Reproductive biology in wildlife conservation: Recent advances, persistent challenges, and knowledge gaps

Introduction

Factors other than the number of individuals left affect the persistence of a wildlife population. The other critical factors for long-term viability include successful reproduction, maintenance and transfer of genetic variation, a suitable age and sex ratio, and the ability of animals to produce offspring under a range of ecological conditions. Infertility, poor nutrition, endocrine disorders, disease, and social incompatibility are the primary causes of reproductive failure, as well as ageing and the lack of genetically appropriate mates. For threatened species these issues are exacerbated as each breeding opportunity may be of disproportionate demographic and genetic importance. Hence, reproductive science plays a role in conservation on two related fronts: understanding when and why animals reproduce and being able to intervene when natural reproduction is inadequate or impractical. Because of the variety of reproductive strategies among vertebrates, there is no universal reproductive protocol that can be expected to be successful for all species, even for closely related groups (Wildt et al. 2010).

The history of wildlife ART is one of technology developed in cattle, horses, laboratory rodents or humans, which does not simply cross over to non-domestic species. Gamete collection to establishment of pregnancy may be affected by differences in ovarian dynamics, sperm structure, gamete membrane composition, ovulatory mechanisms, seasonality, embryonic diapause, placentation, reproductive tract anatomy and response to exogenous gonadotropins. The principle was stated by Pukazhenthi and Wildt (2004) from the outset of the development of conservation ART and continues to be a major constraint on the activity twenty years later and beyond. The bottom line is that basic reproductive physiology is not a prerequisite for biotechnology, but the foundation for the success of biotechnology.Over the past few years however, there have been significant developments. Semen, embryos, gonadal tissue and somatic cells are now stored in conservation biobanks; the recovery of oocytes and in vitro embryo production has been achieved in white rhino programs with repeatable success; the preservation of stem and germline cells have evolved from concept to a pipeline of experimental tests; and genetic rescue by cloning has been accomplished using cells that were stored for decades. Simultaneously, investigators have been more explicit about the uncertainties they confront in the realm of cryptic species, developmental plasticity, epigenetic effects, transferability of protocols, and long-term fitness of the offspring of intensive intervention (Holt and Comizzoli, 2026). The advances make it worth a modern reappraisal of the topic, one that links reproductive biology, technology maturity with conservation decision-making rather than thinking of ART as an end goal without linkage to the field.

2. Review approach and scope

This article is a narrative review. Literature was located using PubMed and publisher databases with a focus on peer reviewed English literature relevant to wildlife reproductive physiology, reproductive monitoring, assisted reproduction, cryobiology, biobanking, cloning and stem-cell technologies. Literature published from 2019 up to August 2026 was prioritized and selected basic literature that was still used in concepts that govern current practice was retained. The review does not seek to be a systematic inventory or meta-analysis of species, but covers mammals, birds, amphibians and reptiles. Rather, it brings together mechanisms, illustrative case studies of conservation, key knowledge gaps and knowledge translation priorities. Due to the nature of the story, examples were chosen that illustrate major technology categories, important taxonomy contrasts and translation issues, rather than to calculate pooled effect sizes.

Figure 1. Integrated reproductive-conservation pathway. Original conceptual figure prepared for this review.

3. Reproductive biology as the foundation of conservation breeding

3.1 Species-specific reproductive phenotypes

The question that's often first tackled in conservation programs is a seemingly simple one: When are individuals fertile? The response may need a longitudinal knowledge of the ovarian cycles, testicular activity, seasonality, sexual maturation, reproductive ageing and social or environmental modulation. The example is aptly illustrated with wild felids. There are species and even individuals that vary in ovulatory pattern, ovarian cyclicity and response to hormonal stimulation, and male fertility can also be affected by low quality of semen, which can be linked to small populations with a low genetic variability. Thongphakdee et al. (2020) conducted a review of reproductive work in wild felids and demonstrated that a decade and a half of research in the physiology of the domestic cat have inspired the progress of AI and embryo production and transfer in the various species rather than the direct adoption of domestic-cat protocols.

