Structural characterization of carangid fish myoglobins

The primary structures of myoglobin (Mb) from the following five carangid species were determined: yellowtail Seriola quinqueradiata, greater amberjack Seriola dumerili, yellowtail kingfish Seriola lalandi, Japanese horse mackerel Trachurus japonicus, and silver trevally Pseudocaranx dentex. The sequences were of varying composition both in the coding and in the noncoding regions, but all contained the open reading frame of 444 nucleotides encoding 147 amino acids. Amino acid sequence identities of carangid Mbs were in the range of 81-99%. The similarity of the heme pocket and associated heme-binding residues of carangid Mbs were evidence of the conservative nature of Mbs. Similar to the other teleost Mbs, carangid Mbs did not contain a D helix and had mostly conserved A and E helices as well as E-F and G-H inter-helical segments. Hydropathy profiles of carangid Mbs showed species-specific variations where silver trevally Mb exhibited generally higher hydrophobicity. Phylogenetic analysis based on the primary structures was in agreement with conventional morphological taxonomy, establishing close proximity of carangid Mbs with those of cichlid and scombroid, the other members of the Perciformes order.     

Analysis of genetic relations between <Emphasis Type="Italic">Broad bean wilt virus 1</Emphasis> and <Emphasis Type="Italic">Broad bean wilt virus 2</Emphasis>

We determined the complete nucleotide sequence of RNA-1 and the 5-terminal region of RNA-2 from Broad bean wilt virus 1 (BBWV-1) isolate PV132. This report is the first analysis of the genome organization of BBWV-1. We also determined the complete nucleotide sequence of RNA-1 from Broad bean wilt virus 2 (BBWV-2) isolate IP and analyzed the genetic relations between BBWV-1 and BBWV-2. Similar to the BBWV-2 isolates, both RNAs of PV132 encoded a single large polyprotein, which was predicted to contain some functional proteins in a manner similar to those of comovirus. With respect to the deduced amino acid sequences of the mature proteins, PV132 and IP had only 20%–40% homology to comovirus. On the other hand, IP was 73%–98% homologous to BBWV-2 isolates, but PV132 was 39%–67% homologous to the isolates. Although the extent of the homologies differed, the homologies were limited between BBWV-1 and BBWV-2 not only for the coat protein but also for the other proteins. These results clearly support the placement of BBWV-1 and BBWV-2 in the genus Fabavirus as distinct species, proposed on the basis of double immunodiffusion tests.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB084450 (RNA-1 of isolate PV132), AB084451 (RNA-2 of isolate PV132), and AB023484 (RNA-1 of isolate IP)     

Counting absolute number of lymphocytes in quail whole blood by flow cytometry

In a previous study, we reported a new method for counting quail blood cells. After quail blood cells were stained with fluorescent lipophilic dye (DiOC6(3)), absolute counts of erythrocytes, granulocytes, and monocytes were obtained by means of flow cytometry (FC). The FC method has the potential for application to avian blood cells count; however, the method was unable to distinguish between lymphocytes and thrombocytes. In the present study, we improved the FC method to obtain separate counts of lymphocytes using DiOC5(3). After quail blood cells were stained with DiOC5(3), the cells were measured with FC. Each blood cell type was distinguished by means of their typical FL-1 (green fluorescence) and SSC (side scatter). Absolute numbers of erythrocytes, granulocytes, monocytes and lymphocytes in whole blood were obtained. The improved FC analysis worked equally well with chicken (Gallus gallus) and goose (Anser cygnoides) blood.     

Optimization of Ca2+ concentrations in fusion and activation media for production of cloned embryos from miniature pig somatic cells

The effects of Ca(2+) concentration in activation medium and cytochalasin B treatment after activation on the parthenogenetic development of pig oocytes were examined. In addition, cloned embryos derived from miniature pig somatic cells were activated under optimal conditions and the effects of Ca(2+) in fusion medium on the development of embryos after activation was examined. When oocytes were activated in 0.1 mM Ca(2+) and then treated with cytochalasin B, the blastocyst formation rate (28.6%) was significantly higher than those activated in 0-0.05 or 1.0 mM (11.0-18.3%). Treatment with cytochalasin B decreased the second polar body extrusion rate of activated oocytes. The presence or absence of Ca(2+) in fusion medium did not affect the fusion rate of miniature pig somatic cells with recipient oocytes. A few cloned embryos developed to the blastocyst stage (2.7-9.0%) without an additional activation treatment. On the other hand, significantly more embryos developed to the blastocyst stage after activation treatment when they were fused in the absence (28.9%) of Ca(2+) rather than the presence (16.5%) of it. These results show that the highest blastocyst formation rate for miniature pig cloned embryos is obtained when donor cells and recipient oocytes are fused in the absence of Ca(2+) and then activated in 0.1 mM Ca(2+) and treated with cytochalasin B.     

