Large Mammalian Species Research Articles

Comparative Analysis of Progenitor Cells Isolated from the Iris, Pars Plana, and Ciliary Body of the Adult Porcine Eye

Photoreceptor loss causes irreversible blindness in many retinal diseases. The identification of suitable donor cell populations is of considerable interest because of their potential use to replace the photoreceptors lost in disease. Stem or progenitor cells that give rise to neurons and glia have been identified in several regions of the brain, including the embryonic retina and the ciliary epithelium of the adult eye, raising the possibility of autologous transplantation. However, there has been little systematic investigation into precisely which regions of the large mammalian adult eye give rise to such cells. Here, we show for the first time using the porcine eye the presence of progenitor cells in additional regions of the adult eye, including the pars plana and iris, regions that, in the human, are readily accessible during routine eye surgery. When cultured in the presence of growth factors, these cells proliferate to form neurospheres comprised of cells expressing retinal progenitor markers. Using an adherent monolayer culture system, these cells could be readily expanded to increase their number more than 1 million-fold and maintain a progenitor phenotype. When grown on the substrate laminin in the presence of serum, cells derived from both spheres and monolayer cultures differentiated into neurons and glia. These results suggest that a population of cells derived from the adult iris, pars plana, and ciliary body of a large mammalian species, the pig, has progenitor properties and neurogenic potential, thereby providing novel sources of donor cells for transplantation studies. Disclosure of potential conflicts of interest is found at the end of this article.

Risk of capture-related mortality in large free-ranging mammals: experiences from Scandinavia

Chemical capture and anaesthesia of free-ranging mammals will always involve some risk of mortality even in healthy animals. Deaths may be directly or indirectly attributable to the anaesthetic event itself (e.g. drug overdose, drowning during induction and dart trauma) or may be caused by secondary effects from the capture (e.g. stress, myopathy, trauma or instrumentation with radio-transmitters). In long-term research projects on five major wildlife species in Scandinavia, the capture-related mortality rates (number of captures) were: moose Alces alces 0.7% (N = 2,816), brown bears Ursus arctos 0.9% (N = 1,079), wolverines Gulo gulo 2.8% (N = 461), Eurasian lynx Lynx lynx 3.9% (N = 380), and gray wolves Canis lupus 3.4% (N = 89). We suggest that wildlife professionals should strive for a zero mortality rate but adopt the standard that a mortality rate of > 2% probably should not be accepted in any large mammalian species. This can be achieved by: 1) using an experienced professional capture team, 2) developing and following a capture protocol specific to each species, and 3) requiring that a mortality assessment be undertaken after any capture-related death. This assessment should re-evaluate the capture protocol, including how changes in anaesthetics and methodological approaches could have prevented the mortality.

Les rhytines de Steller (Mammalia, Sirenia, Dugongidae) du Muséum d'Histoire naturelle de Lyon

Collections of the Natural History Museum of Lyon include an almost complete adult skeleton of the gigantic sea-cow Hydrodamalis gigas, together with two skulls and two mandibles of the same species. These osteological pieces were acquired during the XIXth century and exhibited since to the public but remained unpublished. These are described here in detail for the first time. This material increases our knowledge of a recently extinct and poorly known large mammalian species relatively rare in the world's scientific collections.

Wildlife tuberculosis in South African conservation areas: Implications and challenges

Tuberculosis, caused by Mycobacterium bovis, was first diagnosed in African buffalo in South Africa's Kruger National Park in 1990. Over the past 15 years the disease has spread northwards leaving only the most northern buffalo herds unaffected. Evidence suggests that 10 other small and large mammalian species, including large predators, are spillover hosts. Wildlife tuberculosis has also been diagnosed in several adjacent private game reserves and in the Hluhluwe-iMfolozi Park, the third largest game reserve in South Africa. The tuberculosis epidemic has a number of implications, for which the full effect of some might only be seen in the long-term. Potential negative long-term effects on the population dynamics of certain social animal species and the direct threat for the survival of endangered species pose particular problems for wildlife conservationists. On the other hand, the risk of spillover infection to neighboring communal cattle raises concerns about human health at the wildlife-livestock-human interface, not only along the western boundary of Kruger National Park, but also with regards to the joint development of the Greater Limpopo Transfrontier Conservation Area with Zimbabwe and Mozambique. From an economic point of view, wildlife tuberculosis has resulted in national and international trade restrictions for affected species. The lack of diagnostic tools for most species and the absence of an effective vaccine make it currently impossible to contain and control this disease within an infected free-ranging ecosystem. Veterinary researchers and policy-makers have recognized the need to intensify research on this disease and the need to develop tools for control, initially targeting buffalo and lion.

