• Title/Summary/Keyword: Amino Acid Requirement

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Nutritional Regulation of GLUT Expression, Glucose Metabolism, and Intramuscular Fat Content in Porcine Muscle

  • Katsumata, M.;Kaji, Y.;Takada, R.;Dauncey, M.J.
    • Asian-Australasian Journal of Animal Sciences
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    • v.20 no.8
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    • pp.1297-1304
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    • 2007
  • We conducted a series of investigations in order to elucidate role of nutritional status in regulating GLUT expression and energy metabolism in porcine muscle. Firstly, the role of mild undernutrition in regulating muscle GLUT gene expression and function was studied in growing pigs (3 wk of age) on a high (H) or low (L) food intake (H = 2L) at $35^{\circ}C$ or $26^{\circ}C$. Low food intake selectively upregulates GLUT1 and GLUT4 gene expression; mRNA levels were elevated in longissimus dorsi (L. dorsi) and rhomboideus muscles but not in diaphragm or cardiac muscles. Our next step was to determine whether dietary lysine, a major primary limiting amino acid in diets for pigs, affects muscle GLUT4 expression. Pigs of 6 wk of age were pair-fed a control or low lysine (LL) diet. The control diet contained optimal amounts of all essential amino acids, including 1.15% lysine. The LL diet was similar but contained only 0.70% lysine. GLUT4 mRNA expression was upregulated by the LL diet in L. dorsi and rhomboideus muscles, whereas that in cardiac muscle was unaffected. GLUT4 protein abundance was also higher in rhomboideus muscle of animals on the LL diet. We conducted another investigation in order to elucidate effects of the LL diet on post-GLUT4 glucose metabolism. Activity of hexokinase was unaffected by dietary lysine levels while that of citrate synthase was higher both in L. dorsi and rhomboideus muscles of pigs fed on the LL diet. Glucose 6-phosphate content was higher in L. dorsi msucle in the LL group. Glycogen content was higher both in L. dorsi and rhomboideus muscles in the LL group. Further, we determined the effects of dietary lysine levels on accumulation of intramuscular fat (IMF) in L. dorsi muscle of finishing pigs. A low lysine diet (lysine content was 0.40%) meeting approximately 70% of the requirement of lysine was given to finishing pigs for two months. IMF contents in L. dorsi of the pigs given the low lysine diet were twice higher than those of the pigs fed on a control diet (lysine content was 0.65%). Finally, we proved that a well known effect of breadcrumbs feeding to enhance IMF of finishing pigs could be attributed to shortage of amino acids in diets including breadcrumbs.

Effects of Dietary Synthetic Amino Acid Supplementation in Korean Rockfish Fry Sebastes schlegeli (치어기 조피볼락에 있어서 사료내 합성아미노산 첨가 효과)

  • 김강웅;박건준;옥임호;배승철;최영준;신인수
    • Journal of Aquaculture
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    • v.15 no.3
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    • pp.157-163
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    • 2002
  • Thirteen groups, each consisting of 25 juveniles (0.64 g) of the Korean rockfish, Sebastes schlegeli were reared in aquaria after period of one week conditioning. Each group was fed with one of the 13 diets at the rate of 7~10% body weight (on a dry-matter basis) 2 times a day for 6 weeks. Each diet was supplemented with one of the following amino acids at 3g/kg diet: Alanine (Ala), Arginine (Arg), Glycine(Gly), Glutamate (Glu), Histidine (His), Isoleusine(Ile), Lysine (Lys), Methionine (Met), Phenylalanine (Phe), Proline (Pro), Threonine (Thr), Tryptophan (Trp) or Valine (Val). Groups fed with Pro, Thr, Met or Gly-supplemented diet showed significantly (p<0.05) higher weight gain and faster specific growth rate than the groups fed on other diets, while those fed Pro, Thr, Met or Gly were not significantly different each other(P>0.05). Feed efficiency (FE) of fish fed Pro was significantly higher than those fed the other diets except that fed Thr (P<0.05). However, there was no significant difference between FE of fish fed Pro and Thr, and among FE of fish fed Thr, Met and Gly (P>0.05). The requirement of rockfish is higher for Met and Thr. Extra Pro and/or Gly may also stimulate biosynthesis of the nucleic acids (IMP, GMP) which are known as the feed stimulant in fish.

