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.

Prospective, randomized, double blind comparison of suture materials with and without triclosan in dogs undergoing tibial plateau leveling osteotomy.

Objectives: To determine if triclosan-impregnated suture decreases surgical site infection rates after tibial plateau leveling osteotomy (TPLO) in dogs. Sample population: There were 116 dogs with naturally occurring cranial cruciate ligament disease presenting for treatment with TPLO. Procedures: Written consent was obtained by all clients in order to be included in this study. Dogs were randomly assigned a suture type immediately before the start of anesthesia. Infection rates were compared between the suture groups, as were the gender, duration of anesthesia, duration of surgery, age of dog, weight, length of incision, and stifle side. Direct examination by a veterinarian was conducted at 24 h, 10 to 14 d, and 8 to 12 wk after surgery. If the dogs did not return for direct examination, owners were contacted by a veterinarian and phone interviews were conducted. Results: Overall, 12.9% of the incisions were diagnosed with a surgical site infection (SSI). The SSI rate for dogs that received the triclosan suture was 5.35% (3/56), and the rate for dogs that received the regular suture was 19.64% (11/56), with P = 0.016. The duration of anesthesia, duration of surgery, age, weight, length of incision, and right versus left stifle did not show a significant difference in infection rates. The suture type did have a significant effect, and triclosan-impregnated suture had a decreased infection rate when compared to regular suture. Gender also had a significant effect, with P = 0.032. Conclusion: Triclosan-impregnated suture decreased SSI when used for closure in dogs undergoing TPLO. Triclosan-impregnated suture may be considered a material of choice to close surgical wounds at risk of SSI when implants are used.

Comparaison prospective, randomisée, en double aveugle des matériaux de suture avec et sans triclosan chez les chiens subissant une ostéotomie de nivellement du plateau tibial. Objectifs: Déterminer si la suture imprégnée de triclosan diminue les taux d'infection du site opératoire après une ostéotomie de nivellement du plateau tibial (TPLO) chez le chien. Échantillon de population: Il y avait 116 chiens avec une pathologie naturelle du ligament croisé crânial se présentant pour un traitement avec TPLO. Procédures: Un consentement écrit a été obtenu par tous les clients afin d'être inclus dans cette étude. Les chiens ont été répartis au hasard à un type de suture immédiatement avant le début de l'anesthésie. Les taux d'infection ont été comparés entre les groupes de suture, de même que le sexe, la durée de l'anesthésie, la durée de la chirurgie, l'âge du chien, le poids, la longueur de l'incision et le côté du grasset. Un examen direct par un vétérinaire a été effectué à 24 h, 10 à 14 j et 8 à 12 semaines après la chirurgie. Si les chiens ne revenaient pas pour un examen direct, les propriétaires étaient contactés par un vétérinaire et des entretiens téléphoniques étaient menés. Résultats: Dans l'ensemble, 12,9 % des incisions ont été diagnostiquées avec une infection du site opératoire (SSI). Le taux de SSI pour les chiens ayant reçu la suture au triclosan était de 5,35 % (3/56) et le taux pour les chiens ayant reçu la suture régulière était de 19,64 % (11/56), avec P = 0,016. La durée de l'anesthésie, la durée de la chirurgie, l'âge, le poids, la longueur de l'incision et le grasset droit versus le gauche n'ont pas montré de différence significative dans les taux d'infection. Le type de suture avait un effet significatif et la suture imprégnée de triclosan avait un taux d'infection réduit par rapport à la suture ordinaire. Le sexe avait également un effet significatif, avec P = 0,032. Conclusion: La suture imprégnée de triclosan a diminué le SSI lorsqu'elle était utilisée pour la fermeture de l'incision chez les chiens subissant une TPLO. La suture imprégnée de triclosan peut être considérée comme un matériau de choix pour fermer les plaies chirurgicales à risque de SSI lorsque des implants sont utilisés.(Traduit par Dr Serge Messier).

Phylogenetic investigation of skin sloughing rates in frogs: relationships with skin characteristics and disease-driven declines.

