Spermic Urine Research Articles

The Effect of Gentamicin on the Motility of Hormonally Induced Spermatozoa of Toad Bufo bufo during Storage at 4°C.

We studied the effect of antibiotic gentamicin at concentrations of 0.05, 0.1, 0.2, 0.4, and 1 mg/ml on the maintenance of sperm motility of the common toad Bufo bufo during cold storage of spermic urine samples at 4°C. Parameters of sperm motility during storage of samples with gentamicin at concentrations of 0.05-0.4 mg/ml did not differ significantly, but were higher (p<0.0001) than in the control (storage without antibiotic). Gentamicin at a concentration of 1 mg/ml had a negative effect on sperm motility. After 2 weeks of storage of toad spermic urine samples with gentamicin, the largest number of sperm was preserved when using antibiotic at a concentration of 0.4 mg/ml.

Characterisation of the urinary microbiome of a frog, and the effect of antibiotics on bacterial abundance and sperm viability during refrigerated storage

Reproductive technologies are increasingly being adopted to improve the conservation management of threatened species. The storage and transport of sperm is a vital aspect of the practical implementation of reproductive technologies, however, bacterial contamination during the storage and transport of sperm samples presents a biosecurity risk and can contribute to a reduction in sperm longevity during storage. The present study: 1) characterised the urinary microbiome (bacterial species composition and abundance) using culture-independent 16S rRNA sequencing; 2) quantified the effect of various doses of gentamicin and streptomycin-penicillin on bacterial abundance (colony-forming units; CFUs) and; 3) quantified the effect of antibiotic supplementation on the sperm viability (proportion live/dead) of spermic urine during a 12-day cold-storage period, using the common eastern froglet, Crinia signifera. Overall, urine samples were found to host a diverse array of bacteria, dominated by the phyla Proteobacteria, Firmicutes, and Bacteroidetes. Bacterial abundance was significantly reduced in all antibiotic treatment groups compared to the control group. Antibiotic supplementation had no effect on sperm viability between day 0 and day 6 of storage, however both antibiotic treatments significantly improved sperm viability from days 9 to 12 of storage compared to the control group. Overall, the results of this study provide novel insight into the urinary microbiome, being the first to use a metagenomics approach to characterise the bacterial community present in the urine of an amphibian. Importantly, this study provides evidence that antibiotic supplementation with either gentamicin or streptomycin-penicillin, minimises bacterial proliferation and improves sperm viability during cold storage. These findings will contribute to the development of biosecurity protocols aimed at reducing the risk of disease transmission and cross-infection from unwanted bacteria and infectious agents in amphibian captive breeding programs.

Effect of Antibiotics Metranidazole, Streptomycin, and Gentamicin on the Maintenance of Sperm Motility of the European Common Frog (&lt;i&gt;Rana temporaria&lt;/i&gt;) During Refrigerated Storage

The problem of asynchrony in the production of gametes in amphibians can be solved by increasing the longevity of spermatozoa during the storage of spermic urine with antibiotics. This study evaluated the effect of metranidazole and streptomycin at concentrations of 0.1, 0.2, 0.4, and 1 mg/ml on the maintenance of motility and the longevity of spermatozoa in the spermic urine of the European common frog (Rana temporaria) during refrigerated storage at 4°C. Metranidazole did not improve the lifespan of spermatozoa, and streptomycin had a positive effect at all analyzed concentrations. In addition, the streptomycin and gentamicin at the same concentrations of 0.05, 0.1, and 0.2 mg/ml were compared for their effect on maintaining sperm motility during storage. In spermic urine samples with significant bacterial contamination, no significant differences were found in the effect of streptomycin and gentamicin on sperm motility. In the absence of any visible signs of significant bacterial contamination of spermic urine samples, the motility of spermatozoa with gentamicin remained significantly longer than that with streptomycin for all analyzed concentrations. In addition, in this study, for the first time, a positive correlation was revealed between the initial concentration of spermatozoa and the longevity of spermatozoa during the refrigerated storage of amphibian spermic urine samples with antibiotics (r = 0.899, P 0.0001). However, no relationship was found between the longevity of spermatozoa and the initial osmolality of spermic urine in the range from 62 to 92 mosmol/kg (r = 0.083, P = 0.768).