The cheetah offers a rather powerful example of how basic biology can influence management. Females can be reproductively inactive for extended periods, but the competence of the oocyte can be relatively high for a wide age range, with age-associated changes to the uterus decreasing the ability to carry pregnancies. Male cheetahs often have high numbers of abnormal sperm in their ejaculate, but these properties do not simply translate to fertility. Crosier et al. (2022) demonstrated the transformative effects of endocrine monitoring, ultrasonography, gamete studies and assisted breeding on ex situ cheetah management. This is a more useful integrated reproductive phenotype for conservation than a single parameter like sperm motility or circulating hormone level.

3.2 Non-invasive reproductive monitoring

In wildlife, repeated endocrine sampling can be challenging, as capture and restraint can induce stress, anesthetic may be needed, or it may not be feasible in free-ranging populations. Longitudinal assessment of reproductive status can be achieved by repeated non-invasive sampling of the same individuals using either feces or urine, or by sampling of hair, feathers or other matrices. These tools can detect ovarian cycles, pregnancy, reproductive suppression and seasonal changes, and can be combined with behavioral and environmental data. Edwards (2026) noted that the number of non-invasive biomarkers is growing rapidly and presents one of the most promising avenues for scaling up the field of reproductive biology in vertebrates in the near future. The biggest limitation for assays is that they need to be biologically validated for the species and matrix target before they can be given physiological significance by cross reactivity only.

3.3 Natural breeding remains a benchmark, not a competitor

Technology should not give rise to the false impression that ART is always better than natural mating. In giant pandas, a study of 21 years' breeding records revealed that natural mating produced significantly more cubs than artificial insemination alone that there was a great importance in the compatibility of the animals' behavior and the correct time and species appropriate breeding. Li et al. (2017) stated that, although AI has its value for certain genetic goals, they do not see it as a substitute for management that allows natural reproduction. This principle is generally applicable: ART is best justified when it addresses a specific problem or constraint (such as geographic isolation, genetic imbalance, reproductive disease, loss of a donor or inability to mate) rather than when it is used only because the technology is available.

Table 1. Reproductive features that influence conservation intervention

4. Cryopreservation and biobanking: preserving future options

Genome-resource banks are among the most useful links between the animals of today and animals that could be used in the future for conservation. Sperm, embryos, gonadal tissues and somatic cells can be stored to increase the genetic life of founders, to move genes without moving animals, to provide additional research material after an animal has died, and for technologies that may not be ready. Bolton et al. (2022) looked at biobanking not as a repository, but as an infrastructure that enables advanced reproductive technologies. This is significant because the conservation value of a sample is dependent on many factors including provenance, identity, health information, reproductive history, genetic representation, processing metadata, quality control and a realistic pathway for future use.

Semen preservation is the most advanced wildlife biobanking use, albeit with variable performance across the board. A recent state-of-the-art review (Nagashima et al., 2025) showed that significant advances have been made at the taxa level and highlighted that routine incorporation of cryopreserved germplasm into population management is underutilized. Oocytes are especially challenging due to their size, lipid content, sensitivity and susceptibility to zona changes and embryos and reproductive tissues need extra developmental and recipient management knowledge. Therefore, the term ‘frozen genetic resource' cannot be thought of as ‘recoverable future offspring’. A biobank should not only monitor storage, but functional use.

4.1 Male germline preservation

Sperm cryopreservation is still attractive, due to the fact that in most cases, sperm can be collected from males at a lower biological cost than female gametes, and a single sample of sperm can be used to produce many offspring. For small ruminants in the wild, the cryoresistance is affected by the source of semen, season, and by the endocrine status and by molecular characteristics such as the membrane water transport. Santiago-Moreno et al. (2023) highlighted that testicles and epididymia may provide extra breeding opportunities for post mortem genetic rescue but quality and fertility after thawing are inconsistent. Conservation lessons are reinforced by these findings, in that a technically successful freeze-thaw cycle does not necessarily reflect reproductive competence, and where possible, fertility endpoints should be included.