The Effects of Supplementing Methionine plus Cystine to a Low-protein Diet on the Growth Performance and Fat Accumulation of Growing Broiler Chicks

  • Bunchasak, C.;Satoso, U.;Tanaka, K.;Ohtani, S.;Collado, Cristino M.
    • Asian-Australasian Journal of Animal Sciences
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    • v.10 no.2
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    • pp.185-191
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    • 1997
  • This experiment was conducted to determine the effects of a low-protein diet supplemented with DL-methionine plus L-cystine (Met + Cys) on the growth performance and fat accumulation of female broiler chicks during the growing period (3-6 wks old). A low-protein diet (17% CP; 3,200 ME kcal/kg) was supplemented with Met + Cys (1.1 : 1.0) at levels 0.75, 0.94, 1.25, 1.31 or 1.50% of diet, respectively. Another diet with 21% CP and 3,200 ME kcal/kg served as the control group. All essential amino acids were adjusted to meet the National Research Council (1984) requirement for chicks. Feed and water were given ad libitum. Body weight of the chicks fed the low-CP diets supplemented with Met + Cys were heavier than those of the control birds. Feed conversion ratio and feed intakes were not significantly different between and among the treatment groups. Similary, abdominal fat content was not significantly different among the various treatments except that of the chicks fed the low CP diet with 1.25% Met + Cys which was higher than that of the control group. Fatty acid synthetase (FAS), acetyl-CoA carboxylase (ACC) activities and carcass protein content were not influenced by dietary treatments. Carcass fat content was lowest in chicks fed low CP diet with 0.75% Met + Cys and highest in the group that received 1.50% Met + Cys supplementation. Liver triglyceride increased as Met + Cys supplementation level increased. Various lipid fraction concentrations (cholesterol ester, free cholesterol, and phospholipid) in the serum went up as Met + Cys increased up to 1.25% after which it levelled off. Results of this experiment suggest that it is possible to reduce dietary protein level from 21% to 17% for growing broiler chicks by the supplementation of Met + Cys when other EAA were sufficient.

Development of Protein-rich Food Mixtures for Infants and Growing Children in Korea (I) Preparation, Chemical Compositions and Rheological Properties of the Mixtures (유유아(乳幼兒) 및 성장기 아동을 위한 영양식품 개발에 관한 연구 (1) 제품의 조제(調製) 및 그 성상(性狀))

  • Cheigh, Hong-Sik;Kwon, Tai-Wan
    • Korean Journal of Food Science and Technology
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    • v.2 no.1
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    • pp.96-103
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    • 1970
  • No foods are available commercially for weanling infants except a limited amount of expensive milk products in Korea. Although the majority of infants are breast-fed, when it is not possible, rice products must usually be substituted which is not sufficient in protein. Therefore, it is urgent to develop low-cost quality protein food mixtures. In order to accomplish this purpose three food mixtures (F-S-2, F-F-3 and F-P-4), consisting of rice (37∼46%), soybean (24∼40%), FPC (3∼7%), vitamins, minerals and other food additives, are developed. The food mixtures are white to light yellow in color; dispersed readily in water with water absorption index 320; viable bacterial population, less than $10^4$ per gram; sedimentation value, 63; Bostwick consistency value, 15cm/30%; and ring test value, 23cm/30%. The products contain 22∼25% protein and ensure reasonably balanced essential amino acids for the requirement of infants compared with FAO provisional pattern, Rao's maximum growth requirements and Holt's amino acid requirements in early life. Although threonine is limiting, protein score of F-P-4 formula is 93 based on the modified FAO provisional pattern (1965). Furthermore, a 100g of the products supplies required amounts of vitamins and minerals by the recommended daily dietary allowances for infants.

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The effect of nanoemulsified methionine and cysteine on the in vitro expression of casein in bovine mammary epithelial cells

