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Non-lethal methods for semen collection from elasmobranchs to better understand species reproduction has accompanied the development of artificial insemination. Ejaculates (n = 82) collected from whitespotted bamboo sharks Chiloscyllium plagiosum (n = 19) were assessed and cold-stored raw or extended at 4 °C. Females (n = 20) were inseminated with fresh or 24–48 h cold-stored raw or extended semen and paternity of offspring determined with microsatellite markers. Insemination of females with fresh semen (n = 10) resulted in 80 hatchlings and 27.6% fertility. Insemination of females with semen cold-stored 24 h (n = 4) and 48 h (n = 1) semen resulted in 17 hatchlings and fertilization rates of 28.1% and 7.1% respectively. Two females inseminated with fresh or cold-stored semen laid eggs that hatched from fertilization and parthenogenesis within the same clutch. Parthenogenesis rate for inseminated females was 0.71%. Results demonstrate artificial insemination with cold-stored semen can provide a strategy for transport of male genetics nationally and internationally, precluding the need to transport sharks. Production of parthenotes in the same clutch as sexually fertilized eggs highlights the prevalence of parthenogenesis in whitespotted bamboo sharks and poses important considerations for population management.

Understanding the fundamental reproductive biology of a species is the first step toward identifying parameters that are critical for reproduction and for the development of assisted reproductive techniques. Ejaculates were collected from aquarium (n = 24) and in situ (n = 34) sand tiger sharks Carcharias taurus. Volume, pH, osmolarity, sperm concentration, motility, status, morphology, and plasma membrane integrity were assessed for each ejaculate. Semen with the highest proportion of motile sperm was collected between April and June for both in situ and aquarium sand tiger sharks indicating a seasonal reproductive cycle. Overall, 17 of 30 semen samples collected from aquarium sharks from April through June contained motile sperm compared to 29 of 29 of in situ sharks, demonstrating semen quality differences between aquarium and in situ sharks. Sperm motility, status, morphology, and plasma membrane integrity were significantly higher (P < 0.05) for in situ compared to aquarium sand tiger sharks. Testosterone was measured by an enzyme immunoassay validated for the species. Testosterone concentration was seasonal for both aquarium and in situ sharks with highest concentrations measured in spring and lowest in summer. In situ sharks had higher (P < 0.05) testosterone concentration in spring than aquarium sharks. This study demonstrated annual reproduction with spring seasonality for male sand tiger sharks through marked seasonal differences in testosterone and semen production. Lower testosterone and poorer semen quality was observed in aquarium sharks likely contributing to the species' limited reproductive success in aquariums. Summary sentence During mating season, in situ sand tiger sharks Carcharias taurus have higher plasma testosterone and better semen quality than aquarium housed sand tiger sharks impairing reproductive success of aquarium populations.

Hyatt, M.W.; Anderson, P.A., and O'Donnell, P.M., 2018. Influence of temperature, salinity, and dissolved oxygen on the stress response of bull (Carcharhinus leucas) and bonnethead (Sphyrna tiburo) sharks after capture and handling. Capture and handling stress can induce acidosis in sharks. This response, endured during commercial bycatch and in catch-and-release recreational fisheries, could be exacerbated in certain environmental conditions. To assess environmental influence on stress response, changes in acid-base, blood gas, and metabolite analytes (pH, pCO2, and lactate) measured with the i-STAT portable clinical analyzer were evaluated immediately after capture and removal from gillnets among wild bull (Carcharhinus leucas) and bonnethead (Sphyrna tiburo) sharks caught in waters of differing temperature (T), salinity (Sal), and dissolved oxygen (DO). Time from capture to blood collection (C-BD) was also recorded. Effects of T, Sal, DO, and C-BD on acid-base physiology were evaluated by modeling their ability to predict pH, pCO2, and lactate concentrations using ordinal logistic regression (OLR). The OLR models suggest that C. leucas sharks experienced a mixed metabolic and respiratory acidosis in warmer waters and at the low end of their salinity tolerance, and that S. tiburo sharks experienced a metabolic acidosis in warmer waters with a potential for respiratory acidosis at the high end of their salinity tolerance. In S. tiburo, capture and handling time exacerbated acidosis. Based on these findings, it is recommended that commercial and catch-and-release fisheries conduct operations cautiously during times of the year when water temperatures are high and salinities are at either extreme, by decreasing soak times, using the strongest proper tackle gear to reduce fight times, and releasing sharks as soon as possible after capture and detection.