4.2 Female germline and gonadal tissues

In general, the collection and cryopreservation of female gametes (oocytes and embryos) have been less developed than the collection and cryopreservation of male gametes (spermatozoa). One factor to change this equation in part is the capability of banking ovarian tissue, which allows for the storage of a larger number of immature follicles from animals that are prepubertal, dead suddenly, or unable to be subjected to repeated collection of oocytes. In Neotropical wildlife, Silva, Pereira and Comizzoli (2022) reviewed the storage of gonadal tissues and demonstrated its potential to be a tool for the conservation of tissues as well as for studying gametogenesis. However, the route of preserved tissue to offspring will require culture and grafting, and in vitro maturation and embryo technologies that are still species specific.

As research advances across disciplines in human reproduction preservation, there are increasingly valuable comparative opportunities. Silva et al. (2025) pointed out that the mechanisms of both ovarian and testicular tissue cryopreservation and culture grafting are shared in humans and wildlife, except for some significant anatomical and physiological differences which require extensive adaptation. The ability to share experience across translation is especially useful for rare species where researchers are not necessarily able to create complex techniques ex nihilo. The following section, therefore, outlines the usefulness of the domestic or biomedical model as a hypothesis generating platform rather than a replacement for target species validation.

Figure 2. Qualitative readiness of selected reproductive technologies across major vertebrate groups. This is an author-derived synthesis, not a quantitative meta-analysis.

5. Assisted reproductive technologies: from artificial insemination to embryo transfer

5.1 Artificial insemination

Artificial insemination is the oldest of the ARTs, and is most likely to be transferred from research to routine management stage when semen collection and preservation and timing of the ovulation are well understood in wildlife. Conservation benefits include decreased need for animal transport, planned institution gene flow, semen stores from males that are far away or dead, and overcoming behavioural incompatibility. But even amongst mammals, only certain species are successful. After decades working with wild felids, Swanson (2023) concluded that the main challenges relate to incomplete knowledge of the reproductive biology, lack of access to research animals, institutional limitations, and the preference to adopt highly complex methods rather than simpler ones that have been tested. One of the most preventable reasons for failure is this ‘technology before biology' mismatch.

5.2 Oocyte recovery, IVF and ICSI

In vitro fertilization or intracytoplasmic sperm injection may be especially useful in cases of small sperm numbers or if donor animals are unable to mate or if there are limited numbers of genetically valuable gametes available. The northern white rhino has been the most prominent example of this approach. A program was reported by Hildebrandt et al. (2023) which involves repeated ovum pick-up (RPUP), in vitro maturation (IVM) and fertilization (IVF) to produce viable white-rhinoceros blastocysts. What is important about the work is that it demonstrates that when species-specific anatomy, anesthesia, ovarian stimulation, oocyte retrieval, laboratory embryology and ethics evolve together over many years, one can achieve what is possible.

5.3 Embryo transfer and recipient biology

Creating an embryo is just a step toward creating a child. Recipient physiology is central to advanced ART and could differ from domestic models based on the requirements for conservation benefit – the need for implantation, pregnancy, birth, and healthy offspring and for luteal support, uterine health, placentation and fetal development. After a forty-year history of wildlife ART, Mastromonaco (2024) concluded that the most successful advances in these technologies have occurred when research institutions, zoos, veterinary practitioners and population managers are united around concrete conservation goals. Embryo transfer is thus best perceived as a systems problem: optimization of the two competencies, the laboratory competence and the recipient competence, is to be achieved simultaneously.

5.4 Species with stronger translational potential

Certain taxa are more conducive to technology transfer because their close relatives exist domestically and have been validated as reproductive models. The case of the bison is one where cattle reproductive knowledge has been developed and led to the development of estrus synchronization, handling of gametes, production of embryos, and cryopreservation. These tools can help to move genetics and minimize biosecurity and transport requirements in conservation herds, argued Acevedo and Barfield (2023). The drawback of this is that even species that are closely related can have different seasons, follicular dynamics and treatment response, so that domestic models do not remove the validation burden, but merely lessen it.

