Effect of volume of non-surgical embryo transfer medium on ability of porcine embryos to survive to term

Non-surgical embryo transfer is a promising method for improving efficiency in the pork industry and also for biotechnology applications, such as in vitro embryo production, transgenesis and cloning. Several groups have reported successful piglet production using an artificial insemination catheter or flexible catheter designed for this procedure; however, the efficiency of the technique is still low. The critical points that need to be addressed in order to improve this procedure are (1) the embryo deposition site and (2) volume of transfer medium associated with the embryos; however, the latter has not yet been examined systematically. In the present study, we evaluated the effect of the volume of non-surgical embryo transfer medium on the ability of porcine embryos to survive to term by using a recently produced flexible catheter. The catheter consists of a guide and an injector. Blastocysts 200-230 mum in diameter were collected from donor gilts and transferred to recipient gilts. The time required for the completion of embryo transfer using this catheter was 14.6 +/- 3.9 min. The tip of the injector was determined by laparotomy to be located in a uterine horn 20-30 cm anterior from the branching point of the uterus body. We transferred 17.0-17.3 embryos with different volumes of medium (1.6, 3.2 and 10 ml) into each of 5, 4 and 4 recipients, respectively, and pregnancy was confirmed in 4, 3 and 1 of these recipients, respectively. Three recipients in the 1.6 ml group farrowed a total of 19 piglets (4, 5 and 10 piglets, respectively). These results suggest that successful non-surgical embryo transfer is affected by the volume of transfer medium.     

Induction of reactive oxygen generation and functional changes in mitochondria and their involvement in the development of bacterial rot in lettuce caused by Pseudomonas cichorii

Pseudomonas cichorii is the major causal agent of bacterial rot disease in lettuce, and apoptosis-like programmed cell death is closely associated with disease development. Depletion of cellular ATP and expression of the alternative oxidase gene was observed in lettuce leaves inoculated with P. cichorii suggesting mitochondrial dysfunction. Cytochemical observation showed production of reactive oxygen species in mitochondria of P. cichorii-inoculated lettuce leaf cells. Release of cytochrome c into the cytosol was also induced by inoculation with the bacterium. Superoxide production was observed in the mitochondrial fraction isolated from P. cichorii-infected leaves much more intense than water-treated leaves. Loss of swelling ability was also observed in the mitochondrial fractions following inoculation with P. cichorii. Intriguingly, inhibitors of the mitochondrial respiratory complex III prevented loss of swelling ability, whereas superoxide generation was scarcely affected by inhibitors of mitochondrial permeability transition in the mitochondrial fractions. Inhibitors of the mitochondrial respiratory complex III and mitochondrial permeability transition delayed not only P. cichorii-induced cell death, but also disease development. In contrast, P. cichorii-induced DNA fragmentation was not inhibited in the presence of either type of inhibitor. The findings suggest that mitochondria may play a crucial role in DNA fragmentation-independent cell death pathway(s).     

Foraging dispersion of Ryukyu flying-foxes and relationships with fig abundance in East-Asian subtropical island forests

Background

Figs are widely distributed key resources to many tropical-subtropical animals, and flying-foxes are major consumers and seed dispersers of figs. Bat-fig interrelationships, however, may vary among species differing in fruiting traits, i.e., bat- versus bird-dispersed figs. We examined Ryukyu flying-fox foraging dispersion and the relationships with tree species composition and fig abundance in forests of Iriomote Island.

Results

Bat foraging dispersion showed no spatial patterns with respect to different areas of the island, and was not explained by heterogeneity, density, or basal area (BA) of total trees, nor by relative density or BA of fruiting trees or total fruiting figs among sites. Instead, bat densities were positively dependent on the relative density of total figs, and particularly the relative BA of bat-dispersed figs Ficus septica and F. variegata. Both species were dominant figs in forests, fruiting asynchronously with long crop seasons, and were used as predominant foods. Bats foraged mostly solitarily and the mean density was in a hump-shaped relationship with crop sizes of the dominant bat-figs. These two species and Ficus benguetensis are larger-sized bat-figs, all contained more seeds, higher dry-pulp mass and water mass, but not necessarily water content. By approximate estimation, higher proportions of seeds of these bat-figs would have been removed from fruits through the bat consumption, than that of small-sized bird-figs like F. virgata, F. superba, and F. microcarpa.

Conclusions

The foraging dispersion of Ryukyu flying-foxes in forests depends on the availability of the most abundant bat-figs that serve as predominant foods. Intermediate levels of crop sizes of theses figs appear most fit with their solitary foraging. Our results suggest that as density and BA coverage of these dominant bat-figs are below a certain level, their effectiveness to attract bats may dwindle and so would their chance of dispersal by bats.

