Development of genetic markers for reproductive management of toucans.

Retention of genetic diversity in successive generations is key to successful ex situ programs and will become increasingly important to restore wild populations of threatened animals. When animal genealogy is partly unknown or gaps exist in studbook records, the application of molecular resources facilitates informed breeding. Here, we apply molecular resources to an ex situ breeding population of toucans (Ramphastidae), a bird family zoos commonly maintain. Toucans face population declines from illegal poaching and habitat degradation. We developed novel microsatellite markers using blood samples from 15 Keel-billed Toucans (Ramphastos sulfuratus Lesson 1830). Parentage of two individuals was known a priori, but possible sibship among 13 putative founders-including the parents-was unknown. We compared available avian heterologous and novel microsatellite markers to recover known relationships and reconstruct sibship. Eight of 61 heterologous markers amplified consistently and were polymorphic, but less so than the 18 novel markers. Known sibship (and three sibling pairs whose relatedness was unknown a priori) and paternity-though not maternity except in one case-were well-recovered using both likelihood and pairwise relatedness methods, when incorporating novel but not heterologous markers. Zoo researchers seeking microsatellite primer sets for their breeding toucan populations will likely benefit from our heterologous markers, which can be leveraged both to assess relatedness and select breeding pairs. We recommend that zoo biologists rely on species-specific primers and not optimize heterologous primers for toucan species without molecular resources. We conclude with a brief discussion of modern genotyping methods of interest to zoo researchers.

Testosterone and semen seasonality for the sand tiger shark Carcharias taurus†.

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.

Reproductive Science in Sharks and Rays.

Sharks and rays make up 96% of the class Chondrichthyes. They are among the most endangered of any taxa, threatened through habitat loss, overfishing and hunting for shark fin soup, traditional medicines or sport, and because many species are slow to mature and produce low numbers of offspring. Sharks and rays are ecologically and reproductively diverse, though basic knowledge of their reproductive physiology is lacking for many species. There has been a move towards non-lethal approaches of data collection in sharks and rays, especially with reproductive technologies such as ultrasound and hormone analysis. Additionally, technologies such as semen collection and artificial insemination are lending themselves to develop tools to manage small or closed populations, with cold-stored sperm being shipped between institutions to maximize genetic diversity in managed populations. The role of steroid hormones in elasmobranch reproduction appears broadly conserved, though heavily influenced by environmental cues, especially temperature. For this reason elasmobranchs are likely at risk of reproductive perturbations due to environmental changes such as ocean warming. Current reproductive technologies including computer assisted sperm assessments to study warming effects on sperm motility and intra-uterine satellite tags to determine birthing grounds will serve to generate data to mitigate anthropogenic changes that threaten the future of this vulnerable groups of fish.

A pilot study evaluating protein abundance in pressure ulcer fluid from people with and without spinal cord injury.

OBJECTIVE: To determine whether the biochemistry of chronic pressure ulcers differs between patients with and without chronic spinal cord injury (SCI) through measurement and comparison of the concentration of wound fluid inflammatory mediators, growth factors, cytokines, acute phase proteins, and proteases. DESIGN: Survey. SETTING: Tertiary spinal cord rehabilitation center and skilled nursing facilities. PARTICIPANTS: Twenty-nine subjects with SCI and nine subjects without SCI (>18 years) with at least one chronic pressure ulcer Stage II, III, or IV were enrolled. OUTCOME MEASURES: Total protein and 22 target analyte concentrations including inflammatory mediators, growth factors, cytokines, acute phase proteins, and proteases were quantified in the wound fluid and blood serum samples. Blood samples were tested for complete blood count, albumin, hemoglobin A1c, total iron binding capacity, iron, percent (%) saturation, C-reactive protein, and erythrocyte sedimentation rate. RESULTS: Wound fluid concentrations were significantly different between subjects with SCI and subjects without SCI for total protein concentration and nine analytes, MMP-9, S100A12, S100A8, S100A9, FGF2, IL-1b, TIMP-1, TIMP-2, and TGF-b1. Subjects without SCI had higher values for all significantly different analytes measured in wound fluid except FGF2, TGF-b1, and wound fluid total protein. Subject-matched circulating levels of analytes and the standardized local concentration of the same proteins in the wound fluid were weakly or not correlated. CONCLUSIONS: The biochemical profile of chronic pressure ulcers is different between SCI and non-SCI populations. These differences should be considered when selecting treatment options. Systemic blood serum properties may not represent the local wound environment.

Analysis of the proteomic profile of chronic pressure ulcers.

