Exploring pathways toward open-hardware ecosystems to safeguard genetic resources for biomedical research communities using aquatic model species.

Development of reliable germplasm repositories is critical for preservation of genetic resources of aquatic species, which are widely utilized to support biomedical innovation by providing a foundational source for naturally occurring variation and development of new variants through genetic manipulations. A significant barrier in repository development is the lack of cryopreservation capability and reproducibility across the research community, posing great risks of losing advances developed from billions of dollars of research investment. The emergence of open scientific hardware has fueled a new movement across biomedical research communities. With the increasing accessibility of consumer-level fabrication technologies, such as three-dimensional printers, open hardware devices can be custom designed, and design files distributed to community members for enhancing rigor, reproducibility, and standardization. The overall goal of this review is to explore pathways to create open-hardware ecosystems among the communities using aquatic model resources for biomedical research. To gain feedback and insights from community members, an interactive workshop focusing on open-hardware applications in germplasm repository development was held at the 2022 Aquatic Models for Human Disease Conference, Woods Hole, Massachusetts. This work integrates conceptual strategies with practical insights derived from workshop interactions using examples of germplasm repository development. These insights can be generalized for establishment of open-hardware ecosystems for a broad biomedical research community. The specific objectives were to: (1) introduce an open-hardware ecosystem concept to support biomedical research; (2) explore pathways toward open-hardware ecosystems through four major areas, and (3) identify opportunities and future directions.

Establishment of a Practical Sperm Cryopreservation Pathway for the Axolotl (Ambystoma mexicanum): A Community-Level Approach to Germplasm Repository Development.

The axolotl (Ambystoma mexicanum) draws great attention around the world for its importance as a biomedical research model, but housing and maintaining live animals is increasingly expensive and risky as new transgenic lines are developed. The goal of this work was to develop an initial practical pathway for sperm cryopreservation to support germplasm repository development. The present study assembled a pathway through the investigation of axolotl sperm collection by stripping, refrigerated storage in various osmotic pressures, cryopreservation in various cryoprotectants, and in vitro fertilization using thawed sperm. By the stripping of males, 25-800 µL of sperm fluid was collected at concentrations of 1.6 × 106 to 8.9 × 107 sperm/mL. Sperm remained motile for 5 d in Hanks' Balanced Salt Solution (HBSS) at osmolalities of 100-600 mOsm/kg. Sperm cryopreserved in 0.25 mL French straws at 20 °C/min in a final concentration of 5% DMFA plus 200 mM trehalose and thawed at 25 °C for 15 s resulted in 52 ± 12% total post-thaw motility. In six in vitro fertilization trials, 20% of eggs tested with thawed sperm continued to develop to stage 7-8 after 24 h, and a third of those embryos (58) hatched. This work is the first report of successful production of axolotl offspring with cryopreserved sperm, providing a general framework for pathway development to establish Ambystoma germplasm repositories for future research and applications.

Transitioning from a research protocol to a scalable applied pathway for Xenopus laevis sperm cryopreservation at a national stock center: The effect of cryoprotectants.

Sperm cryopreservation is a critical tool for safeguarding and managing valuable genetic resources. Protocols for cryopreservation of Xenopus laevis sperm were available but lacking sperm quality evaluation and scalability and the outcomes were inconsistent. The goal of this study was to begin developing a center-level cryopreservation pathway for this species by integrating French straws as containers that would facilitate germplasm repository development. The objectives were to analyze the effect of: (1) three sperm concentrations (33, 50, and 100 × 106 sperm/mL) on post-thaw fertilization, (2) three final concentrations (2.5%, 5%, and 10%) of dimethyl sulfoxide, methanol, and dimethylformamide (DMFA) on sperm membrane integrity of fresh and frozen samples, (3) two concentrations (5% and 10%) of DMFA with and without 5% sucrose at four cooling rates (5, 10, 20, and 40°C/min) on sperm membrane integrity and motility, and (4) egg exposure to different concentrations of DMFA on fertilization. Few differences in sperm viability were found among fresh samples incubated in cryoprotectants, but thawed samples frozen in methanol or DMFA presented higher membrane integrity. Samples frozen in 10% DMFA at 20°C/min showed higher membrane integrity (60 ± 7%) than other DMFA concentrations and cooling rates, and the same total motility (30 ± 7%) as at 10°C/min. Higher DMFA concentrations (10%-13%) were detrimental for embryo development compared to lower concentrations (<6%). This study provided a reliable protocol for sperm cryopreservation in Xenopus laevis to yield an application pathway with potential for high throughput that can be used as a roadmap for work with other species.