Table 2. Major reproductive technologies in wildlife conservation

6. Emerging technologies and genetic rescue

6.1 Cloning and somatic cell nuclear transfer

Somatic cell nuclear transfer (SCNT) is a technique that is different from standard ART, in that it can derive a genome from a stored somatic cell. This opens an exceptional opportunity for conservation of historical cell banks. Swegen et al. (2023) discussed the different cloning techniques and found that SCNT is relevant to particular scenarios of genetic rescue but is limited by low efficiency, requirement of oocytes, surrogate compatibility and developmental abnormalities. These restrictions restrict the general use of cloning as breeding and its only real justification is the recovery of genetic variation not found in the living population.

This selective function was recently shown in a demonstration with Przewalski's horse. Novak et al. (2025) introduced the concept of cloning from historically cryopreserved cells of an underrepresented lineage with the goal of expanding the representation of rare genetic material from the living population. This case brings up the utility of long-term somatic-cell banking from a theoretical discussion to a viable genetic-management choice. It also emphasizes an important planning concept: Planning opportunities which occurred many years ago can open up opportunities that were not available at collection time.

6.2 Spermatogonial stem cells and testicular germline rescue

Another option for preserving and perhaps propagating male genetic material is via sperm stem cells. Such testicular tissue can be removed from immature and/or adult animals and, in principle, transplanted or grafted or transferred into in vitro culture systems to produce sperm. Damyanova et al. (2024) summarized the potential transferability of technologies in development for human fertility preservation to the wildlife and highlighted the lack of characterization of basic SSC markers and niche biology and differentiation pathways in most non-model species. They might therefore benefit most in the near term from being able to store viable testicular tissue for use at a later time, and continue to work on a parallel track to develop retrieval methods.

6.3 Induced pluripotent stem cells and synthetic reproductive pathways

Somatic cells could be a renewable source of pluripotent germ cells, differentiated via induced pluripotent stem cells (iPSCs) for embryo models or other applications involving regenerator. Lou et al. (2025) summarized the swift advances in the field of research on iPSCs derived from endangered animals, such as non-integrating methods for reprogramming and future methods for directing them toward germ line. The promise of conservation is significant as somatic cells are more readily collected and banked than mature gametes for many species. However, the critical issues of germline differentiation, genomic stability, and epigenetic fidelity, imprinting, and developmental competence still exist. Reproduction using iPSCs should be viewed as a research investment to be made, not as a means of population management.

7. Comparative progress beyond mammals

7.1 Amphibians

The use of hormone induction, sperm cryopreservation and artificial fertilization has been integrated to amphibians, and it is a key testbed for these applications. They are urgently in need of conserving, and some reproductive technologies are relatively low cost for them compared with large mammals. Successful hormone therapies and gamete storage and fertilization methods were summarized, with a focus on the need for technical capability to be followed by adoption in conservation breeding (Silla and Byrne, 2019). Reproduction-biobanking-conservation systems, rather than just single procedures, are now increasingly being discussed, and capacity and geographic equity are necessary if technology is to be made available to those areas of highest amphibian diversity and threat (Browne et al., 2024).

7.2 Reptiles

Although similar to other amphibians and mammals, Reptile ART is still less advanced. The protocol can be complicated due to reproductive anatomy, seasonal cycles, semen collection, sperm storage, long retention period in the female and different types of oviparity or viviparity. Perry and Mitchell (2022) reviewed the advancements in AI, hormonal control, gamete collection, and genome conservation, and highlighted key limitations in embryo manipulation, ICSI and cloning. Similarly, a prior cross-taxon analysis by Clulow and Clulow (2016) highlighted the need for different technical solutions for amphibians and reptiles due to their different reproductive biology and thus distinct evolutionary endpoints. The field is therefore enriched with development pathways that are parallel and do not follow one another.

7.3 Neotropical wildlife and underrepresented taxa

Geographic and taxonomic bias is a serious constraint on conservation reproductive science. The detailed knowledge of reproduction is disproportionately derived from charismatic species kept in well-resourced institutions. Silva et al. (2026) discussed the recent advances of ART in Neotropical mammals, noting improvements and the lack of progress in its deployment at scale in conservation practice. They argue that there is a need to move towards regional biobanks, domestic or model relatives with care and long-term capacity development in countries with high biodiversity. This change is critical, if not, technology development would become focused on the species that was easiest to study and not the one that requires the most conservation attention.

