New strategies of boar sperm cryopreservation: Development of novel freezing and thawing methods with a focus on the roles of seminal plasma

Cryopreservation of boar spermatozoa offers an effective means of long‐term storage of important genetic material. Many researchers have investigated how to improve reproductive performance by artificial insemination (AI) using cryopreserved boar spermatozoa. Recently, we and other groups reported that high conception rates (70–80%) can be achieved by AI with frozen‐thawed boar spermatozoa using a modified temperature program during freezing, or a novel cryopreservation extender to improve sperm quality (including sperm survivability, motility, membrane status and fertilization ability) after thawing, or a novel sperm infusion method, deep intra uterine insemination. However, these techniques have not yet been used for commercial pig production. The variation in sperm freezability among boars or among ejaculations in an identical boar is one of the main reasons for this problem. In our previous study, it was revealed that some components of seminal plasma have a negative effect on the freezability of boar sperm. One of these factors is bacteria‐released endotoxin (lipopolysaccharide: LPS). LPS binds to Toll‐like receptor‐4 (TLR‐4) expressed on the sperm surface, resulting in induction of apoptosis. On the other hand, seminal plasma suppresses cryo‐capacitation induced by thawing stress. On the basis of these findings, we designed a novel protocol of AI using frozen‐thawed boar sperm.     

Collie eye anomaly in Hokkaido dogs: case study

Objective To describe a Hokkaido dog, one of the traditional Japanese breeds that was affected by Collie eye anomaly (CEA), and to report the genotype of this dog and the Hokkaido dog allelic frequency of the CEA‐associated mutation. Case A nine‐month‐old intact female Hokkaido dog without any obvious visual disturbance was diagnosed ophthalmoscopically with CEA. Severe choroidal hypoplasia was observed in the bilateral temporal area adjacent to the optic nerve head, appearing as whitish areas. Therefore, the dog was suspected of possessing the CEA‐associated mutation that was previously reported as an intronic 7.8‐kilo base deletion in the canine NHEJ1 gene. Procedures SYBR Green‐based real‐time PCR with a melting curve analysis, conventional PCR with agarose gel electrophoresis, and direct DNA sequencing were carried out to determine the genotype of the dog. Furthermore, a preliminary genotyping survey was carried out in 17 Hokkaido dogs from three kennels using the real‐time PCR method, and the pedigree relationships were analyzed using their pedigree papers. Results The Hokkaido dog affected by CEA was proven to possess the CEA‐associated mutation. Of these 17 Hokkaido dogs, 12 dogs were heterozygous carriers and five dogs were affected by this mutation. The preliminary genotyping survey and pedigree analysis demonstrated that the allelic frequency of the CEA‐associated mutation is very high in Hokkaido dogs. Conclusion These data suggest that the Hokkaido breed is highly susceptible to CEA because of the known CEA‐associated mutation much like the Collie‐related breeds.     

Impaired postnatal development in C/EBPbeta-deficient mice

Tail DNA genotyping of fetal and neonatal mice from C/EBPbeta heterozygous parents was performed to determine whether the decreased number of surviving C/EBPbeta mutants was caused by prenatal or postnatal death. Eighty-four 3-week-old mice born of heterozygous parents had significantly lower numbers of C/EBPbeta-deficient offspring than the expected Mendelian ratio (29.8%+/+, 65.5%+/-, 4.76%-/-, P<0.05). The genotypes of 72 fetal mice from intercrossed heterozygotes showed approximately the expected 1:2:1 Mendelian ratio (18.1% +/+, 52.8% +/-, 29.2% -/-, P>0.05). No difference in the proportions by sex could be detected in these perinates. This data indicates that C/EBPbeta-deficient mice have unknown lethal problems between the embryonic stage and weaning.     

Development of green onion and cabbage certified reference materials for quantification of organophosphorus and pyrethroid pesticides

Green onion and cabbage certified reference materials for the analysis of pesticide residues were issued by the National Metrology Institute of Japan, part of the National Institute of Advanced Industrial Science and Technology. Green onion and cabbage samples were grown so as to contain several kinds of organophosphorus and pyrethroid pesticides, and those were collected from a field in the Kochi Prefecture in Japan. The certification was carried out by using multiple analytical methods to ensure the reliability of analytical results; the values of target pesticides (diazinon, fenitrothion, cypermethrin, etofenprox, and permethrin for green onion and chlorpyrifos, fenitrothion, and permethrin for cabbage) were obtained by isotope dilution mass spectrometry. Certified values of target pesticides were 0.96-13.9 and 2.41-6.9 mg/kg for green onion and cabbage, respectively. These are the first green onion and cabbage powder certified reference materials in which organophosphorus and pyrethroid pesticides are determined.     