     

Phagocytosis of bovine blood and milk polymorphonuclear leukocytes after ozone gas administration in vitro

To determine the effects of ozone on the phagocytosis of bovine polymorphonuclear leukocytes (PMNs), ozone gas was administered in vitro on the blood and milk of healthy lactating cows, cows with acute mastitis, and cows with milk fever. In the blood of healthy dairy cattle, although there was no significant effect of ozone gas on the viability of the leukocytes, phagocytosis of PMNs significantly decreased. In contrast, ozone gas administration in vitro significantly increased phagocytosis of PMNs from the blood of cows with acute mastitis and milk fever, and from mastitic milk. These findings showed that ozone administration in vitro has positive and negative effects on bovine PMN phagocytosis, depending on the health status of the animal.     

Significance of PWT4–Rwt4 interaction in the species specificity of Avena isolates of Magnaporthe oryzae on wheat

We examined whether PWT4, an avirulence gene of Avena isolates of Magnaporthe oryzae toward wheat, corresponded to Rwt4, a resistance gene identified in wheat cultivar Norin 4, in a one-to-one manner. Twelve wheat cultivars were inoculated with 65X1, an F₁ culture with PWT4 derived from a cross between an Avena isolate (Br58) and a Triticum isolate (Br48). Three wheat cultivars (Norin 26, Shin-chunaga, Cheyenne) were resistant and therefore selected as possible carriers of Rwt4. The three cultivars were then inoculated with a population derived from a backcross of 61M2 carrying PWT4 with Br48 carrying pwt4. Segregation analyses revealed that PWT4 operates against the three cultivars. If PWT4 corresponds to Rwt4 in a one-to-one manner, all three cultivars should carry Rwt4. To test if this is the case, the three cultivars were crossed with Chinese Spring (a noncarrier of Rwt4) and Norin 4. When F₂ seedlings from Chinese Spring × Norin 26, Chinese Spring × Shin-chunaga, and Chinese Spring × Cheyenne were inoculated with 61M2, resistant and susceptible seedlings segregated in a 3 : 1 ratio. On the other hand, crosses between the three cultivars and Norin 4 yielded no susceptible F₂ seedlings. These results indicate that all three cultivars carry Rwt4. Considering all results, we concluded that PWT4 corresponds to Rwt4 in a one-to-one manner. An inoculation test with Chinese Spring–Cheyenne chromosome substitution lines indicated that Rwt4 is located on chromosome 1D.     

An Antibody Against the SecA Membrane Protein of One Phytoplasma Reacts with Those of Phylogenetically Different Phytoplasmas

ABSTRACT Antisera raised against phloem-limited phytoplasmas generally react only with the phytoplasma strain used to produce the antigen. There is a need for an antiserum that reacts with a variety of phytoplasmas. Here, we show that an antiserum raised against the SecA membrane protein of onion yellows phytoplasma, which belongs to the aster yellows 16S-group, detected eight phytoplasma strains from four distinct 16S-groups (aster yellows, western X, rice yellow dwarf, and elm yellows). In immunoblots, approximately 96-kDa SecA protein was detected in plants infected with each of the eight phytoplasmas. Immunohistochemical staining of thin sections prepared from infected plants was localized in phloem tissues. This antiserum should be useful in the detection and histopathological analysis of a wide range of phytoplasmas.     

Demonstration of the absence of intervening sequences (IVSs) within 16S rRNA genes of Taylorella equigenitalis and Taylorella asinigenitalis isolates

A total of 57 Taylorella equigenitalis (n=22) and Taylorella asinigenitalis (n=35) isolates was shown not to carry any intervening sequences (IVSs) within 16S rRNA gene sequences. By contrast, we have already shown the genus Taylorella group to carry several kinds of IVSs within the 23S rRNA gene sequences.     

Analysis of a pair of END+ and END? viruses derived from the same bovine viral diarrhea virus stock reveals the amino acid determinants in Npro responsible for inhibition of type I interferon production

The Exaltation of Newcastle disease virus (END) phenomenon is induced by the inhibition of type I interferon in pestivirus-infected cells in vitro, via proteasomal degradation of cellular interferon regulatory factor (IRF)-3 with the property of the viral autoprotease protein N^pro. Reportedly, the amino acid residues in the zinc-binding TRASH motif of N^pro determine the difference in characteristics between END-phenomenon-positive (END⁺) and END-phenomenon-negative (END⁻) classical swine fever viruses (CSFVs). However, the basic mechanism underlying this function in bovine viral diarrhea virus (BVDV) has not been elucidated from the genomic differences between END⁺ and END⁻ viruses using reverse genetics till date. In the present study, comparison of complete genome sequences of a pair of END⁺ and END⁻ viruses isolated from the same virus stock revealed that there were only four amino acid substitutions (D136G, I2623V, D3148G and D3502Y) between two viruses. Based on these differences, viruses with and without mutations at these positions were generated using reverse genetics. The END assay, measurements of induced type I interferon and IRF-3 detection in cells infected with these viruses revealed that the aspartic acid at position 136 in the zinc-binding TRASH motif of N^pro was required to inhibit the production of type I interferon via the degradation of cellular IRF-3, consistently with CSFV.