Cryopreservation and in vitro culture of primary cell types from lung tissue of a stranded pygmy sperm whale (Kogia breviceps)

Current models for in vitro studies of tissue function and physiology, including responses to hypoxia or environmental toxins, are limited and rely heavily on standard 2-dimensional (2-D) cultures with immortalized murine or human cell lines. To develop a new more powerful model system, we have pursued methods to establish and expand cultures of primary lung cell types and reconstituted tissues from marine mammals. What little is known about the physiology of the deep-sea diving pygmy sperm whale (PSW), Kogia breviceps, comes primarily from stranding events that occur along the coast of the southeastern United States. Thus, development of a method for preserving live tissues and retrieving live cells from deceased stranded individuals was initiated. This report documents successful cryopreservation of PSW lung tissue. We established in vitro cultures of primary lung cell types from tissue fragments that had been cryopreserved several months earlier at the stranding event. Dissociation of cryopreserved lung tissues readily provides a variety of primary cell types that, to varying degrees, can be expanded and further studied/manipulated in cell culture. In addition, PSW-specific molecular markers have been developed that permitted the monitoring of fibroblast, alveolar type II, and vascular endothelial cell types. Reconstitution of 3-D cultures of lung tissues with these cell types is now underway. This novel system may facilitate the development of rare or disease-specific lung tissue models (e.g., to test causes of PSW stranding events and lead to improved treatments for pulmonary hypertension or reperfusion injury in humans). Also, the establishment of a "living" tissue bank biorepository for rare/endangered species could serve multiple purposes as surrogates for freshly isolated samples.     

The diet of pygmy sperm whales, Kogia breviceps, stranded in New Zealand: implications for conservation

The stomach contents of 27 pygmy sperm whales, Kogia breviceps, stranded on New Zealand beaches between 1991 and 2003 are reported. These individuals comprise 16 males, 10 females, and one for which no sex information is available. The diet was found to include fish and crustaceans, but is comprised primarily of cephalopods, with 0–526 lower beaks, representing an estimated maximum of c. 60 kg of cephalopod prey consumed by any one whale. Cephalopod prey is attributed to 23 species from 13 families, and is dominated by juvenile individuals of the families Histioteuthididae and Cranchiidae (adults of which usually occur at depths exceeding 400 m). Perceived threats to this whale, particularly those affecting distribution and abundance of prey species, are also discussed. These are the first data reporting the diet of this whale species in New Zealand waters. A comparison of the diet of K. breviceps is made with that of the sperm whale, Physeter macrocephalus from New Zealand waters, and with the diet of Kogia known elsewhere.     

Anisakid nematodes from stranded pygmy sperm whales, Kogia breviceps (Kogiidae), in three localities of the Yucatan Peninsula, Mexico

The present paper reports the presence of 3 adult and juvenile anisakid nematode species: Anisakis simplex, A. brevispiculata, and Pseudoterranova ceticola, which were recovered from the digestive tract of stranded pygmy sperm whales (Kogia breviceps) from 3 localities along the coast of the Yucatan Peninsula. The presence of these anisakid adult nematodes suggests that larval stages may occur in cephalopods or fishes used for human consumption, which represents a potential danger to public health. The occurrence of the 3 anisakid species in coastal waters of the Yucatan Peninsula represents new geographical records for Mexico.     

A new species of Isocyamus (Amphipoda: Cyamidae) from Kogia breviceps (De Blainville, 1838) in Australian waters

Isocyamus kogiae n. sp. is described from a pygmy sperm whale Kogia breviceps (De Blainville, 1838) stranded on Moreton Island, Queensland, Australia. It belongs in the genus Isocyamus because of its white appearance, epaulette-like processes at the anterior dorsum of pereon segment 2, lateral spines on segments 3 and 4, reduced maxilliped, and absence of maxilliped palps, large accessory gills and dentate comb on the dactyle of gnathopod 1. It differs from Isocyamus delphini (Guerin-Meneville, 1836), the only other member of this genus, in that the accessory gills are not equal in length to the main gills, it has no ventral spines, and the dactylus of percopod 1 has a double rather than a single dentate comb. This is the first report of a cyamid from whales of the genus Kogia.     