Genetic characterization of H5N1 influenza A viruses isolated from zoo tigers in Thailand

The H5N1 avian influenza virus outbreak among zoo tigers in mid-October 2004, with 45 animals dead, indicated that the avian influenza virus could cause lethal infection in a large mammalian species apart from humans. In this outbreak investigation, six H5N1 isolates were identified and two isolates (A/Tiger/Thailand/CU-T3/04 and A/Tiger/Thailand/CU-T7/04) were selected for whole genome analysis. Phylogenetic analysis of the 8 gene segments showed that the viruses clustered within the lineage of H5N1 avian isolates from Thailand and Vietnam. The hemagglutinin (HA) gene of the viruses displayed polybasic amino acids at the cleavage site, identical to those of the 2004 H5N1 isolates, which by definition are highly pathogenic avian influenza (HPAI). In addition, sequence analyses revealed that the viruses isolated from tigers harbored few genetic changes compared with the viruses having infected chicken, humans, tigers and a leopard isolated from the early 2004 H5N1 outbreaks. Sequence analyses also showed that the tiger H5N1 isolated in October 2004 was more closely related to the chicken H5N1 isolated in July than that from January. Interestingly, all the 6 tiger H5N1 isolates contained a lysine substitution at position 627 of the PB2 protein similar to the human, but distinct from the original avian isolates.

Ageing and the extinction of large animals

In the modulation of longevity by natural selection there is a trade-off between the investment of resources in the maintenance of the body, or soma, and the investment in reproduction. There is accumulating evidence that long-lived mammalian species have much more efficient maintenance than short-lived ones. It is also clear that short-lived ground-living mammalian species reproduce very much more quickly than larger long-lived species, and in all mammals there is an inverse relationship between maximum reproductive potential and maximum longevity. These features of life-history strategies very strongly support the disposable soma theory of the evolution of ageing. Slow development and large size are associated with delayed ageing. If the environment changes, for whatever reason, small rapidly breeding species are able to adapt and survive much more easily that large slow breeding species. This can explain the very well documented extinction of many large mammalian species during the Pleistocene. In an environment which remains constant for a long period of time, selection favours the evolution of larger species, but these are put at risk if the environment becomes less favorable. Fluctuating environments are more likely to promote the evolution of small short-lived species with high fecundity.

Agroforestry, elephants, and tigers: balancing conservation theory and practice in human-dominated landscapes of Southeast Asia

Large mammal populations theoretically are best conserved in landscapes where large protected areas are surrounded by buffer zones, connected by corridors, and integrated into a greater ecosystem. Multi-use buffer zones, including those containing complex agroforestry systems, are promoted as one strategy to provide both economic benefits to people and conservation benefits to wildlife. We use the island of Sumatra, Indonesia to explore the benefits and limitations of this strategy. We conclude that conservation benefits are accrued by expanding the habitat available for large mammals but more attention needs to be focused on how to reduce and respond to human–wildlife conflict that is likely to occur in these multiple use areas. Agroforestry systems are likely to play an increasingly valuable role in the conservation of large mammalian species. We believe this value can be increased still further if the agroforestry community decides to assume a leadership role in addressing the issue of human–wildlife conflict, which is fast becoming a central threat to the survival of many large endangered species like tigers and elephants. Both people and wildlife can benefit enormously if appropriate methods are developed to more rigorously define the distribution and frequency of conflict between tigers and elephants with people along the edge of protected areas. Sharp forest–agriculture boundaries may reduce tiger–human conflicts, but not elephant–human conflict according to the data we currently have. © 2004 Elsevier B.V. All rights reserved.