Table 3. Representative conservation case studies and lessons

8. Biological and technical challenges that still limit translation

8.1 The small-sample problem

The sample size of wildlife reproductive research is sometimes small enough that it is not considered large enough in domestic-animal or biomedical studies. There may be few reproductively available individuals of endangered species and conditions may be set up to take advantage of situations, and ethical or husbandry limitations may preclude experimental replication. This means that there is a large uncertainty in apparent treatment effects and long optimization times. Along with technology, limited contribution of fertility-preservation technologies to species conservation is also due to the lack of biological knowledge, facilities, expertise and funding, as argued by Comizzoli (2015). These limitations may be as significant as creating a new protocol and collaborative standardization and data sharing can be as crucial.

8.2 Cryobiological diversity

The sensitivity of the cell to cooling and freezing is related to the type of lipid composition of the cell, the size of the cells, their permeability, osmotic tolerance and developmental stage. Therefore, the optimum concentration of a cryoprotectant in one species may be detrimental or sub-optimal to another. Multi cellular structure, intracellular lipid and diffusion gradients make the problem worse for oocytes, embryos and tissues. Pukazhenthi et al. (2006) highlighted the significance of emerging technology for endangered species, and the critical role that fundamental biology plays in achieving the effectiveness of the tools. Although there are many possible solutions in modern cryobiology, optimization on the species level still cannot be avoided.

8.3 Reproductive ageing, disease and hidden pathology

A conservation program can identify valuable genetic individuals and leave their reproductive organs to get old and out of shape. Species that live long in captivity can outlive their reproductive age and thus be demographically valuable while of little reproductive value. This is one of the reasons why biobanking is better than salvage in times of crisis. Comizzoli and Holt (2019) emphasized the critical importance of understanding these "latent" physiological limitations before it is too late for them to be able to help effect breakthroughs in the reproductive biology of rare species. Monitoring of reproductive health should therefore be a part of genetic management, and not just veterinary care.

8.4 From laboratory endpoints to conservation endpoints

Laboratory endpoints such as motility, integrity of the membrane, the rate of cleavage and blastocyst formation are proxies. Conservation outcomes are population-level: retained heterozygosity, representation of founders, reduced inbreeding, viable pregnancies, healthy offspring, reproductive competence of descendants and, where applicable, successful reinforcement or reintroduction. The study of reproductive science has the potential to provide valuable insights but also its own constraints, when cast outside the ecological and demographic framework of the species being studied, wrote Holt and Comizzoli (2022). The reproductive intervention should thus be chosen in relation to the program's anticipated conservation goal.

9. Ethics, welfare, governance and social legitimacy

Anesthetic, multiple hormonal treatments, surgery, transrectal procedures, embryo manipulation, surrogates and intensive veterinary treatment may be part of ART. Allowing these procedures could be ethically justified in the case of high priority conservation objectives, but the technical availability of a procedure does not mean that it is ethically acceptable. Biasetti et al. (2022) have created an ethical-analysis model for ovum pick-up of white rhinos, taking into account the welfare of the animals, the conservation value, the quality of the procedure and the interests of the stakeholders. In contrast to a post-study assessment for ethical approval, prospective assessment can influence the study design, stopping rules, frequency of procedures and choice of alternatives from the start.

There is also a strategic element to ethical evaluation. Expensive reproductive technologies could be pitted against habitat management, disease management, anti-poaching efforts, and community outreach or less complicated breeding strategies. The correct comparison is then 'this intervention versus the best alternative use of conservation resources. Ethical considerations are particularly relevant for those populations that are functionally extinct or very small, where ambitious ART can yield large scientific gains and recovery is not certain. Clear goals, self-evaluation of well-being, and fixed standards of success and elimination will help maintain public and scientific credibility.

Figure 3. Knowledge landscape and priority actions for wildlife reproductive science.

