• Asian-Australasian Journal of Animal Sciences
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• v.17 no.2
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• pp.164-167
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• 2004

This study was carried out to investigate in vitro fertilization and development of in vitro matured pig oocytes inseminated with the Duroc boar sperm by different sperm washing media after thawing of the 5 ml frozen straws. Immature follicular oocytes (30-40) were transferred into each well of a Nunc 4-well multidish containing $500{\mu}l$ mTCM199 maturation medium. The sperm rich portion of ejaculates was collected into a 250 ml insulated vacuum bottle and gradually cooled 22 to $24^{\circ}C$ over a 2 h period. Semen was centrifuged at 800 g for 10 min and the seminal plasma discarded. Sperm were esuspended in a lactose-egg yolk and N-acetyl-Dglucosamine (LEN) diluent to contain $1{\times}10^{9}$ sperm/ml and cooled to $5^{\circ}C$ over a 2 h period. Immediately before freezing, semen was rediluted with an equal volume of LEN+4% glycerol and packed into 5 ml straws. After thawing of the 5 ml straw, the 5 ml semen was diluted with 20 ml Beltsville thawing solution (BTS) at room temperature. Oocytes were inseminated with untreated (unwashed and nonpreincubated) or treated sperm (washed two times in BTS, mTLP-PVA and mTBM media, respectively and nonpreincubated) with $2{\times}10^{7}$ sperm concentration. Oocytes were coincubated for 6 h in $500{\mu}l$ mTBM fertilization. At 6 h after IVF, oocytes were transferred into $500{\mu}l$ NCSU-23 culture medium for further culture of 6 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. Sperm penetration rate, male pronuclear formation and rate of cleaved embryos were higher in the BTS, mTLP-PVA and mTBM treatments than the unwashed treatment (p<0.05). The rate of blastocysts from the cleaved oocytes (2-4 cell stage) were higher in the mTLP-PVA treatment than in the unwashed, BTS and mTBM treatments. In conclusion, we recommend the washing of frozen-thawed sperm with mTLP-PVA medium before in vitro fertilization of oocytes in mTBM medium.

• Korean Journal of Animal Reproduction
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• v.16 no.4
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• pp.335-340
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• 1993

The development of efficient method for the production of transgenic mice has been investigated in our laboratory. This study was conducted to develop the artificial insemination in the mouse. Spermatozoa were collected from the cauda epididymis of ICR males(age:12~15 weeks, Body weight : 30g) and artificially inseminated into the intrauterine via cervix of hormone-primed ICR females(age: 6~8 weeks, body weight: 25g) using the capillary tube, 200~300 $\mu$m in inner diameter. The effect of concentration of sperm(80$\times$104, 40$\times$104, 20$\times$104, 10$\times$104, 5$\times$104, 3$\times$104, 1$\times$104/20${mu}ell$. The artificial insemination was succeeded but fertilization rate was very low(5~15%) compared to the natural mating and 59 normal youngs born from 60 females. Therefore, our findings suggest that it is possible to produce the great number of mice from the same orgin of male by artificial inseminatin. However, the lower pregnancy rate has to be solved to used broadly the artificial insemination in mouse.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.7
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• pp.1035-1039
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• 1999

Four ruminally cannulated buffalo bulls were fed mottgrass diets using a $4{\times}4$ Latin square design. Treatments were arranged factorially and consisted of mottgrass fertilized with 0 for Kg nitrogen (N) per acre and harvested at 40 and 60 days of age. Nitrogen fertilization improved the concentrations of neutral detergent fiber (NDF), and acid detergent fiber (ADF) in early-cut mottgrass (ECM) but, the acid detergent lignin (ADL) contents were higher in the late-cut mottgrass (LCM). The crude protein (CP) contents of the mottgrass decreased with advancing maturity, but N fertilization increased CP at both maturities. The intake of dry matter (DM), organic matter (OM), CP, NDF and ADF were higher by buffalo bulls fed ECM than those fed LCM. The ruminal pH increased in first 6 hours post feeding in animals fed N fertilized mottgrass and may be due to higher concentration of ruminal ammonia. The digestibilities of DM, OM, CP, NDF and ADF were higher by buffalo bulls fed ECM than those fed LCM. However, the application of N fertilizer did not affect the digestibilities of these nutrients.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.8
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• pp.1127-1133
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• 2008

