tubes) has been reported to improve post-thaw sperm survival parameters compared to conventional methods (in 0.5 ml straws). However, the biophysical properties that increase post-thaw survival are poorly understood. Therefore, the aim for the current study was to investigate the effect of ice morphology on the post-thaw survival of domestic boar spermatozoa directionally and conventionally cryopreserved in 0.5 ml straws. Ice morphology was quantitatively analyzed using a combination of cryo-scanning electron microscopy and Fiji Shape Descriptors. Multivariate analysis found a significant, non-linear effect (p < 0.05) of interface velocity on ice morphology, with an increase in both ice-lake size, as indicated by area and in aspect ratio, at an interface velocity of 0.2 mm/s. By contrast, post-thaw sperm survival (defined as spermatozoa with both intact plasma membranes and acrosomes) was biphasic, with peaks of survival at interface velocities of 0.2 mm/s (54.2 ± 1.9%), and 1.0 or 1.5 mm/s (56.5 ± 1.5%, 56.7 ± 1.7% respectively), and lowest survival at 0.5 (52.1 ± 1.6%) and 3.0 mm/s (51.4 ± 1.9%). Despite numerical differences in Shape Descriptors, there was no difference (p > 0.05) in the post-thaw survival between conventionally and directionally cryopreserved samples at optimal interface velocities of 1.0 or 1.5 mm/s. These findings suggest that: 1) ice morphology has little impact on post-thaw survival of boar spermatozoa, and 2) directional freezing in 0.5 ml straws (rather than 2 or 10 ml hollow glass tubes) may attenuate benefits of directional freezing.

Conservation Biology and Reproduction in a Time of Developmental Plasticity.

The objective of this review is to ask whether, and how, principles in conservation biology may need to be revisited in light of new knowledge about the power of epigenetics to alter developmental pathways. Importantly, conservation breeding programmes, used widely by zoological parks and aquariums, may appear in some cases to reduce fitness by decreasing animals' abilities to cope when confronted with the 'wild side' of their natural habitats. Would less comfortable captive conditions lead to the selection of individuals that, despite being adapted to life in a captive environment, be better able to thrive if relocated to a more natural environment? While threatened populations may benefit from advanced reproductive technologies, these may actually induce undesirable epigenetic changes. Thus, there may be inherent risks to the health and welfare of offspring (as is suspected in humans). Advanced breeding technologies, especially those that aim to regenerate the rarest species using stem cell reprogramming and artificial gametes, may also lead to unwanted epigenetic modifications. Current knowledge is still incomplete, and therefore ethical decisions about novel breeding methods remain controversial and difficult to resolve.

How can mating systems inform future biobanking strategies? An illustration using two Indonesian bovids, banteng (Bos javanicus) and lowland anoa (Bubalus depressicornis).

Storing cryopreserved spermatozoa in a genome resource bank safeguards against the loss of heterozygosity in endangered species and provides opportunities to reincorporate genes into populations through the application of assisted reproductive technologies. The aim of this study was to demonstrate the effect of breeding strategy on ejaculate characteristics to illustrate how this information may be used to select appropriate methods for the storage and use of cryopreserved sperm. In the present study, ejaculates from a polygynous bovid, banteng (Bos javanicus), were characterized (motility 72.7 ± 4.3%; total sperm count 2,702 ± 764 ×106 sperm; morphologically normal sperm 87.9 ± 3.0%), as well as ejaculates from a monogamous bovid, lowland anoa (Bubalus depressicornis; motility 47.5 ± 5.4%; total sperm count 279 ± 84 ×106 sperm; morphologically normal sperm 69.0 ± 6.1%). As banteng produce an ejaculate with characteristics similar to domestic cattle, translating assisted reproductive technologies from domestic cattle is feasible. By contrast, lowland anoa produce smaller quantities of sperm with a higher prevalence of morphologically abnormal sperm; thus, alternative protocols, optimized for the storage and use of ejaculates containing lower quantities of sperm, is necessary. Sperm tail length was more conserved in banteng (CV 2.7%) than lowland anoa (CV 6.4%) and could be due to differences in levels of sperm competition between species. Additionally, the use of three different diluents (Biladyl, TES-Tris yolk buffer, and whole milk) were investigated for banteng sperm cryopreservation. Sperm cryopreserved in Biladyl and whole milk diluents produced significantly higher post-thaw survival parameters then TES-Tris yolk buffer.

