Reproductive biotechnologies for conservation of endangered mammalian species

Introduction

Endangered species are defined as organisms facing an exceptionally high probability of extinction in the near future according to global conservation assessments. Present-day extinction rates greatly exceed speciation, largely as a consequence of anthropogenic pressures including habitat degradation, excessive hunting and fishing, and poaching. India, recognized as a mega-diverse nation, harbors approximately 8.6% of the world's mammalian species, 13.7% of birds, 12.2% of fishes, and 6.0% of flowering plants in 2025. At a global scale, extinction risk threatens nearly 12% of birds, 27% of mammals, and 41% of amphibians (IUCN, 2025). India alone supports around 813 globally threatened animal species, representing about 1.9% of the world's total threatened fauna (IUCN Red List Year, 2025). The accelerating erosion of biodiversity is not only an ecological crisis but also a socioeconomic one, influencing food security, public health, climate regulation, and social equity (WHO, 2015). In addition to species-specific diseases (Shome et al. 1999; Rai et al., 2010; Srivastava et al., 2017) [94,84,99] that contribute to population decline, the pace of environmental change driven by human activity often outstrips the capacity of organisms to adapt, precipitating extinction events (Ceballos and Ehrlich, 2018) [17] . These realities underscore the urgent need for comprehensive conservation initiatives. Effective animal conservation aims to maintain populations of sufficient size and genetic variability to ensure long-term viability (Comizzoli et al., 2019) [22] . This objective requires complementary use of in situ strategies, which preserve species within their native habitats, and ex situ approaches implemented through managed programs in captivity such as zoos, Ramsars, and ecological parks. While in situ conservation maintains natural adaptations (Mestanza-Ramón et al., 2020) [62] , it may be insufficient for very small or fragmented populations where genetic diversity is rapidly eroded (Felix et al., 2007) [28] . Captive breeding, though successful in certain cases, can suffer from restricted gene pools, resulting in inbreeding depression and reduced fertility (Saragusty et al., 2020) [92] . Increased homozygosity further compromises adaptive capacity and reproductive performance (Wildt et al., 2010) [113] . For species in which conventional breeding programs have failed (Lees and Wilcken, 2009) [53] , the preservation of genetic material through cryopreservation and cryobanking storage of viable cells and tissues at -196 °C in liquid nitrogen or in ultra-low freezers combined with ARTs and aARTs provides an alternative means of safeguarding genotypes that would otherwise be lost (Mitchell & Williams, 2022) [63] . ARTs encompass techniques that utilize oocytes and spermatozoa to generate offspring, including artificial insemination (AI), in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), embryo transfer (ET), and cloning.

Assisted Reproductive Technologies for Endangered Species Conservation

ARTs markedly enhance reproductive efficiency and represent powerful tools for conserving both domestic and wild animal populations at risk of extinction (Bolton et al., 2022) [10] . These technologies help overcome limitations such as poor semen quality, low sperm output, infertility, small population size, and loss of genetic diversity in threatened species (Swanson, 2023) [101] . ART development is often described in successive generations. Early applications focused on gamete and embryo cryopreservation and AI, while later advancements incorporated ultrasonography and multiple ovulation and embryo transfer (MOET). Subsequent progress enabled ovum pick-up (OPU) and IVF, culminating in advanced ARTs (aARTs) that include cloning by nuclear transfer, transgenesis, stem cell technologies, and marker-assisted selection (MAS) (Bertolini and Bertolini, 2009) [7] . A wide array of reproductive interventions like estrus synchronization, superovulation, non-surgical embryo recovery and transfer, oocyte retrieval, in vitro maturation, cloning, and nanotechnology are now applied toward conservation goals (Bisla et al., 2021; Poonia et al., 2023) [8,76] . Continuous refinement of in vitro embryo production and cryopreservation protocols has further improved outcomes (Mohammed et al., 2005; Arav, 2014) [64,3] . Techniques such as IVF, subzonal sperm injection, and ICSI have expanded fertilization options (Gordon, 2003) [32] . Nevertheless, the effectiveness of both ARTs and aARTs is constrained by limited knowledge of reproductive physiology in many species; detailed understanding exists for only about 250 species (Herrick, 2019) [36] .