Terranova ceticola n. sp. (Nematoda: Anisakidae) from the dwarf sperm whale; Kogia simus (Owen), in the Gulf of Mexico

Summary Terranova ceticola n.sp. is described from specimens in the stomachs of an individual dwarf sperm whale, Kogia simus, which stranded on the beach at Biloxi, Mississippi. It is characterized by possessing a single pair of medial preanal papillae, 38 to 40 pairs of lateral preanal papillae, five pairs of postanal papillae, and spicules 1 to 2% of the body length and by lacking cuticular plates at the posterior anal lip of males. ac]19800820     

Unusual multisystemic pathology in a sperm whale bull

This report describes an unusual combination of lesions and apparent behavioral abnormalities in a sexually mature sperm whale bull taken in whaling operations off Iceland. Lesions included heavy combative scarring of the head, grossly roughened and thickened skin on the lower left flank, cutaneous maculae, genital papillomatosis, partial duodenal obstruction by plastic debris, colo-rectal obstruction by ambergris, cystic degeneration of the right kidney, and a deeply ulcerative gastric nematodiasis. Sealskin was found in the stomach. Gross and histopathologic observations suggested that the disease complex in this animal may have been related both to habitat degradation and health risks naturally associated with its ecology and age.     

Characterization of 18 SNP markers for sperm whale (Physeter macrocephalus)

We report the characterization of 18 new single nucleotide polymorphism (SNP) markers for an endangered species, the sperm whale (Physeter macrocephalus), developed using a targeted gene approach. SNP markers were derived from autosomal regions of the genome using primers originally characterized for genome mapping in other mammals. These SNP markers are the first to be designed for genotyping sperm whale populations and will provide a necessary addition to the genetic tools employed for understanding population structure on a global scale and for developing a conservation management strategy for this endangered species.     

Genital papillomatosis in sperm whale bulls

Examination of 31 male sperm whales (Physeter catodon) caught off the western coast of Iceland revealed three cases of genital papillomatosis involving the unsheathed penis. One subadult and two sexually mature bulls were affected. Gross lesions resembled papillomas common in terrestrial mammalian species. Transmission electron microscopy of these lesions revealed nonenveloped intranuclear virus particles 28-40 nm in diameter and round to hexagonal in shape. In two cases immunoperoxidase staining was negative for group-specific papillomavirus antigen. These findings indicate that the spectrum of animal species affected with virus-associated genital papillomatosis includes at least one globally distributed species of the order Cetacea (whales, dolpins, and porpoises).     

The cardiac ventricles of a baleen whale (Balaenoptera acutorostrata: Minke whale) and a toothed whale (Hyperoodon ampullatus: Bottlenose whale)

Fifteen minke whale (Balaenoptera acutorostrata: Mysticeti) and three bottlenose whale (Hyperoodon ampullatus: Odontoceti) hearts were obtained from Norway. Only the ventricular mass and the base of the great arteries were available for study. The shape of the heart, the double apex, the narrowness of the interventricular septum in the dorsiventral plane, the side-to-side position of the ventricles, the relative thickness of the wall of the right ventricle, and the heavy trabeculation agree with other, brief reports of whale heart morphology. Coronary artery tortuosity, which has been described in sperm whales by others, was found in the bottlenose but not in the minke whales. It is suggested that the shape of the heart is associated with depth and duration of dives and that the pattern of trabeculation may be related to the size of the animal.     

In vitro sperm characterization and development of a sperm cryopreservation method using directional solidification in the killer whale (Orcinus orca)

Research was conducted to characterize seminal traits and to develop a sperm cryopreservation method using directional freezing (DF) for the killer whale (Orcinus orca). Experiments evaluated effects of: (i) freezing rate (SLOW, MED, FAST) by diluent (BF5F, Biladyl®, EYC) in 0.5 mL straws; and (ii) freezing method (straw or DF) by glycerol (3, 6, or 9% final concentration, v:v) on in vitro sperm quality. Fresh ejaculates (n = 161) were (mean ± SD) 7.8 ± 7.4 mL at 740 × 10⁶ sperm/mL with 92.2 ± 6.3% total motility (TM), 85.4 ± 6.9% progressive motility (PM), 89.6 ± 9.0% viability and 89.8 ± 9.2% acrosome integrity. Samples frozen using straws by the MED or SLOW method were improved (P < 0.05) over FAST across all diluents. At 3 h post thaw (PT), TM, PM, Rapid motility (RM), VAP, VCL, ALH and viability for 3% and 6% glycerol were improved (P < 0.05) over 9% glycerol. Directional freezing samples at 0 h and 3 h PT, at all glycerol concentrations, displayed higher (P < 0.001) TM, PM, RM, VAP, VSL, VCL and viability /intact acrosomes (PI/FITC-PNA) than straw. These data provided the first information on ejaculate characteristics and the development of a semen cryopreservation method using DF in the killer whale.     