Amphibian skin is highly variable in structure and function across anurans, and plays an important role in physiological homeostasis and immune defence. For example, skin sloughing has been shown to reduce pathogen loads on the skin, such as the lethal fungus Batrachochytrium dendrobatidis ( Bd), but interspecific variation in sloughing frequency is largely unknown. Using phylogenetic linear mixed models, we assessed the relationship between skin turnover rate, skin morphology, ecological traits and overall evidence of Bd-driven declines. We examined skin sloughing rates in 21 frog species from three continents, as well as structural skin characteristics measured from preserved specimens. We found that sloughing rate varies significantly with phylogenetic group, but was not associated with evidence of Bd-driven declines, or other skin characteristics examined. This is the first comparison of sloughing rate across a wide range of amphibian species, and creates the first database of amphibian sloughing behaviour. Given the strong phylogenetic signal observed in sloughing rate, approximate sloughing rates of related species may be predicted based on phylogenetic position. While not related to available evidence of declines, understanding variation in sloughing rate may help explain differences in the severity of infection in genera with relatively slow skin turnover rates (e.g. Atelopus).

Hormone-induced sperm-release in the critically endangered Booroolong frog (Litoria booroolongensis): effects of gonadotropin-releasing hormone and human chorionic gonadotropin.

Research into the development of reproductive technologies for amphibians has increased in recent years due to the rapid decline of amphibian species globally. Reproductive technologies have great potential to overcome captive breeding failure and improve the propagation and genetic management of threatened species. However, the incorporation of these technologies into conservation breeding programs has been protracted, primarily as a result of trial-and-error approaches to the refinement of hormone therapies. The present study investigated the effects of: (1) GnRH-a dose (0, 0.5, 1, 2, 4, 8 or 16 µg g-1), and (2) hCG dose (0, 2.5, 5, 10, 20 or 40 IU g-1), on the sperm-release response of the critically endangered Booroolong frog. Administration of GnRH-a at a dose of 0.5 µg g-1 resulted in the greatest number of sperm released (mean total sperm = 3.5 ×106, n = 11). Overall, hCG was more effective at eliciting spermiation in Booroolong frogs, with peak sperm release (mean total sperm = 25.1 ×106, n = 10) occurring in response to a dose of 40 IU g-1. Sperm output in response to 40 IU g-1 hCG was greatest between 1 and 6 h and steadily declined between 8 and 24 h post-hormone administration. Percent sperm motility peaked between 4 and 10 h (58.1-62.7%), and sperm velocity between 4 and 12 h (24.3-27.2 µm s-1). Booroolong frogs join a small, but growing number of amphibian species that exhibit improved spermiation in response to hCG. Further research is required to identify optimal hormone-induction protocols for threatened amphibians and expedite the incorporation of reproductive technologies into CBPs.

Dose and life stage-dependent effects of dietary beta-carotene supplementation on the growth and development of the Booroolong frog.

Carotenoids are known for their antioxidant capacity and are considered to play an important role in vertebrate growth and development. However, evidence for their beneficial effects remains limited, possibly because very few studies have tested for dose effects across different life stages. The present study investigated the effect of various doses of dietary beta-carotene supplements on the growth and development of larval and post-metamorphic Booroolong frogs (Litoria booroolongensis). Larval and post-metamorphic basal diets (containing 0.015 and 0.005 mg g-1 total carotenoids, respectively) were supplemented with beta-carotene at one of four concentrations: 0 mg g-1, 0.1 mg g-1, 1 mg g-1 and 10 mg g-1. Each treatment included 72 replicate individuals, and individuals remained on the same diet treatment over both life stages (spanning 53 experimental weeks). Our results show that larvae receiving an intermediate (1 mg g-1) beta-carotene supplement dose grew faster than unsupplemented larvae (0 mg g-1), and metamorphosed earlier. After metamorphosis, there was no effect of the lowest supplement dose (0.1 mg g-1) on growth and development. However, juveniles fed the highest supplement dose (10 mg g-1) displayed significantly smaller body mass and lower body condition, compared to all other supplement doses, from 4-months through to sexual maturity (7-months). These findings indicate that beta-carotene supplementation has positive effects on growth and development, but only at intermediate doses, and only in the larval life stage. This knowledge may assist with amphibian conservation by expediting the rate that metamorphs can be generated in captive breeding programmes. More broadly, this is the first study to demonstrate both dose and life stage-dependent effects of dietary beta-carotene supplementation on vertebrate growth and development.