Birds living near airports do not show consistently higher levels of feather corticosterone.

Noise represents a threat to human and wildlife health, triggering physiological and behavioral challenges to individuals living close to sources of extreme noise. Here, we considered airport environments as sources of potentially stressful stimuli for birds and tested if those living near airports are under higher physiological stress than birds living in quiet sites. We used measurements of CORT in feathers (CORTf) as a proxy of chronic stress. We evaluated 14 passerine and 1 non-passerine species, living near three Brazilian airports. We found that, across species, individuals with a better body condition had lower CORTf concentration. At the species level, we found that CORTf concentration was not consistently affected by airport noise. Comparing individuals living in quiet sites with those living near airports, we found that 2 species had higher and 2 had lower CORTf concentrations near airports, while 11 species presented no significant differences between sites. At the population level, model selection indicated that the direction and strength of these differences are weakly related to species' song frequency (peak frequency), as lower-frequency singers tended to present higher CORTf levels at airport-affected sites. In summary, we were unable to find a consistent response among species, probably due to species-specific differences in their response to anthropogenic disturbances. Instead, we found that species might be affected differently according to their singing spectral frequency and that individuals in good body condition show lower CORTf, suggesting that this measure is consistent with lower physiological stress.

Gamete Collection, Artificial Fertilization and Captive-Rearing of Eggs in a Terrestrial-Breeding Anuran with Parental Care: Alytes obstetricans.

The genus Alytes consists of six primitive terrestrial-breeding species (five of them endangered or present in restricted distributions) with unique male parental care. Alytes obstetricans was used as a model for the development of assisted reproductive technologies for gamete collection, artificial fertilization and captive-rearing of embryos. Sperm collection was achieved with human chorionic gonadotropin (hCG), but only 17 to 50% of males responded to the hormone. In addition, an effect of captivity on sperm quality was observed. A combination of gonadotropin-releasing hormone agonist (GnRHa) and hCG was tested for induction of ovulation, and 100% of females responded to the treatment. Recently collected sperm was used in artificial fertilization, and 10% of eggs showed complete embryo development. To design a method for captive-rearing of embryos, natural clutches (n = 13) were removed from males and divided into two sub-clutches. One was kept with all eggs together and in the other, the egg string was cut and eggs were split individually. All sub-clutches with eggs kept separated presented embryo survival (ranging from 27 to 61%) that hatched normally, while only one sub-clutch with embryos maintained together had some embryo survival (36%). These results may contribute to the management of this and related species.

Ciliate Microtubule Diversities: Insights from the EFBTU3 Tubulin in the Antarctic Ciliate Euplotes focardii.

Protozoans of the Phylum Ciliophora (ciliates) assemble many diverse microtubular structures in a single cell throughout the life cycle, a feature that made them useful models to study microtubule complexity and the role of tubulin isotypes. In the Antarctic ciliate Euplotes focardii we identified five ß-tubulin isotypes by genome sequencing, named EFBTU1, EFBTU2, EFBTU3, EFBTU4 and EFBTU5. By using polyclonal antibodies directed against EFBTU2/EFBTU1 and EFBTU3, we show that the former isotypes appear to be involved in the formation of all microtubular structures and are particularly abundant in cilia, whereas the latter specifically localizes at the bases of cilia. By RNA interference (RNAi) technology, we silenced the EFBTU3 gene and provided evidence that this isotype has a relevant role in cilia regeneration upon deciliation and in cell division. These results support the long-standing concept that tubulin isotypes possess functional specificity in building diverse microtubular structures.

Cryopreservation of Hydractinia symbiolongicarpus Sperm to Support Community-Based Repository Development for Preservation of Genetic Resources.

