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Bedford N.M.,University of Cincinnati | Winget G.D.,Center for Conservation and Research of Endangered Wildlife | Srikoundinya P.,University of Cincinnati | Steckl A.J.,University of Cincinnati
Biomacromolecules | Year: 2011

Electrospun fibers consisting of poly(3,4- ethylenedioxythiophene)/ poly(styrene sulfonate) (PEDOT/ PSS) and poly(ethylene oxide) (PEO) have been used to successfully encapsulate and stabilize thylakoid membrane vesicles isolated from spinach. Light-driven electronic properties were measured. Fibers with immobilized thylakoids show higher electrical conductivity compared with fibers without thylakoids under white light conditions. This is attributed to the electron-generating photosynthetic reactions from the thylakoids. Electron and optical microscopy show the presence of thylakoid vesicles within the fibers using lipid-specific stains. After electrospinning into fibers, the thylakoid vesicles still exhibit an ability to produce a light-driven electron gradient, indicating that activity is preserved during the electrospinning process. These electrospun fibers provide an excellent example of incorporating photosynthetic function into an artificial system © 2011 American Chemical Society.


Roth T.L.,Center for Conservation and Research of Endangered Wildlife | Szymanski D.C.,Center for Conservation and Research of Endangered Wildlife | Keyster E.D.,Center for Conservation and Research of Endangered Wildlife
Theriogenology | Year: 2010

The goals of this study were to test the effects of exogenous hormones and hibernation on breeding behavior and gamete release by boreal toads (Bufo boreas boreas). Each year, a subset of 77 toads was hibernated and then paired with hibernated or nonhibernated mates and treated with luteinizing hormone releasing hormone analogue (LHRHa), human chorionic gonadotropin (hCG), or left untreated. Amplexus and egg and sperm production were recorded. At 1 yr of age, only 19% of pairs exhibited amplexus, and no sperm or eggs were produced. At 2 and 3 yr of age, most male toads treated with LHRHa exhibited amplexus (56.9% and 100%, respectively). Among 2-yr-old males, amplexus was more prevalent (P < 0.05) in those that were hibernated than in those that were nonhibernated (54.0% and 33.3%, respectively), but most males in each group (93.3% and 75%, respectively) produced sperm in response to LHRHa treatment. Only one 2-yr-old and two 3-yr-old females produced eggs. At 4 yr of age, eight females produced eggs, but two died from egg retention. More nonhibernated than hibernated females developed eggs (7 of 10 vs. 1 of 10, P < 0.05). Mean (±SD) weight of female toads producing eggs (58.9 ± 11.9 g) was greater (P < 0.05) than that of nonproducing females (43.6 ± 7.0 g). Similarly, four of seven nonhibernated females (58.8 ± 8.3 g) produced eggs at 5 yr of age. All eggs were produced by females treated once with LHRHa. Number of eggs per female varied (141 to 3307), and development to tadpoles was low (0 to 36.5%), although tadpoles did become toadlets. In conclusion, male and female boreal toads matured at 2 and 4 yr of age, respectively, and heavier females were more likely to produce eggs. To enhance breeding success, males should be hibernated and treated with LHRHa. In contrast, female productivity was enhanced by improving their body condition instead of subjecting them to hibernation prior to LHRHa treatment. © 2010 Elsevier Inc. All rights reserved.


Swanson W.F.,Center for Conservation and Research of Endangered Wildlife
Reproduction in Domestic Animals | Year: 2012

Contents: Embryo transfer (ET) and artificial insemination (AI) are potentially invaluable techniques for the propagation and management of genetically valuable domestic cat and endangered nondomestic cat populations. Many of the challenges that impair the effective application of ET and AI in felids may be overcome by using laparoscopic oviductal (LO) approaches. LO-ET and LO-AI are minimally-invasive procedures, requiring only two small skin incisions for insertion of a laparoscope and grasping forceps into the abdominal cavity to permit visualization and catheterization of the oviduct for embryo or semen deposition. With concurrent improvements in embryo culture systems and ovarian synchronization protocols, LO-ET has proven effective over the past decade for propagation of laboratory cats, cat models of hereditary disease and nondomestic cats. To date, viable offspring have been produced following LO-ET of non-frozen and frozen-thawed IVF-derived embryos in eight cat hereditary disease models and two nondomestic cat species, the ocelot and sand cat. LO-AI with low sperm numbers (c. 2-8 million motile) has shown similar efficacy to LO-ET, resulting in high pregnancy percentages (50-70%) following insemination of gonadotropin-treated domestic cats. Multiple kittens also have been produced in two hereditary disease models following LO-AI with frozen semen, and both ocelot and Pallas' cat kittens have been born after LO-AI with freshly-collected semen. The application of LO-ET and LO-AI to felids has resulted in substantial improvement in the efficiency of assisted reproduction for genetic management of these invaluable domestic cat and wild cat populations. © 2012 Blackwell Verlag GmbH.


