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Age data are essential for estimating life history parameters and are thus critical for population assessment, management, and conservation. Traditional vertebrae-based age estimation in elasmobranchs can be costly, time intensive, of low accuracy, and is by necessity lethal. Herein, epigenetic clocks were developed for an elasmobranch, the cownose ray ( Rhinoptera bonasus ), using aquarium-born individuals ( n  = 42) with known dates of birth (age range: 7−7,878 days or 0−21 years) and two tissue types (fin clips and whole blood) that can be sampled in a relatively non-invasive manner. Enzymatically-converted restriction site-associated DNA sequencing (ECrad-seq) was used to identify CpG sites that exhibited age-correlated DNA methylation. The epigenetic clocks developed were highly accurate (mean absolute error, MAE, < 0.75 years) and precise ( R 2  > 0.98). Age-associated CpG sites were identified across tissues, and a multi-tissue clock was also highly accurate (MAE < 1 year) and precise ( R 2  = 0.97). Using the developed fin clip clock, three wild-caught individuals of unknown age but managed in aquariums for > 22 years were predicted to be 22.10−23.49 years old. Overall, the results have important implications for future epigenetic clock development and noninvasive age estimation in elasmobranchs.

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.

The reproductive system of the red stingray Hemitrygon akajei was described from 1,418 specimens, 682 males and 736 females, sampled year-round (2003–2014) from Ariake Bay to provide reproductive life history information for conservation and management of the species. Females reach sexual maturity at a larger size than males with the size at 50% sexual maturity 522.2 mm disc width (DW) and 321.5 mm DW, respectively. Male stingrays had semen in the seminal vesicles year-round. Dental sexual dimorphism was aseasonal. The mating period is protracted, spanning 7 consecutive months October–April but ovulation occurred during May. This suggests female sperm storage, which has not been described for dasyatid stingrays. Females have a single functional ovary and uterus (left) and reproduce via aplacental viviparity with lipid histotrophy. Gestation required 3 months with parturition during late July and early August. Uterine eggs without macroscopic embryos were observed during the first half of gestation suggesting a short period of arrested development or diapause. Developmental cohorts based on morphological features were described for embryos and can be used to characterize embryo growth and development for other stingray species. Observations of foetal mortality (1.25%) and morphologically abnormal embryos (0.72%) were uncommon. Pregnancy rate was 90% and reproduction was annual and synchronous. Hemitrygon akajei fecundity ranged from 7 to 25 and increased with female size. Although H. akajei is a medium-sized dasyatid ray, it has the highest fecundity reported for any batoid species. Elasmobranchs life histories usually are associated with a low degree of productivity that results in rapid population decline with increased fishing pressure. Producing high numbers of small young is a successful reproductive strategy for H. akajei that may be partly responsible for their resilience to fishing pressure and continued abundance in Ariake Bay. This study is the first to describe reproduction of H. akajei, an economically important top predator inhabiting coastal ecosystems throughout Asia.

Background Heterotopic ossification (HO) is a significant problem for wounded warriors surviving high-energy blast injuries; however, currently, there is no biomarker panel capable of globally characterizing, diagnosing, and monitoring HO progression. The aim of this study was to identify biomarkers for HO using proteomic techniques and blood serum. Methods Isobaric tags for relative and absolute quantitation (iTRAQ) was used to generate a semi-quantitative global proteomics survey of serum from patients with and without heterotopic ossification. Leveraging the iTRAQ data, a targeted selection reaction monitoring mass spectrometry (SRM-MS) assay was developed for 10 protein candidates: alkaline phosphatase, osteocalcin, alpha-2 type I collagen, collagen alpha-1(V) chain isoform 2 preprotein, bone sialoprotein 2, phosphatidate phosphatase LPIN2, osteomodulin, protein phosphatase 1J, and RRP12-like protein. Results The proteomic survey of serum from both healthy and disease patients includes 1220 proteins and was enriched for proteins involved in the response to elevated platelet Ca +2 , wound healing, and extracellular matrix organization. Proteolytic peptides from three of the ten SRM-MS proteins, osteocalcin preprotein, osteomodulin precursor, and collagen alpha-1(v) chain isoform 2 preprotein from serum, are potential clinical biomarkers for HO. Conclusions This study is the first reported SRM-MS analysis of serum from individuals with and without heterotopic ossification, and differences in the serum proteomic profile between healthy and diseased subjects were identified. Furthermore, our results indicate that normal wound healing signals can impact the ability to identify biomarkers, and a multi-protein panel assay, including osteocalcin preproprotein, osteomodulin precursor, and collagen alpha-1(v) chain isoform 2 preprotein, may provide a solution for HO detection and monitoring.

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.