  • Kim, Tae-Il;Kim, Tae-Gyun;Lim, Dong-Hyun;Kim, Sang-Bum;Park, Seong-Min;Lim, Hyun-Joo;Kim, Hyun-Jong;Ki, Kwang-Seok;Kwon, Eung-Gi;Kim, Young-Jun;Mayakrishnan, Vijayakumar
    • Asian-Australasian Journal of Animal Sciences
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    • v.32 no.2
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    • pp.257-264
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    • 2019
  • Objective: Dairy cattle nutrient requirement systems acknowledge amino acid (AAs) requirements in aggregate as metabolizable protein (MP) and assume fixed efficiencies of MP used for milk protein. Regulation of mammary protein synthesis may be associated with AA input and milk protein output. The aim of this study was to evaluate the effect of nanoemulsified methionine and cysteine on the in-vitro expression of milk protein (casein) in bovine mammary epithelial cells (MAC-T cells). Methods: Methionine and cysteine were nonionized using Lipoid S 75 by high-speed homogenizer. The nanoemulsified AA particle size and polydispersity index were determined by dynamic light scattering correlation spectroscopy using a high-performance particle sizer instrument. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine the cytotoxicity effect of AAs with and without nanoionization at various concentrations (100 to $500{\mu}g/mL$) in mammary epithelial cells. MAC-T cells were subjected to 100% of free AA and nanoemulsified AA concentration in Dulbecco's modified Eagle medium/nutrient mixture F-12 (DMEM/F12) for the analysis of milk protein (casein) expression by the quantitative reverse transcription polymerase chain reaction method. Results: The AA-treated cells showed that cell viability tended to decrease (80%) in proportion to the concentration before nanogenesis, but cell viability increased as much as 90% after nanogenesis. The analysis of the expression of genetic markers related to milk protein indicated that; ${\alpha}_{s2}$-casein increased 2-fold, ${\kappa}$-casein increased 5-fold, and the amount of unchanged ${\beta}$-casein expression was nearly doubled in the nanoemulsified methionine-treated group when compared with the free-nanoemulsified methionine-supplemented group. On the contrary, the non-emulsified cysteine-administered group showed higher expression of genetic markers related to milk protein ${\alpha}_{s2}$-casein, ${\kappa}$-casein, and ${\beta}$-casein, but all the genetic markers related to milk protein decreased significantly after nanoemulsification. Conclusion: Detailed knowledge of factors, such nanogenesis of methionine, associated with increasing cysteine and decreasing production of genetic markers related to milk protein (casein) will help guide future recommendations to producers for maximizing milk yield with a high level of milk protein casein.

Quality Evaluation of Domestic and Foreign Extruded Pellets and Moist Pellet Based on Biochemical Analyses for Juvenile Olive Flounder, Parazichthys Olivaceus (시판용 넙치(치어)사료의 성분 비교분석을 통한 품질평가)

  • 최세민;한경민;왕소길;이승형;배승철
    • Journal of Aquaculture
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    • v.17 no.2
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    • pp.144-150
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    • 2004
  • This experiment was conducted to evaluate the parameters such as nutrient requirements, POY, AnV, Totox, VBN, total plate count, dietary fatty acids and amino acids composition, that are not included in the registered standard composition items required by the Ministry of Agriculture and Forestry, of a moist pellet (MP), three domestic extruded pellets (DEP-1, DEP-2, DEP-3), and two foreign extruded pellets (FEP-1, FEP-2) that are utilized by domestic flounder farms at present. The crude protein was added in excess of the dietary protein requirement in 6 kinds of feeds. When considering the proper PH ratio, it is obvious that protein was added in excess, especially in MP and FEP-2. Crude fat was also added in excess, especially in FEP-1. MP contained a higher dietary phosphorus content than formulated feeds, surpassing the dietary phosphorus requirement and greatly increasing the possibility for causing water pollution. The oxidation of fatty acid and decomposition of protein in MP were higher than in formulated feeds, and may also cause problems on fish farms. Also, it is difficult to store and manage MP, Among the fatty acids, EPA and DHA contents in MP were higher than those in formulated feeds. It is necessary to conduct further studies of EPA and DHA contents in formulated feeds. Lysine content in MP and FEP-2 could meet the dietary lysine requirement of flounder, however, the possibility of insufficient lysine content in the other formulated feeds was high and we considered that extra supplementation was necessary. Therefore, it is necessary to set up quality control standards according to fish species and sizes while considering the specific character of aquatic formulated feeds to restore the confidence of feed companies and aquaculturists to these feeds. This may be an opportunity to make an earlier change from MP to formulated feeds.

