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Calatayud, N. E.

Publications and source records attributed to Calatayud, N. E..

7 recordsLinked to original sources

A Cell Viability and Utility Index (CVUI) for wildlife fibroblast biobanking: framework development and preliminary empirical validation

Wildlife biobanking depends on consistent, reproducible cell culture outcomes, yet no standardised framework exists for assessing culture quality across taxa or for integrating quality metrics into institutional collections management systems. Here, we present the Cell Viability and Utility Index (CVUI), a scored pipeline framework validated in a pilot study of 154 culture rounds from 46 species held in the Ian Potter Australian Wildlife Biobank at Museums Victoria Research Institute. The CVUI was adapted from the Wildlife Sperm Index (Jacobs et al., 2026) and assigns weighted scores encompassing four sequential culture stages -- establishment, first passage, expansion, and cryobanking -- with a continuous viability modifier applied at the cryobanking stage. Survival analysis identified individual animal identity as the primary source of variance in culture establishment, whereas taxon was the primary predictor of first-passage success. Beyond first passage, failure rates were too low to reliably assess either effect, with critical attrition concentrated at the earliest pipeline stages. These findings informed the differential weighting of CVUI components and have direct implications for workflow prioritisation and banking strategy. Monitoring can now be targeted at establishment and first passage, the stages where intervention has the greatest impact, with taxon-specific attention warranted at first passage and individual-level attention at establishment, while the robustness of the late-stage protocol means that banking yield is largely determined before a culture reaches P1. Integration of CVUI scores into collections management platforms such as Axiell EMu (Electronic Museum) would transform passive record-keeping into active decision support, enabling real-time quality tracking, early flagging of at-risk cultures, and longitudinal benchmarking of biobank performance across species, collectors, and tissue types. This framework provides a foundation for optimising wildlife fibroblast biobanking protocols, developing taxon-specific culture standards, and expanding standardised quality assessment across wildlife biobanks nationally and internationally.

zoology↗

Chytridiomycosis infection and heat compromises sperm quality in a threatened frog

Environmental change is reshaping wildlife reproduction through increasing temperatures and the spread of emerging infectious diseases, yet the physiological consequences of managing these stressors remain poorly understood. Amphibians are particularly vulnerable due to their ectothermy and high susceptibility to chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd). Here, we examine how Bd infection and thermal treatment interact to influence sperm quality and reproductive investment in male green and golden bell frogs (Ranoidea aurea), a species that has suffered severe population declines. Moderate Bd infection was associated with elevated sperm concentration relative to uninfected and heavily infected males, consistent with increased short-term reproductive investment under elevated mortality risk. However, severe infection led to pronounced reductions in sperm concentration and motility. Thermal treatment successfully eliminated Bd infection but imposed substantial reproductive costs: sperm concentration declined following treatment and remained significantly reduced six months later, despite partial recovery of sperm motility and membrane integrity. These results indicate persistent impairment of spermatogenic capacity rather than transient suppression. Our findings reveal that disease and thermal stress jointly shape amphibian reproductive outcomes through context-dependent trade-offs between immune defence and gamete production. While mild infection may trigger short-lived increases in reproductive output, both severe infection and pathogen clearance via thermal exposure impose lasting constraints on fertility. These results highlight an underappreciated cost of disease mitigation and suggest that increasing thermal extremes associated with climate change may further limit amphibian reproductive resilience, with important implications for conservation management and population persistence.

physiology↗

Hormonal and Environmental Drivers of Spermiation in the Endangered Mountain Yellow-Legged Frog (Rana muscosa): Toward Biologically Informed Assisted Reproductive Technologies.

Amphibians are among the most threatened vertebrates, yet assisted reproductive technologies (ARTs) remain underutilised in their conservation. We developed and evaluated a biologically relevant, non-lethal sperm collection protocol for Rana muscosa, a critically endangered frog in a long-term conservation breeding program. Thirteen hormone treatments were tested across six post-injection time points, and sperm quality was assessed via concentration, motility, osmolality, and pH. Generalised linear mixed models revealed that gonadotropin-releasing hormone (GnRHa) alone significantly outperformed hCG-based regimens. The 3 {micro}g/g GnRHa dose yielded the highest sperm concentration and sustained motility from 3 to 24 hours post-injection. Motility was highest under moderately acidic (pH 6.5-7.0) and hypoosmotic (75-100 mOsm/kg) conditions. To support decision-making, we applied a refined Wildlife Sperm Index (WSI) that weights motility and concentration as primary functional-yield traits, with motility given greater relative weight given its direct role in fertilisation, and treats pH and osmolality as secondary environmental-compatibility traits. Under this revised composite score, 3 {micro}g/g GnRHa achieved the highest overall WSI score, outperforming 2 {micro}g/g and 4 {micro}g/g GnRHa, which ranked second and third, respectively, despite their more favourable pH and osmolality profiles. These findings provide the first evidence-based ART protocol for R. muscosa and offer a transferable framework for optimising gamete collection, IVF, and cryopreservation in line with other amphibian species, advancing both genetic management and species recovery goals.