Hydractinia symbiolongicarpus is an emerging model organism in which cutting-edge genomic tools and resources are being developed for use in a growing number of research fields. One limitation of this model system is the lack of long-term storage for genetic resources. The goal of this study was to establish a generalizable cryopreservation approach for Hydractinia that would support future repository development for other cnidarian species. Specific objectives were to: (1) characterize basic parameters related to sperm quality; (2) develop a generalizable approach for sperm collection; (3) assess the feasibility of in vitro fertilization (IVF) with sperm after refrigerated storage; (4) assess the feasibility of IVF with sperm cryopreserved with various sperm concentrations; (5) evaluate feasibility of cryopreservation with various freezing conditions, and (6) explore the feasibility of cryopreservation by use of a 3-D printed open-hardware (CryoKit) device. Animal husbandry and sperm collection were facilitated by use of 3-D printed open hardware. Hydractinia sperm at a concentration of 2 × 107 cells/mL stored at 4 °C for 6 d were able to achieve 50% fertilization rate. It appeared that relatively higher sperm concentration (>5 × 107 cells/mL) for cryopreservation could promote fertilization. A fertilization rate of 41−69% was observed using sperm equilibrated with 5, 10, or 15% (v/v) cryoprotectant (dimethyl sulfoxide or methanol) for 20 min, cooled at a rate of 5, 10, or 20 °C/min from 4 °C to −80 °C, at a cell concentration of 108/mL, in 0.25 mL French straws. Samples cryopreserved with the CryoKit produced a fertilization rate of 72−82%. Establishing repository capabilities for the Hydractinia research community will be essential for future development, maintenance, protection, and distribution of genetic resources. More broadly, these generalizable approaches can be used as a model to develop germplasm repositories for other cnidarian species.

Niche differentiation drives microbial community assembly and succession in full-scale activated sludge bioreactors.

Network models and community phylogenetic analyses are applied to assess the composition, structure, and ecological assembly mechanisms of microbial communities. Here we combine both approaches to investigate the temporal dynamics of network properties in individual samples of two activated sludge systems at different adaptation stages. At initial assembly stages, we observed microbial communities adapting to activated sludge, with an increase in network modularity and co-exclusion proportion, and a decrease in network clustering, here interpreted as a consequence of niche specialization. The selective pressure of deterministic factors at wastewater treatment plants produces this trend and maintains the structure of highly functional and specialized communities responding to seasonal environmental changes.

Fertilization potential of cold-stored Fowler’s toad (Anaxyrus fowleri) spermatozoa: temporal changes in sperm motility based on temperature and osmolality.

Asynchrony of gamete release is problematic in amphibian captive breeding programs but can be overcome by short-term storage of spermatozoa. Hormonally induced sperm from the model species Anaxyrus fowleri were used to determine storage conditions for optimal fertilisation capacity. Sperm motility was measured over time, as a function of storage temperature (4°C or 22°C) and solution osmolality (7-40mOsm/kg). Sperm at 40mOsm/kg (spermic urine) stored at 4°C exhibited higher motility compared to 22°C. Also, sperm stored at 40mOsm/kg retained higher motility compared to sperm stored below 15mOsm/kg at both temperatures. Under optimal storage conditions (40mOsm and 4°C) a 30% decrease in sperm motility occurred within 24h, however, subsequent loss of sperm motility was lower (<10%/day) for days2-8 thereafter. Sperm samples stored for 1-8days under optimal conditions were tested for fertilising capacity by conducting in vitro fertilisation trials. Sperm stored for 8days yielded 48% neurula development, similar to sperm stored for 1day, which produced 60% neurula development. Overall, sperm stored for up to 8days at 4°C as spermic urine retained fertilising capacity and thus can be used to circumvent asynchronous gamete release in assisted breeding efforts for amphibians.

Determining the effects of sperm activation in anuran cloaca on motility and DNA integrity in Epidalea calamita (Bufonidae).

The effect of time inside the animal's cloaca on sperm quality after hormone-induced spermiation is unknown. However, this knowledge is critical for the development of assisted reproductive biotechnologies in amphibians. Out-of-season spermatozoa were collected from Epidalea calamita for 4h after injection of 10IU g-1 human chorionic gonadotrophin either hourly (Group I (n =10); four samples per male) or every 2h (Group II (n =9); two samples per male). Sperm samples were assessed for motility and DNA integrity using the sperm chromatin dispersion (SCD) test and the sperm chromatin structure assay (SCSA). The collection strategy affected total motility (mean (±s.e.m.) 84.4±9.9% vs 73.6±16.7% in Group I and II respectively; P =0.014) and the sperm motility index (67.6±17.7% vs 57.6±16.3% in Group I and II respectively; P =0.034). There was a significant effect of the male in Group II, but not in Group I. In Group I, the quality of the first samples collected was lower than that of samples collected thereafter (P ≤ 0.032). No significant correlations were found between the results of the SCD test and SCSA, showing that these techniques provide different information in this species. In conclusion, collecting spermatozoa every hour resulted in better sperm quality and may be more efficient. However, the between-male differences were considerable and collection of spermatozoa at just 1h after hormone treatment produced lower-quality spermatozoa.

