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In Vitro Fertilization of Pig Oocytes Matured In­Vitro by liquid Boar Spermatozoa (체외성숙 돼지 난포란의 액상정액을 이용한 체외수정)

  • 박창식;이영주
    • Korean Journal of Animal Reproduction
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    • v.26 no.1
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    • pp.17-23
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    • 2002
  • The present study was carried out to investigate the effects of the maturation media such as a modified TCM-199 (mTCM-199) medium, modified Waymouth MB 752/1 (mWaymouth MB 752/1) medium or NCSU-23 medium on penetrability of pig oocytes by liquid boar sperm. Oocytes (30~40) were transferred into each well of a Nunc 4-well multidish containing 0.5 $m\ell$ maturation medium. When immature pig oocytes were cultured in mTCM-199, mWaymouth MB 752/1 and NCSU-23 maturation media for 44 h in 5% $CO_2$, in air at 38.5$^{\circ}C$, the germinal vesicle breakdown (CVBD) rates of the oocytes were 95.6, 94.1 and 94.9%, respectively, and the maturation rates (metaphase II) of oocytes were 92.5, 90.1 and 91.1%, respectively. No differences were observed among the maturation media. The spermrich portion of ejaculates with greater than 90% motile sperm were used in the experiment. The semen was cooled 22 to 24$^{\circ}C$ over 2 h period. The semen was diluted with Beltsville Thawing Solution (BTS) extender at room temperature to give 2$\times$10$^{8}$ sperm/$m\ell$ in 100 $m\ell$ plastic bottle. Liquid boar semen of 30 $m\ell$ in 100 $m\ell$ plastic bottle was kept at 17$^{\circ}C$ for 5 days. The sperm with greater than 70% motility after day 5 of storage were used for in-vitro fertilization (IVF). After 44 h maturation of immature oocytes, cumulus cells were removed and oocytes (30~40) coincubated far 6 h in 0.5 $m\ell$ mTCM-199 and mTBM fertilization media with 2$\times$1061$m\ell$ sperm concentration. At 6 h after IVF, oocytes were transferred into 0.5 $m\ell$ mTCM-199 and NCSU-23 culture media for further culture 6 or 42 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF, and developmental ability of oocytes at 48 h after IVF were evaluated. The oocytes in combination with NCSU-23 medium for maturation and mTBM medium for IVF increased male pronuclear formation (48.0%) compared to those in combination with mTCM-199 media for maturation and IVF, and mWaymouth MB 752il medium for maturation and mTCM-199 medium far IVF. The rates of cleaved embryos (2~4 cell stage) at 48 h after IVF were 24.1% in combination with mTCM-199 media for maturation, IVF and culture, 43.6% in combination with mWaymouth MB 75211 medium fur maturation and mTCM-199 media for IVF and culture, and 71.2% in combination with NCSU-23 medium for maturation, mTBM medium for IVF and NCSU-23 medium for culture. In conclusion, we found out the oocytes matured in vitro were fertilized by liquid boar sperm stored in BTS extender at 17$^{\circ}C$ for 5 days. We recommend the simple defined NCSU-23 medium for nuclear maturation, mTBM medium and liquid boar sperm for IVF, and NCSU-23 medium for embryo culture.

Self-purification Mechanisms in Natural Environments of Korea: I. A Preliminary Study on the Behavior of Organic/Inorganic Elements in Tidal Flats and Rice Fields (자연 정화작용 연구: I. 갯벌과 농지 상층수중 유 ${\cdot}$ 무기 원소의 거동에 관한 예비 연구)