Of Mice and Dogs

Large animal models have provided much of the descriptive data regarding the cellular and molecular events in myocardial infarction and repair. The availability of genetically altered mice may provide a valuable tool for specific cellular and molecular dissection of these processes. In this report we compare closed chest models of canine and mouse infarction/reperfusion qualitatively and quantitatively for temporal, cellular, and spatial differences. Much like the canine model, reperfused mouse hearts are associated with marked induction of endothelial adhesion molecules, cytokines, and chemokines. Reperfused mouse infarcts show accelerated replacement of cardiomyocytes by granulation tissue leading to a thin mature scar at 14 days, when the canine infarction is still cellular and evolving. Infarcted mouse hearts demonstrate a robust but transient postreperfusion inflammatory reaction, associated with a rapid up-regulation of interleukin-10 and transforming growth factor-beta. Unlike canine infarcts, infarcted mouse hearts show only transient macrophage infiltration and no significant mast cell accumulation. In correlation, the growth factor for macrophages, M-CSF, shows modest and transient up-regulation in the early days of reperfusion; and the obligate growth factor for mast cells, stem cell factor, SCF, is not induced. In summary, the postinfarction inflammatory response and resultant repair in the mouse heart shares many common characteristics with large mammalian species, but has distinct temporal and qualitative features. These important species-specific differences should be considered when interpreting findings derived from studies using genetically altered mice.

Conservation genetics of carnivores in Italy

Pleistocene climatic changes shaped the patterns of biodiversity in Europe and around the Mediterranean. Describing the phylogeographic structure of animal populations and inferring past population dynamics is essential to develop a framework for conservation biology in Europe. Direct persecution, habitat loss, population fragmentation and hybridization with domesticated conspecifics, are the main threats to the survival of large mammalian species. In this paper I will summarize the available information on phylogeography and population genetics of brown bear, wolf, wildcat and otters in Italy and in Europe. To cite this article: E. Randi, C. R. Biologies 326 (2003).

Rabies DNA vaccine in the horse: strategies to improve serological responses

In order for DNA vaccines to become a practical alternative to conventional vaccines their ability to induce antibody responses in large mammals needs to be improved. We used DNA vaccination against rabies in the horse as a model to test the potential of two different strategies to enhance antibody responses in a large mammalian species. The administration of the DNA vaccine in the presence of aluminum phosphate improved both the onset and the intensity of serological responses but was not potent enough to achieve seroconversion in all vaccinated ponies. However, when the DNA vaccine was formulated with the cationic lipid DMRIE–DOPE instead of aluminum phosphate, a very strong impact on both onset and intensity of serological responses was observed. This latter strategy ensured excellent seroconversion in all vaccinated ponies after a primary course of two injections, demonstrating a clear improvement of the homogeneity of the induced responses. These data indicate that rabies DNA vaccination is feasible in horses and further suggests that properly formulated DNA vaccines can generate immune responses in large veterinary species at a level comparable to the responses achieved with conventional vaccines.

Large mammalian herbivores and woody plant species diversity in Zimbabwe

Study sites from various tree savannas and miombo woodland with adjacentareas with different herbivore species (game and cattle) and stocking rates wereselected from around Zimbabwe. Using the fence-lines separating the adjacentareas, 60 m2 plots were placed on both sides of thefence at 18 randomly chosen positions. The identity and number of plants of eachwoody species were recorded in paired 60 m2 plots. Therewere no significant differences in the woody species richness between pairedplots for any of the fence-line contrasts. For only two of the fence-linecontrasts were there significant differences (P < 0.05)in the relative abundance of woody species. For the first fence-line contrast,the stocking rates were the same, but the herbivore species present weredifferent, while for the second fence-line contrast, the numbers of herbivorespecies present were the same, but the stocking rates differed. Even thoughthere were no differences in the numbers of woody plant species present, therewere differences in the woody plant species and the numbers of plants of eachspecies: the second most common species was different for three fence-linecontrasts and the third most common species was different for four fence-linecontrasts.

Distribution and numbers of mucous glands in the horse trachea.