Cryopreservation Cooling Rate Impacts Post-Thaw Sperm Motility and Survival in Litoria booroolongensis.

The cryopreservation and storage of gametes (biobanking) can provide a long-term, low-cost option for the preservation of population genetic diversity and is particularly impactful when applied to manage selective breeding within conservation breeding programs (CBPs). This study aimed to develop a sperm cryopreservation protocol for the critically endangered Booroolong frog (Litoria booroolongensis) to capture founder genetics within the recently established (est. 2019) CBP for this species. Hormone-induced sperm release was achieved using established protocols, and spermic urine samples were collected over a 6-h period. Pooled spermic urine samples (n = 3 males) were divided equally between two cryoprotectant (CPA) treatments and diluted by 1:5 (sperm:CPA) with either 15% (v/v) dimethyl sulfoxide + 1% (w/v) sucrose in simplified amphibian Ringer's (SAR; CPAA) or 10% (v/v) dimethylformamide + 10% (w/v) trehalose dihydrate in SAR (CPAB). The samples were cryopreserved in 0.25 mL straws using either a programmable freezer (FrA) or an adapted dry shipper method (FrB). The thawed samples were activated via dilution in water and assessed for viability and motility using both manual assessment and computer-assisted sperm analysis (CASA; 0 h, 0.5 h post-thaw). Upon activation, the survival and recovery of motility (total motility, forward progression and velocity) of cryopreserved sperm suspensions were higher for sperm preserved using FrB than FrA, regardless of CPA composition. This work supports our long-term goal to pioneer the integration of biobanked cryopreserved sperm with population genetic management to maximize restoration program outcomes for Australian amphibian species.

Recovery and Characterization of Spermatozoa in a Neotropical, Terrestrial, Direct-Developing Riparian Frog (Craugastor evanesco) through Hormonal Stimulation.

The Vanishing Rainfrog (Craugastor evanesco) is an endemic and critically endangered frog species of Panama. It is suspected that 90% of the population has disappeared from the wild. Frogs were collected from the wild and brought to a Captive Breeding Program; however, accomplishing regular reproductive events for this species has been difficult. The objective of this study was to determine the effect of hormonal stimulation on the production and quality of C. evanesco spermatozoa, aiming to develop an efficient and safe sperm collection protocol as a tool to help reproduce this endangered species. Mature males received intra-peritoneal injections with one of six hormone treatments, including des-Gly10, D-Ala6, Pro-NHEt9-GnRH-A, Amphiplex or hCG. Urine samples were collected at 10 different time points post-injection. Quality assessments included sperm concentration, percentage motility, percentage forward progressive motility (FPM), osmolality, pH and morphology analysis. Our results indicate that the optimal treatment for the collection of highly concentrated sperm samples of C. evanesco is 4 µg/gbw GnRH, followed by Amphiplex and 2 µg/gbw GnRH as sub-optimal treatments and finally, 6 µg/gbw GnRH and 5 and 10 IU/gbw hCG as non-optimal treatments. GnRH-A at 4 μg/gbw and Amphiplex stimulated the production of samples with the highest sperm concentrations and quality, despite Amphiplex producing lower percentages of intact acrosome and tail. In contrast, hCG concentrations were not reliable inducers of sperm production, consistently showing lower concentrations, higher percentages of sperm abnormalities and more acidic spermic urine than that induced by Amphiplex and GnRH-A. Morphological assessments revealed that C. evanesco spermatozoa have a filiform shape with a large acrosome on the anterior part of an elongated head, a small midpiece and a long tail with two filaments joined together by an undulating membrane.

Applying sperm collection and cryopreservation protocols developed in a model amphibian to three threatened anuran species targeted for biobanking management