Hyatt, M.W.; Anderson, P.A., and O'Donnell, P.M., 2016. Behavioral release condition score of bull and bonnethead sharks as a coarse indicator of stress. Capture and handling stress can induce acidosis and sometimes mortality in sharks. To approximate physiological condition after capture, fisheries researchers may use a behavioral health assessment at release. The goal of this study was to assess the efficacy of the behavioral release condition score (BRCS) in estimating the physiological stress response. The score was tested against changes in acid-base, blood gas, and metabolite analytes (pH, partial pressure of CO2, and lactate) and factors known to influence those analytes (species, capture and handling time, and water temperature) among wild-caught bull (Carcharhinus leucas) and bonnethead (Sphyrna tiburo) sharks. After gill net capture, sharks were processed for tagging, morphometrics, and blood sampling. Blood was sampled immediately before release. At release, a BRCS was assigned as good, fair, poor, or moribund. BRCS was modeled as a response to changes in blood analytes and putative stressors using ordinal logistic regression (OLR). Effects of significant main factors were further explored graphically and in chi-square tests or (multivariate) analyses of variance (MANOVAs/ANOVAs). Linear discriminant analyses with cross-validation were used to assess the ability of those factors to discriminate among BRCS on a case-by-case basis. The OLR models suggest that BRCS responds in species-specific ways to all three blood analytes and putative stressors. However, the broad overlaps in ranges of these parameters among BRCS lend prediction of BRCS by either of these two sets of predictors to be challenging to utilize. Given the coarse relationship of BRCS to acid–base status in these species, further investigation of this and other behavioral assessment methods is recommended.

Aspergillosis is a common respiratory fungal disease in African penguins (Spheniscus demersus) under managed care, and treatment failures with itraconazole due to drug resistance are increasingly common, leading to recent use of voriconazole. Empirical dosing with voriconazole based on other avian studies has resulted in adverse clinical drug effects in penguins. The objective of this study was to determine oral voriconazole pharmacokinetics (PK) in African penguins (n = 18). Single and once daily multiple oral doses of 5 mg/kg voriconazole were evaluated with a 4-mo washout period between trials. Plasma voriconazole concentrations were determined via high-performance liquid chromatography. Data was modeled using 3-compartamental population methodologies that supported first-order elimination. Observed mean peak concentration (1.89 μg/ml) after single dosing PK analysis was determined within the first hour following voriconazole administration. In the multiple-dose trial average plasma voriconazole concentrations were significantly higher on days 4 and 7 as compared with day 2. The mean estimates for volume of distribution (V/F) and clearance (Cl/F) for the multiple-dose study were 3.34 L and 0.18 L/hr, respectively. Monte Carlo simulations determined the median area under the curve (AUC0–24) at 84 hr was 37.7 μg·h/ml. As this assessment was comparable with the average AUC in humans receiving the recommended human oral dosage 200 mg b.i.d., it suggests that 5 mg/kg p.o. s.i.d. could be a safe and effective regimen in African penguins for treatment of aspergillosis. However, due to potential drug accumulation and subsequent toxicity, therapeutic drug monitoring with dosage adjustments is recommended to individualize dosing.

Fundamental characteristics of the reproductive biology of female sand tiger sharks Carcharias taurus are needed to understand the periodicity, seasonality and environmental factors essential for reproduction in this iconic species. Animals in managed care, such as aquariums, provide the unique opportunity for longitudinal study in contrast to in situ sharks that are examined opportunistically, and at a single point in time. Additionally, comparison of reproductive observations from successfully reproducing in situ sharks and aquarium sharks may help elucidate reasons for lack of reproduction among aquarium sharks and aid the development of assisted reproductive techniques for managed populations. Reproductive status of in situ and aquarium female sharks was assessed using ultrasonography and plasma hormone (17 β -estradiol, testosterone, and progesterone) monitoring. The reproductive cycle was divided into eight stages based on ovarian activity and uterine contents. In situ sharks were sampled from Delaware Bay ( n = 29), North Carolina ( n = 39) and South Carolina ( n = 11) during April-November from 2015–2020. Nineteen aquarium females from five aquaria were examined longitudinally for two or more consecutive years. Reproductive regionalization was observed among in situ females with the majority (83%) of North Carolina females in an active state of reproduction and all Delaware females in a resting reproductive state. All aquarium females had a pattern of reproductive cycling that was consistent with alternating years of activity and rest with confirmed biennial ( n = 7) or triennial ( n = 3) reproductive cycles with spring seasonality. In contrast to in situ females, aquarium females often retained uterine eggs for 9-20 months after ovulation in the absence of a developing embryo(s). Pre-ovulatory aquarium females had significantly higher concentrations of 17 β -estradiol, testosterone and progesterone than other reproductive stages. For females in the ovulatory stage, in situ females had higher testosterone than aquarium females. Endocrine differences between successfully reproducing in situ females and aquarium females likely contribute to the limited reproductive success observed for this species in managed care and may be a reflection of diminished seasonal cues and environmental differences.

