Only an optimum number of viable spermatozoa in a frozen-thawed insemination dose can ensure conception at artificial insemination (AI). We report here the percentages of normal, abnormal and viable spermatozoa present in the frozen-thawed semen of 20 Black Bengal bucks used for commercial AI. Bucks in this experiment were of 19.3~46.1 months old and 25~42 kg body weight. Four semen straws (0.25 ml) from each buck were collected for evaluation of their kinetic parameters. Scrotal circumference was measured by using a scrotal tape, sperm motility was estimated on eye estimation and sperm concentration was determined by using a haemocytometer. Sperm morphology was studied in paraformaldehyde fixed spermatozoa under differential interference contrast (DIC) microscope. To determine the proportion of live (plasma membrane intact) spermatozoa, semen was stained with SYBR-14 and propidium iodide and examined under fluorescent microscope. Scrotal circumference, post-thaw sperm motility, sperm concentration per insemination dose and proportion of normal spermatozoa were , , million and , respectively. The percentages of spermatozoa with head shape and acrosome abnormalities were lower ( and , respectively), whereas higher percentages of abnormalities () were observed in mid piece and tail portion. The proportion of live spermatozoa was . It is concluded that although a good number of morphologically normal spermatozoa are present in the insemination dose, the proportion of live spermatozoa is low, which warrants further improvements of buck semen freezing procedures to ensure good quality at AI.

The standard does much to improve merchantable quality, distribution efficiency and fair dealings by shipping of the standard agricultural products. Mushrooms notified as the standard are five items; Pleurotus ostreatus, Pleurotus eryngii, Flammulina velutipes, Agaricus bisporus and Ganoderma lucidum. But many farmers are suffering from a strict standards. So these standard is required modification to fit farmhouse situations. This study was carried out to investigate mushroom quality of farm shipping and quality change during preservation at various treatment. Flammulina velutipes and Agaricus bisporus preserved at different temperature( 4℃, 10℃, 20℃) to investigate quality changes. Pileus diameters of Flammulina velutipes was 67% of first grade and 33% of second grade at the early stage. After two weeks, pileus diameters was not signigicant changes; 50% of first grade, 50% of second grade at 4℃ and 50% of second grade, 50% of third grade at 10℃. Although there were no significant changes of diameter at 20℃, most of the fruit bodies were decayed. In case of Agaricus bisporus, pileus diameter was 15% of first grade, 81% of second grade and 4% of third grade at the early stage. The more storage period longed, the more the color of the fruit body was browned. But pileus diameters was not signigicant changes. Hardness and chromaticity of these mushroom was declined as time passed. Now we are carrying out more research on some mushroom’s standards for improve consumer confidence.

Neurotrophic factors are essential to maintain and organize neurons functionally; thereby neurotrophic factor-like substances or their inducers are expected to be applied to the treatment of neurodegenerative diseases such as stroke. In the present study, we firstly examined the effects of ethanol extracts of Hericium erinaceus (HE, Yamabushitake), on nerve growth factor expression in neuronal cells. HE extract promoted NGF expression in a brain tissue. Here we assessed neuroprotective effects of HE with a transient global cerebral ischemia model. Global cerebral ischemia was induced by occluding both common carotid arteries. Treatment with HE was initiated after ischemia induction and given once a day for 7 consecutive days. Neuronal cell loss in CA1 of hippocampus was significantly decreased and the performance in the Morris water maze was significantly improved in rats administered HE. We conclude that treatment with HE attenuated learning and memory deficits, motor functional disability, and neuronal cell loss induced by global cerebral ischemia. These results suggest that HE may be a potential candidate for the treatment of vascular dementia.

Mushrooms including Mycoleptodonoides aitchisonii are used as foods and employed as folk remedies for diabetes and inflammatroy diseases. This study analyzed anti-diabetic effects of Mycoleptodonoides aitchisonii, which grows in some areas such as Gangwon-do and Jeju-do in Korea, as insulin-derived phosphorylation. When 100 ng/ml of IL-6, inflammatory cytokine was given to SK-hep1, HepG2, Akt phosphorylation by insulin was found to be remarkably reduced. In addition, metformin, Antidiabetics serving as positive control in liver was used. Mycoleptodonoides aitchisonii used for analyzing anti-diabetic effects in this test didn’t give a great impact on the decrease in Akt phosphorylation by IL-6 at high concentration. However, fruit body in Mycoleptodonoides aitchisonii inhibited the decrease in Akt phosphorylation by IL-6 according to the concentration. At the highest concentration 100㎍/ml, it had an effect of increasing Akt phosphorylation to 77%, which was decreased by 50% compared to the insulin treated group by IL-6. Therefore, Mycoleptodonoides aitchisonii fruit body used in this test activated Akt phosphorylation inhibited by IL-6 and showed a possibility of significant anti-diabetic effects compared to positive control(metformin).

