Isolation of <Emphasis Type="Italic">Moraxella bovis</Emphasis> from frozen bovine semen and determination of microbial load

In the present study, isolation of Moraxella bovis was done from the microbiological examination of frozen semen straws from clinically healthy twelve Jersey and one Jersey-cross bull supplied by a semen laboratory. The organism was identified on the basis of colonial morphology and biochemical characteristics. Further, viable cell count of the bacterium was studied in detail in the semen sample.     

Reproductive potential of domestic Ovis aries for preservation of threatened Ovis orientalis isphahanica: in vitro and in vivo studies

Although the potential use of reproductive biotechnology for safeguarding of endangered wildlife species is undoubted, initial evaluation of the genetic and reproductive relationship between the endangered mammals and those closely related species is indispensable. Isfahan mouflon Ovis orientalis isphahanica is now considered as a threatened species by International Union for the Conservation of Nature. Therefore, little is known about the biology of this species. This study was carried out to investigate the possible reproductive potential of domestic sheep for ex situ conservation of the Isfahan mouflon. Somatic cell cultures were taken from ear biopsies of the wild and domestic sheep and were used for karyotype analysis. Semen samples were collected by electroejaculator from the wild and domestic rams. The spermatological characteristics of the collected semen samples were determined and used for both cryopreservation and cross-insemination of the synchronized wild and domestic female sheep. To establish a cryobank for the threatened species biomaterials, freezed samples of the somatic cells and semen were transferred to a cryotank. The result suggested that Isfahan mouflon has conserved its chromosomal integrity as previously observed and contains the same chromosomal number as the domestic sheep (2n = 54). The semen samples of both species revealed similar cryoviability (>35% gross motility postthawing). Cross-insemination of both species resulted in successful pregnancy. It was suggested that domestic sheep possesses the required biological characteristics to be considered for safeguarding of the Isfahan mouflon.     

Morphology,ultrastructure and taxonomy of the raphidophycean algaHeterosigma akashiwo

Heterosigma akashiwo and its related algae were re-examined by light and electron microscopes using cultured materials including type cultures of bothH. akashiwo andH. inlandica as well as specimens referred to asOlisthodiscus luteus maintained in CCAP and UTEX. All the strains examined were similar to one another in appearance and ultrastructural features. They can be accommodated in a single species,H. akashiwo, which has been invalidly published. In this paper, the genus is validly desribed by providing a Latin diagnosis and designating a type species. Ultrastructural characteristics are also given for the genus.     

The communicative life of a social carnivore: acoustic repertoire of the ring-tailed coati (Nasua nasua)

The coati is a highly social mammal that features sophisticated cognitive and social abilities. We hypothesized that the ring-tailed coati, Nasua nasua, uses an extensive acoustic repertoire that correlates to their diverse range of social interactions. We tested this hypothesis by observing and recording a free-ranging managed population of N. nasua in Tietê Ecological Park (PET), in the municipality of São Paulo, State of São Paulo, Brazil. Of 404 h of sampling, 47 h of coati vocalizations were recorded over 3 years. Additional records were obtained opportunistically on other free-living populations at PET by using passive acoustic monitoring. We describe here an acoustic repertoire composed of 15 calls (12 basic calls, 2 rhythmic calls and the non-random complex calls composed of three or four different units). This diverse repertoire of signals was used in contact/cohesion regulation, foraging activities, alert or potential threat situations, playing and fighting interactions and during social isolation and acute distress. The contact call (chirp) is produced through biphonation, and other non-linear phenomena are present. Our study found a complex vocal repertoire that encourages further studies to describe the evolution of the cognitive characteristics and social abilities of ring-tailed coatis.     