Artificial Insemination

Artificial insemination was the earliest widely adopted reproductive technology, involving semen collection from males followed by deposition into females during the receptive phase of the reproductive cycle (Manafi, 2011) [59] . Owing to its relative simplicity and minimal invasiveness, AI is the most broadly applicable ART for wildlife (Comizzoli, 2015) [21] . A major conservation benefit of AI lies in mitigating genetic deterioration caused by population fragmentation. In small or isolated populations, temporary capture of females for insemination with semen from genetically valuable males maintained in captivity can facilitate gene flow, after which females may return to their natural habitats to give birth (Pukazhenthi and Wild, 2004) [82] . AI has resulted in live births across a wide spectrum of wildlife species, including Blackbuck (Holt et al., 1988) [67] , African lion (Bowen et al., 1982) [14] , Black-footed ferret (Howard et al., 1991) [43] , Ocelot and Eld's deer (Monfort et al., 1993) [66] , Cheetah (Wildt et al., 1997) [114] , Marmoset (Morrell et al., 1998) [67] , Poitou donkey (Trimeche et al., 1998) [109] , Asian and African elephants (Olson and Wiese, 2000; Brown et al., 2009) [74,39] , Oryx (Morrow et al., 2000) [ 68] , and Giant panda (Masui et al., 1989) [61] . Despite more than 50 reported species successes, routine AI application remains largely confined to commercially managed wildlife such as Red deer, Fallow deer, White-tailed deer, and Reindeer. Further investigation is required to address constraints, including the effects of anesthesia used during electro-ejaculation on semen quality (Hildebrandt et al., 2000) [37] .

Embryo Transfer and In vitro Fertilization

Embryo transfer enables the production of multiple offspring from genetically superior females (Gordon, 2003; Mohammed et al., 2011) [32,65] . Embryos are recovered from the oviduct or uterus using surgical or non-surgical techniques and can be transferred to recipient females or cryopreserved for future use (Mohammed et al., 2011) [65] . IVF involves fertilizing oocytes with fresh or cryopreserved spermatozoa in a controlled culture environment (Mohammed et al., 2005) [64] . Prior to fertilization, spermatozoa undergo in vitro capacitation commonly achieved via swim-up or Percoll gradient methods to acquire the ability to penetrate the zona pellucida (Gordon, 2003 [32,64,47] . Capacitation induces physiological changes such as enhanced motility, calcium influx, and protein interactions (Donà et al., 2020) [27] . Following coincubation of gametes for approximately 8-22 hours, fertilized oocytes (zygotes) are formed (Greda et al., 2006) [33] . Mature oocytes used for IVF may be collected from slaughterhouse ovaries and matured in vitro or retrieved from live females via OPU (Mohammed et al., 2005; Aller et al., 2010) [65,1] . IVF is particularly valuable for producing embryos from terminal or compromised females and for conservation breeding. While AI primarily facilitates dissemination of male genetics, embryo-based techniques like IVF, ET, and cryopreservation enable representation of female genetic contributions. These methods are, however, more invasive, technically demanding, and costly. Interspecies embryo transfer has therefore been explored as a conservation strategy, wherein embryos from endangered species are transferred to surrogate females of related species. Landmark successes include a gaur calf born to a Holstein cow (Stover et al., 1981) [100] , Armenian red sheep gestated by domestic sheep (Coonrod et al., 1994) [25] , and Spanish ibex carried by domestic goats (Fernandez-Arias et al., 1996) [29] . Subsequent achievements have involved banteng, zebra, Indian desert cat, and European mink embryos carried by surrogate species (Loskutoff, 2003; Amstislavsky et al., 2004) [58,2] . Another application of IVF is in vitro maturation (IVM) of oocytes recovered from females that die unexpectedly, a strategy termed "genetic recovery" (Galli et al., 2003; Pukazhenthi and Wildt, 2004) [30,82] . This approach has yielded live offspring in species such as gaur (Johnston et al., 1994) [50] , Armenian red sheep, llama, and European mouflon (Ptak et al., 2002) [81] . Success depends on donor age, health, and culture conditions (Christensen et al., 1993) [20] . IVF and in vitro production have also been applied in Armenian red sheep, Sika deer, European mouflon, African elephant, minke whale, gorilla, and zebra (Comizzoli et al., 2001; Ptak et al., 2002; Tetsuka et al., 2004) [23,81,104] .