We have purified and characterized of metalloprotease metalloprotease from Nomuraea atypicola. N. atypicola was cultured in Sabouraud medium supplemented with powdered pupae. The metalloprotease from culture supernatant was purified to electrophoretically homogeneous state. The molecular mass of metalloprotease from N. atypicola was 50 kDa. The enzyme was most active at pH 8.5 and 40oC and stable at pH 5.0-7.0 and up to 40oC. The activity was inhibited by o-phenanthroline and EDTA. The N-terminal amino acid sequence of the enzyme showed a similarity to those of proteases (Metallo peptidase M36 family (Fungalysin)) from Coccidioides posadasii and Aspergillus fumigatus. The enzyme was found to be Fungalysin-like metalloprotease. cDNA encoding metalloprotease from N. atypicola was amplified by PCR using oligonucleotides deduced from the N-terminal endo peptide sequence, 5’- and 3’-RACE. Predicted enzyme structure consists of 637 amino acids with pro- and signal sequences. The mature enzyme had 391 amino acids and its deduced amino acid sequence coincided completely with the N- terminal amino end (20 amino acids) of metalloprotease purified from N. atypicola. We are studying on expression of the metalloprotease gene in Escherichia coli.

This study was carried out to decrease the royalties through the dissemination of domestic mushroom varieties by following UPOV (Union Internationale Pour la Protaection des Obtentions Vegetablue: the international union for the protection of new varieties of plants). Although RDA has bred various mushrooms of 25 species of 78 varieties since 1990, most of these were not cultivated in mushroom farms due to two reasons. One is the weaknees of spawn supply system caused by increasing own spawn production and the other is the difficulty in replacing a new variety due to the year-round production in bottle cultivation. Therefore, we made a program to disseminate domestic mushroom varieties. In 2010, five species of 14 varieties were provided as a form of spawn for 43 farms that do not have their own cultivation facility and 11 species of 18 varieties were provided for 45 farms that were equipped with their own cultivation facility. In addition, the breeders explained the cultural characteristics of their own varieties and consulted cultural farms. Since these activities were helpful to mushroom farmers, we plan to open the presentation for mushroom instructor in February 2011 with the consideration of the opinion of mushroom farmers to fully disseminate the spawn of domestic mushroom varieties for the bottle cultivation farms.

This study was carried out investigate to availability of commercial microbial pesticide and antibacterial activity of isolates isolated from different mushroom media. Ten commercial microbial pesticide and EM liquid were collected from different company. EM of these one has been used for control of mushroom disease and growth promotion at the mushroom farms. The density of bacteria and yeast in EM cultural liquid was higher than those of the crude liquid. The pH values of EM showed the low acid levels(pH 3.5~3.9) by organic acid secreted from microorganism. The kinds of organic acid was acetic acid, lactic acid and succinic acid. The dominant bacteria isolated from EM liquid was Lactobactillus sp.(21 strains), Acetobacter sp.(9 strains), PaeniBacillus sp.(9 strains) and others(12 strains). The organic acid bacteria isolated from fermentation foods( was 92 strains and the dominant genus was Weissella sp.(41 strains), Leuconostoc sp.(21 strains), Enterococcus sp.(9 strains), Lactococcus sp.(9 strains) and others(12 strains). And we isolated 2,500 bacteria from oyster mushroom and button mushroom cultural media for selection of antagonistic bacteria. Thirty five strains of these isolates showed very strong antagonistic activity. These strains were identified Bacillus amyloliquefaciens, Bacillus subtilis, Brevibacterium halotolerans, Pseudomonas libanensis, Stenotrophomonas maltophilia and Alcaligenes faecalis by 16S rDNA analysis.