Analysis of the proteomic profile of pressure ulcers over time is a critical step in the identification of biomarkers of healing or nonhealing in pressure ulcers. The wound fluid from 32 subjects with 42 pressure ulcers was evaluated over 6 weeks at 15 time points. Samples specific to both the interior and the periphery of the wound bed were collected. Antibody screening arrays, isobaric tags for relative and absolute quantitation with mass spectrometry and multiplexed microarrays were used to characterize wound fluid and results were correlated with clinical outcome. Twenty-one proteins were found to distinguish between healed and chronic wounds and 19 proteins were differentially expressed between the interior and periphery of wounds. Four proteins, pyruvate kinase isozymes M1/M2, profilin-1, Ig lambda-1 chain C regions, and Ig gamma-1 chain C region, were present in lower levels for periphery samples when compared to interior samples and six proteins, keratin, type II cytoskeletal 6A (KRT6A), keratin, type I cytoskeletal 14, S100 calcium binding proteins A7, alpha-1-antitrypsin precursor, hemoglobin subunit alpha, and hemoglobin subunit beta, were present in higher levels in periphery samples when compared with interior samples. S100 calcium binding protein A6, S100 calcium binding protein A7, and soluble receptor for advanced glycation end-products had higher levels in the periphery of chronic wounds vs. the interior in planar arrays. A significant temporal trend was noted for monokine induced by gamma interferon (MIG), synonomous with chemokine (C-X-C motif) ligand 9 (CXCL9), which increased as wounds healed and remained nearly constant for ulcers that were not approaching closure.

Granulation tissue of chronic pressure ulcers as a predictive indicator of wound closure.

OBJECTIVE: : To describe the temporal relationship between the quantity of granulation tissue in a chronic pressure ulcer (PrU) and its clinical outcome. DESIGN: : Study participants were seen on days 0, 1, 2, 3, 4, 7, 8, 9, 10, 11, 14, 21, 28, 35, and 42. On each visit, the wounds were digitally photographed with a 3-cm calibration target. Images were analyzed using VeV MD (version 1.1.14; VERG Inc, Winnipeg, Manitoba, Canada) and Adobe Photoshop CS3 Extended (version 10.0.1; Adobe Systems Inc, San Jose, California). Granulation tissue was selected from calibrated digital images by 1 of 2 methods: manual selection and automated selection. Granulation tissue area was expressed as a percentage of total wound area. SETTING: : Academic research laboratory. PARTICIPANTS: : Thirty-one chronic PrUs were observed in 27 subjects. MAIN OUTCOME MEASURES: : Quantitative measure of granulation tissue area. MAIN RESULTS: : There was no relationship between the amount of granulation tissue expressed as a percentage of the total PrU area and wound outcome. CONCLUSIONS: : This study is the first to both quantitatively measure the amount of granulation tissue in a chronic PrU and attempt to correlate it to wound outcome. Although counterintuitive, the amount of granulation tissue was not predictive of outcome, and no temporal trends could be described.

Artificial insemination and parthenogenesis in the whitespotted bamboo shark Chiloscyllium plagiosum.

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.

Analysis of pressure ulcer wound fluid using two-dimensional electrophoresis.

The incidence rate of pressure ulcers in the USA ranges from 0.4% to 38% in acute care settings and from 2.2% to 23.9% in long-term care settings, and their treatment costs are in the billions of dollars yearly. The proteome of wound fluid may contain early indicators or biomarkers associated with healing in pressure ulcers that would enable treatment regimes to be optimised for each individual. Wound fluid was collected from the interior and periphery of 19 chronic pressure ulcers at 15 time points during 42 days for an analysis of protein expression. Proteins were fractionated using two-dimensional polyacrylamide gel electrophoresis. A comparison of the spot distributions indicates a biochemical difference between the interior and the periphery of wounds. Pressure ulcers that healed show a greater number of spots for interior and peripheral locations combined over time when compared with wounds that did not heal. Using this technique, protein S100A9 was identified as a potential biomarker of wound healing. The identification of differences within the proteome of healing versus non healing pressure ulcers could have great significance in the use of current treatments, as well as the development of new therapeutic interventions.

Ultrasonographic and hormonal characterization of reproductive health and disease in wild, semiwild, and aquarium-housed southern stingrays (Hypanus americanus).