Genome Resource Banking (GRB) or Biobanking

Given the rapid pace of extinction, it is impractical to design tailored conservation programs for all threatened species. Genome resource banking offers a strategic means of preserving biodiversity by storing genetic materials like DNA, RNA, semen, oocytes, embryos, tissues, and cells at ultra-low temperatures near -196 °C (Wisely et al., 2015) [116] . GRBs function as interfaces between in situ and ex situ efforts, facilitating exchange of genetic diversity and creation of global gene pools (Hanks, 2001; Holt and Pickard, 2004) [35,41] . Worldwide, 22 institutions currently maintain biological repositories for wildlife ART applications. The Frozen Zoo at the San Diego Zoo Institute for Conservation Research houses more than 10,000 living cell cultures and gametes from nearly 1,000 taxa. In India, CCMB-LaCONES hosts the National Wildlife Genetic Resource Bank, the country's sole dedicated wildlife biobanking facility.

Semen Cryopreservation

Semen cryopreservation preserves spermatozoa at sub-zero temperatures, commonly -196 °C in liquid nitrogen, enabling long-term storage (Murray and Gibson, 2022) [71] . Cryoprotective agents are essential to maintain sperm viability during freezing and thawing (Tamburrino et al., 2023) [103] . This approach underpins numerous conservation and breeding applications, including AI and IVF (Prieto et al., 2014) [80] . Cryopreserved spermatozoa form the basis of living cryobanks that support genetic management across taxa (Browne et [15,17,104,86,16] . Because sperm are haploid and continuously produced, they allow broad genetic representation. Advances in cryopreservation protocols (Pande et al., 2015; Katiyar et al., 2022) [75,51] support the use of well-studied domestic species as models for related wildlife, including equine protocols for rhinoceros (Reid et al., 2009) [85] , bovine models for gazelles (Saragusty et al., 2006) [93] , and domestic ferrets for Blackfooted ferrets (Howard et al., 2016) [42] . Comparable adaptations have been applied to Asian elephants, giant pandas, bees, killer whales, and bottlenose dolphins (Saragusty et al., 2009 [94,60,22,87] .

Oocyte Cryopreservation

Oocytes represent another valuable genetic resource for conservation. However, their large size and low surface areato-volume ratio make them particularly susceptible to intracellular ice formation during freezing (Borini and Bianchi, 2012) [13] . Variable membrane permeability to cryoprotectants further complicates preservation, often resulting in reduced fertilization rates after thawing (Arav, 2014; Tharasanit and Thuwanut, 2021) [3,105] . Despite these challenges, progress has been achieved in multiple species (Felix et al., 2007) [28] . In felids, oocyte cryopreservation remains largely experimental, with post-thaw developmental competence still limited (Jewgenow and Zahmel, 2020) [48] . Domestic cats are therefore used as models to refine protocols applicable to wild felids (Fernandez-Gonzalez et al., 1996) [29] .

Gonadal Tissue Cryopreservation

Preservation of gonadal tissue provides an alternative to storing mature gametes, enabling retention of the full spectrum of germ cell stages (Comizzoli et al., 2012) [24] .

Cryopreservation of Testicular Tissue

Testicular tissue cryopreservation enables recovery of genetic material from valuable males that die unexpectedly. In domestic cats, viable spermatozoa retrieved from cryopreserved testicular tissue have been used via ICSI to produce live offspring (Tharasanit et al., 2012) [106] . Testicular sperm often exhibit higher post-thaw viability (Chatdarong, 2011) [18] . Techniques such as in vitro culture of thawed tissue and ultra-rapid freezing show promise (Sato et al., 2011; Pothana et al., 2017; da Silva et al., 2020) [95,78,11] . Reports span numerous wildlife species, including jungle cat, lion, leopard, Rusa deer, Fea's muntjac, Sumatran serow, and Eld's deer (Thuwanut, 2013) [107] . Comparable success has been observed in nonhuman primates using slow-freezing protocols (Pothana et al., 2016) [94] .