The winter mushroom, Flammulina velutipes, is one of the major economical crops cultivated in Korea. The total production have steadily increased approximately 40,161 M/T in 2005 to 61,057 M/T in 2009. Several bacteria have been known as the causal agents of certain diseases of cultivated button mushroom(Agaricus bisporus) oyster mushroom(Pleurotus ostreatus) and winter mushroom(Flammulina velutipes). It is well known as bacterial diseases of the cultivated mushroom such as brown blotch, mummy disease, bacterial pit, bacterial rot, weeping disease, ginger blotch, and drippy gill. Black rot has been recognized as a major problem within the mushroom industry. Pseudomonas tolaasii has been shown to be associated with a black disorder of the caps and stipe of the mushroom. Recently, P. tolaasii was isolated from disease cultivated winter mushrooms grown in Korea. Its symptom appeared as dark brown and sunken lesions on the caps and stipes of affected mushrooms. Inoculation of bacterial isolates into mushroom caps and stipes showed characteristic black rot symptoms and sunken lesions. Results of Gram stain, staining of flagella and biochemical tests identified these isolates as P. tolaasii. This was confirmed by pathogenicity, physiological and biochemical characteristics, and results of an analysis of the 16S rRNA gene sequences.

Agaricus bisporus grows on a substrate known as compost, which is a product of aerobic fermentation by various microorganisms. These organisms convert and degrade the straw and form lignin humus complex which is utilized later on by the population of organisms. Theses microflora play a key role in the process of composting and can be regarded as the active agents in the preparation of nutrient medium as many of them may ultimately contribute themselves to the nutrition of A. bisporus. The diversity of microflora according to growing farmhouse and fruiting body of Agaricus bisporus were investigated. The aerobic bacteria and Bacillus as longer of turning stage of compost pile were increased. And, thermophilic actinomycetes and fluorescent Pseudomonas sp. showed high density after the pasteurization stage. But Tricoderma sp. was decreased toward the end of turning stage of compost pile. Ten mushroom farms was selected to research of microflora of fruiting body of button mushroom. The microflora showed significant difference according to mushroom farms. The bacteria density was 0.4~41.6×105 cfu/ml and the fungus was 1.3~3.9×103 cfu/ml. But The microorganism density was not significant change for the storage periods. These isolates were classified into Chryseobacterium indologenes(6 strains), Pseudomonas agarici(5 strains), Sphingobacterium multivorum(2 strains), Flavobacterium anhuiense(2 strains), Microbacterium sp.(10 strains), Pseudomonas sp.(13 strains) on the basis of 16 rDNA analysis. The most dominants of these species were Chryseobacterium indologenes and Pseudomonas agarici.

Mushroom is cultivated as one of the major economical crops in many areas of the Korea. The total production have steadily increased approximately 151,913 M/T in 2000 to 186,400 M/T in 2007. This study was carried out to investigate applicability of mushroom production using various organic media resources within the country. Eight organic resources were collected from various areas. Pleurotus ostreatus and Fulammulina velutipes showed the highest growth at the media of 10% red ginseng marc, 20% lacquer tree, 20% Juglans mandshurica, 10% Cudrania tricuspidata, 10~20% Acer pensylvanicum, 10% Lindera glauca. Mushroom mycelial growth at red ginseng marc media was slower than that of the control. But the sponin of the red ginseng was not detected at the fruiting body grown from red ginseng marc media, And three organic resources(barly powder, sweet potato powder, potato powder) was used to substitute rice bran used in mushroom cultivation. Pleurotus ostreatus and Fulammulina velutipes showed the highest growth at 10~30% sweet potato powder, 20% barly powder and 10% potato powder.