Recent Progress in Spermatology Contributing to the Knowledge and Conservation of Rare and Endangered Species.

There is a remarkable diversity in the animal kingdom regarding mechanisms underlying the production, maturation, structure, and function of sperm cells. Spermatology studies contribute to the knowledge of species diversity and also provide information about individual or population fitness. Furthermore, this fundamental research is required before collected spermatozoa can be used for conservation breeding, including assisted reproduction and cryobanking. This article aims to (a) review the most recent knowledge on sperm morphology and function in wild animal species, (b) analyze how this knowledge can be used to save species in their natural habitat or ex situ, and (c) propose future scientific directions in wildlife spermatology that could positively impact animal conservation. Variations in sperm structure and performance within and between species have multiple origins and significance. This collective body of knowledge enables the design and implementation of conservation strategies and action plans that integrate several disciplines.

Sperm transport and male pregnancy in seahorses: An unusual model for reproductive science.

The Syngnathidae (seahorses and pipefishes) are a group of teleost fishes in which, uniquely, developing embryos are hosted throughout pregnancy by males, using a specialized brood pouch situated on the abdomen or tail. Seahorses have evolved the most advanced form of brood pouch, whereby zygotes and embryos are intimately connected to the host's circulatory system and also bathed in pouch fluid. The pouch is closed to the external environment and has to perform functions such as gaseous exchange, removal of waste and maintenance of appropriate osmotic conditions, much like the mammalian placenta. Fertilization of the oocytes occurs within the brood pouch, but unlike the mammalian situation the sperm transport mechanism from the ejaculatory duct towards the pouch is unclear, and the sperm: egg ratio (about 5:1) is possibly the least of any vertebrate. In this review, there is highlighting of the difficulty of elucidating the sperm transport mechanism, based on studies of Hippocampus kuda. The similarities between seahorse pouch function and the mammalian placenta have led to suggestions that the pouch provides important nutritional support for the developing embryos, supplementing the nutritional functions of the yolk sac provided by the oocytes. In this review, there is a description of the recent evidence in support of this hypothesis, and also emphasis, as in mammals, that embryonic development depends on nutritional support from the placenta-like pouch at important stages of the gestational period ("critical windows").

Opportunities and Limitations for Reproductive Science in Species Conservation.

Reproductive science in the context of conservation biology is often understood solely in terms of breeding threatened species. Although technologies developed primarily for agriculture or biomedicine have a potentially important role in species conservation, their effectiveness is limited if we regard the main objective of animal conservation as helping to support populations rather than to breed a small number of individuals. The global threats facing wild species include the consequences of climate change, population growth, urbanization, atmospheric and water pollution, and the release of chemicals into the environment, to cite but a few. Reproductive sciences provide important and often unexpected windows into many of these consequences, and our aim here is both to demonstrate the breadth of reproductive science and the importance of basic knowledge and to suggest where some of the insights might be useful in mitigating the problems.

Extracellular vesicles in the male reproductive tract of the softshell turtle.

Extracellular vesicles (EVs) are a heterogeneous group of cell-derived membranous structures comprising exosomes and microvesicles that originate from the endosomal system or are shed from the plasma membrane respectively. As mediators of cell communication, EVs are present in biological fluids and are involved in many physiological and pathological processes. The role of EVs has been extensively investigated in the mammalian male reproductive tract, but the characteristics and identification of EVs in reptiles are still largely unknown. In this review we focus our attention on EVs and their distribution in the male reproductive tract of the Chinese softshell turtle Pelodiscus sinensis, mainly discussing the potential roles of EVs in intercellular communication during different phases of the reproductive process. In softshell turtles, Sertoli-germ cell communication via multivesicular bodies can serve as a source of EVs during spermatogenesis, and these EVs interact with epithelia of the ductuli efferentes and the principal cells of the epididymal epithelium. These EVs are involved in sperm maturation, transport and storage. EVs are also shed by telocytes, which contact and exchange information with other, as well as distant interstitial cells. Overall, EVs play an indispensable role in the normal reproductive function of P. sinensis and can be used as an excellent biomarker for understanding male fertility.

Collective dynamics of sperm cells.