The challenge of replicating environmental cues in amphibian captive assurance colonies has been shown to be one of the contributing factors leading to limited breeding success. To circumvent this challenge, assisted reproductive technologies (ART) have been developed in model organisms and can now be applied to at-risk species. This study tested the transferability of cryopreservation protocols developed in the Fowler's toad (Anaxyrus fowleri) to three threatened anurans, the Chiricahua leopard frog (Lithobates chiricahuensis), Puerto Rican crested toad (Peltophryne lemur), and Houston toad (Anaxyrus houstonensis). Males were administered human chorionic gonadotropin and/or gonadotropin releasing hormone-analog to elicit spermiation, and spermic urine was collected and analyzed for motility. Samples were cryopreserved with 10 % N,N-dimethylformamide + 10 % trehalose + 0.25 % bovine serum albumin and frozen at two rates, −32 to −45 °C/min and − 20 to −29 °C/min. Results show that sperm frozen at −32 to −45 °C/min recovered significantly higher post-thaw sperm motility than −20 to −29 °C/min for P. lemur (p = 0.04) and L. chiricahuensis (p = 0.03). When compared across species, L. chiricahuensis had significantly higher post-thaw motility at both freezing rates (p < 0.05) than the other species, yet there was no difference between sperm motility between the three bufonid species for either freezing rate (p > 0.06). These results indicate that transferability of a cryopreservation protocol between species is possible, and differences in sensitivity and response to cryopreservation may exist between genera.

The impact of time and environmental factors on the mitochondrial vesicle and subsequent motility of amphibian sperm

For amphibian species that display external fertilization in an aquatic environment, hypoosmotic shock to sperm cells can quickly result in damage to cellular structure and function. This study sought to determine how fertilization media osmolality, temperature, and time impact the stability of the mitochondrial vesicle's association with the sperm head and thus motility and quality of forward progression. The presence of the mitochondrial vesicle and its relationship with sperm motility and quality of forward progression were analyzed in sperm samples from the Fowler's toad (Anaxyrus fowleri) (n = 10) when held for six hours under two temperatures and four osmolalities. Results indicated that the presence of the mitochondrial vesicle is needed for sperm motility over time (p < 0.001), where higher osmolalities (p < 0.001) and lower temperatures (p < 0.001) correlated with maintaining the vesicle attachment to the spermatozoa. The higher osmolality of spermic urine was the most important factor for maintaining higher quality of forward progressive motility (p < 0.01) of spermatozoa. Sperm samples held at 4 °C and 40 mOsm/kg had the longest half-life for motility (540 min) and quality of forward progression (276 min), whereas sperm held at 22 °C and 2.5 mOsm/kg had the shortest half-life for motility (7 min) and quality of forward progression (18 min). Special attention should be given to the osmolality and temperature of fertilization solutions, or breeding tank water, when developing cold storage protocols for toad sperm or reproducing animals to ensure the retention of the mitochondrial vesicle for maximum fertilization capability.

Antibiotics for the refrigerated storage at 4 °C of hormonally induced European сommon frog (Rana temporaria) spermatozoa

A valuable reproduction technology for the sustainable management of amphibian biodiversity is the refrigerated storage of spermatozoa at 4 °C. Antibiotics offer the potential to increase the storage life of refrigerated spermatozoa by inhibiting bacterial growth. In the present study, the effect of antibiotics lincomycin, enrofloxacin and gentamicin, alone or in combination, on the retention of motility, and longevity of spermatozoa in the spermic urine of the European common frog ( Rana temporaria ) during a 36-day storing period in the refrigerator at 4 °C was investigated. The percentage of motile spermatozoa declined in each treatment group over time; however, in samples supplemented with three antibiotics in combination or gentamicin alone, the sperm motility was significantly higher (from 17 to 52%) on Day 30 after sperm collection than that in samples in control and other antibacterial groups. Lincomycin did not improve the spermatozoa longevity during refrigerated storage, and enrofloxacin had a less pronounced beneficial effect than gentamicin did. Also, the effect of gentamicin at concentrations of 15, 30, 60, 100, 200, 400 µg/mL, and 1, 2, 4 mg/mL for maintaining sperm motility during refrigerated storage was evaluated. Gentamicin at concentrations of 30 to 400 µg/mL had a positive effect on the retention of sperm motility, with the highest rates of motility during storage at a concentration of 100 µg/mL. Gentamicin at a concentration of 15 µg/mL had no effect on maintaining sperm motility. The sperm motility was partially inhibited by gentamicin at concentrations of above 400 µg/mL (1 and 2 mg/mL) and stopped completely at 4 mg/mL. Moreover, gentamicin effects on the cleavage of fertilized eggs and subsequently on the hatch rates were evaluated. Аfter 30 days of refrigerated storage with 30 to 120 μg/mL gentamicin, the motility of the spermatozoa in more than half of the samples was 50% and above. The cleavage rate of eggs (55.0 ± 5.9%) and the hatching rate of cleaved eggs (68.2 ± 2.9%) did not differ significantly from those obtained for fresh spermatozoa (P < 0.05). Over all storage periods of spermatozoa in the presence of 30 to 120 μg/mL gentamicin, the cleavage rate and hatch rate of cleaved eggs were high and consistent, which may indirectly indicate the preservation of genetic material in the fertilizing spermatozoa of treated spermic urine samples.