Hormone treatment does not reliably induce spermiation or mating in Hamilton’s frog from the archaic leiopelmatid lineage.

Amidst a global amphibian decline, captive breeding has become an important component of many conservation management programs. Some species fail to readily reproduce in captivity, including leiopelmatid frogs, an archaic lineage endemic to New Zealand. Assisted reproductive technologies can improve the reproductive potential and genetic management of captive programs. Herein we describe efforts to induce spermiation and mating in the threatened Hamilton's frog Leiopelma hamiltoni using a gonadotrophin-releasing hormone agonist and human chorionic gonadotrophin. The spermiation response was poor, with few individuals releasing spermatozoa and a low sperm concentration (mean (±s.e.) 27.4±8.5×103 spermatozoa mL-1 ) when spermiation was stimulated. The sperm concentration peaked 12-24h after hormone administration, suggesting this species may be slower to react than other anurans. Spermic urine was present in five males before hormone treatment. The sperm concentration was higher in this group (128.7±54.9×103 spermatozoa mL-1 ) than in the hormone-induced males. All spermatozoa observed exhibited no motility or, at most, a limited swaying movement with no forward progression. In separate hormone induction trials with 60 individuals (two males to one female per trial), no mating was exhibited during the 12h after treatment. Further studies to hormonally stimulate gamete release and mating are needed in leiopelmatids.

Effect of Season and Social Environment on Semen Quality and Endocrine Profiles of Three Endangered Ungulates (Gazella cuvieri, G. dorcas and Nanger dama).

Knowledge of factors affecting semen quality could be of great importance for the collection and preservation of semen from threatened animals. To assess the effect of seasonality, sperm parameters and testosterone levels were examined throughout the year and compared with the distribution of conceptions. Cuvier's gazelle showed higher sperm quantity in April, coinciding with one peak of conceptions. In dorcas gazelle, sperm parameters showed a drop in October. However, percentage of conceptions increased during that month. In Mohor gazelle, sperm quality was best in April and August, in agreement with higher conception rates and high testosterone levels. Percentage of conceptions was correlated with photoperiod and rainfall in Cuvier's gazelle and with temperature in Mohor gazelle. To assess the effect of social environment, semen quality, testosterone and cortisol levels were quantified in males housed alone, in bachelor groups or with females. No differences were seen in Cuvier's and Mohor gazelles' semen traits, whereas dorcas males housed with females showed lower semen quality than males kept alone or with other males. Overall, ejaculate quality is influenced by seasonal factors in the three gazelle species, while social factors only appear to affect that of dorcas gazelle.

Tuning up microbiome analysis to monitor WWTPs' biological reactors functioning.

Wastewater treatment plants (WWTPs) are necessary to protect ecosystems quality and human health. Their function relies on the degradation of organic matter and nutrients from a water influent, prior to the effluent release into the environment. In this work we studied the bacterial community dynamics of a municipal WWTP with a membrane bioreactor through 16S rRNA gene sequencing. The main phyla identified in the wastewater were Proteobacteria, Bacteroidetes, Chloroflexi, Planctomycetes and Actinobacteria. The WWTP is located in Spain and, like other studied WWTP in temperate climate zones, the temperature played a major role in community assembly. Seasonal community succession is observed along the two years sampling period, in addition to a continual annual drift in the microbial populations. The core community of the WWTP bioreactor was also studied, where a small fraction of sequence variants constituted a large fraction of the total abundance. This core microbiome stability along the sampling period and the likewise dissimilarity patterns along the temperature gradient makes this feature a good candidate for a new process control in WWTPs.

Hormonal induction of spermiation in a Eurasian bufonid (Epidalea calamita).