Pence V.C.,Center for Conservation and Research of Endangered Wildlife
Annals of the Missouri Botanical Garden | Year: 2013

For the majority of plant species of conservation concern, seed banking and traditional propagation methods are the most efficient ways of meeting the ex situ and recovery conservation goals of Global Strategy for Plant Conservation (GSPC) Target 8. However, there are estimated to be 5000 or more endangered species for which these methods will not be adequate conservation tools. These "exceptional" species are those with recalcitrant seeds or those that produce few or no seeds. In vitro methods can provide alternative procedures for propagating and preserving germplasm in the long term for these species. Research at the Center for Conservation and Research of Endangered Wildlife (CREW) with several U.S. endangered species has shown the potential of these methods. In vitro propagation can provide plants for reintroduction and research when traditional propagation methods are not adequate. Phytotissue banking can be used for long-term ex situ conservation when seed or embryo banking is not possible. In vitro methods are also needed for recovery when embryo banking of recalcitrant seeds is possible. The full implementation of in vitro methods is constrained by information, scientific, and economic challenges, but the need for its use in meeting the needs of exceptional species should provide impetus for overcoming these challenges and making these methods an integral part of an overall ex situ conservation strategy.


Pence V.C.,Center for Conservation and Research of Endangered Wildlife
International Journal of Plant Sciences | Year: 2014

Cryopreservation of nonseed plant tissues can provide a method for preserving plant genetic diversity ex situ when seeds or spores cannot be adapted to traditional methods of preservation. Shoot tips, somatic embryos, and gametophytes can be used as propagules for long-term storage in liquid nitrogen. Tissue cryo-preservation is particularly important in dealing with "exceptional species," i.e., species that have unbankable seeds or that are producing few or no seeds. In vitro methods can be applied to collecting tissues and establishing cultures as a source of tissue for cryopreservation. The applicability of vitrification/dehydration cryoprocedures has been demonstrated for a wide range of species and types of tissues, and the choice and modifications of methods can be informed by the natural adaptations of the species. While tissues in culture are clonally propagated, preserving tissues from multiple genotypes can provide genetic diversity to a cryopreserved collection. Such collections will ultimately serve to provide materials for restoration if wild populations are lost or severely threatened. There is great potential for the use of in vitro methods and cryopreservation in plant conservation strategies, but there are also both scientific and practical challenges to their application. Whereas in vitro propagation and preservation methods are well established, these methods remain highly empirical in their application to new species. This can be a challenge for conservation, which deals with rare and often unstudied species, which may also have unusual natural adaptations. Because of the labor involved, tissue cryopreservation is also more costly than more traditional ex situ conservation methods, which can limit its use in traditionally underfunded work with endangered plants. However, it is suggested that collaborative efforts can help meet these challenges, bringing together expertise and infrastructure from several sources to facilitate the application of tissue cryopreservation to the ex situ conservation of endangered exceptional species. © 2013 by The University of Chicago. All rights reserved.


Pence V.C.,Center for Conservation and Research of Endangered Wildlife
In Vitro Cellular and Developmental Biology - Plant | Year: 2011

In vitro methods provide opportunities for propagating and preserving endangered plant species when seed-based methods are not adequate. Such species include those that produce few or no seeds, as well as species with recalcitrant seeds. Tissue culture propagation methods can be used to produce such plants for reintroduction, research, education, display, and commerce. They can also be the basis for tissue banking as a way to preserve genetic diversity when seeds cannot be banked. With some recalcitrant species, embryo banking, a method which also utilizes in vitro culture for recovery germination, is possible. The number of endangered species that will require in vitro methods is estimated to be at least 5,000 worldwide. Further information is needed to identify these species, and the ongoing collection of information into databases on endangered species and recalcitrant species will help provide this. The costs of these methods are higher than for traditional propagation and preservation, but they may be necessary for species under higher threat. The multiplication rate of a culture, as well as the rates of rooting and acclimatization, has a major effect on the number of transfers needed for producing plants or tissue for banking, and improvements that will increase the efficiency of these steps can help lower costs. Further research into factors affecting the growth of tissues in vitro, as well as coordination of efforts among institutions with infrastructure for in vitro work, should facilitate the application of in vitro methods to the endangered species that cannot be propagated or preserved using seeds. © 2010 The Society for In Vitro Biology.


Pence V.C.,Center for Conservation and Research of Endangered Wildlife
Kew Bulletin | Year: 2010

Summary: Seed-based methods are generally the most efficient for propagating and storing plant germplasm, but these methods are not always adequate, and some species can benefit from in vitro methods for conservation. For species that produce few or no seeds in the wild, plants may be propagated in vitro, and in vitro shoot tips can provide material for cryostorage when seeds are not available or are recalcitrant. In vitro propagated plants may also serve as subjects for research, without depleting the genetic resources of the species. Clonal plants can be used to test out suitable habitat and can be used for basic research on endangered species, without disturbing the wild population. Despite the effectiveness of widely used techniques, however, there are still species that resist initiation into culture or that may be difficult to root or acclimatise. Similarly, tissue cryopreservation methods may be restrained by cost, particularly in maintaining multiple genotypes of many species. Maintaining such genotypes in vitro is also costly and runs the risk of loss or change over time. Examples of the successful use of in vitro methods will illustrate the variety of applications of these techniques, but costs and specific challenges will also be discussed to help define areas where further research is needed to realise the potential of in vitro methods as a tool for conservation. © 2011 The Board of Trustees of the Royal Botanic Gardens, Kew.