Public aquaria showcase aquatic life while raising awareness and educating the public on biodiversity and the need for conservation. Recently, aquaria have followed in the footsteps of zoos by taking more directed approaches in species conservation as well as leveraging animals in their collection to fill biological knowledge gaps through research. Similar to zoos, aquaria are able to house animals that are not feasible to care for in traditional academic settings, allowing important life history information to be gained. In particular, our understanding of reproductive aspects of large migratory fishes such as elasmobranchs (sharks, skates and rays) have benefited from information gleaned in aquarium settings that would otherwise be difficult or impossible to study in the natural environment. For example, the ability of elasmobranchs to reproduce parthenogenetically was discovered through observation of sharks maintained in public aquaria. Since its discovery, parthenogenesis has been observed for many ex situ shark and ray species. Aquaria have made other important contributions to characterizing elasmobranch reproductive biology such as understanding reproductive cycling and trialing assisted reproductive techniques, among others. Here, we review the role aquaria have played in the field of elasmobranch reproductive biology and discuss how zoos and aquariums can continue to contribute to this field in the future.

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.

Clearnose skates, Raja eglanteria, are a common species of skate found seasonally along the Atlantic coast of North America from Cape Cod to mid-Florida and in the Gulf of Mexico from mid-Florida to eastern Texas. Clearnose skates inhabit the west central coast of Florida during winter months, when Gulf temperatures are conducive to mating and egg-laying (approximately 16°C-22°C). Specimens collected during this time will breed in captivity and, if maintained at 20°C, mated females will store sperm and continue to lay fertile eggs for up to six months. With fertile eggs available from day of laying until hatching, the first complete description of batoid elasmobranch embryonic development with a timetable has been established and is presented here. If eggs are maintained at 20°C, appearance and progression of embryonic events proceed over a reproducible timeline. The timeline has been divided into discrete time periods into which developmental events have been grouped. These include cleavage and expansion of the blastodisc (day 1-day 4), embryonic axis and neural tube formation (day 4-day 7), pharyngeal pouches and gill filament development (day 10-day 28), expansion of fins and transition from external filaments to internal gills (week 4-week 7), and epidermal pigmentation and yolk absorption (week 8-week 12). Fully formed offspring hatch after an incubation period of approximately 12 weeks (85 ± 6 days). To examine possible mechanisms of transport and storage of sperm in the reproductive tract of females following copulation, experiments resulting in the first artificial insemination for any elasmobranch fish are described. These experiments provide evidence for functional roles of the alkaline gland, whose secretions stimulate sperm motility and may enhance migration of sperm to the oviducal glands, and the clasper gland, whose secretions may provide nutrition during storage of viable sperm in the oviducal gland.

Growth is frequently described as weight gain over time. Researchers have used this information in equations to predict carcass composition and estimate fat deposition. Diet, species, breed, and gender all influence fat deposition. Alterations in diets result in changes in fat deposition as well as the fatty acid profile of meat. Additionally, the amount and composition of the fat can affect lipid stability and flavor development upon cooking. Fat functions not only as a storage of energy and contributor of flavor compounds, but also participates in signaling that affects many aspects of the physiological functions of the animal. Transcription factors that are upregulated in response to excess energy to be stored are an important avenue of research to improve the understanding of fat deposition and thus, the efficiency of production. Additionally, further study of the inflammation associated with increased fat depots may lead to a better understanding of finishing animals, production efficiency, and overall health.

Batoids comprise five of the seven most threatened families of sharks and rays. The East Asian endemic Naru eagle ray Aetobatus narutobiei is a large bodied ray whose estuarine habitat overlaps with an economically valuable bivalve fishery. In response to decreased bivalve yields, the government initiated a predator control program and as a result, Naru eagle rays have faced intense and targeted fishing pressure during the last two decades. The long-term impacts of the predator control program on the population of rays and bivalves and their balance in the ecosystem are unknown because the life history of the Naru eagle ray has not been characterized. To begin to fill these critical knowledge gaps, the reproductive life history of the Naru eagle was described. Females mature at a larger size than males and require nearly twice as many years to reach maturity (DW50, 952.0 mm vs. 764.2 mm; Age50, 6.0 years vs. 3.5 years). Both males and females reproduce annually and their reproductive cycles are synchronized and seasonal. Females have a single ovary and paired uteri, are viviparous, and reproduce via matrotrophic histotrophy. Mating occurs in August and September and gestation lasts approximately 12 months including a 9.5-month diapause that begins soon after mating and ends in June of the following year, leaving 2.5 months for embryos to complete development. Fecundity ranged from 1 to 7 embryos per brood ( n = 158, mean ± SD = 3.36 ± 1.26) and was positively correlated with female disc width (linear regression; F = 105.73, d.f. = 151, P < 0.05). Naru eagle rays are vulnerable to overfishing because of their low fecundity, long reproductive cycle and long time to reach sexual maturity. Obligate embryonic diapause during overwintering and seasonal migrations is a survival strategy that benefits the adults and neonates. This research is a valuable resource to help guide science-based management, conservation and protection of the endemic Asian A . narutobiei and its nursery areas.