Horses commonly suffer from respiratory diseases associated with excess secretions in the airway lumen, some of which are presumably derived from airway mucous glands. However, these structures have been little investigated in the horse. Accordingly, we describe here the number, distribution and size of equine tracheal mucous glands, and compare the data with similar information for other mammalian species. Two types of gland acini were present. In the thick connective tissue, up to 400 microm beneath the epithelium, gland acini were grouped in thin sheets that, in cross-section, averaged 20 microm thick and were up to 4.0 mm in length. However, it is probable that most sheets had maximal diameters much less than 4.0 mm. Between 400 to 900 microm below the epithelium, the connective tissue was much more diffuse, and glands were larger and more globular. Gland volume in the ventral portion was approximately 1.7 microl/cm2 of mucosal surface, and approximately 1.1 microl/cm2 in the dorsal portion. Glands were somewhat more abundant between, rather than over, the cartilaginous rings, but the difference between the 2 locations was not marked. Mucous gland openings were small (20 microm diameter) and very unevenly distributed, generally occurring about 100 microm apart in longitudinal rows of about 5. Average frequency of openings in the ventral portions of 3 tracheas was approximately 1.0/mm2 of mucosal surface. The volume of individual glands was therefore approximately 17 nl. Although the frequency of gland openings in the horse trachea is similar to that for the tracheas of other large mammalian species, horse tracheal gland volume was only about 15% that of the other species. Therefore, the excess 'mucous' secretions seen in equine recurrent airway obstruction and other respiratory diseases are unlikely to be caused by comparatively high levels of airway mucous gland secretion. Instead, they may be caused mainly by hyperplasia of the mucus-producing cells of the surface epithelium or by vascular transudation.

A review of terrestrial mammalian and reptilian carnivore ecology in Australian fossil faunas, and factors influencing their diversity: the myth of reptilian domination and its broader ramifications

The notion that Australia’s large, terrestrial carnivore faunas of the middle Tertiary to Pleistocene were dominated by reptiles has gained wide acceptance in recent decades. Simple but sweeping hypotheses have been developed seeking to explain this perceived ecological phenomenon. However, a review of the literature does not support these interpretations, which are based on largely speculative and, in many cases, clearly erroneous assumptions. Few size estimates of fossil reptilian taxa are based on quantitative methodology and, regardless of method, most are restricted to maximum dimensions. For species of indeterminate growth, this practice generates misleading perceptions of biological significance. In addition to misconceptions with respect to size, much speculation concerning the lifestyles of large extinct reptiles has been represented as fact. In reality, it has yet to be demonstrated that the majority of fossil reptiles underpinning the story of reptilian domination were actually terrestrial. No postcranial evidence suggests that any Australian mekosuchine crocodylian was less aquatic than extant species, while a semi-aquatic habitus has been posited for madtsoiid snakes and even the giant varanid, Megalania. Taphonomic data equivocally supports the hypothesis that some Australian mekosuchines were better adapted to life on land than are most extant crocodylians, but still semi-aquatic and restricted to the near vicinity of major watercourses. On the other hand, the accelerating pace of discovery of new large mammalian carnivore species has undermined any prima facie case for reptilian supremacy regarding pre-Pleistocene Australia (that is, if species richness is to be used as a gauge of overall impact). However, species abundance and consumption, not richness, are the real measures. On this basis, even in Pleistocene Australia, where species richness of large mammalian carnivores was relatively low, available data expose the uncommon and geographically restricted large contemporaneous reptiles as bit players. In short, the parable of a continent subject to a Mesozoic rerun, wherein diminutive mammals trembled under the footfalls of a menagerie of gigantic ectotherms, appears to be a castle in the air. However, there may be substance to some assertions. Traditionally, erratic climate and soil-nutrient deficiency have been invoked to explain the perception of low numbers or relatively small sizes of fossil mammalian carnivore taxa in Australia. But these arguments assume a simple and positive relationship between productivity, species richness and maximum body mass and either fail to recognise, or inappropriately exclude, other factors. Productivity has undoubtedly played a role, but mono-factorial paradigms cannot account for varying species richness and body mass among Australia’s fossil faunas. Nor can they explain differences between Australian fossil faunas and those of other landmasses. Other factors that have contributed include sampling bias, a lack of internal geographic barriers, competition with large terrestrial birds and aspects of island biogeography unique to Australia, such as landmass area and isolation, both temporal and geographic.

Electrically induced static exercise elicits a pressor response in the decerebrate rat.