OBJECTIVE: To characterize physical examination, plasma biochemical, and ultrasonographic findings in aquarium-housed, managed semiwild, and wild southern stingrays (Hypanus americanus) with and without reproductive disease. ANIMALS: Southern stingrays from aquarium (n = 48), lagoon (managed semiwild; 34), and wild (12) habitats. PROCEDURES: Limited, opportunistic prosections were performed of presumed anatomically normal wild southern stingrays and compared with findings for aquarium-housed stingrays with reproductive disease. Ultrasonographic video data from both groups were used to assign a score (1 to 5) indicating increasing severity of ovarian and uterine reproductive disease. Plasma total 17ß-estradiol, estrone, progesterone, and testosterone concentrations were measured with enzyme immunoassays validated for use in southern stingrays. RESULTS: Ultrasonographic ovarian scores were significantly correlated with uterine scores. No reproductive disease was detected in semiwild or wild stingrays, but 65% (31/48) of aquarium-housed stingrays had developing or advanced reproductive disease (ie, ultrasonographic ovarian or uterine score of 4 or 5). Significant correlations were identified between ovarian and uterine disease status and plasma concentrations of all steroid hormones except testosterone. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggested that ultrasonography and plasma hormone concentrations may be useful in the identification of reproductive disease and determination of disease severity in southern stingrays.

A Systems Biology Approach for Studying Heterotopic Ossification: Proteomic Analysis of Clinical Serum and Tissue Samples.

Heterotopic ossification (HO) refers to the abnormal formation of bone in soft tissue. Although some of the underlying processes of HO have been described, there are currently no clinical tests using validated biomarkers for predicting HO formation. As such, the diagnosis is made radiographically after HO has formed. To identify potential and novel biomarkers for HO, we used isobaric tags for relative and absolute quantitation (iTRAQ) and high-throughput antibody arrays to produce a semi-quantitative proteomics survey of serum and tissue from subjects with (HO+) and without (HO-) heterotopic ossification. The resulting data were then analyzed using a systems biology approach. We found that serum samples from subjects experiencing traumatic injuries with resulting HO have a different proteomic expression profile compared to those from the matched controls. Subsequent quantitative ELISA identified five blood serum proteins that were differentially regulated between the HO+ and HO- groups. Compared to HO- samples, the amount of insulin-like growth factor I (IGF1) was up-regulated in HO+ samples, whereas a lower amount of osteopontin (OPN), myeloperoxidase (MPO), runt-related transcription factor 2 (RUNX2), and growth differentiation factor 2 or bone morphogenetic protein 9 (BMP-9) was found in HO+ samples (Welch two sample t-test; P < 0.05). These proteins, in combination with potential serum biomarkers previously reported, are key candidates for a serum diagnostic panel that may enable early detection of HO prior to radiographic and clinical manifestations.

A survey of proteomic biomarkers for heterotopic ossification in blood serum.

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.

In vivo exposure of clearnose skates, Raja eglanteria, to ionizing X-radiation: acute effects on the thymus.

To investigate for the first time the effects of ionizing radiation on thymus of a representative cartilaginous fish, juvenile clearnose skates, Raja eglanteria, were exposed to 0-75 Gy of X-radiation and sacrificed after 12 days. Morphometrics (weight, disc width and total length) and thymus and thymic cyst area were compared to controls using ANOVA. Thymus area declined logarithmically and medullary cysts increased as a function of dose (P < or = 0.05). To assess thymic recovery, skates were exposed to 0, 9, 13.5 or 18 Gy of X-radiation and sacrificed when moribund or on days 10, 20, 30 and 40 post-irradiation. Complete restoration of the thymus was not achieved during the 40-day observation period, although repopulation with pro-thymocytes and partial recovery of thymic architecture were evident histologically. The observed high radiosensitivity of R. eglanteria thymocytes was similar to responses of other vertebrates, but recovery time was prolonged.

Principles and Techniques of Electron Microscopy: Biological Applications, Fourth Edition, by M. A. Hayat.

FATAL ERROR -- Missing abstract.

In vivo exposure of clearnose skates, Raja eglanteria, to ionising X-radiation: acute effects on the peripheral blood, spleen, and epigonal and Leydig organs.

The effects of ionising radiation on the peripheral blood, spleen, and epigonal and Leydig organs of cartilaginous fishes were investigated using juvenile clearnose skates, Raja eglanteria. Skates (N = 80) were sacrificed 12 days after exposure to 0-75 Gy of X-radiation, and morphometrics (body mass, disc width, total length), mass of spleens and epigonal organs, and peripheral blood leucocyte (PBL) counts were compared to controls using ANOVA. Spleen and epigonal organ mass and PBL counts declined logarithmically as a function of radiation dose. To assess recovery from X-radiation, skates (N = 40) were exposed to 0, 9 or 18 Gy and sacrificed when moribund or on days 10, 20, 30 and 40 post-irradiation. Partial recovery of Leydig organ and splenic red pulp was evident after 40 days in skates exposed to 9 Gy, but no indication of recovery was apparent at higher doses. Median lethal dose by 30 days (LD50/30) was calculated to be 9-18 Gy, similar to that determined for other fishes.