Gleitflug des FlugbeutlersPetaurus breviceps papuanus (Thomas)

Summary Experiments with models made on thin flat plates were made in front of a wind tunnel in order to determine the relationship between their coefficients of lift and drag (Fig. 2) and the angle of attack as well as their aspects of flow (Fig. 8). The following results were achieved. When the plates were covered on one side with the fur from aPetaurus' flight skin, coefficients of lift at average angles of attack were improved, the critical angle of attack was increased and the characteristics of flow separation were flattened. However, these positive effects were only achieved by physiological orientation of the fur (i.e. placed on the upper surface with fur lying backwards; Fig. 3). The gliding number is improved at very high angles of attack only (Fig. 6). Thus the fur of aPetaurus acts as a lift generator within high (critical) angles of attack, during gliding flight (Nachtigall, 1979). Other natural and synthetic furs show a qualitatively similar, but quantitatively less distinct effect (Fig. 7). The aerodynamic efficiency of a fur coating is due to the boundary layer effects provided by the individual hairs which seem to act as miniature back-flow breaks (Fig. 8,9). The bionic transferability of this effect to technical wings is discussed.

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Mit Unterstützung der Deutschen Forschungsgemeinschaft

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Für melßtechnische Mitarbeit danke ich Frfiulein Hedwig Reichel und Herrn Rainer Grosch. Frau Lore Dinnendahl danke ich für Literaturhinweise.     

Struvite calculus in the vagina of a bottlenose dolphin (Tursiops truncatus)

On 27 January 2000, a struvite calculus was observed in the vagina during necropsy of a 138-cm-long female bottlenose dolphin (Tursiops truncatus) collected from the Stono River, South Carolina (USA). Vaginal calculi have been reported in other species of cetaceans but not in bottlenose dolphins. Urinary tract infection might have been an underlying cause of the calculus. While urinary tract inflammation was not detected by light microscopic evaluation of sections of the urinary tract, it is conceivable that sufficient time had lapsed following voiding of the calculus through the urethra for urinary tract infection to have resolved. To further define the prevalence and significance of urolithiasis, prosectors of dead stranded marine mammals are encouraged to closely observe their urinary and genital tracts for calculi and to submit them for quantitative analysis.     

Nucleotide sequence of the D-loop region of the sperm whale (Physeter macrocephalus) mitochondrial genome

We have amplified, by the polymerase chain reaction, and have sequenced the D-loop region of the mitochondrial DNA from the sperm whale (Physeter macrocephalus). The sperm whale D-loop was aligned with D- loop sequences from four other cetaceans (Commerson's dolphin, orca, fin whale, and minke whale) and an out-group (cow). This alignment showed the sperm whale sequence to be larger than that of other cetaceans. In addition, some sequence blocks were highly conserved among all six species, suggesting roles in the functioning of mitochondrial DNA. Other blocks that were previously reported to be well conserved among cetaceans showed little sequence conservation with the sperm whale D-loop, which argues against the functional importance of these sequence blocks in cetaceans.      

An attempt at intracytoplasmic sperm injection of frozen-thawed minke whale (Balaenoptera bonaerensis) oocytes.

Little is known about the characteristics of fertilisation events in minke whales. Cryopreserved minke whale oocytes and spermatozoa do not fertilise in a standard IVF. This study was conducted to investigate the pronucleus formation ability of cryopreserved minke whale oocytes and their subsequent development following intracytoplasmic sperm injection (ICSI). In experiment 1, frozen-thawed minke whale immature oocytes were cultured for in vitro maturation (IVM) in a maturation medium (TCM199) supplemented with either porcine follicle stimulating hormone (pFSH)/estradiol-17beta (E2) or pregnant mare's serum gonadotropin (PMSG)/human chorionic gonadotropin (hCG). After 120 h of IVM, oocyte survival was examined before ICSI, and showed no significant difference in morphological normality (24-36%) between the two IVM media. Two-cell embryos (two oocytes from 21 sperm-injected oocytes) were obtained when the maturation medium was supplemented with pFSH/E2 or PMSG/hCG. In experiment 2, cryopreserved maturing oocytes were investigated for the effects of repeat-culture (2 h or 24 h) on survival before ICSI. Pronuclear formation and development were examined for the effects of sperm pretreatment with dithiothreitol (DTT) and oocyte activation with ethanol at ICSI. A frequency of 49-69% of frozen-thawed maturing oocytes was used for ICSI. Although oocyte activation did not produce a significant difference in survival, pronucleus formation and embryonic development, 2- and 4-cell cleaved oocytes were observed after injection of sperm pretreated with DTT.