Stoops M.A.,Center for Conservation and Research of Endangered Wildlife | MacKinnon K.M.,Center for Conservation and Research of Endangered Wildlife | Roth T.L.,Center for Conservation and Research of Endangered Wildlife
Theriogenology | Year: 2012

The objective was to identify suitable enzyme immunoassays to monitor gonadal and placental function in the female polar bear. Immunoreactive progesterone, progesterone metabolite (PdG), estrogen, and androgen metabolite (T) concentrations were measured in fecal samples collected over 24 mo from captive female bears (N = 20). Whereas fecal extracts produced displacement curves parallel to the standard curve for each respective steroid, T and PdG more accurately reflected reproductive events. Concentrations of fecal T increased (P < 0.05) during the breeding season, and brief spikes were associated with estrus and mating. A postovulatory increase in PdG was not always detected, but sustained baseline T after mating appeared consistent with ovulation. Parturient bears excreted higher PdG concentrations (P < 0.05) during expected time of embryo implantation in Fall, and a late gestational rise in fecal T occurred 30 days prepartum. Many nonparturient bears also had a PdG rise in the Fall, suggesting they experienced either pregnancy loss or a pseudopregnancy. Differentiating pregnant and pseudopregnant states was not achieved using fecal PdG alone, but when combined with fecal T, comprehensive diagnoses could be made. Nonparturient bears demonstrated elevated (P < 0.05) fecal T during summer months, whereas parturient bears did not. In summary, noninvasive hormone monitoring techniques were established for the female polar bear. Although this study was directed at facilitating management and breeding efforts of captive polar bears, the methods could be applied to studies of reproductive function in wild populations. © 2012 Elsevier Inc.


Pence V.C.,Center for Conservation and Research of Endangered Wildlife
Acta Horticulturae | Year: 2014

Cryopreservation and in vitro culture of plant tissues are two tools that can be brought into service for ex situ plant conservation. They are especially important for the long-term preservation of genetic diversity of species for which traditional seed banking is not workable. Such species have been termed "exceptional" species, and they include species with recalcitrant seeds, as well as species that produce few or no seeds. The ability to propagate and cryopreserve tissues from multiple genotypes can bring endangered species into stable, long-term storage and provide material that can be used for restoration. Several species under study at CREW provide examples, including Asimina tetramera, Deeringothamnus pulchellus and D. rugelii, three endemic Florida pawpaws that have seeds and have been reported as recalcitrant, Hedeoma todsenii and Mespilus canescens, species from New Mexico and Arkansas, respectively, that are not producing seed, and Crotalaria avonensis and Ranunculus aestivalis, endemic species from Florida and Utah, respectively, for which few seeds have been available. These species also illustrate some of the challenges of propagating and maintaining multiple genotypes, of genotypic variation in propagation and survival through cryopreservation, and of survival through acclimatization and outplanting. However, they also illustrate the potential of these techniques for providing tissues for storage and plants for restoration when traditional methods are not adequate. As these tools are applied to more species, these methods should become more efficient and effective and more widely applied for the propagation and preservation of exceptional species as a resource for the future.


Stoops M.A.,Center for Conservation and Research of Endangered Wildlife | O'Brien J.K.,Center for Conservation and Research of Endangered Wildlife | Roth T.L.,Center for Conservation and Research of Endangered Wildlife
Theriogenology | Year: 2011

Mortality rates are high among captive African black rhinoceroses (Diceros bicornis), due to increased susceptibility to disease. The ability to rescue genetic material from individuals that die unexpectedly represents a practical approach to assist ex situ conservation efforts. The objectives of the present study were to attempt postmortem oocyte recovery from ovaries of African black rhinoceroses (N = 6) and to test the efficacy of equine protocols for rhinoceros oocyte IVM and IVF using cryopreserved rhinoceros sperm. The interval from ovary removal to oocyte recovery was 25.3 ± 13.9 h (mean ± SD). Ovaries were transported at 4 °C or 22 °C and effects of temperature on postmortem oocyte competence was evaluated. Numbers of oocytes collected per female averaged 15.8 ± 6.9. In total, 95 oocytes were recovered. Of these, 85 were inseminated using homologous sperm and 10 were inseminated using heterologous sperm. Overall, substantial numbers of viable oocytes were retrieved from African black rhinoceros ovaries 1 to 2 days postmortem from ovaries stored at ambient temperature. A proportion of these oocytes matured and underwent penetration and fertilization by heterologous or homologous frozen-thawed rhinoceros sperm. The reproductive competence of postmortem oocytes was further demonstrated by development of a single two-cell embryo. Despite the need for further refinements, gamete rescue in the rhinoceros has promise for producing rhinoceros embryos, as well as testing sperm functions in vitro. © 2011 Elsevier Inc.

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