Effect of Harpin from Four Pathovars of Pseudomonas syringae on Pea Defense Responses

To investigate the role of the proteinaceous elicitor, harpin, on host and nonhost plants, we isolated the harpin-coding gene, hrpZ, from Pseudomonas syringae pvs. pisi, glycinea, tabaci and tomato. Effects of the recombinant harpin proteins on pea plants were analyzed and compared with the effects of the corresponding bacterial treatment. After inoculation of pea with pea pathogen P. syringae pv. pisi, the bacterial population increased and the accumulation of PAL-mRNA and pisatin was inhibited. The nonpathogenic pathovars, glycinea, tabaci and tomato induced both defense responses in pea. However, none of the harpins induced the hypersensitive reaction or accumulation of PAL-mRNA and pisatin in pea. Harpins from P. syringae pvs. glycinea, tomato and pisi did induce these defense responses in tobacco, however, suggesting that externally applied harpins either are not recognized or are nonfunctional in pea plants. Received 27 June 2000/ Accepted in revised form 21 February 2001     

Properties and classification of selected volcanic ash soils from Abashiri,Northern Japan—transition of Andisols to Mollisols

Abstract

Properties and classification of four selected volcanic ash soils from Abashiri, Hokkaido were studied and the transition of Andisols to Mollisols was discussed. Two of the four pedons (Brown Andosol and Cumulic Andosol)¹ showed morphological, clay mineralogical, physical, and chemical properties common to most Andisols in Japan. However, the properties of the other two pedons (Acid Brown Forest soil and Brown Forest soil)¹ were considerably different from those of common Andisols in Japan. It was found that the changes in the andic soil properties or transition of Andisols to Mollisols was closely related to the progression of clay weathering, mainly the transformation of noncrystalline clay materials to halloysite. One of the four pedons (Brown Forest soil)¹had the clay fraction dominated by halloysite from the uppermost horizon down to the bottom of the profile and satisfied both andic and mollic requirements. Thus we concluded that the pedon is a transitional soil between Andisols and Mollisols and that the transition is closely related to the duration of surface weathering under relatively weak leaching conditions. The four pedons were classified according to the Andisol Proposal (Leamy et al. 1988, New Zealand Soil Bureau) as follows:

Pedon 1: Medial, amorphic (allophane/imogolite), frigid Typic Hapludand (Brown Andosol).¹ Pedon 2: Medial, amorphic (allophane/imogolite), frigid Typic Melanudand (Cumulic Andosol).¹ Pedon 3: Medial, amorphic (allophane/ imogolite), over kandic, frigid Typic Melanudand (Acid Brown Forest soil).¹ Pedon 4: Medial, kandic, frigid Typic Hapludand (Brown Forest soil).¹     

Characterization of the major components in bark from five Japanese tree species for chemical utilization

Summary Characterization of the major components in bark from five Japanese tree species, Pinus densiflora, Larix leptolepis, Cryptomeria japonica, Fagus crenata, Quercus mongolica, was performed from a viewpoint of chemical utilization. Relatively high yields of formaldehyde-condensable polyphenols were obtained from the bark of P. densiflora (22.5%) and L. leptolepis (20.8%) by 1% NaOH extraction. The alkali insoluble residue contained large amounts of neutral sugars (26.1%–40.7%) in comparison with untreated bark. Glucose was generally the dominant sugar in the residue, although the major neutral sugar content in F. crenata was xylose. By successive treatment with 1% NaOH and acid chlorite, a marked decrease in lignin content was observed in P. densiflora. In the other tree species, bark lignin was resistant to acid chlorite treatment to a certain extent. Received 20 December 1996     

Lateral resistance of anchor-bolt joints between timber sills and foundations II. Effective lateral resistance of multiple anchor-bolt joints

Monte Carlo simulations were conducted to estimate the effective lateral resistance of multiple anchor-bolt joints with ordinary specifications of Japanese post and beam constructions. Basic lateral load-slip curves of single anchor-bolt joints required in the simulations were determined from the test results of our earlier report. The effective lateral resistance of multiple anchor-bolt joints was estimated for some combinations of loading direction, length/diameter ratio of anchor bolts, lead-hole clearance, and number of anchor bolts. The principal results of the simulations are: (1) anchor-bolt joints loaded perpendicular to lateral forces are not recommended to be counted as supplementary resisting elements because their supplementary shares are far less than those expected from their allowable lateral resistance; (2) multiple anchor-bolt joints with small length/diameter ratios have comparatively lower effective resistance ratios than multiple anchor-bolt joints with large length/diameter ratios; (3) the effective resistance of multiple anchor-bolt joints is affected not only by lead-hole clearance or number of bolts but also variance of load-slip characteristics of single anchor-bolt joints. Part of this work was presented at WCTE2004, Lahti, June 2004