The status of dwarf carnivores on Cozumel Island, Mexico

Cozumel Island in the Mexican Caribbean is inhabited by four carnivores, of which two, the Cozumel coati Nasua nelsoni and pygmy raccoon Procyon pygmaeus, are endemic species. The taxonomic status of a third carnivore, a dwarf gray fox Urocyon cinereoargenteus, is undetermined, but may deserve subspecific or species-level recognition. The fourth species, the kinkajou (Potos flavus), may be a recent introduction. We review the status of these carnivores, report our field observations and results of line transect and trapping efforts, discuss current threats to these taxa, and make recommendations for their conservation. A population density of 0.43 ± 0.27 coatis/km², and a total island population size of 150 ± 95 individuals, was estimated from 386 km of line transects in 1994–1995. Intensive trapping efforts (1479 trap-nights) in 2001 at multiple localities were unsuccessful. Pygmy raccoons were observed in the mangrove and coastal wetland areas of the island and in 2001 we captured 11 individuals with the same sampling efforts as for coatis (8.8 raccoons/1000 trap-nights). The gray fox is also apparently very rare on the island. While a few observations of the animals have been made (1984, 1994 and 2001), no animals were seen along transects and none were trapped. The primary threats to the persistence of these taxa include introduced congeners, introduced predators, parasite and disease spill-over from exotic animals, habitat fragmentation, hunting and collection as pets, and hurricanes. We suggest that the Cozumel coati, pygmy raccoon, and the Cozumel population of the gray fox be considered as Critically Endangered according to the IUCN classification system. Current conservation actions focusing on Cozumel carnivores are extremely limited. We recommend eradication of introduced species, maintenance of habitat connectivity, ex situ conservation programs, explicit public policies on land-use and sustainable development, public awareness campaigns, and continuous scientific research and monitoring.     

Infestation of arboreal nests of coatis by triatomine species,vectors of Trypanosoma cruzi,in a large Neotropical wetland

The coati (Nasua nasua, Carnivora) is a medium‐sized mammal common in the Pantanal of Brazil. Unlike most mammals, coatis construct arboreal nests used for resting and reproduction. In this region, the coati is an important host of Trypanosoma cruzi, the causative agent of Chagas disease. There are two possible routes through coatis can be infected by T. cruzi: the oral route or the vectorial route. However, the relative importance of each of these routes in the infection of coatis and its role in the sylvatic cycle of the parasite are unknown. Our objectives were to investigate: (i) whether coati nests were infested by triatomine bugs, (ii) what species were frequent in the nests, (iii) whether the triatomines in nests were infected by T. cruzi, and (iv) what were the food resources of these triatomines. Eight of the 24 nests sampled were infested with triatomines, a total of 37 specimens of at least two species (Rhodnius stali and Triatoma sordida). In one nest, R. stali and T. sordida co‐occurred and both fed on multiple resources, including coatis. This is the first report of triatomines occurring in arboreal nests of coatis. The co‐occurrence of two different genera of triatomine vectors and coatis within the limited space of the coati nests provide multiple opportunities for the exchange of the protozoan parasite through both the vectorial and oral transmission routes.     

Characterization of oligosaccharides in milk of a mink, Mustela vison

Carbohydrates were extracted from a sample of milk from a mink, Mustela vison (Family Mustelidae). Free neutral and acidic oligosaccharides were isolated from the carbohydrate fraction and their chemical structures were compared with those of white-nosed coati (Nasua narica, Procyonidae) and harbour seal (Phoca vitulina, Phocidae) that we had studied previously. The ratio of free lactose to milk oligosaccharides was similar to that in milk of the white-nosed coati; in both species, this ratio was much lower than that in the milk of most eutherians. The neutral oligosaccharides of mink milk had alpha(1-3)-linked Gal or alpha(1-2)-linked Fuc residues at their non-reducing ends, as in the neutral oligosaccharides of white-nosed coati milk. Some of the neutral and acidic oligosaccharides, determined here, had been found also in harbour seal milk, but the harbour seal oligosaccharides did not contain alpha(1-3)-linked Gal residues.     

Morphology and ultrastructure of the cyanobacterium Mastigocladus laminosus growing under nitrogen-fixing conditions

The morphological and ultrastructural characteristics of the cyanobacterium Mastigocladus laminosus growing under N₂-fixing conditions were examined with light and electron microscopy. Vegetative cells in narrow filaments contained randomly arranged segments of thylakoid membrane, centrally located carboxysomes (polyhedral bodies), peripherally located lipid bodies, and large numbers of polysaccharide granules in addition to nuclear material and ribosomes. The ultrastructural characteristics of cells in wide filaments were similar, except for increased numbers of carboxysomes and lipid bodies. Heterocytes and proheterocysts developed at a variety of locations in narrow filaments, wide filaments, and the lateral branches off of wide filaments. Akinetes were not observed in any of the filaments. The morphological characteristics of heterocysts and proheterocysts were variable and depended on those of the vegative cells from which the heterocysts and proheterocysts developed. Mature M. laminosus heterocysts were somewhat similar to those formed in other cyanobacterial genera, but they possessed a number of distinct and unique ultrastructural characteristics, including (i) the absence of a fibrous and, possibly, a laminated wall layer, (ii) the presence many closely packed membranes throughout most of the cytoplasm, and (iii) the presence of unidentified, spherical inclusion bodies of variable electron density.     