Efficacy of hormone stimulation on sperm production in an alpine amphibian (Anaxyrus boreas boreas) and the impact of short-term storage on sperm quality

The Southern Rocky Mountain boreal toad (Anaxyrus boreas boreas) has disappeared from much of its range in the alpine regions of Central and Western North America, and restoration efforts are compromised by limited knowledge of this species’ reproductive biology. This study aimed to establish whether assisted reproductive techniques could be used to improve breeding output in captive boreal toads by determining the most effective concentration of human chorionic gonadotropin (hCG) for induction of spermiation and viability of sperm during cold storage. Male toads (n = 21) were treated with a Low (3 IU g-1), Medium (10 IU g-1), or High (15 IU g-1) concentration of hCG and spermic urine samples were collected over 24 hrs. Treatment effectiveness was evaluated by measuring the response rate, Total Motility (TM), Forward Progressive Motility (FPM), Quality of FPM (QFPM), and concentration. For short-term cold storage, spermic urine samples (n = 13) were stored at 4 °C for 14 days and sperm TM and FPM monitored daily. All treatments induced spermiation; however, a greater number of toads produced sperm in the Medium and High treatments compared to the Low. Overall, TM, FPM, QFPM and sperm concentration were similar across all three treatments, but variation existed in the timing and duration of peak sperm production. Sperm motility was maintained for up to 14 days in cold storage, although the quality slowly decreased over time. An effective reproduction strategy for the boreal toad will provide a means to improve captive breeding efforts and increase our understanding of the reproductive physiology of alpine Bufonids.

Short-term storage of tiger salamander (Ambystoma tigrinum) spermatozoa: The effect of collection type, temperature and time

The aims of this project were to characterize tiger salamander (Ambystoma tigrinum) spermatozoa motility over time, when excreted as either milt or spermic urine prior to packaging into a spermatophore, and to determine the effect of temperature on sperm motility. A split-plot design was utilized to assess the motility of the two pre-spermatophore sample types at two temperatures, 0°C and 20°C (n = 10 for each treatment). Spermiation was induced through exogenous hormone treatment of luteinizing hormone releasing hormone analog in order to collect both milt and spermic urine, which were evaluated for motility, divided into two separate aliquots, and subsequently stored in either an ice-bath (0°C) or on the benchtop (20°C). The decay rate of sperm motility was assessed by reevaluating subsamples at 0.5, 1, 2, 3, 5, 7, and 24 hours following the initial assessment. Results showed that sperm stored at 0°C had significantly higher progressive, non-progressive, and total motility for both sperm collection types over time. An interaction was found between collection type and time, with milt exhibiting lower initial motility that was more sustainable over time, compared to spermic urine. For both milt and spermic urine, motility decreased rapidly with storage duration, indicating samples should be used as soon as possible to maximize motility for in-vitro fertilization and cryopreservation. This is the first study to describe the differences in sperm motility between milt and spermic urine from an internally fertilizing caudate and demonstrates the benefits of near freezing temperatures on sperm longevity.

Fertilization potential of cold-stored Fowler&#x2019;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.

Spermiation response to exogenous hormone therapy in hibernated and non-hibernated boreal toads (Anaxyrus boreas boreas).

Conservation programs for threatened high- elevation amphibian species rely on hibernation to trigger appropriate male reproductive behaviours and gametogenesis. Although common practice and anecdotal observations have supported the practice of hibernation, there is limited empirical evidence documenting the effects on reproduction in these species. In this study, the effect of hibernation on sperm quantity and quality was evaluated for the alpine species Anaxyrus boreas boreas . Hibernated (n =19) and non-hibernated (n =21) male toads were administered 10IUg-1 body weight (BW) human chorionic gonadotropin (hCG) and spermic urine was collected over 24h. Hibernation had no effect on the number of males undergoing spermatogenesis, but hibernated males produced sperm in higher concentrations. Sperm quality was measured in terms of total motility, forward progressive motility and quality of forward progression. Although there was no difference in the total sperm motility of samples from hibernated and non-hibernated toads, the percentage of sperm exhibiting forward progressive motility and the quality of forward progression was significantly greater from hibernated toads. These results support our hypothesis that hibernation impacts both sperm quantity and quality in male boreal toads. This study will better inform captive breeding management decisions for threatened alpine species, in imminent danger of extinction.