In vitro formation of holes on the inner perivitelline layer of quail ovum by chicken spermatozoa

The aim of the present study was to examine whether chicken semen can be substituted for quail semen to conduct in vitro experiments on fertilization. Chicken spermatozoa was incubated with the inner perivitelline layer (IPL) isolated from the largest follicle in the quail ovary under in vitro conditions. The perforation of chicken and quail spermatozoa were assessed by counting the number of all visible holes in the pieces of IPL. No difference was found in the number of holes formed by the chicken sperm and quail IPL interaction compared with that between intraspecies. In addition, the number of holes in the IPL was significantly increased with the increase in sperm concentration from 1 × 10⁶ to 8 × 10⁶ sperm/mL (P < 0.05). Interestingly, after treatment of chicken spermatozoa with 2.5 mg/mL of the solubilized quail IPL followed by incubation with the intact chicken IPL, the number of holes in the intact chicken IPL was significantly decreased as compared with that of spermatozoa treated without the solubilized IPL (P < 0.05). This indicates that sperm receptors in the solubilized quail IPL and binding ligands on the chicken spermatozoa would find each other and bind, forming complexes and these complexes blocked the interaction between chicken spermatozoa and intact chicken IPL. These results show that: (i) chicken spermatozoa possess the penetrability into quail IPL; and (ii) a high degree of affinity via the receptor interactions exists between chicken spermatozoa and quail IPL. Therefore, it appears that the substitution of chicken semen for quail semen is possible to use as an in vitro technique to examine the sperm‐IPL interaction during fertilization in quail.     

Phylogenetic and morphological analyses of Pythium graminicola and related species

Isolates of Pythium graminicola and related species were differentiated using restriction fragment length polymorphism (RFLP) analyses of the internal transcribed spacer (ITS) regions of rDNA and the cytochrome c oxidase subunit II (COX II) gene. These sequences were used in subsequent phylogenetic analyses. Finally, the phylogenetic placement of species was compared to that determined from morphological characteristics. The 62 isolates tested were divided into seven groups, A–G, based on RFLP analysis of the rDNA-ITS region. In the RFLP analysis of the COX II gene, isolates were divided into groups similar to those based on ITS-RFLP. Groups A and B were each separated into two additional subgroups. Grouping of isolates based on RFLP analyses agreed with the morphological differentiation. Groups A, B, D, E, F, and G were identified as P. graminicola, P. arrhenomanes, P. aphanidermatum, P. myriotylum, P. torulosum, and P. vanterpoolii, respectively. Group C was closely related to group B based on phylogenetic analysis of the rDNA-ITS region and the COX II gene and is similar to P. arrhenomanes. Each of the other species occupied their own individual clades. Although P. arrhenomanes is morphologically similar to P. graminicola, our phylogenetic analyses revealed that it was evolutionarily distant from P. graminicola and more closely related to P. vanterpoolii. Our analysis also revealed that P. torulosum with smaller oogonia is more closely related to P. myriotylum with large oogonia than to P. vanterpoolii, which forms smaller oogonia and is morphologically similar to P. torulosum. P. aphanidermatum with large oogonia and aplerotic oospores was not related to the morphologically similar species P. myriotylum. Results suggest that P. graminicola and related species are phylogenetically distinct, and molecular analyses, in addition to morphological analyses, are necessary for the accurate taxonomic placement of species in this complex.     

Effects of lauric acid on physical, chemical and microbial characteristics in the rumen of steers on a high grain diet

The effects of being fed lauric acid on rumen characteristics were evaluated in a double 3 × 3 Latin square design using six Holstein steers with ruminal cannulas on a high grain diet. The steers were fed commercial concentrate (8.7 kg/day/steer) with one of three levels of lauric acid (0, 25 or 50 g/day/steer) and timothy hay (1.8 kg/day/steer). The feed intake and digestibility were determined. Ruminal fluid was collected at 3 h after feeding to determine chemical, physical and microbial parameters. An in vitro pure culture study was performed to determine the effects of lauric acid on Streptococcus bovis, a potent bloat‐ and acidosis‐promoting rumen bacterium. There were no differences in feed intake and digestibility among the treatments. The proportion of butyrate and the viscosity of the rumen fluid tended to be lowered (P < 0.08 and P < 0.09, respectively) and the stable ingesta volume increase was significantly decreased (P < 0.01) by the lauric acid feed. The abundance of protozoa and bacteria did not differ among the treatments. In the in vitro study, the growth of S. bovis was inhibited by the lauric acid (100 nmol/L) but it showed an adaptive growth to lauric acid in long‐term subculturing. The S. bovis that had adapted to lauric acid showed decreased viscosity and lactate production (P < 0.01) in culture with sucrose. These results indicate that supplemental lauric acid added to a high grain diet improves physical properties, possibly by altering the metabolic activity of S. bovis, and it may prevent the occurrence of feedlot bloat and acidosis in beef cattle.