Improved ultrastructural preservation ofPetunia andBrassica ovules and embryo sacs by high pressure freezing and freeze substitution

Summary In order to improve the ultrastructural preservation of the female gametophyte ofPetunia x hybrida andBrassica napus we tested several cryofixation techniques and compared the results with those of conventional chemical fixation methods. Ovules fixed with glutaraldehyde and osmium tetroxide in the presence or absence of potassium ferrocyanide showed poor cell morphological and ultrastructural preservation. In ovules cryo-fixed by plunging into liquid propane, the cell morphology was well preserved. However, at the ultrastructural level structure-distorting ice crystals were detected in all tissues. Due to the large size of the ovules, cryofixation by plunging in liquid propane is not adequate for ultrastructural studies. In contrast,P. x hybrida andB. napus ovules cryo-fixed by high pressure freezing showed improved cell morphological as well as ultrastructural preservation of the embryo sac and the surrounding integumentary tissues. The contrast of the cellular membranes after freeze substitution with 2% osmium tetroxide and 0.1% uranyl acetate in dry acetone was high. At the ultrastructural level, the most prominent improvements were: straight plasma membranes which were appressed to the cell walls; turgid appearing organelles with smooth surface contours; minimal extraction of cytoplasmic and extracellular substances. In contrast to the chemically fixed ovules, in high pressure frozen ovules numerous microtubules and multivesicular bodies could be distinguished.     

Effects of cooling and freezing on the motility of Ostrea edulis (L., 1758) spermatozoa after thawing

The aim of this work was to evaluate the effects of temperature, cryoprotectant agents and freezing curves on sperm motility of Ostrea edulis. All phases of cryopreservation were studied (evaluation of semen motility pattern, choice of cryoprotectants and freezing rates) to restore after thawing the motility characteristics distinctive of fresh semen.To assess the temperature effects on sperm motility, semen was activated using four different temperatures (25, 18, 10 and 3 °C). Sperm aliquots were maintained inactive at these temperatures for 1 and 3 h, then activated with FSW at same temperature of conservation. Sperm was activated and incubated to 3 °C with dimethylsulfoxide (Me₂SO), ethylene glycol (EG), 1–2 propylene glycol (PG) (5%, 7%, 10% and 15% final concentrations), glycerol (GlOH; 5%, 10% and 15% final concentrations) and methanol (MetOH; 4% and 10% final concentrations) for 10, 20 and 30 min. A first evaluation of freezing rates was made by testing four freezing curves: −1, −3, −6 and −10 °C/min. Then, an optimization was made by testing four freezing curves: −2.5, −3.0, −3.5 and −4 °C/min.The selected temperature for short term conservation has been 3 °C, because only this temperature has allowed good sperm motility conservation after 3 h of dry-storage; this is a time sufficient to conduct cryopreservation procedures. The sperm showed a particular sensitivity to GlOH and PG to all tested concentrations and to 15% Me₂SO. EG and MetOH to all concentrations and Me₂SO to concentrations lower than 15% have not shown significant toxic effects. The freezing rate −3 °C/min using 15% EG has shown an highest percentage of RVF (rapid, vigorous and forward) spermatozoa (class 3, about 75% of fresh semen) and an highest sperm motility duration.     

Epididymal histoplasmosis diagnosed by isolation ofHistoplasma capsulatum from semen

An autochthonous case of epididymal histoplasmosis masquerading as tuberculosis in a 55-year-old male patient is reported from India. It was diagnosed by culture ofHistoplasma capsulatum from semen and by demonstration of the fungus upon re-examination of epididymal biopsy sections previously misinterpreted as tuberculous granuloma. The patient's main complaints were painful epididymal swelling, occasional fever and cough. He was treated successfully by excision of epididymis and vas deferens combined with amphotericin B therapy. This is believed to be the first case of epididymal histoplasmosis to be reported outside the American continent and the fourth of its type reported in the English literature. The case is also noteworthy in thatH. capsulatum was isolated for the first time from semen, and it underlines the importance of mycological culture of semen specimens for diagnosis of genitourinary infections of obscure etiology.Presented at the XII Congress of the International Society for Human and Animal Mycology, Adelaide, Australia, March 13–18, 1994.     