While only a single sperm may fertilize the egg, getting to the egg can be facilitated, and possibly enhanced, by sperm group dynamics. Examples range from the trains formed by wood mouse sperm to the bundles exhibited by echidna sperm. In addition, observations of wave-like patterns exhibited by ram semen are used to score prospective sample fertility for artificial insemination in agriculture. In this review, we discuss these experimental observations of collective dynamics, as well as describe recent mechanistic models that link the motion of individual sperm cells and their flagella to observed collective dynamics. Establishing this link in models involves negotiating the disparate time- and length scales involved, typically separated by a factor of 1000, to capture the dynamics at the greatest length scales affected by mechanisms at the shortest time scales. Finally, we provide some outlook on the subject, in particular, the open questions regarding how collective dynamics impacts fertility. This article is part of the theme issue 'Multi-scale analysis and modelling of collective migration in biological systems'.

Premature birth stunts early growth and is a possible driver of stress-induced maternal effects in the guppy Poecilia reticulata.

We tested the effects of gestational stress, principally in the form of alarm cue extracted from the skin of conspecifics, on reproduction in female guppies (Poecilia reticulata) and the growth and behaviour of their offspring. Offspring from mothers exposed to alarm cue exhibited stunted growth in the first few days post-partum, which appeared to be mediated by shortening of the gestation period, the length of which directly correlated with growth rate within the first 6 days post-partum. Mature offspring did not differ in behaviour or stress responses compared with controls and so the effects of maternal predation stress did not appear to persist into adulthood. A different form of gestational stress, dietary restriction, did not significantly affect offspring growth, though brood size was reduced, suggesting that the effects of predation stress were not mediated by differences in resource demand or consumption.

Advances in understanding mechanisms of long-term sperm storage-the soft-shelled turtle model.

Long-term sperm storage is a special reproductive strategy, which can extend the time window between mating and fertilization in some animal species. Spermatozoa of the soft-shelled turtle, Pelodiscus sinensis, can be stored in the epididymis and oviduct for at least six months and one year, respectively. How spermatozoa can be stored in vivo for such a prolonged period is yet to be explained. We analyze the mechanisms that contribute to long-term sperm storage in P. sinensis, and compare them with other species from three different perspectives: the spermatozoon itself, the storage microenvironment and the interaction between the spermatozoon and microenvironment. Characteristics of soft-shelled turtle spermatozoa itself, such as the huge cytoplasmic droplet with its content of several large lipid droplets (LDs) and onion-like mitochondira, facilitate long-term sperm storage. The microenvironment of reproductive tract, involving in the secretions, structural barriers, exosomes, androgen receptors, Toll-like receptors and survival factor Bcl-2, are important for the maintenance of spermatozoa long-term storage. Sperm heads are always embedded among the oviductal cilia and even intercalate into the apical hollowness of the ciliated cells, indicating that the ciliated cells support the stored spermatozoa. RNA seq is firstly used to detect the molecular mechanism of sperm storage, which shows that autophagy, apoptosis and immune take part in the long-term sperm storage in this species.

Reproductive Science as an Essential Component of Conservation Biology: New Edition.

The previous edition of this book mainly provided a snapshot of the state of the art in terms of species-specific reproductive biology and emerging technologies. The influence of environmental changes on reproductive fitness was introduced but not fully explored. The objectives of this second edition were to (1) emphasize the need for holistic and global efforts to understand and sustain reproduction in a constantly changing environment and (2) provide more knowledge in the reproductive physiology of different taxa. The first section of the book is dedicated to survival and adaptation of species in a changing environment (including chapters on environmental impacts in different taxa, as well as the role of microbiomes). The second section focuses on progress in understanding, assisting or even suppressing reproduction in wild species, keeping in mind the influence of environmental factors as well. It contains chapters from the previous edition that were updated (reproduction in elephants, koalas, marsupials, amphibians, and corals), new chapters on species such as sharks and rays, and contributions about the increasing role of reproductive manipulations, such as assisted reproduction and contraception. While the present book emphasizes the overarching issue of environmental impacts on reproduction (resulting in infertility, subfecundity, or fitness), it also highlights the challenges of maintaining wild species in captivity, including those associated with ensuring good welfare. Captive environments can influence reproduction in a multitude of ways, some unexpected, such as the selection of unwanted genetic traits, an essential dimension to be considered to ensure the success of conservation breeding programs. Lastly, new approaches, such as the use of allostatic load indexes and reproductive microbiome analyses also will be closely examined for the first time in rare and endangered species to address conservation issues.

Using the Koala (Phascolarctos cinereus) as a Case Study to Illustrate the Development of Artificial Breeding Technology in Marsupials: an Update.