BACKGROUND: Amphibian diversity is declining at an alarming rate due to habitat loss, invasive species, climate change and diseases. Captive assurance colonies have been established for some species at risk; however, many species do not breed well in captivity and the development of assisted reproductive technologies (ART) is critical to help sustain genetic diversity. To date, the majority of the work has been accomplished in species from the American continent and Australia, and there is a need to address similar breeding challenges in Eurasian and African species of amphibians. METHODS: The aim of this study was to develop a hormone protocol for stimulation of spermiation in Epidalea calamita as a model for Eurasian bufonids. Hence, the effect on sperm production and quality of three doses of chorionic gonadotropin hormone (5, 10 and 15 IU hCG/g) over time (1 to 24 h) was evaluated. In addition, cold storage (at 5 °C) of sperm for 24 and 48 h and three frequencies for hormonal treatment (weekly, biweekly and monthly) were examined. RESULTS: Hormone concentrations of 10 or 15 IU of hCG induced spermiation in 100% of males and produced sperm of comparable quality, while 5 IU hCG stimulated spermiation in only 40% of males. Total motility peaked between 1 to 4 h post-treatment with 10 IU hCG, whereas treatment with 15 IU hCG peaked between 2 to 6 h. After 24 h of cold storage total motility dropped by 20% and forward motility dropped by 10% for both the 10 and 15 IU treatments. Weekly hormone administration resulted in higher variation between trials in all motility parameters and a lower overall Total Motility and Forward Movement. Furthermore, the effect of exogenous hormone treatment overlapped between the last two trials in the weekly frequency. Sperm concentration was higher in the first trial for all frequencies but showed no differences among other trials. CONCLUSIONS: Overall, these results show that hormone concentration, time after treatment, frequency of hormone treatment and cold storage should be borne in mind when developing a hormone stimulation protocol for Eurasian amphibian species.

Unravelling mechanisms of bacterial quorum sensing disruption by metal-based nanoparticles.

Nanoparticles are released in the environment causing a negative impact in several ecosystems such as microbial communities. To adapt to environmental changes some bacteria use a collective behaviour ruled by a cell-to-cell communication process called quorum sensing (QS). In this study, the impact of some of the most employed metal-based nanoparticles, such as zinc oxide nanoparticles (ZnONPs), titanium dioxide nanoparticles (TiO2NPs) and silver nanoparticles (AgNPs) on bacterial QS has been assessed by using two different strains of the model organism Chromobacterium violaceum and by employing different experimental conditions. TiO2NPs were tested with and without applying a previous step of UV-irradiation while the effect of AgNPs of two diameter sizes (40 and 60 nm) and two different coating agents (PVP and citrate) was evaluated. Results evidenced that all nanoparticles produced a significant effect on violacein production and therefore, in the QS system. ZnONPs mainly disrupted the QS steps related to signal perception and response whereas TiO2NPs and AgNPs affected the autoinducer biosynthesis. AgNPs with the smallest size and citrate as capping agent produced the most deleterious effect while the impact of TiO2NPs was not affected by UV irradiation. The present study provides new insights into the mechanisms by which these commonly employed metal-based nanoparticles disturb bacterial QS-based communication and clearly evidences the potential risk of releasing nanoparticles to the environment, especially for microbial communities which play a key role in many environmental and technological processes.

Selenium and tellurium-based nanoparticles as interfering factors in quorum sensing-regulated processes: violacein production and bacterial biofilm formation.

A cell-to-cell communication system called quorum sensing (QS) promotes the transcription of certain target genes in bacterial cells leading to the activation of different cellular processes, some of them related to bacterial biofilm formation. The formation of bacterial biofilms favours antibiotic resistance, which is nowadays a significant public-health problem. In this study, the effect of selenium (SeNPs) and tellurium (TeNPs) nanoparticles was examined in two bacterial processes mediated by QS: violacein production by Chromobacterium violaceum and biofilm formation by Pseudomonas aeruginosa. For this purpose, quantification of the pigment production in the presence of these nanoparticles was monitored using the C. violaceum strain. Additionally, a combination of different microscopical imaging techniques was applied to examine the changes in the 3D biofilm structure of P. aeruginosa, which were quantified through performing architectural metric calculations (substratum area, cell area coverage and biovolume). SeNPs produce an 80% inhibition in the violacein production by C. violaceum and a significant effect on the P. aeruginosa biofilm architecture (a reduction of 80% in the biovolume of the bacterial biofilm was obtained). TeNPs similarly affect violacein production and the P. aeruginosa biofilm structure but at lower concentration levels. The results obtained suggest an important disruption of the QS signalling system by SeNPs and TeNPs, supporting nanotechnology as a promising tool to fight against the emerging problem of bacterial resistance related to bacterial biofilm formation.