Survival, gene and metabolite responses of Litoria verreauxii alpina frogs to fungal disease chytridiomycosis.

The fungal skin disease chytridiomycosis has caused the devastating decline and extinction of hundreds of amphibian species globally, yet the potential for evolving resistance, and the underlying pathophysiological mechanisms remain poorly understood. We exposed 406 naïve, captive-raised alpine tree frogs (Litoria verreauxii alpina) from multiple populations (one evolutionarily naïve to chytridiomycosis) to the aetiological agent Batrachochytrium dendrobatidis in two concurrent and controlled infection experiments. We investigated (A) survival outcomes and clinical pathogen burdens between populations and clutches, and (B) individual host tissue responses to chytridiomycosis. Here we present multiple interrelated datasets associated with these exposure experiments, including animal signalment, survival and pathogen burden of 355 animals from Experiment A, and the following datasets related to 61 animals from Experiment B: animal signalment and pathogen burden; raw RNA-Seq reads from skin, liver and spleen tissues; de novo assembled transcriptomes for each tissue type; raw gene expression data; annotation data for each gene; and raw metabolite expression data from skin and liver tissues. These data provide an extensive baseline for future analyses.

Evolution of resistance to chytridiomycosis is associated with a robust early immune response.

Potentiating the evolution of immunity is a promising strategy for addressing biodiversity diseases. Assisted selection for infection resistance may enable the recovery and persistence of amphibians threatened by chytridiomycosis, a devastating fungal skin disease threatening hundreds of species globally. However, knowledge of the mechanisms involved in the natural evolution of immunity to chytridiomycosis is limited. Understanding the mechanisms of such resistance may help speed-assisted selection. Using a transcriptomics approach, we examined gene expression responses of endangered alpine tree frogs (Litoria verreauxii alpina) to subclinical infection, comparing two long-exposed populations with a naïve population. We performed a blinded, randomized and controlled exposure experiment, collecting skin, liver and spleen tissues at 4, 8 and 14 days postexposure from 51 wild-caught captively reared infection-naïve adult frogs for transcriptome assembly and differential gene expression analyses. We analysed our results in conjunction with infection intensity data, and the results of a large clinical survival experiment run concurrently with individuals from the same clutches. Here, we show that frogs from an evolutionarily long-exposed and phenotypically more resistant population of the highly susceptible alpine tree frog demonstrate a more robust innate and adaptive immune response at the critical early subclinical stage of infection when compared with two more susceptible populations. These results are consistent with the occurrence of evolution of resistance against chytridiomycosis, help to explain underlying resistance mechanisms, and provide genes of potential interest and sequence data for future research. We recommend further investigation of cell-mediated immunity pathways, the role of interferons and mechanisms of lymphocyte suppression.

Interventions for reducing extinction risk in chytridiomycosis-threatened amphibians.

Wildlife diseases pose an increasing threat to biodiversity and are a major management challenge. A striking example of this threat is the emergence of chytridiomycosis. Despite diagnosis of chytridiomycosis as an important driver of global amphibian declines 15 years ago, researchers have yet to devise effective large-scale management responses other than biosecurity measures to mitigate disease spread and the establishment of disease-free captive assurance colonies prior to or during disease outbreaks. We examined the development of management actions that can be implemented after an epidemic in surviving populations. We developed a conceptual framework with clear interventions to guide experimental management and applied research so that further extinctions of amphibian species threatened by chytridiomycosis might be prevented. Within our framework, there are 2 management approaches: reducing Batrachochytrium dendrobatidis (the fungus that causes chytridiomycosis) in the environment or on amphibians and increasing the capacity of populations to persist despite increased mortality from disease. The latter approach emphasizes that mitigation does not necessarily need to focus on reducing disease-associated mortality. We propose promising management actions that can be implemented and tested based on current knowledge and that include habitat manipulation, antifungal treatments, animal translocation, bioaugmentation, head starting, and selection for resistance. Case studies where these strategies are being implemented will demonstrate their potential to save critically endangered species.

Knot security and tensile strength of suture materials.