The Effects of Dietary Lysine Deficiency on Muscle Protein Turnover in Postweanling Pigs

  • Chang, Yi-Ming;Wei, Hen-Wei
    • Asian-Australasian Journal of Animal Sciences
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    • v.18 no.9
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    • pp.1326-1335
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    • 2005
  • The main purpose of this study is to investigate the effects of dietary lysine deficiency on protein turnover of porcine muscles. There were 18 LYD three-breed-crossing postweanling barrows from six litters cannulated with gastric tubes through the esophagus at approximate 10 kg of body weight and allocated into three treatment groups. When their body weights reached over 12 kg, one group was sacrificed for determining the initial protein masses of m. masseter, m. longissimus dorsi, m. adductor and m. biceps femoris from the right body side. The others received a diet containing 100% or 61.4% (calculated values) of the lysine requirement (NRC, 1998) multiplied by 1.103 for a period of 17 days. Daily feed provision was computed for each pig according to body weight at the same day. All pigs were infused a flooding dose of $^2$H$_5$-phenylalanine to determine the fractional protein synthesis rates (FSR) of the aforementioned muscles in the end. Their four muscles from the right body side were also dissected for measuring the fractional rates of protein accretion (FAR). As for protein degradation, fractional rates (FDR) were calculated by differences between synthesis and accretion. Results showed that the lysine deficiency resulted in, significantly (p<0.05), lighter body weights, smaller muscles and a slower growth rate. The protein mass, accreted by the muscles, of the deficient group was only 54% averaged of the pigs fed adequately (p<0.05). The FAR of these muscles in the deficient group was significantly lower (p<0.05) and only achieved 61.1% averaged of the control; there was no significant difference (p>0.05), nevertheless, in the amino-acid composition of muscles between two groups. The lysine deficiency reduced significantly (p<0.05) the FSR of m. longissimus dorsi but did not influence its FDR. The m. biceps femoris also presented an inhibited FSR while its FDR reduced only exhibited a very high tendency (p = 0.055) compared to the adequately-fed pigs. As for the m. masseter and m. adductor, both of the FSR and FDR were depressed significantly (p<0.05) by the lysine deficiency, and changes in the FSR were severer than those in the FDR, so that their FAR were significantly slower (p<0.05) in comparison with the control group. The lysine deficiency also inhibited the RNA translation activity of the muscles while the effects on RNA capacity were not significant (p>0.05). In conclusion, the FAR of muscle protein was changed by the current lysine deficiency through the alterations in the FSR and/or FDR.

A Study on the Nutritive Value and Utilization of Powdered Seaweeds (해조의 식용분말화에 관한 연구)