Hormone treatment does not reliably induce spermiation or mating in Hamilton&#x2019;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.

Nasal administration of gonadotropin releasing hormone (GnRH) elicits sperm production in Fowler\u2019s toads (Anaxyrus fowleri)

BackgroundDeclining amphibian populations around the world necessitate the establishment of captive assurance colonies as a hedge against extinction. For species that are difficult to breed in captivity, assisted reproductive techniques, such as treatment with exogenous hormones, are necessary for successful reproduction. The purpose of this study was to determine whether intranasal administration of a gonadotropin releasing hormone analog (GnRHa) elicits sperm production in anurans.MethodsMale Anaxyrus fowleri (n = 15/trt) were nasally administered GnRHa (1, 5, 10, and 20 μg) in phosphate buffered saline (PBS) using a pipette. Spermic urine was collected from animals over an 8 h period. Samples were assessed for sperm presence, motility, and concentration.ResultsTreatment of male toads with a PBS control and 1 μg of GnRHa did not elicit sperm production. Nasal administration of GnRHa resulted in sperm production from 60, 93, and 80% of males in the 5, 10, and 20 μg treatment groups, respectively. Sperm motilities averaged 70, 63, and 52% within the 5, 10, and 20 μg treatment groups, respectively, with the highest (p < 0.05) sperm motility observed using 5 μg of GnRHa. Significantly higher sperm concentrations were observed in males treated with 10 μg of GnRHa compared with 5 or 20 μg of GnRHa.ConclusionNasal administration of GnRHa was successful in eliciting spermiation from male Anaxyrus fowleri, which typically began less than 3 h after treatment. Nasal administration of GnRHa may provide a novel non-invasive method of hormone delivery for at-risk amphibian species that have low reproductive output.

Cryopreservation and hormonal induction of spermic urine in a novel species: The smooth-sided toad (Rhaebo guttatus)

Global amphibian declines have fueled an increased interest in amphibian assisted reproductive technologies. Within the genus Rhaebo, half of the species are experiencing decreasing population trends; however, insufficient information is available on many of these species’ reproductive biology. Using the smooth-sided toad, Rhaebo guttatus, we present effective methods for collecting and cryopreserving an example of Rhaebo sperm. Specifically, our findings show that administering 10 IU/g body weight of hCG (human chorionic gonadotropin) yields the most motile and concentrated sperm and that cryopreserving spermic urine in a solution of 5% DMFA (N,N-Dimethylformamide) and 10% trehalose returns sperm with a 33 ± 3% average post-thaw motility. These findings may represent an important step forward in developing techniques that can be safely applied to other, more vulnerable species within the Rhaebo genus.