Semen characteristics of the quaker parakeet (Myiopsitta monachus)

This report describes characteristics of semen samples collected from eight captive quaker parakeets (Myiopsitta monachus) using the massage technique. Overall, semen characteristics were (mean±SE, range in parentheses, n=8 males): ejaculate volume 1.96±0.26μl (1.02–2.96), sperm concentration 346.6±64.6 million/ml (74.8–579.3), and number of sperm per ejaculate 0.71±0.16 million (0.09–1.53). Significant differences were observed between males for all three semen characteristics. Semen pH for the eight males ranged from 8.05 to 8.5. The semen samples were collected early in the breeding season, so the data reported may not be representative of ejaculates from males in peak breeding condition. However, this study provides the first rigorous semen data from this species and demonstrates that good‐quality semen samples, suitable for artificial insemination, can be collected regularly from quaker parakeets using the massage technique. Zoo Biol 21:507–512, 2002. © 2002 Wiley‐Liss, Inc.     

Variations in seminal parameters over a 12-month period in captive bonnet monkeys

Semen samples were collected from adult fertile bonnet monkeys twice a month by penile electroejaculation for twelve consecutive months. Various parameters like semen volume, weight of ejaculate and coagulum, sperm count, sperm motility, sperm morphology, and functional parameters e.g. plasma membrane integrity,in vitro nuclear chromatin decondensation and acrosomal status were evaluated to assess within and between animal variations. Effects of seasonality, if any, on quantity and quality of semen were also studied. Considerable intra- and inter-individual variations in the geometric mean values were observed for semen volume, weights of ejaculate and coagulum, and sperm counts during the study period. On the other hand, sperm motility, morphology, and functional parameters showed less within and between animal variations. Results on motility, morphology, and functional parameters indicated that good semen quality was maintained throughout the year. Various routine and functional parameters did not show any annual variations. The diurnal rhythmicity in circulatory testosterone levels was observed throughout the year. The study shows lack of seasonality in exocrine and endocrine testicular functions and further suggests that motility, morphology, and functional parameters are better indicators of semen quality in captive bonnet monkeys.     

Seasonal changes in semen quality and correlation with plasma hormone profiles in Karan Fries bulls

The aim of the study was to assess the influence of summer and winter seasons on semen quality and plasma hormone concentrations in cross-bred bulls. Semen was collected by an artificial vagina from eight bulls and microscopically evaluated for quality parameters. Semen volume was higher in summer season (p < 0.05) than winter season, whereas nonsignificant variation (p > 0.05) was observed in mass motility, individual motility, sperm viability, sperm concentration and percentage of membrane-intact and acrosome-intact spermatozoa. Plasma prolactin and testosterone concentration were significantly (p < 0.01) higher in summer season than winter season. Plasma testosterone levels were positively correlated with semen volume and negatively correlated with individual motility (p < 0.05). Prolactin showed a significant positive correlation with semen volume. A well-defined seasonal pattern in semen characteristics was not observed and few correlations existed between plasma hormone levels and semen characteristics in Karan Fries bulls.     

Cryobanking of lemur spermatozoa

Artificial inseminations using fresh semen were successful inLemur fulvus mayottensis. Inseminations with frozen semen are in progress.     

Effects of extender composition, cooling rate and freezing on the fertilisation viability of spermatozoa of the Pacific oyster (Crassostrea gigas)

The aim of this research was to optimise protocols for freezing spermatozoa of the Pacific oyster. All the phases of the cryopreservation procedure (choice of cryoprotectant, cooling, freezing, and thawing) were studied in relation to the species of spermatozoa to restore on thawing the morphological and physiological characteristics of fresh semen. The choice of type and concentration of cryoprotectant in which semen is incubated before freezing is fundamental for a successful cryopreservation: the cryoprotectants (dimethylsulfoxide--Me(2)SO, ethylene glycol--EG, propylene glycol-PG, and glycerol in concentrations between 5 and 15%) were tested for their toxicity on the semen exposed up to 30 min at +26 degrees C (room temperature) by evaluating its ability to fertilise and the embryo development to the regular D larval stage. The best cryoprotectants, Me(2)SO, EG, and PG 5, 10, and 15% respectively, were used for the pre-cooling (adaptation/cooling) tests. Two different adaptation/cooling procedures were tested: (A) from +26 degrees C to 0-2 degrees C (2.6 degrees C/min) and (B) at +26 degrees C for 15 min. Lastly, using the cryoprotectants and the adaptation procedure (B) that had given the best results in the preceding stages of the experiment, four cooling rates were tested: 6, 11, 16, and 21 degrees C/min. It was seen that the semen that was incubated with EG 10%, adapted at +26 degrees C for 15 min, and then cooled at a rate of 6 degrees C/min showed a percentage of regular D larvae on thawing comparable to that of fresh semen (p > 0.05).     