Several bacteria have been known as the causal agents of certain diseases of the cultivated button mushroom (Agaricus bisporus) and oyster mushroom (Pleurotus ostreatus). It is well known as bacterial diseases of the cultivated mushroom such as brown blotch, mummy disease, bacterial pit, bacterial rot and weeping disease, ginger blotch, and drippy gill. Brown blotch is the most critical cause of crop loss in the commercial mushroom industry. The classical bacterial blotch disease of mushrooms is caused by a fluorescent pseudomonad, Pseudomonas tolaasii. Affected mushrooms show lesions which become dark chocolate-brown, are wet, and deeply pit the caps and stalks. Although Pseudomonas tolaasii has been known as the casual agent of bacterial blotch, much controversy exists regarding the identification of this bacterium and whether blotch may be caused by more than one organism. This study was carried out to investigate characterization and biological control of Pseudomonas tolaasi and other possible browning pathogens isolated from cultivated mushrooms. One hundred seventy four bacteria were isolated from the cultivated mushroom and collected from main producing districts throughout the country. The isolates were classified into Pseudomonas tolaasii(20 strains), Pseudomonas gingeri(1 strains), Pseudomonas agarici(4 strains), Pseudomonas putida(11 strains), Pseudomonas sp.(46 strains), Ewingella americana(14 strains), Stenotrophomonas sp.(4 strains), and others(74 strains) on the basis of 16 rDNA analysis. The most dominants of these species were Pseudomonas tolaasii and Ewingella americana. Pseudomonad isolates were mainly divided into two groups in white line test and a sharply defined white line of precipitate forms in Pseudomonas agar F(Difco) between the opaque white colonies of P. tolaasii and translucent colonies of certain unidentified pseudomonads. The white line test was positive when 20 isolates of P. tolaasi from different countries were examined, whereas 62 isolates of pseudomonads did not give the white line reaction with a reacting translucent colony Pseudomonas. All the isolates tested for white line forming bacteria including P. tolaasi were highly pathogenic to mushroom tissue. Although browning of mushrooms in host tests does not perfectly help in the identification of P. tolaasi, a conspicuous pitting produced at the cut surface of mushroom tissue is as specific as the white line test in detecting P. tolaasii in suspension in distilled water. URP2F primers of 20-mer were used to assess the genetic diversity of white line forming bacteria. The phylogenetic tree was constructed by using the neighbor-joining method. In the analysis of RAPD pattern, all isolates of white line precipitate have some of the different genetic traits as collected districts. Phylogenetic analysis of 16S rDNA revealed that twenty isolates including white line forming bacteria were closely related to P. tolaasii and showed high similarity. To biological control on bacterial browning disease of cultivated mushrooms, six hundreds plant extracts (332 EtOH extracts, 268 water extracts) was used for control of mushroom disease. Thirty plant extracts in bacterial disease(Pseudomonas tolaasii, P. agarici, B. gladioli, E. americana) and thirty three in fungus disease(T. harzianum, C. mycophilum, V. fungicola) showed strong anti-microbes activity. They showed stronger anti-microbes activity at ethanol extracts than water extracts. MIC of extract BCW128 on Pseudomonas tolaasii was 700ppm and HDE17 was 330ppm. MIC of extract YCE107 on P. agarici was 330ppm, JGE96 was 330ppm and BCW128 was 700ppm. The bacteria inhibit tolaasin secreted by Pseudomonas tolaasii was selected three genus(Bacillus sp. etc). Now we are carrying out more research on these bacteria.

This study was carried out to investigate characteristic pattern of fruiting body of Ganoderma lucidum and their antioxidant activity. Mycelia of all strains were firstly inoculated into potato dextrose agar(PDA) and then transfered to a media of saw dust which contained 20% rice bran. These mycelia of saw dust were then inoculated into oak tree in polyethylene bags which has been sterilized for 8h at 120℃. The polyethylene bags were sent to a growth room for growth of fruit bodies. Antioxidant activities of each fruiting body were investigated by DPPH method.

The price of mushrooms harvested by bottle cultivation is rapidly dropping and the income of farm households is also rapidly decreasing due to the increase of production cost. Although some of these mushroom farms have employed the systems of mass production, they are in financial difficulty because of the investment they need to do for facilities such as cultivation room and automated systems. In the other hands, some retailers want to buy a small volume of mushrooms of many different mushrooms produced by the small farms and these small farms was required to investigate the common cultural condition for mushroom production of many different mushrooms. In this study, we investigated the common cultural conditions for production of many different mushrooms (e.g., Pleurotus eryngii and Agrocybe cylindracea) at the bottle cultivation farms. The cultural period of Pleurotus eryngii and Agrocybe cylindracea was 30~35 days. The optimum temperature of the mycelial growth was 25~28℃ with the growth room being maintained about 20~23℃ with the consideration of the respiration heat of the mycelium. The temperature of mushroom growth room was 16~18℃ for all growth periods. In our results, Pleurotus eryngii and Agrocybe cylindracea produced the highest yields at the substrate formulation of sawdust 75%, rice brain 20%, soybean cake wastes 5%, water contents 70% and 850 ml P.P. In the long run, our results will result in the development of new automatic cultivation model and increase the income of small production farms.