1. The purpose of this investigation was to determine if activation of the exercise pressor reflex in the decerebrate rat induced circulatory responses comparable to those reported in large mammalian species. 2. To activate both mechanically and metabolically sensitive afferent fibres, static hindlimb contractions were induced by stimulating the cut ends of L4 and L5 spinal ventral roots in Sprague-Dawley rats (300-400 g). To selectively stimulate mechanically sensitive receptors, hindlimb muscles were passively stretched. 3. In intact halothane-anaesthetized animals (n = 10), static contraction and passive stretch induced a decrease in mean arterial pressure (Delta MAP = -17 +/- 3 and -8 +/- 1 mmHg for contraction and stretch, respectively) and heart rate (HR). In contrast, MAP increased 23 +/- 2 mmHg during contraction and 19 +/- 3 mmHg during stretch in decerebrate rats (n = 10). These pressor responses were accompanied by a significant tachycardia. In decerebrate animals, the reintroduction of halothane attenuated the increase in MAP and HR caused by both contraction and stretch. 4. In both anaesthetized and decerebrate rats, sectioning the spinal dorsal roots innervating the activated skeletal muscle eliminated responses to contraction and stretch. This finding indicated that an intramuscular neural reflex mediated the response to each stimulus. 5. The results demonstrate that a decerebrate preparation in the rat is a reliable model for the study of the exercise pressor reflex. Development of the model would enable the study of this reflex in a variety of pathological conditions and allow investigation of the mechanisms controlling cardiovascular responses to exercise in health and disease.

Challenges implicit to gene discovery research in the control of ventilation during hypoxia.

Appointing physiological function to specific genetic determinants requires a systems physiologist to consider ways of assessing precise phenotypic mechanisms. The integration of ventilation, metabolism and thermoregulation, for example, is very complex and may differ among small and large mammalian species. This challenge is particularly applicable to the study of short- and long-term adaptation of these systems to hypoxic exposure associated with high altitude. Our laboratory has initiated a research effort to dissect the complexity of hypoxic adaptation using traditional quantitative genetic analysis and contemporary DNA genotyping techniques. Although the current evidence in murine models demonstrates that specific genes influence control of hypoxic ventilatory responses (HVR), the relevance of these determinants to human adaptation to altitude remains open to exploration. Our review discusses the progress and uncertainties associated with assigning a genetic basis to variation in acute and chronic HVR.

Differential depressant effects of general anesthetics on the cardiovascular response to cocaine in mice.

In recent years, murine models have gained increasing importance for studies of cardiovascular physiology and pharmacology, largely due to the development of transgenic strains with specific alterations in phenotype. Differential effects of general anesthetic agents on the cardiovascular responses to cocaine have been reported in larger mammals; therefore, we studied the effects of commonly used anesthetics on heart function and on blood pressure responses to cocaine in Swiss Webster mice. We positioned a polyethylene catheter (PE-10) in the right carotid artery or left ventricle of mice anesthetized with equivalent anesthetic dose of either ketamine-xylazine (KX, 40 mg/kg + 5 mg/kg), pentobarbital (PEN, 40 mg/kg) or alpha-chloralose-urethane (CU, 80 mg/kg + 100 mg/kg). Cocaine (0.3 mg/kg, 1 mg/kg and 3 mg/kg) was administrated via the left jugular vein by bolus injection. In the KX group, the basal mean arterial pressure (MAP) and systolic left ventricular pressure (LVP) were 110 +/- 12 and 120 +/- 13 mmHg, respectively, close to conscious values. However, PEN and CU significantly decreased the basal parameters (P < 0.01 compared to the KX group). The lowest dose of cocaine (0.3 mg/kg) elicited minimal changes. Significant responses were obtained with a 1-mg/kg dose of cocaine (P < 0.01 compared to baseline). However, at 3 mg/kg, a toxic effect of cocaine appeared in all three anesthetic groups. Compared to published conscious animal data, anesthetic agents attenuated the cardiovascular effects of cocaine. Taken together, our results indicate that minimally effective doses of general anesthetics may significantly alter the basal hemodynamic state and the responses to sympathomimetic agents in the murine model, as has been reported in larger mammalian species. We concluded that anesthesia with ketamine-xylazine provides baseline hemodynamic values close to reported values in conscious animals, but also attenuates the hemodynamic response to cocaine.

Preparation of animals with a high degree of chimerism by one-step coculture of embryonic stem cells and preimplantation embryos.