The successful development and application of an assisted breeding program in any animal relies primarily on a thorough understanding of the fundamental reproductive biology (anatomy, physiology and behaviour) of the species in question. Surely, the ultimate goal and greatest hallmark of such a program is the efficacious establishment of a series of reliable techniques that facilitate the reproductive and genetic management of fragmented populations, both in captivity and in the wild. Such an achievement is all that more challenging when knowledge of the reproductive biology of that species is essentially rudimentary and without adequate models to compare to. Using the koala (Phascolarctos cinereus) as a case study, this chapter provides insights into the development of a concept that began as small undergraduate student project but that subsequently evolved into the first-ever successful artificial insemination of a marsupial. Apart from this historical perspective, we shall also provide a brief review of the current reproductive biology of the koala, discuss technical elements of current assisted breeding technology of this species, its application to the closely related wombat, and the potential role it might play in helping to conserve wild koala populations in the form of a live koala genome bank. There is little doubt that the unique reproductive biology and tractability of the koala has been a benefit rather than a hindrance to the success of artificial breeding in this species.

From the Ivory Tower to Reality! Conclusions of the New Edition.

While many of the traditional scientific disciplines have developed over centuries, animal conservation is a relative newcomer. It relies on multiple specialties with different levels of expertise that, eventually, generate vast amounts of data. More specifically, conservation physiology is an emerging area that can be defined as 'an integrative scientific discipline applying physiological concepts, tools, and knowledge to characterizing biological diversity and its ecological implications; understanding and predicting how organisms, populations, and ecosystems respond to environmental change and stressors; and solving conservation problems across the broad range of taxa, including microbes, plants, and animals' (Cooke et al. 2013). Reproductive biology is more focused, given that it mainly deals with the physiology underlying the production of gametes, embryos, and offspring, and the many associated processes that control these events. However, it is integrated into the different components of conservation physiology. In bringing together the various contributors for this book, the editors' purpose was to provide readers with a new perspective about the complexity behind reproduction and the role it plays in species conservation. Chapters highlight the diversity of reproductive mechanisms across taxa, and provide insight into how they may have evolved, and likely will continue to evolve in a changing environment. To conservation physiologists, the hope is that this information will be applied to sustain populations in both natural habitats and managed facilities. Ultimately, a major goal is to forecast and mitigate negative impacts of environmental change or anthropogenic pressures on animal fitness, which will only follow once we have acquired a solid understanding of reproductive processes.

Integrated Approach Reveals Role of Mitochondrial Germ-Line Mutation F18L in Respiratory Chain, Oxidative Alterations, Drug Sensitivity, and Patient Prognosis in Glioblastoma.

Glioblastoma is the most common and malignant primary brain tumour in adults, with a dismal prognosis. This is partly due to considerable inter- and intra-tumour heterogeneity. Changes in the cellular energy-producing mitochondrial respiratory chain complex (MRC) activities are a hallmark of glioblastoma relative to the normal brain, and associate with differential survival outcomes. Targeting MRC complexes with drugs can also facilitate anti-glioblastoma activity. Whether mutations in the mitochondrial DNA (mtDNA) that encode several components of the MRC contribute to these phenomena remains underexplored. We identified a germ-line mtDNA mutation (m. 14798T > C), enriched in glioblastoma relative to healthy controls, that causes an amino acid substitution F18L within the core mtDNA-encoded cytochrome b subunit of MRC complex III. F18L is predicted to alter corresponding complex III activity, and sensitivity to complex III-targeting drugs. This could in turn alter reactive oxygen species (ROS) production, cell behaviour and, consequently, patient outcomes. Here we show that, despite a heterogeneous mitochondrial background in adult glioblastoma patient biopsy-derived cell cultures, the F18L substitution associates with alterations in individual MRC complex activities, in particular a 75% increase in MRC complex II_III activity, and a 34% reduction in CoQ10, the natural substrate for MRC complex III, levels. Downstream characterisation of an F18L-carrier revealed an 87% increase in intra-cellular ROS, an altered cellular distribution of mitochondrial-specific ROS, and a 64% increased sensitivity to clomipramine, a repurposed MRC complex III-targeting drug. In patients, F18L-carriers that received the current standard of care treatment had a poorer prognosis than non-carriers (373 days vs. 415 days, respectively). Single germ-line mitochondrial mutations could predispose individuals to differential prognoses, and sensitivity to mitochondrial targeted drugs. Thus, F18L, which is present in blood could serve as a useful non-invasive biomarker for the stratification of patients into prognostically relevant groups, one of which requires a lower dose of clomipramine to achieve clinical effect, thus minimising side-effects.