23 Viability staining techniques for cryopreserved spermatozoa in 3 caudata species

Salamanders are the most threatened vertebrate taxa; thus, conservation-based research including spermatozoa cryopreservation and other assisted reproductive technologies is essential to their survival. To determine the effectiveness of sperm cryopreservation, methods for evaluating sperm quality are necessary but underdeveloped in caudate research. Evaluating motility has been the primary analysis for sperm viability but is difficult to perform due to the scythe-like morphology, slow rotating progression, and minute undulations of the tail membrane. Estimating apoptosis is a new approach to evaluating caudate spermatozoa survival through cryostress. Fluorescent dyes, such as SYBR-14, annexin-V, and propidium iodide (PI), are valuable tools for identifying degrees of cell viability, apoptosis, and necrosis. Annexin-V marks the externalization of phosphatidylserine on the cell membrane indicating early steps in the apoptosis signalling cascade. Compromised membranes allow PI, a nucleic acid stain, access to DNA, marking cellular necrosis. The SYBR-14 is a nucleic acid stain that permeatesssss intact membranes, labelling live cells. These fluorescent stains were assessed for marking viability and stages of cell death in post-thaw spermatozoa across 3 caudate species: the Eastern tiger salamander (Ambystoma tigrinum), Kweichow Emperor newt (Tylototriton kweichowensis), and black-spotted newt (Notophthalmus meridionalis). For each species, spermic urine samples were acquired by hormone treatment and frozen based on protocols developed in A. tigrinum, yielding an average of 18.2% relative motility recovered at thaw. Straws were thawed for 5min at 20°C. Viability was tested by staining 5μL 1:1 with a 1:50 dilution of SYBR-14 and 2μL of PI. Stages of cell death were evaluated by staining 10μL with 2μL of annexin-V and 2μL of PI. Cell viability was assessed immediately under a fluorescence microscope. For each of the 3 species, 2 samples were stained with both assays in triplicate. Sperm stained with SYBR-14 alone were considered viable, and sperm stained with any annexin-V or PI were considered not viable. Visible dynamic shifting from SYBR-14 to PI staining was observed in real time, indicating rapid necrosis. Morphological abnormalities, not observed in unstained samples, were prevalent across all species following staining, signifying a possible cytotoxic effect of the dyes. High mortality and abnormality rates suggest that fluorescent dyes have elevated toxicity and permeability in caudate sperm. Caudate spermic urine has a very low osmolality, implying high permeability, which could lead to rapid staining and toxicity effects. Shorter incubation times may be required for accurate staining. Results may also indicate that cryopreservation protocols need to be species specific and do not transfer well across taxa. This is one of the earliest studies to evaluate the use of fluorescent stain protocols on measuring cell viability in caudate sperm and indicates that further refinement is required.

Influence of extracellular environment on the motility and structural properties of spermatozoa collected from hormonally stimulated Panamanian Golden Frog (Atelopus zeteki)

A better understanding of the factors influencing the biology of amphibian spermatozoa after release from the testis is a prerequisite to the development of sperm preservation methods. The objective of the study was to determine the effect of extracellular conditions (exposure to water and different temperatures) over time on the sperm motility and structural properties (including morphology and DNA integrity) collected from hormonally stimulated Atelopus zeteki. Following intraperitoneal injection of gonadotropin-releasing hormone agonist (des-Gly10, D-Ala6, Pro-NHEt9 GnRH; 4 μg/g of body weight), human chorionic gonadotropin (hCG, 10 IU/gbw), or Amphiplex™ (0.4 μg/gbw GnRH-A + 10 μg/gbw metoclopramide hydrochloride), spermic urine samples from 27 males were collected and analyzed for sperm motility, morphology and DNA integrity while maintained at room temperature (23 °C), 4 °C, or diluted in water (hypo-osmotic environment) over a period of 46 min post-collection. Percentages of sperm motility and forward progressive motility remained high (>60%) when spermic urine was kept at room temperature or at 4 °C for 46 min regardless of the hormonal stimulation method. Dilution in water at room temperature greatly reduced the percentage of motile spermatozoa and forward progression (<50%) as well as DNA integrity (32.8% of intact cells) after 23 min while morphology did not differ (30.4% of normal cells), regardless of the hormone stimulation. This is the first systematic study on the effect of extracellular environment over time on A. zeteki sperm quality. This will contribute to the development of sperm handling protocols and reproductive technologies for this and other endangered Atelopus species.

Effects of hormonal stimulation on the concentration and quality of excreted spermatozoa in the critically endangered Panamanian golden frog (Atelopus zeteki)