  • Yu, Jong-Yull;Lee, Ki-Yull;Kim, Sook-Hee
    • Journal of Nutrition and Health
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    • v.8 no.1
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    • pp.15-37
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    • 1975
  • I. Subject of the study A study on the nutritive value and utilization of powdered seaweeds. II. Purpose and Importance of the study A. In Korea the shortage of food will be inevitable by the rapidly growing population. It will be very important study to develop a new food from the seaweeds which were not used hitherto for human consumption. B. The several kinds of seaweeds have been used by man in Korea mainly as side-dishes. However, a properly powdered seaweed will enable itself to be a good supplement or mixture to certain cereal flours. C. By adding the powdered seaweed to any cereals which have long been staple foods in this country the two fold benefits; saving of cereals and change of dietary pattern, will be secured. III. Objects and scope of the study A. Objects of the study The objects will come under four items. 1. To develop a powdered seaweed as a new food from the seaweeds which have been not used for human consumption. 2. To evaluate the nutritional quality of the products the analysis for chemical composition and animal feeding experiment will be conducted. 3. Experimental cocking and accepability test will be conducted for the powdered products to evaluate the value as food stuff. 4. Sanitary test and also economical analysis will be conducted for the powdered products. B. Scope of the study 1. Production of seaweed powders Sargassum fulvellum growing in eastern coast and Sargassum patens C.A. in southern coast were used as the material for the powders. These algae, which have been not used for human consumption, were pulverized through the processes of washing, drying, pulverization, etc. 2. Nutritional experiments a. Chemical composition Proximate components (water, protein, fat, cellulose, sugar, ash, salt), minerals (calcium, phosphorus, iron, iodine), vitamins (A, $B_1,\;B_2$ niacin, C) and amino acids were analyzed for the seaweed powders. b. Animal feeding experiment Weaning 160 rats (80 male and 80 female rats) were used as experimental animals, dividing them into 16 groups, 10 rats each group. Each group was fed for 12 weeks on cereal diet (Wheat flour, rice powder, barley powder, potato powder, corn flour) with the supplementary levels of 5%, 10%, 15%, 20% and 30% of the seaweed powder. After the feeding the growth, feed efficiency ratio, protain efficiency ratio and ,organs weights were checked and urine analysis, feces analysis and serum analysis were also conducted. 3. Experimental cooking and acceptability test a. Several basic studies were conducted to find the characteristics of the seaweed powder. b. 17 kinds of Korean dishes and 9 kinds of foreign dishes were prepared with cereal flours (wheat, rice, barley, potato, corn) with the supplementary levels of 5%, 10%, 15%, 20% and 30% of the seaweed powder. c. Acceptability test for the dishes was conducted according to plank's Form. 4. Sanitary test The heavy metals (Cd, Pb, As, Hg) in the seaweed powders were determined. 5. Economical analysis The retail price of the seaweed powder was compared with those of other cereals in the market. And also economical analysis was made from the nutritional point of view, calculating the body weight gained in grams per unit price of each feeding diet. IV. Results of the study and the suggestion for application A. Chemical composition 1. There is no any big difference in proximate components between powders of Sargassum fulvellum in eastern coast and Sargassum patens C.A. in southern coast. Seasonal difference is also not significant. Higher levels of protein, cellulose, ash and salt were found in the powders compared with common cereal foods. 2. The levels of calcium (Ca) and iron (Fe) in the powders were significantly higher than common cereal foods and also rich in iodine (I). Existence of vitamin A and vitamin C in the Powders is different point from cereal foods. Vitamin $B_1\;and\;B_2$ are also relatively rich in the powders.'Vitamin A in ·Sargassum fulvellum is high and the levels of some minerals and vitamins are seemed4 to be some influenced by seasons. 3. In the amino acid composition methionine, isoleucine, Iysine and valine are limiting amino acids. The protein qualities of Sargassum fulvellum and Sargassum patens C.A. are seemed to be .almost same and generally ·good. Seasonal difference in amino acid composition was found. B. Animal feeding experiment 1. The best growth was found at.10% supplemental level of the seaweed Powder and lower growth rate was shown at 30% level. 2. It was shown that 15% supplemental level of the Seaweed powder seems to fulfil, to some extent the mineral requirement of the animals. 3. No any changes were found in organs development except that, in kidney, there found decreasing in weight by increasing the supplemental level of the seaweed powder. 4. There is no any significant changes in nitrogen retention, serum cholesterol, serum calcium and urinary calcium in each supplemental level of the seaweed powder. 5. In animal feeding experiment it was concluded that $5%{\sim}15%$ levels supplementation of the seaweed powder are possible. C. Experimental cooking and acceptability test 1. The seaweed powder showed to be utilized more excellently in foreign cookings than in Korean cookings. Higher supplemental level of seaweed was passible in foreign cookings. 2. Hae-Jo-Kang and Jeon-Byung were more excellent than Song-Pyun, wheat cake, Soo-Je-Bee and wheat noodle. Hae-Je-Kang was excellent in its quality even as high as 5% supplemental level. 3. The higher levels of supplementation were used the more sticky cooking products were obtained. Song-Pyun and wheat cake were palatable and lustrous in 2% supplementation level. 4. In drop cookie the higher levels of supplementation, the more crisp product was obtained, compared with other cookies. 5. Corn cake, thin rice gruel, rice gruel and potato Jeon-Byung were more excellent in their quality than potato Man-Doo and potato noodle. Corn cake, thin rice gruel and rice gruel were excellent even as high as 5% supplementation level. 6. In several cooking Porducts some seaweed-oder was perceived in case of 3% or more levels of supplementation. This may be much diminished by the use of proper condiments. D. Sanitary test It seems that there is no any heavy metals (Cd, Pb, As, Hg) problem in these seaweed Powders in case these Powders are used as supplements to any cereal flours E. Economical analysis The price of the seaweed powder is lower than those of other cereals and that may be more lowered when mass production of the seaweed powder is made in future. The supplement of the seaweed powder to any cereals is also economical with the criterion of animal growth rate. F. It is recommended that these seaweed powders should be developed and used as supplement to any cereal flours or used as other food material. By doing so, both saving of cereals and improvement of individual's nutrition will greatly be achieved. It is also recommended that the feeding experiment for men would be conducted in future.