Quorum Sensing versus Quenching Bacterial Isolates Obtained from MBR Plants Treating Leachates from Municipal Solid Waste.

Quorum sensing (QS) is a mechanism dependent on bacterial density. This coordinated process is mediated by the synthesis and the secretion of signal molecules, called autoinducers (AIs). N-acyl-homoserine lactones (AHLs) are the most common AIs that are used by Gram-negative bacteria and are involved in biofilm formation. Quorum Quenching (QQ) is the interference of QS by producing hydrolyzing enzymes, among other strategies. The main objective of the present study was to identify QS and QQ strains from MBR wastewater treatment plants. A total of 99 strains were isolated from two Spanish plants that were intended to treat leachate from municipal solid waste. Five AHL producers were detected using AHL biosensor strains (Chromobacterium violaceum CV026 and Agrobacterium tumefaciens NT1). Fifteen strains of seventy-one Gram-positive were capable of eliminating or reducing at least one AHL activity. The analysis of 16S rRNA gene sequence showed the importance of the Pseudomonas genus in the production of biofilms and the relevance of the genus Bacillus in the disruption of the QS mechanism, in which the potential activity of lactonase or acylase enzymes was investigated with the aim to contribute to solve biofouling problems and to increase the useful lifespan of membranes.

Unravelling the interactions among microbial populations found in activated sludge during biofilm formation.

Microorganisms colonize surfaces and develop biofilms through interactions that are not yet thoroughly understood, with important implications for water and wastewater systems. This study investigated the interactions between N-acyl homoserine lactone (AHL)-producing bacteria, yeasts and protists, and their contribution to biofilm development. Sixty-one bacterial strains were isolated from activated sludge and screened for AHL production, with Aeromonas sp. found to be the dominant AHL producer. Shewanella xiamenensis, Aeromonas allosaccharophila, Acinetobacter junii and Pseudomonas aeruginosa recorded the highest adherence capabilities, with S. xiamenensis being the most effective in surface colonization. Additionally, highly significant interactions (i.e. synergic or antagonistic) were described for dual and multistrain mixtures of bacterial strains (P. aeruginosa, S. xiamenensis, A. junii and Pseudomonas stutzeri), as well as for strongly adherent bacteria co-cultured with yeasts. In this last case, the adhered biomass in co-cultures was lower than the monospecific biofilms of bacteria and yeast, with biofilm observations by microscopy suggesting that bacteria had an antagonist effect on the whole or part of the yeast population. Finally, protist predation by Euplotes sp. and Paramecium sp. on Aeromonas hydrophila biofilms not only failed to reduce biofilm formation, but also recorded unexpected results leading to the development of aggregates of high density and complexity.

Membrane bioreactor wastewater treatment plants reveal diverse yeast and protist communities of potential significance in biofouling.

The yeast community was studied in a municipal full-scale membrane bioreactor wastewater treatment plant (MBR-WWTP). The unexpectedly high diversity of yeasts indicated that the activated sludge formed a suitable environment for them to proliferate, with cellular concentrations of 2.2 ± 0.8 × 10(3) CFU ml(-1). Sixteen species of seven genera were present in the biological reactor, with Ascomycetes being the most prevalent group (93%). Most isolates were able to grow in a synthetic wastewater medium, adhere to polyethylene surfaces, and develop biofilms of variable complexity. The relationship between yeast populations and the protists in the MBR-WWTP was also studied, revealing that some protist species preyed on and ingested yeasts. These results suggest that yeast populations may play a role in the food web of a WWTP and, to some extent, contribute to membrane biofouling in MBR systems.

Xenografting of testicular tissue pieces: 12 years of an in vivo spermatogenesis system.

Spermatogenesis is a dynamic and complex process that involves endocrine and testicular factors. During xenotransplantation of testicular tissue fragments into immunodecifient mice, a functional communication between host brain and donor testis is established. This interaction allows for the progression of spermatogenesis and recovery of fertilisation-competent spermatozoa from a broad range of mammalian species. In the last few years, significant progress has been achieved in testis tissue xenografting that improves our knowledge about the factors determining the success of grafting. The goal of this review is to provide up to date information about the role of factors such as donor age, donor species, testis tissue preservation or type of recipient mouse on the efficiency of this technique. Applications are described and compared with other techniques with similar purposes. Recent work has demonstrated that testicular tissue xenografting is used as a model to study gonadotoxicity of drugs and to obtain sperm from valuable young males.