Knowledge of basic gamete biology is critical to better protect and propagate endangered amphibian species and also to develop reproductive technologies combined with germplasm cryopreservation. The objectives of the study were to test different hormonal stimulations and then characterize the quantity and quality of Panamanian golden frog (Atelopus zeteki) spermatozoa. Following intraperitoneal injection of the gonadotropin-releasing hormone agonist (des-Gly10, D-Ala6, Pro-NHEt9--GnRH 1, 2 or 4 μg/g of body weight), human chorionic gonadotropin (hCG; 5 or 10 IU/gbw), or Amphiplex™ (0.4 μg/gbw GnRH-A + 10 μg/gbw metoclopramide hydrochloride), spermic urine samples from 29 males were collected at different time points (from 0.5 to 24 h post-injection) to analyze the concentration, motility, and morphology of the spermatozoa. Peak of sperm concentration was observed at 3.5 h post injection for all hormonal treatments. Amphiplex™ led to the highest sperm concentrations (4.45 ± 0.07 × 106 cells/mL) followed by 4 μg/gbw GnRH-A (2.65 ± 0.21 × 106 cells/mL). Other stimulation protocols and doses induced sperm production, but at lower levels (ranging from 1.34 to 1.70 × 106 cells/mL). More than 60% of spermatozoa were motile following all treatments but the highest motility (>90%) was obtained from the 10 IU/gbw hCG treatment. Spermic urine samples obtained with all hormone treatments had higher pH (ranging from 7.1 to 7.8) than the urine alone (6.7–6.8). Spermatozoa were filiform and elongated with an apical acrosome, a mitochondrial sheath, a small midpiece and a long tail with an undulating membrane. More than 80% of cells were morphologically normal and 50–70% had intact DNA. These sperm characteristics were not influenced by hormonal treatments. This first comprehensive characterization of sperm samples following optimized hormonal stimulations in A. zeteki lays the foundation for more fundamental studies, reproductive technologies, and future preservation strategies.

Variation in male spermiation response to exogenous hormones among divergent populations of Red-eyed Treefrogs.

BackgroundThe non-lethal collection of sperm from live males is an important component for multiple captive-breeding techniques, including assisted reproductive technology (ART) protocols, sperm cryopreservation and in vitro fertilization. However, in amphibians, the type and amount of hormone necessary to induce spermiation can be highly variable, even among closely related species. We are unaware of any studies that have examined the spermiation response to exogenous hormones across highly differentiated populations within a species.MethodsWe examined variation in sperm viability and production in response to the hormone LHRH among four divergent populations of the Red-eyed Treefrog (Agalychnis callidryas). We hypothesized that these highly differentiated populations would show variability in sperm count and viability in response to two dosages, 2 μg/g and 4 μg/g, of the hormone LHRH. We collected spermic urine 3 h post injection (PI). We then examined variation in spermiation at 3, 7, 12, and 24 h PI of LHRH for two allopatric populations that previously showed evidence of premating behavioral isolation.ResultsOne population of Red-eyed Treefrog exhibited reduced sperm viability but not count in response to the hormone LHRH compared to all other populations. In addition, we found peak viability at 3 h PI for the allopatric population comparison. There was no difference in sperm production within or between populations at 3, 7, 12, or 24 h PI. For both studies, intrapopulation variation was high.ConclusionART often focuses on threatened species with small, isolated populations, which could evolve localized differences due to the evolutionary process of drift and selection. The high variation in response and the population-level differences in sperm viability we observed demonstrate that practitioners of ART should consider the possibility of divergent responses to hormones which may affect study design and animal receptivity to ART protocols.Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-016-0216-3) contains supplementary material, which is available to authorized users.

The effect of gentamicin on sperm motility and bacterial abundance during chilled sperm storage in the Booroolong frog

Antibiotics can inhibit bacterial contamination and extend sperm longevity during storage; a primary goal of captive facilities conducting biobanking and artificial fertilisation (AF). This study evaluated the effects of gentamicin on the short-term storage of Booroolong frog sperm. Sperm suspensions were obtained via either testis maceration, or as spermic urine, following hormonal induction of sperm-release. The effect of 0, 1, 2, 3 or 4mgmL−1 gentamicin on bacterial abundance (CFUmL−1) was determined and sperm motility assessed. In both testis macerate samples and spermic urine samples, gentamicin administered at intermediate-to-high doses (2, 3 & 4mgmL−1) eliminated, or significantly reduced, bacterial abundance. Sperm samples obtained via testis maceration exhibited significantly lower sperm motility at the highest doses (3 & 4mgmL−1). All remaining treatments (0, 1 & 2mgmL−1) were statistically similar and maintained sperm motility >55%. Sperm samples obtained as spermic urine exhibited no difference in sperm motility or velocity when treated with gentamicin at any dose. While antibiotic treatment did not improve sperm longevity as predicted, this is the first study to demonstrate that antibiotic treatment can reduce bacterial abundance without compromising sperm motility in an anuran amphibian. Antibiotic supplementation may be an important tool for reducing pathogen transmission where sperm samples are transferred between captive institutions for biobanking and AF.