In order to develop of practical model of the sawdust cultivation of Lentinula edodes, we conducted experiments of the sawdust cultivation in different conditions of cultivation environment and shapes of sawdust medium. For different types of cultivation environments, we provided environmental control system facility, vinyl house and forest cultivation. We also chose the 2.6kg cylindrical type and the 1.5kg cylindrical type for shapes of medium. We decided cultivation on shelves for the 2.6kg cylindrical type sawdust medium and cultivation on the ground for the 1.5kg cylindrical type sawdust medium to cultivate the mushroom. The result showed that forest cultivation was the first place cultivated mushroom followed by environmental control system facility and cultivation vinyl house. Among 2 types of sawdust medium, the 1.5kg cylindrical type showed better quantity in terms of a fruit body of mushroom.

Lipoxygenase (LOX) is considered to be a key enzyme in the biosynthetic pathways of the most important mushroom aroma, 1-octen-3-ol. In previous work, we purified and characterized a LOX from Pleurotus ostreatus (probably H1 strain) fruit bodies [1] and also determined its partial amino acid sequence. In this study, to clarify the biosynthetic mechanism of 1-octen-3-ol, we isolated cDNA and genomic DNA corresponding to a LOX (Polox1) gene of P. ostreatus H1, and analyzed the expression of the gene in the fruit bodies. A commercial P. ostreatus H1 strain (Onuki kinjin, Utsunomiya, Japan) was used in this study. To isolate the Polox1 cDNA, RT-PCR was done using degenerate primers designed from the partial amino acid sequence. This approach generated a single DNA band of approximately 1.1 kbp, which was cloned and sequenced. The deduced amino acid sequence showed high similarity to LOXs of some ascomycetes fungi. To obtain the full-length cDNA of Polox1, clones corresponding to the Polox1 gene were isolated by plaque hybridization from a cDNA library of the P. ostreatus H1 fruit body. DNA sequences of all clones were determined. The 5’ end of the Polox1 cDNA was amplified by the 5’ RACE method and cloned. The full-length cDNA of Polox1 is 2,031 bp long and contains 640 amino acid residues. The deduced amino acid sequence contains LOX iron-binding catalytic domain signature sequences. Next, to determine the genomic DNA sequence of the Polox1 gene, inverse PCR and PCR was done with P. ostreatus H1 genomic DNA. After inverse PCR and PCR, 3.3 and 1.9 kbp DNA fragments, respectively, were amplified and sequenced. Sequence comparison between cDNA and genomic DNA showed that Polox1 gene contained one intron. To investigate expression of the Polox1 gene, northern blot analysis and measurement of LOX activity were performed. P. ostreatus fruit bodies were produced in a sawdust medium containing beech sawdust and rice bran and separated into pileus and stipe. Two transcripts were detected by northern blot analysis in both pileus and stipe. The band intensities were relatively higher in the stipe than in the pileus. The level of LOX activity in the stipe was 3.8 times higher than that in the pileus. By Southern blot analysis, several major bands were detected after the digestion of 4 restriction enzymes. These blot analyses suggest that the Polox1 gene is probably a member of a small gene family. [1] T. Kuribayashi et al., J. Agric. Food Chem., 50, 1247 (2002).