  • Choi, Kang-Won;Cho, Yeong-Gil;Choi, Man-Sik;Lee, Bok-Ja;Hyun, Jung-Ho;Kang, Jeong-Won;Jung, Hoi-Soo
    • The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
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    • v.5 no.3
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    • pp.195-207
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    • 2000
  • Organic and inorganic characteristics including bacterial cell number, enzyme activity, nutrients, and heavy metals have been monitored in twelve acrylic experimental tanks for two weeks to estimate and compare self-purification capacities in two Korean wet-land environments, tidal flat and rice field, which are possibly different with the environments in other countries because of their own climatic conditions. FW tanks, filled with rice field soils and fresh water, consist of FW1&2 (with paddy), FW3&4 (without paddy), and FW5&6 (newly reclaimed, without paddy). SW tanks, filled with tidal flat sediments and salt water, are SW1&2 (with anoxic silty mud), SW3&4 (anoxic mud), and SW5&6 (suboxic mud). Contaminated solution, which is formulated with the salts of Cu, Cd, As, Cr, Pb, Hg, and glucose+glutamic acid, was spiked into the supernatent waters in the tanks. Nitrate concentrations in supernatent waters as well as bacterial cell numbers and enzyme activities of soils in the FW tanks (except FW5&6) are clearly higher than those in the SW tanks. Phosphate concentrations in the SW1 tank increase highly with time compared to those in the other SW tanks. Removal rates of Cu, Cd, and As in supematent waters of the FW5&6 tanks are most slow in the FW tanks, while the rates in SW1&2 are most fast in the SW tanks. The rate for Pb in the SW1&2 tanks is most fast in the SW tanks, and the rate for Hg in the FW5&6 tanks is most slow in the FW tanks. Cr concentrations decrease generally with time in the FW tanks. In the SW tanks, however, the Cr concentrations decrease rapidly at first, then increase, and then remain nearly constant. These results imply that labile organic materials are depleted in the FW5&6 tanks compared to the FW1&2 and FW3&4 tanks. Removal of Cu, Cd, As from the supernatent waters as well as slow removal rates of the elements (including Hg) are likely due to the combining of the elements with organic ligands on the suspended particles and subsequent removal to the bottom sediments. Fast removal rates of the metal ions (Cu, Cd, As) and rapid increase of phosphate concentrations in the SW1&2 tanks are possibly due to the relatively porous anoxic sediments in the SW1&2 tanks compared to those in the SW3&4 tanks, efficient supply of phosphate and hydrogen sulfide ions in pore wates to the upper water body, complexing of the metal ions with the sulfide ions, and subsequent removal to the bottom sediments. Organic materials on the particles and sulfide ions from the pore waters are the major factors constraining the behaviors of organic/inorganic elements in the supernatent waters of the experimental tanks. This study needs more consideration on more diverse organic and inorganic elements and experimental conditions such as tidal action, temperature variation, activities of benthic animals, etc.

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Studies on the Nitrogenous Utilization and Basal Metabolism of Korean Native Goat (한국(韓國) 재래산양(在來山羊)의 질소대사(窒素代謝) 및 기초대사량(基礎代謝量)에 관(關)한 연구(硏究))

  • Oh, Hong Rock
    • Korean Journal of Agricultural Science
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    • v.9 no.2
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    • pp.546-555
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    • 1982
  • To evaluate the digestibility and absorbability of proteins, and the rates of energy and nitrogen(N) metabolism of the Korean native goats, studies were carried out with open type respiration apparatus based on the nitrogen-carbon method. The results on the nitrogen retention and the metabolic rate of energy, which was obtained with one male (10-month-old) and one female (24-month-old) goats, both weighing ${\simeq}20kg$, are summarized as follows. 1. When the goats were fed ad libitum the medium quality orchard grass hay, they consumed hay about 0.66 to 0.92% of body weight per day. The hay intake was remained the same even when high quality hay was provided. This amount of hay intake was relatively lower than that of dairy goat and sheep. It was believed to be partly due to the change in feeding enviroment. When fed with hay and soybean meal together, the goats ate hay about 1.06% and soybean meal about 0.60% of body weight, corresponding to 1.66% of body weight as fed basis. 2. The $CO_2$ gas produced from the goat in the open type respiration chamber and absorbed with KOH solution was estimated to be 99~117g/day. The difference in feed intake did not influence the $CO_2$ production; however, these seems to be a linea relationship between body weight and $CO_2$ production. 3. When fed orchard grass hay only, the goats showed protein digestibility of 24~41%. The protein digestibility incresed to 58.2% when fed hay and soybean meal together. A negative nitrogen balance(-0.16g N/day) was observed with goats fed 11.53g N originated from 212g hay and 150g soybean meal. Converting that nitrogen ingested to a crude protein, the amount of crude protein intake by the goats per day was 77.9g compared to 40~45g N known to be required in a day by goat weighing 20kg, indicating that the extra protein ingested was metabolized to provide energy. 4. When the male and female goats comsumed 624 kcal gross energy and 824 kcal gross energy by consuming 158g and 213g of hay, respectively, the digestible energy intake was calculated to be 260kcal for the male and 199kcal for the female goat. The daily heat production of male and female goats were 338kcal and 334kcal, respectively, when fed hay only. However, the female goat fed 212g hay and 150g soybean meal produced about 591kcal per day. Consequently, the energy requirment of the Korean native goats weighing ${\simeq}20kg$ was concluded to be $${\geq_-}$$600kcal net energy per day. 5. The fasting heat product ion of a male goat weighing 27.7kg was 412kcal per day when fasted for 2~3 days. When fasted for 3~4 days, the value decresed to 240kcal. The enviromental temperatures during the expreimental period were ranged from 19 to $34.5^{\circ}C$. The goats seemed to be panting when the chamber temperature rose to $32^{\circ}C$ or above. 6. When fed low levels of dietary protein, serum protein levels of the goats were decresed slightly ($${\leq_-}$$10%); however, urea content in the serum was observed to decrese to a great extent (3X).

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