10. The major unknowns

10.1 How transferable is a successful protocol?

A recurring unknown is the biological distance across which a protocol remains valid. Transfer between domestic and wild relatives can accelerate research, but subtle differences in follicular waves, sperm membrane composition, oocyte lipid content, embryonic timing or placentation may still cause failure. Even populations currently classified as the same species may differ genetically or physiologically enough to alter treatment response. This uncertainty becomes more important as genomics reveals cryptic diversity and previously unrecognized population structure. Reproductive protocols should therefore be treated as population-informed rather than universally species-fixed whenever geographic or genetic divergence is substantial.

10.2 What are the long-term effects on offspring fitness?

Most wildlife ART studies are necessarily evaluated through short-term endpoints such as fertilization, pregnancy or birth. Far less is known about lifetime health, behavior, reproductive performance and ecological competence of ART-derived offspring, especially after release. Advanced procedures that involve prolonged culture, nuclear transfer or stem-cell manipulation raise additional questions about epigenetic programming. These uncertainties do not make the technologies unacceptable, but they do justify long-term follow-up and biobanking of offspring-derived samples so that delayed effects can be detected rather than assumed absent.

10.3 When is advanced ART actually better than simpler management?

The most important unknown may be strategic rather than mechanistic. A sophisticated technique can solve a laboratory problem while leaving the population problem unchanged. For example, creating embryos from overrepresented founders may add individuals without adding meaningful genetic diversity. Conversely, one successful insemination from an underrepresented, deceased founder may substantially improve genetic representation. Technology selection should therefore be linked to pedigree or genomic models, demographic forecasts and explicit marginal benefit. The question is not whether a procedure can produce an offspring, but whether that offspring measurably improves the conservation trajectory.

11. Future directions

The next phase of wildlife reproductive science should focus less on isolated technical milestones and more on reproducible conservation systems. Several priorities emerge from the current literature.

1. Build species-specific reproductive baselines before crisis points. Routine endocrine, ultrasonographic, semen and reproductive-health data should be collected across life stages so that interventions are not developed only after fertility has already declined.

2. Bank broadly, but bank intelligently. Cryobanks should prioritize genetically informative donors and include standardized identity, health, provenance, processing and quality-control metadata. Somatic and gonadal tissues should complement not replace gamete banking.

3. Integrate genomics with reproductive decision-making. Genomic data can help identify underrepresented lineages, confirm taxonomic identity, avoid inadvertent inbreeding and determine which stored samples have the greatest potential value.

4. Validate technologies across institutions. A protocol that works in one laboratory on a few animals should be independently reproduced before it is considered management-ready. Shared standard operating procedures and common outcome metrics are essential.

5. Treat welfare and ethics as design variables. Procedural burden, anesthesia frequency, hormonal exposure, surrogate welfare and opportunity costs should be evaluated prospectively, with explicit thresholds and alternatives.

6. Measure descendants and population impact. Follow-up should include neonatal survival, growth, fertility, health and where possible reintroduction performance. Population models should quantify whether ART actually changes genetic or demographic outcomes.

7. Invest in underrepresented taxa and regions. Technical capacity should expand beyond a small number of charismatic mammals and high-income institutions. Regional biobanks and training networks will determine whether ART contributes to global rather than selective biodiversity conservation.

Table 4. Knowledge gaps and research priorities

Conclusion

Reproductive biology has become an integral part of conservation planning for some threatened species and a supporting tool. The most successful progress is not just technological but also the cooperation of systems where reproductive physiology, veterinary care, genomics, biobanking and population management feed into each other. Semen cryopreservation and AI are now being increasingly applied in select mammals and amphibians, intensive species-specific embryo technologies are advancing; gonadal tissues expand the scope of bankable life stages; and the future potential for genetic variation recovery is being extended from somatic-cell archives by cloning or stem-cell technologies. However, the field is not yet fully ready and has significant uncertainties. There are limitations in translation, including small sample size, cryobiological diversity, recipient physiology, reproductive ageing, welfare constraints, regulatory barriers and long-term uncertainty of offspring fitness. The most fruitful future direction is thus not technological escalation, but strategic matching of least intrusive effective intervention to a well defined conservation goal. Protection of habitat and healthy wild populations and natural reproduction are the cornerstone. Reproductive technologies are most effective when they are used to maintain options, restore broken lines of descent and offer soundly argued solutions to issues that are not addressed by traditional management.

Declarations