This study investigated physiological characteristics and genetic relationship of 30 strains of Lentinula edodes, collected from the Europe, Asia, North America and preserved in the Forest Mushroom Research Institute(FMRI). In physiological characteristics, Papua New Guinea strain was excellent mycelium growth in 25℃ for 7 days on PDA media. For all strains, the optimal temperature for mycelial growth, their tunicate and color of hypha were observed. It surveyed their mycelial growth on oak sawdust media in test tube and independence with inter-strains and cultivar developed in FMRI by strain's anastomosis culture. It was carried by RAPD using operon primers as molecular genetic methods, investigated genetic relationships among strains using UPGMA in NYSYSpc(2.1) according to the presence or absence of bands. <This research was supported by Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries>

The genes encoding cellulases, which belong to glycosyl hydrolase families have been cloned from the basidiomycetous mushrooms. The transcripts of cellulase genes are strongly induced when the mycelia are grown in medium containing crystalline cellulose, and they are not expressed in medium containing glucose, but how insoluble substrates such as microcrystalline cellulose are recognized by these fungal cells is not clear. The polypore mushroom Polyporus arcularius is a wood-decomposing basidiomycete that produces at least three types (I, II, and IIIa) of carboxymethyl cellulase (CMCase) when the medium contains crystalline cellulose as the sole carbon source and produced mainly cellobiose in the medium. The genomic and cDNA clones encoding the family 12 endoglucanase (CMCase IIIa) gene (cel3A) of P. arcularius have been sequenced, and Cel3A has been expressed as an active enzyme in Escherichia coli. To determine the role and function of each type of cellulase in the degradation of crystalline cellulose by basidiomycetous mushrooms, the structure of all of the cellulase genes should be investigated, but the nucleotide sequences of the other cellulase genes in P. arcularius have not yet been reported. In the current study, the genomic and cDNA clones encoding the endoglucanases (cel4), and the two cellobiohydrolases (cel1 and cel2) of P. arcularius sequenced and characterized. The predicted amino acid sequence of Cel1 Cel2, Cel3a and Cel4 are similar to glycosyl hydrolase family 7, 6 12 and 5 protein, respectively. The expressions of the all cellulase genes (cel1 cel2, cel3a and cel4) were induced by Avicel (microcrystalline cellulose) and cellopentaose but repressed by glucose, cellobiose, cellotriose, and cellotetraose. There was a low level of transcription of both genes regardless of the carbon source. These results suggest that P. arcularius cells constitutively express a very low level of cellulase that can degrade insoluble crystalline cellulose and that the transcription of celluases in the cells is induced by products produced by these endoglucanases such as cellooligosaccharides. From our findings, we propose a possible mechanism for the recognition and degradation of insoluble crystalline cellulose by fungal cells.

Hundreds of Steller sea lions, Eumetopias jubatus, migrate from Sakhalin and the northern Kuril Islands to Hokkaido every winter. During this migration, they may use their roaring sounds to navigate and to maintain their groups. We recorded the roars of wild Steller sea lions that had landed on reefs on the west coast of Hokkaido, and those of captive sea lions, while making video recordings. A total of 300 roars of wild sea lions and 870 roars of captive sea lions were sampled. The fundamental frequency (F0), formant frequency (F1), pulse repetition rate (PRR), and duration of syllables (T) were analyzed using a sonagraph. F0, F1, and PRR of the roars emitted by captive sea lions increased in the order male, female, and juvenile. By contrast, the F1 of wild males was lower than that of females, while the F0 and PRR of wild males and females did not differ statistically. Moreover, the F0 and F1 frequencies for captive sea lions were higher than those of wild sea lions, while PRR in captive sea lions was lower than in wild sea lions. Since there was a linear relationship between body length and the F0 and F1 frequencies in captive sea lions, the body length distribution of wild sea lions could be estimated from the F0 and F1 frequency distribution using a regression equation. These results roughly agree with the body length distribution derived from photographic geometry. As the volume of the oral cavity and the length of the vocal cords are generally proportional to body length, sampled roars can provide useful information about a population, such as the body length distribution and sex ratio.

Pacta sunt servanda - agreements must be obeyed - is a peremptory principle of modern international law of treaty. What are the origin and nature of this doctrine? Some say, its Latin wording indicates that it is of Roman origin. And this doctrine is a must for the social good; without it the society will be a chaos. But how does Islam perceive the notion? This paper finds that this doctrine came into being with the very beginning of the creation of human souls by Allaah Whom they accepted as their Sole Lord and made a commitment to follow His commands. And it is a principal tenet for Muslims to adhere to because it is an inseparable part of their faith (Iman), a command from their Lord and a practice (Sunnah) of their Prophet Muhammad (peace be upon him). It is, therefore, obligatory not merely because it is good for the society, but is also rooted in the teachings of Islamic doctrine and a Muslim’s accountability in the life hereafter.