A single-step coculture procedure has been developed that can generate chimeric mice with high efficiency and reproducibility. The procedure involves culture of embryonic stem (ES) cells with 8- to 16-cell embryos in microwells to provide conditions for effective cell-cell-embryo contact. A suspension of ES cells is layered over the microwells, followed by transfer of an embryo without zona pellucida into each microwell. Following overnight culture, the blastocysts are transferred into pseudopregnant recipients. The method has several advantages due to its simplicity and reproducibility: (i) Over 90% of ES-cell contribution in new-borns can be obtained frequently and most of the chimeras display germ-line transmission. (ii) The procedure does not require specialized skills or expensive instruments. (iii) All the steps of embryo manipulation can be completed in a relatively short period of time; therefore, a large number of embryos can be manipulated simultaneously. The method was tested with three independent ES cell lines and two different strains of mice with similar results. The technique may be an alternative to microinjection of DNA into zygotes to prepare transgenic lines of small and large mammalian species.

Meniscal ossification

Examination of knee menisci of Bengal tigers revealed ossicles within the cartilaginous anterior horn of each medial meniscus. This ossification was not evident in the neonatal animal, but was present in animals aged 20 months or older. The ossicle appeared prior to the completion of skeletal maturation at the knee, and was composed of normal remodeling trabecular bone. While most animals had a single, variably sized ossicle, multiple ossicles also occurred. The meniscal cartilage apposed to the femoral articulation exhibited a distinct columnar pattern in the region of the ossicle, in contrast to the non-columnar pattern throughout the bulk of the meniscus, including the ossicle side apposed to the tibial plateau. In this particular large mammalian species medial meniscal ossification appears to be a normal anatomical variation that progressively develops following birth, and may serve as a model for the phylogenetic (developmental) theory of etiology.

Enteric neuropathy in horses with grass sickness.

The degeneration of enteric neurones has been recorded in grass sickness, but the distribution of the lesions in the gut and their possible relationship with the severity of the clinical signs has not been established. Samples obtained from 11 anatomically defined sites along the gastrointestinal tract of eight control horses without gastrointestinal disease, five horses with acute grass sickness and three horses with chronic grass sickness were examined histologically. The organisation of the enteric ganglia was similar to that in other large mammalian species. Minor exceptions included the presence of fundic mucosal nerve cell bodies and blood vessels within submucosal ganglia. All the horses with grass sickness had severe enteric neuropathy, widespread in the acute cases but localised to the distal small intestine in the chronic cases. These neuronal lesions are consistent with, and may explain the intractable gastrointestinal propulsive deficit that characterises grass sickness.

Autologous transplantation of canine long-term marrow culture cells genetically marked by retroviral vectors

Retroviral infection of bone marrow cells in long-term marrow cultures (LTMCs) offers several theoretical advantages over other methods for gene transfer into hematopoietic stem cells. To investigate the feasibility of this approach in a large animal model system, we subjected LTMCs from nine dogs to multiple infections with retrovirus containing the neomycin phosphotransferase gene (neo) during 21 days of culture. Feeder layers, cocultivation, polycations, and selection were not used. The in vitro gene transfer efficiency was 70% as determined by polymerase chain reaction amplification of neo sequences in colony- forming unit granulocyte-macrophage (CFU-GM) obtained from day-21 LTMCs. Day-21 LTMC cells were infused into autologous recipients with (four dogs) and without (three dogs) marrow-ablative conditioning. At 3 months posttransplant, up to 10% of marrow cells contained the neo gene. This percentage declined to 0.1% to 1% at 10 to 21 months posttransplant. Neo was also detected in individual CFU-GM, burst- forming unit-erythroid (BFU-E), and CFU-Mix progenitors derived from marrow up to 21 months postinfusion and in cultures of peripheral blood- derived T cells up to 19 months postinfusion. There was no difference in the percentage of neo-marked cells present when dogs that received marrow ablative conditioning were compared with dogs receiving no conditioning. Detection of neo-marked marrow cells almost 2 years after autologous transplantation in a large mammalian species shows that retroviral infection of marrow cells in LTMCs is a potentially nontoxic and efficient protocol for gene transfer. Further, our results suggest that marrow conditioning and in vivo selection pressure to retain transplanted cells may not be absolute requirements for the retention of genetically marked cells in vivo.