zoology↗

One Number to Rule Them All: The Wildlife Sperm Index for Standardized Gamete Assessment

In wildlife conservation, breeding programs focused on reintroduction are critical to recovering endangered species. Assisted reproductive technologies (ARTs) and biobanking play pivotal roles in these efforts but depend on high-quality gametes. Spermatozoa, due to their structural simplicity and well documented quality assessments are often considered the preferred cell type for cryopreservation and ARTs, especially in rare or exotic species. However, inconsistent retrieval of sperm samples with sufficient volume and concentration, especially for critically endangered species, can pose major limitations to assessing overall sperm quality. To address this information gap, we have developed the Wildlife Sperm Index (WSI), a standardized scoring system that integrates key sperm quality metrics--such as motility, viability, and DNA integrity--into a weighted composite score. While this study applies the WSI to sperm, the framework is adaptable to other cell types used in biobanking and reproductive technologies, broadening its potential utility. Unlike traditional sperm assessments that rely solely on microscopy, the WSI provides a scalable and quantitative framework for evaluating samples across species, including those that yield low-volume or concentration sperm samples. We validated the WSI against traditional means of sperm quality assessments, demonstrating its reliability as both a quality assessment and decision-making tool, enabling users to evaluate sample suitability for cryopreservation, ARTs, or downstream applications. By enabling standardized quality tracking, the WSI provides a foundation for more informed reproductive decisions to support global conservation efforts. Summary sentenceWe present the Wildlife Sperm Index (WSI), a standardized, composite scoring system that integrates key sperm quality metrics to improve gamete assessment and supports assisted reproduction and biobanking in wildlife conservation.

cell biology↗

Hormonal induction, quality assessments and the influence of seasonality on male reproductive viability in a long-term managed ex situ breeding colony of Southern Rocky Mountain Boreal toads, Anaxyrus boreas boreas.

The Southern Rocky Mountain Boreal Toad (Anaxyrus boreas boreas) is an ex situ managed species which relies heavily on the use of assisted reproductive technologies to augment existing in situ populations. Despite the use of ARTs to manipulate reproduction of this species, the ex situ program continues to face challenges with annual reproduction. Human chorionic gonadotropin (hCG) at 10 IU/gbw singly or in combination with 0.4 ug/gbw GnRH-A have been successfully reported for this species, however, with a number of other available hormones, it is not clear if these are the most effective protocols for this species. Moreover, there is no information on how exogenous hormone administration is affected by other factors such as seasonality. Therefore, in the first part of this study, we compare the efficacy of the 10 IU/g hCG and 0.4 Lg/g Gonadotropin Releasing Hormone agonist (GnRH-A) administered singly or in combination, as well as GnRH-Apd + Amphiplex (0.4 Lg/g GnRH-A + 10 Lg/g Metoclopramide [MET] - a dopamine antagonist), or 10 Lg/g MET alone. Spermiation responses and sperm viability were compared across treatments with hormonal curves then correlated to seasonality. The results of this study suggest that the optimal hormonal stimulation protocol, across all treatments, in terms of sperm quality was 10 IU/g hCG + 0.4 Lg/g GnRH-A. Further optimization is required, in particular the exploration of higher doses of GnRH-A. Lastly, we observed that the effect of seasonality influenced the hormonal efficacy and magnitude of the spermiation response. As was expected, quality and concentration parameters were affected by the month in which hormone administration occurred. HIGHLIGHTSO_LISpermiation in the Southern Rocky Mountain Boreal toad, Anaxyrus boreas boreas, is elicited most effectively by hCG singly or in combination with GnRH-A. C_LIO_LISpermiation responses lasted up to 96 hours post injection (hpi) though quantity and quality parameters were highest in the first 12 hpi. C_LIO_LITotal motility, forward progressive motility and percentage live cells all indicated A. b. boreas sperm quality was in a good range. C_LIO_LIAcrosome integrity requires more research as it was comparatively lower than expected. C_LIO_LIResponses to hormone inductions are seasonally affected in this species but suggest semi-discontinuous cycling. C_LIO_LIEx situ breeding should perform better sperm assessments before designing breeding strategies. C_LI

zoology↗

Refrigerated storage and cryopreservation of hormone induced sperm in a threatened frog