OBJECTIVE: To evaluate knot security and tensile failure load of suture tied in simple interrupted, beginning continuous, and ending continuous patterns for 11 suture materials commonly used in small animal surgery. STUDY DESIGN: Mechanical study. METHODS: For each of 11 suture material types, and 5 knot sizes (2, 3, 4, 5, and 6 throws) 2 surgeons each tied 6 knots (n = 12 for each knot size in 11 suture materials). Three types of patterns were evaluated: a simple interrupted square knot, a square knot beginning a simple continuous pattern, and the knot ending a simple continuous pattern. All knots were incubated in healthy canine donor plasma at 40°C for a minimum of 24 hours. Sutures were evaluated for knot security (knots untied, suture failed by breaking, suture slipped from the clamps, or suture untied before testing) and maximum load carried before knot slippage or knot failure (termed tensile failure load). RESULTS: Significant differences were found in knot security and tensile failure load among suture types. There was no significant difference between the simple interrupted knots and the knots at the beginning of a simple continuous pattern; however, both were significantly less likely to fail than the knots tied at the end of a simple continuous pattern. The number of throws per knot had a significant effect for knot security and tensile failure load. Surgeon experience had a significant effect on failure mode and tensile failure load. CONCLUSIONS: Suture type, number of throws per knot (knot size), suture pattern, and surgeon experience play an important role in knot security and should be considered when performing surgery.

Evaluation of the histologic reactions to commonly used suture materials in the skin and musculature of ball pythons (Python regius).

OBJECTIVE: To evaluate histologic reactions to 8 suture materials and cyanoacrylate tissue adhesive (CTA) in the musculature and skin of ball pythons. ANIMALS: 30 hatchling ball pythons. PROCEDURES: In each snake, ten 1-cm skin incisions were made (day 0). At 8 sites, a suture of 1 of 8 materials was placed in the epaxial musculature, and the incision was closed with the same material. One incision was closed by use of CTA. No suture material was placed in the tenth incision, which was allowed to heal by second intention (negative control). Snakes (n = 5/group) were euthanized for harvest of treatment-site tissues at days 3, 7, 14, 30, 60, and 90. Skin and muscle sections were examined microscopically and assigned a subjective score (0 to 4) for each of the following: overall severity of inflammation, fibrosis, number of macrophages, number of granulocytes, number of perivascular lymphocytes, and degree of suture fragmentation. RESULTS: Subjective score analysis revealed that CTA did not cause a significant inflammatory response, compared with the negative control. All suture materials caused significantly more inflammation over all time points; for all suture materials, inflammatory response scores were significantly higher than values for the negative control 90 days after implantation. No sutures were completely absorbed by the end of the study period, and several sutures appeared to be in the process of extrusion. CONCLUSIONS AND CLINICAL RELEVANCE: In snakes, CTA can be used to close small superficial incisions or lacerations with minimal inflammatory response, and sutures may undergo extrusion from tissues prior to complete absorption.

Clinical and histologic effects of intracardiac administration of propofol for induction of anesthesia in ball pythons (Python regius).

OBJECTIVE: To assess the clinical differences between induction of anesthesia in ball pythons with intracardiac administration of propofol and induction with isoflurane in oxygen and to assess the histologic findings over time in hearts following intracardiac administration of propofol. DESIGN: Prospective randomized study. ANIMALS: 30 hatchling ball pythons (Python regius). PROCEDURES: Anesthesia was induced with intracardiac administration of propofol (10 mg/kg [4.5 mg/lb]) in 18 ball pythons and with 5% isoflurane in oxygen in 12 ball pythons. Induction time, time of anesthesia, and recovery time were recorded. Hearts from snakes receiving intracardiac administration of propofol were evaluated histologically 3, 7, 14, 30, and 60 days following propofol administration. RESULTS: Induction time with intracardiac administration of propofol was significantly shorter than induction time with 5% isoflurane in oxygen. No significant differences were found in total anesthesia time. Recovery following intracardiac administration of propofol was significantly longer than recovery following induction of anesthesia with isoflurane in oxygen. Heart tissue evaluated histologically at 3, 7, and 14 days following intracardiac administration of propofol had mild inflammatory changes, and no histopathologic lesions were seen 30 and 60 days following propofol administration. CONCLUSIONS AND CLINICAL RELEVANCE: Intracardiac injection of propofol in snakes is safe and provides a rapid induction of anesthesia but leads to prolonged recovery, compared with that following induction with isoflurane. Histopathologic lesions in heart tissues following intracardiac injection of propofol were mild and resolved after 14 days.