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Comparison of Feed Efficiency Between Rotifers Enriched Lipid-contents to Enrichment and Enhanced Digestive Enzymes Activity to Starch (영양강화 Rotifer와 효소활성 향상 Rotifer의 먹이효율 비교)

  • Kwon, O-Nam;Park, Heum-Gi
    • Journal of Aquaculture
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    • v.22 no.1
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    • pp.105-111
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    • 2009
  • In this study, we carried out an experiment for estimation the larval digestibility in aspects which digestive enzymatic activities and nutrition of the rotifers, Brachionus rotundiformis. Thus we enhanced the digestive enzymatic activity through the addition of starch for the increase of digestibility of rotifer (starch-rotifer), and compared with the feed efficiency through rearing of the olive flounder, Paralichthys olivaceus used rotifer lipid-enriched with Algamac $2000^{(R)}$ (CE-rotifer). The digestive enzyme activities (except for TG-lipase), total protein contents, total essential amino acid, essential amino acids (methionin and phenylalanine) of starch-rotifer (the rotifer used a starch as additive, and enriched not) was assayed significantly higher than CE-rotifer (P<0.05). And total lipid, lipid classes (except for sterol) and fatty acids as DHA and EPA showed higher in CE-rotifer than starch-rotifer (P<0.05). But, sterol contents and ST/TG ratio were shown significantly higher in starch-rotifer (P<0.05). The flounder larvae supplied the two rotifers showed standard length and body weight that not significantly differed with ranges $3.72{\sim}3.79\;mm$ and $32.9{\sim}37.8\;mg$/larva on 6 days after hatching (DAH), respectively (P>0.05). However, these of 12 DAH showed the values of significantly higher to $5.94{\pm}0.249\;mm$, $144.0{\pm}23.86\;mg$/larva and $26.2{\pm}12.13%$ in standard length, body weight and survival in CE-flounder than that of starch-flounder (P<0.05). The hydrolytic enzymatic activities of flounder larvae severally supplied the two rotifers showed the significantly higher activities in acidic -amylase, neutral -amylase, TG-lipase, lysozyme and acidic phosphatase in starch-flounder on 5 DAH (P<0.05). But neutral $\alpha$-amylase, three proteases and two phosphatases of CE-flounder on 11 DAH showed the significantly higher activities than that of starch-flounder (P<0.05). Therefore, for the flounder, Paralichthys olivaceus larvae just depleted yolk was more beneficial to supply the feed, rotifer, enhanced the digestibility than to supply the feed lipid-enriched for aspect of larval digestibility up to 6 DAH, thereafter nutrition of absorption due to the development of digestive organs suggested that enrichment effect appeared with larval somatic growth. Consequently, investigation more detailed about the larval digestive physiological and nutritional requirement variations after 6 DAH will be necessary, thereafter.

Detection of Auxotrophic Mutants form Valsa ceratosperma, the Causal Fungus of Apple Canker (사과나무 부란병균(腐爛病菌) Valsa ceratosperma에서의 Auxotrophic Mutants의 검출(檢出))

  • Hong, Yeon Gyu;Uhm, Jae Youl
    • Current Research on Agriculture and Life Sciences
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    • v.5
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    • pp.119-126
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    • 1987
  • This study was conducted to elucidate the most appropriate method to obtain auxotrophic mutants from Valsa ceratosperma, the causal fungus of apple canker, which may be used as a gene marker in detecting the transfer of the factors of avirulent strains to virulent strains. Among the 3 kinds of synthetic media tested, each have two formula for minimal and complete, the medium which has been used in study of Endothia parasitica (E. P medium) was turned out to be most appropriate for the growth of V. ceratosperma. A medium for single colony formation from pycnidiospore of this fungus was developed by adding 0.5% L - sorbose to the E. P minimal medium. The period of incubation in dark for preventing the photoreactivation after U. V irradiation was estimated as about 60hrs at which most of the spores become binucleate. Largest number of putative auxotrophs were obtained at about 50second of irradiation to the spores smeared on the medium for single colony formation, at which the survival rate of spores was 5 to 6 percent. With these method developed in this experiment, 161 isolates of putative auxotrophs were detected among which the nutrient requirement for 10 isolates were determined. Five out of 10 mutants were still virulent to apple tree and all but one could not sporulate.

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