Ultrastructure of leaves in C4 Cyperus iria and C3 Carex siderosticta

The ultrastructural aspects ofCyperus iria leaves showing the C₄ syndrome and the typical C₃ species,Carex siderosticta, in the Cyperaceae family were examined.C. iria exhibited the chlorocyperoid type, showing an unusual Kranz structure with vascular bundles completely surrounded by two bundle sheaths. The cellular components of the inner Kranz bundle sheath cells were similar to those found in the NADP-ME C₄ subtype, having centrifugally arranged chloroplasts with greatly reduced grana and numerous starch grains. Their chloroplasts contained convoluted thyla-koids and a weakly-developed peripheral reticulum, although it was extensive mostly in mesophyll cell chloroplasts. The outer mestome bundle sheath layer was sclerenchymatous and generally devoid of organelles, but had unevenly thickened walls. Suberized lamellae were present on its cell walls, and they became polylamellate when traversed by plasmodesmata. Mesophyll cell chloroplasts showed well-stacked grana with small starch grains. InC. siderosticta, vascular bundles were surrounded by the inner mestome sheath and the outer parenchymatous bundle sheath with intercellular spaces. The mestome sheath cells degraded in their early development and remained in a collapsed state, although the suberized lamellae retained polylamellate features. Plastids with a crystalline structure, sometimes membrane-bounded, were found in the epidermal cells. The close interveinal distance was 35–50 μm inC. iria, whereas it was 157–218 μm inC. siderosticta. These ultrastructural characteristics were discussed in relation to their photosynthetic functions.     

Cystobasidiopsis nirenbergiae, a new agaricostilbomycete (Pucciniomycotina)

A new genus, Cystobasidiopsis, and a new species, Cystobasidiopsis nirenbergiae, are described for a fungus isolated from an arable loess soil in Ahlum near Braunschweig, Niedersachsen, Germany. An integrated analysis of morphological, ecological, ultrastructural and molecular data indicates that the new species belongs to the Chionosphaeraceae within the Agaricostilbales. Relevant characteristics of the new species are discussed and compared with those of related taxa.     

Biochemical,morphological, and genetic variations in <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> due to colony disaggregation

Microcystis aeruginosa commonly occurs as large colonial morph under natural conditions, but disaggregates and exists as single cells in laboratory cultures. To demonstrate the adaptive changes, differentiation of carbohydrates, pigments, and protein between colonial and disaggregated M. aeruginosa were examined. Their morphological and ultrastructural characteristics were subsequently observed by scanning electron microscopy and transmission electron microscopy. Results showed that chlorophyll a and phycocyanin in cells, soluble carbohydrate produced in the culture medium, and total carbohydrate in cells and sheath of colonial M. aeruginosa are significantly higher (p < 0.05) compared with those in disaggregated cells. No significant change was found in protein concentration per cell (p > 0.05) between them. Their morphological and ultrastructural characteristics were evidently different, and by morphological criteria they could be separated into two morphotypes. In addition, the genetic diversity of 16S–23S internal transcribed spacer of them were examined and compared with reference strains of M. aeruginosa. The alignment of two sequences revealed that genetic identity level was extremely high (96.94%) and no significant difference was found in the nucleotide diversity (0.014 ± 0.008). This suggested that similar genotypes could present distinct morphotypes in M. aeruginosa. The tree topologies and analysis of molecular variance of the two sequences and reference sequences from GenBank database indicated that the genotypes of M. aeruginosa strains were not always related to their localities and exhibit heterogeneity within a species.     

Cone photopigments in nocturnal and diurnal procyonids

Summary Procyonids are small, New World carnivores distributed among some 6 genera. Electroretinogram (ERG) flicker photometry was used to measure the spectra of the cone photopigments for members of two nocturnal species, the raccoon (Procyon lotor) and the kinkajou (Potos flavus), and a diurnal species, the coati (Nasua nasua). Each of the 3 has a class of cone photopigment with maximum sensitivity in the middle to long wavelengths. The spectral positioning of this cone is different for the three. Whereas the raccoon and kinkajou are monochromatic, the diurnal coati is a dichromat having an additional class of cone photopigment with peak sensitivity close to 433 nm.Abbreviations ERG electroretinogram - SWS short wavelength sensitive