Cryopreservation of Ovarian Tissue

Ovarian tissue cryopreservation preserves immature follicles that would otherwise never contribute to reproduction, offering a means to safeguard fertility from neonatal, prepubertal, or non-breeding females (Santos et al., 2010) [91] . This technique has been applied across species such as Whitetailed deer, domestic cat, collared peccary, yellow-toothed cavy, donkey, and cattle (Vasconcelos et [110,79,57,54,31,69] . Following death or neutering, cortical tissue containing follicles is dissected, sectioned, and cryopreserved (Hinkle et al., 2021) [38] . Projects such as the Rhino Fertility Project are advancing in vitro culture systems for critically endangered species like the northern white rhinoceros, although challenges related to survival and developmental competence remain.

Advanced Assisted Reproductive Technology Reproductive Cloning

Reproductive cloning via somatic cell nuclear transfer (SCNT) involves introducing a somatic cell nucleus into an enucleated oocyte to generate an embryo genetically identical at the nuclear level to the donor. The reversibility of cellular differentiation was first demonstrated in Xenopus laevis (Gurdon et al., 1975) [34] , a discovery later recognized with the Nobel Prize in 2012. Mammalian cloning was achieved with the sheep (Wilmut et al., 1997) [115] . SCNT can be performed by microinjection or electrofusion, with species-specific optimization of oocyte maturation and activation protocols (Wakayama et al., 1999; Liu et al., 2015; Qu et al., 2020) [11,56,83] . For endangered species, limited availability of oocytes necessitates interspecific SCNT (iSCNT), yielding offspring with donor nuclear DNA but recipient mitochondrial DNA (Lagutina et al., 2013) [52] . Such individuals are regarded by IUCN as functional proxies capable of restoring ecological roles, though they are not genetically identical to extinct species (IUCN, 2016). Live progeny via iSCNT have been reported in yak, African wildcat, sand cat, bucardo, Bactrian camel, grey wolf, coyote, mouflon, river buffalo, and caracal (Lagutina et al., 2013) [52] . SCNT has supported rescue efforts for species such as the Black-footed ferret and Przewalski's horse (Sandler et al., 2021; Novak et al., 2023) [90,72] . Complications including increased pregnancy loss have been linked to immunological incompatibilities, prompting exploration of alternative strategies such as inner cell mass transfer (Rutigliano et al., 2022; Saragusty et al., 2020) [88,92] .

Stem Cell Technology

Stem cell approaches offer additional avenues for preserving genetic diversity. While embryonic stem cells can generate germ cells (Toyooka et al., 2003) [108] , their derivation is unsuitable for endangered species. Induced pluripotent stem cells (iPSCs), reprogrammed from adult somatic cells (Takahashi and Yamanaka, 2006) [102] , provide a viable alternative, although differences from embryonic stem cells may affect immunogenicity and differentiation (Yamanaka, 2012; Okita et al., 2012) [102,71] [56] . iPSCs derived from frozen fibroblasts of the Drill and the northern white rhinoceros exemplify the promise of this technology for conservation (Ben-Nun et al., 2011) [5] . [6,119,44,89] . Microfluidics also enhances optimization of cryopreservation protocols and reduces contamination risk. Integrated within broader conservation strategies, these tools contribute to a versatile biotechnological toolkit for biodiversity preservation (Corlett, 2017) [26] .

Microfluidics

Conclusion

ARTs and aARTs provide substantial opportunities for conserving endangered mammals, with significant progress achieved over recent decades. Incorporation of tissue cryobanking into conservation planning is increasingly important as a reservoir of genetic diversity for future use (Comizzoli & Holt, 2019) [21] . Effective strategies should combine in situ and ex situ approaches with reproductive biotechnologies such as AI, gamete micromanipulation, cryopreservation, in vitro culture, and tissue grafting. Beyond facilitating reproduction, these techniques also generate valuable insights into the reproductive biology of wild species. Although ethical considerations and potential risks remain, continued research, collaboration, and public engagement can maximize the benefits of these technologies for safeguarding global biodiversity.

Future Prospects

Integration of advanced ARTs with conventional conservation measures is expected to strengthen biodiversity protection for future generations. Further development of alternative longterm biomaterial storage methods is required where existing technologies are insufficient. A major limitation remains the paucity of reproductive physiological data for many wild species, hindering optimization of reproductive interventions. Emerging advances, particularly in SCAT combined with gene-editing technologies, hold promise for establishing genetically robust, self-sustaining populations of critically endangered mammals.

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