Preventative health care of elasmobranchs is an important but understudied field of aquatic veterinary medicine. Evaluation of inflammation through the acute phase response is a valuable tool in health assessments. To better assess the health of bonnethead sharks (Sphyrna tiburo) under managed care, normal reference intervals of protein electrophoresis (EPH) and the acute phase proteins, C-reactive protein (CRP) and haptoglobin (HP), were established. Blood was collected from wild caught, captive raised bonnethead sharks housed at public aquaria. Lithium heparinized plasma was either submitted fresh or stored at −80°C prior to submission. Electrophoresis identified protein fractions with migration characteristics similar to other animals with albumin, α-1 globulin, α-2 globulin, β globulin, and γ globulin. These fractions were classified as fractions 1–5 as fractional contents are unknown in this species. Commercial reagents for CRP and HP were validated for use in bonnethead sharks. Reference intervals were established using the robust method recommended by the American Society for Veterinary Clinical Pathology for the calculation of 90% reference intervals. Once established, the diagnostic and clinical applicability of these reference intervals was used to assess blood from individuals with known infectious diseases that resulted in systemic inflammation and eventual death. Unhealthy bonnethead sharks had significantly decreased fraction 2, fraction 3, and fraction 3:4 ratio and significantly increased fraction 5, CRP, and HP. These findings advance our understanding of elasmobranch acute phase inflammatory response and health and aid clinicians in the diagnosis of inflammatory disease in bonnethead sharks.

Aspergillosis is a common respiratory fungal disease in penguins managed under human care. Triazole antifungal drugs, including itraconazole, are most commonly used for treatment; however, itraconazole treatment failures from drug resistance are becoming more common, requiring newer treatment options. Voriconazole, a newer triazole, is being used more often. Until recently, no voriconazole pharmacokinetic studies had been performed in penguins, leading to empiric dosing based on other avian studies. This has led to increased anecdotal reporting of apparent voriconazole toxicity in penguins. This report describes 18 probable and 6 suspected cases of voriconazole toxicity in six penguin species from nine institutions: 12 African penguins (Spheniscus demersus), 5 Humboldt penguins (Spheniscus humboldti), 3 Magellanic penguins (Spheniscus magellanicus), 2 gentoo penguins (Pygoscelis papua papua), 1 macaroni penguin (Eudyptes chrysolophus), and 1 emperor penguin (Aptenodytes forsteri). Observed clinical signs of toxicity included anorexia, lethargy, weakness, ataxia, paresis, apparent vision changes, seizure-like activity, and generalized seizures. Similar signs of toxicity have also been reported in humans, in whom voriconazole therapeutic plasma concentration for Aspergillus spp. infections is 2–6 μg/ml. Plasma voriconazole concentrations were measured in 18 samples from penguins showing clinical signs suggestive of voriconazole toxicity. The concentrations ranged from 8.12 to 64.17 μg/ml, with penguins having plasma concentrations above 30 μg/ml exhibiting moderate to severe neurologic signs, including ataxia, paresis, and seizures. These concentrations were well above those known to result in central nervous system toxicity, including encephalopathy, in humans. This case series highlights the importance of species-specific dosing of voriconazole in penguins and plasma therapeutic drug monitoring. Further investigation, including pharmacokinetic studies, is warranted. The authors recommend caution in determining voriconazole dosages for use in penguin species.

We determined the short- and long-term effects of a ketogenic diet (KD) or ketone salt (KS) supplementation on multi-organ oxidative stress and mitochondrial markers. For short-term feedings, 4 month-old male rats were provided isocaloric amounts of KD (n = 10), standard chow (SC) (n = 10) or SC + KS (~1.2 g/day, n = 10). For long-term feedings, 4 month-old male rats were provided KD (n = 8), SC (n = 7) or SC + KS (n = 7) for 8 months and rotarod tested every 2 months. Blood, brain (whole cortex), liver and gastrocnemius muscle were harvested from all rats for biochemical analyses. Additionally, mitochondria from the brain, muscle and liver tissue of long-term-fed rats were analyzed for mitochondrial quantity (maximal citrate synthase activity), quality (state 3 and 4 respiration) and reactive oxygen species (ROS) assays. Liver antioxidant capacity trended higher in short-term KD- and SC + KS-fed versus SC-fed rats, and short-term KD-fed rats exhibited significantly greater serum ketones compared to SC + KS-fed rats indicating that the diet (not KS supplementation) induced ketonemia. In long term-fed rats: (a) serum ketones were significantly greater in KD- versus SC- and SC + KS-fed rats; (b) liver antioxidant capacity and glutathione peroxidase protein was significantly greater in KD- versus SC-fed rats, respectively, while liver protein carbonyls were lowest in KD-fed rats; and (c) gastrocnemius mitochondrial ROS production was significantly greater in KD-fed rats versus other groups, and this paralleled lower mitochondrial glutathione levels. Additionally, the gastrocnemius pyruvate-malate mitochondrial respiratory control ratio was significantly impaired in long-term KD-fed rats, and gastrocnemius mitochondrial quantity was lowest in these animals. Rotarod performance was greatest in KD-fed rats versus all other groups at 2, 4 and 8 months, although there was a significant age-related decline in performance existed in KD-fed rats which was not evident in the other two groups. In conclusion, short- and long-term KD improves select markers of liver oxidative stress compared to SC feeding, although long-term KD feeding may negatively affect skeletal muscle mitochondrial physiology.