Breakthroughs and new horizons in reproductive biology of rare and endangered animal species.

Because of higher extinction rates due to human and natural factors, more basic and applied research in reproductive biology is required to preserve wild species and design proper strategies leading to sustainable populations. The objective of the review is to highlight recent, inspiring breakthroughs in wildlife reproduction science that will set directions for future research and lead to more successes in conservation biology. Despite new tools and approaches allowing a better and faster understanding of key mechanisms, we still know little about reproduction in endangered species. Recently, the most striking advances have been obtained in nonmammalian species (fish, birds, amphibians, or corals) with the development of alternative solutions to preserve fertility or new information about parental nutritional influence on embryo development. A novel way has also been explored to consider the impact of environmental changes on reproduction-the allostatic load-in a vast array of species (from primates to fish). On the horizon, genomic tools are expected to considerably change the way we study wildlife reproduction and develop a concept of "precision conservation breeding." When basic studies in organismal physiology are conducted in parallel, new approaches using stem cells to create artificial gametes and gonads, innovations in germplasm storage, and more research on reproductive microbiomes will help to make a difference. Lastly, multiple challenges (for instance, poor integration of new tools in conservation programs, limited access to study animals, or few publication options) will have to be addressed if we want reproductive biology to positively impact conservation of biodiversity.

Making the most of sperm activation responses: experiments with boar spermatozoa and bicarbonate.

Attempting to extract useful and reliable information about semen quality and its fertility potential remains a difficult exercise, partly because the sperm heterogeneity within samples often renders simple statistical analyses rather meaningless. In fact, a mean and standard deviation may reflect neither the very fast swimming activities of the most active cells nor the slow and sluggish activities of others. Herein we propose that the information value within semen samples can be maximised if current knowledge about sperm activation mechanisms is exploited before undertaking the measurements. We explain, using boar semen as an example, that estimating and defining relative sperm subpopulation sizes, after activation by bicarbonate, provides a means of quantifying sperm quality. Although such estimates may indeed be related to in vivo fertility, the general approach also suggests potential new avenues that could be exploited for the elaboration of novel in vitro tests for the characterisation of toxic environmental chemicals and, indeed, to reduce the number of animals used in such testing programs.

Composition of marsupial zona pellucida: a molecular and phylogenetic approach.

The zona pellucida (ZP) is an extracellular matrix that surrounds mammalian oocytes. In eutherians it is formed from three or four proteins (ZP1, ZP2, ZP3, ZP4). In the few marsupials that have been studied, however, only three of these have been characterised (ZP2, ZP3, ZP4). Nevertheless, the composition in marsupials may be more complex, since a duplication of the ZP3 gene was recently described in one species. The aim of this work was to elucidate the ZP composition in marsupials and relate it to the evolution of the ZP gene family. For that, an in silico and molecular analysis was undertaken, focusing on two South American species (gray short-tailed opossum and common opossum) and five Australian species (brushtail possum, koala, Bennett's wallaby, Tammar wallaby and Tasmanian devil). This analysis identified the presence of ZP1 mRNA and mRNA from two or three paralogues of ZP3 in marsupials. Furthermore, evidence for ZP1 and ZP4 pseudogenes in the South American subfamily Didelphinae and for ZP3 pseudogenes in two marsupials is provided. In conclusion, two different composition models are proposed for marsupials: a model with four proteins (ZP1, ZP2 and ZP3 (two copies)) for the South American species and a model with six proteins (ZP1, ZP2, ZP3 (three copies) and ZP4) for the Australasian species.

Introduction: A Brief Guide to the Periconception Environment.

Definition of the periconception period is not an exact science and is probably somewhat arbitrary. One can define it as spanning the period from the final stages of gamete maturation until formation of the embryo and the stages of embryonic development and implantation. Hence, the periconception period includes periods when spermatozoa are in the female reproductive tract, oocytes are matured and ovulated into the oviduct, fertilization occurs and the embryo undergoes development. By definition the implantation process and the early stages of placenta formation are also regarded as a part of the periconception period. In this article we highlight a few of the major advances which have transformed this topic over the last two decades. It is now clear that the fitness and wellbeing of developing mammalian embryos, including the human, are highly dependent on the health status, diet and habits of both parents especially in the months and weeks that precede the formation of oocytes and spermatozoa.