There are strong potential benefits of incorporating assisted reproductive technologies (ARTs) into conservation programs for the management of threatened amphibians as the global amphibian decline continues. As sperm cryopreservation and other ARTs advance in common species, focus on non-lethal sperm collection methods for threatened amphibians is imperative. We aimed to realise this goal by testing various doses of exogenous hormones for non-lethal induction of spermiation in a threatened frog (Litoria aurea) and develop cold storage and cryopreservation protocols following the recovery of urinic sperm. Our major findings include: (1) that sperm release could be induced in high concentrations with 20 IU/g bodyweight of human chorionic gonadotrophin (hCG); (2) high levels (>50%) of live, motile sperm could be recovered post-cryopreservation by treating the sperm with 15% v/v DMSO and 1% w/v sucrose pre-freeze; and (3) urinic sperm stored at 5{degrees}C retained motility over a 14-day period. Our findings demonstrate that it is possible to obtain and store large quantities of quality sperm from a threatened amphibian via non-lethal means, representing an important step forward for the use of ARTs in conservation programs for rare and threatened species. Lay SummaryAmphibians are undergoing an extinction crisis unparalleled in any other taxa. The development of assisted reproductive technologies, such as sperm cryopreservation and IVF have an important role to play in the conservation management of amphibians globally. Here we report non-lethal methods of sperm collection and storage in a highly threatened amphibian. Major findings include successfully induced sperm release in high concentrations, retention of [~]50% live, motile sperm after 7 days storing it at 5{degrees}C, and successful recovery of of more than 50% live, motile sperm following cryopreservation. Our results demonstrate the viability of obtaining and storing sperm from rare and threatened species via non-lethal means, providing an important step forward for the use of ARTs in conservation programs around the globe.

cell biology↗

Induction of Reproductive Behaviors by Exogenous Hormones in Captive Southern Rocky Mountain Boreal Toads, Anaxyrus boreas boreas

Loss of reproductive viability, physiologically and/or behaviorally, can have profound effects on the fitness of a captive population and conservation efforts. The southern rocky mountain (SRM) population of the boreal toad has declined over the past 35 years, making captive breeding necessary to protect and augment the species in the wild. In recent years, a notable reduction in the incidence of amplexus and viable offspring from the captive breeding population has been observed. Hormone treatment protocols to stimulate gamete release in males and females are established in this species and in vitro fertilization has been performed successfully. However, successful hormone stimulation of reproductive behaviors and natural fertilization has not been well documented. During the breeding season of 2012, 24 males and 24 female toads were selected from a population of over 600 captive animals. Both sexes were treated with Human chorionic gonadotropin (hCG) and Gonadotropin Releasing Hormone (GnRH) or phosphate buffered saline (PBS). Females were primed twice with 3.7IU/g hCG and then injected with an ovulatory dose (OvD) of 13.5 IU/ g BW (Body weight) hCG and 0.4 g/ g BW GnRH. Males were injected a single time with 10 IU/g BW hCG and 0.4 g/ g BW GnRH, 12 h after females received their OvD. In 2013, knowing the approximate time when females oviposited after hormone treatments, we tested the best time to induce amplexus and spermiation. Males were divided into 4 groups and injected at 4 different times: (a) 12 h before females OvD; (b) at the same time as OvD; (c) 12 h after OvD; (d) control injected with PBS. Results from 2012 indicated that oviposition was solely dependent on females receiving hormone treatments not males. However, in 2013 we found that the duration of amplexus significantly influenced oviposition (P>0.05), and males injected 12 h prior to females spent more time in amplexus than males injected at the same time or 12 h after the females received hormones. Promoting reproductive behaviors and synchronizing gamete deposition continues to be imprecise and may require more than exogenous hormones. The complexity of promoting breeding behaviors may require a closer assessment of the captive environment.

zoology↗