This study was designed to develop a method of liquid storage of boar sperm at $4^{\circ}C$ by using the modified Beltsville F5 (BF5) diluent with bovine serum albumin (BSA) and N-acetyl-D-glucosamine. Boar sperm were stored in lactose-egg yolk and N-acetyl-D-glucosamine (LEN), BF5 and Golden-Pig liquid 4 (GPL4) diluents at $4^{\circ}C$ for 5 days and were examined for sperm viability, adenosine triphosphate (ATP) and in vitro fertilization (IVF). The percentage of sperm viability in GPL4 diluent was higher than in LEN and BF5 diluent from 1 to 5 days of storage at $4^{\circ}C$. The percentage of sperm viability steadily declined from 1 to 5 days of storage in the three different diluents. Sperm ATP in GPL4 diluent was higher than in LEN and BF5 diluents from 1 to 5 days of storage. Sperm ATP rapidly declined after 5 days of storage in the three different diluents. Porcine oocytes matured in vitro were inseminated with different sperm concentrations of liquid semen stored for 3 days in GPL4 diluent. The percentage of monospermic oocytes did not show any differences from 2.5 to $20{\times}10^5$ sperm/ml. However, the percentage of polyspermic oocytes in the sperm concentration of $2.5{\times}10^5$ sperm/ml was lower than in concentrations of 5, 10 and $20{\times}10^5$5 sperm/ml. The percentage of blastocysts from the cleaved oocytes at $2.5{\times}10^5/ml$ sperm concentration was significantly lower than at 5, 10 and $20{\times}10^5sperm/ml$ concentrations. In conclusion, GPL4 diluent can be stored at $4^{\circ}C$ for 5 days and showed higher sperm viability and sperm ATP concentration compared with LEN and BF5 diluents. Also, we found that GPL4 diluent can be used for IVF of porcine oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.257-263
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• 1999

It is well known that discard of seminal plasma from the semen and separation of motile sperm should be preceded before insemination for IUI or IVF. Till now, more than ten kinds of semen treatment methods have been developed. Of those, swim-up and Percoll methods have been used widely in ART laboratories as a routine semen treatment methods because of its advantages. However, there are reports that Percoll can make a genetic trouble because of its chemical structure and therefore the necessity has been arisen to substitute Percoll for other equivalent materials. This study was performed to evaluate the effects of three different sperm preparation methods (swim-up, Percoll and Sil-Select) on sperm motility, sperm recovery rate and fertilization rate. Also, the feasibility of using Sil-Select instead of Percoll in ART was evaluated. Each semen samples were divided into three fractions and motile sperm were recovered by swim-up, Percoll and Sil-Select gradient centrifugation methods. Normal and sub-normal criteria of fifteen semen samples and seventeen IVF cycles were included in these study. As results, no significant difference was found in sperm recovery rate in normal semen treated by a Swim-up, Percoll and Sil-Select method ($13.2{\times}10^6,\;17.5{\times}10^6\;and\;17.7{\times}10^6$ respectively). The initial sperm motility was 61.9% and this increased to 87.1%, 92.6% and 89.5% through Swim-up, Percoll and Sil-Select treatment, respectively. Higher motility was observed in Percoll and Sil-Select treated groups (81.5%, 79.2%, respectively) than swim-up group (66.8%) after incubation for 24hrs. In sub-normal group, sperm recovery rates were higher in Sil-Select group $(2.9{\times}10^6)$ than Percoll gradients group $(1.8{\times}10^6)$. In IVF cycles, the outcomes of fertilization using sperm treated by swim-up and Sil-Select group were similar (82.2%, 79.7% respectively). In conclusion, our results indicate that Sil-Select can be used as a substitute material for sperm preparation instead of Percoll.

• Journal of Korean Society of Forest Science
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• v.85 no.1
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• pp.96-106
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• 1996

Red alder(Alnus rubra Bong.) seedlings inoculated with Frankia only or both Frankia and spores of Alpova diplophloeus(Zeller & Dodge) Trappe & Smith were grown in a greenhouse for ten weeks. The ten-week-old seedlings were fertilized with six nitrogen(N) and phosphorus(P) fertility regimes (no fertilization, 10mM $NH_4NO_3$, 50mM $NH_4NO_3$, 5mM $KH_2PO_4$, 10mM $NH_4NO_3+5mM$ $KH_2PO_4$, and 50mM $NH_4NO_3+5mM$ $KH_2PO_4$) three times a week for ten weeks. The higher N-fertilization significantly increased mycorrhiza formation by greenhouse contaminant mycorrhizal fungi, but decreased N-fixation and P concentration in nodule tissues. P-fertilization significantly increased nodule and shoot dry weight, and P concentration in plant tissues. When N was highly fertilized, however, the P-fertilization effect disappeared in nodule P concentration but doubled in leaf P concentration. A. diplophloeus inoculation significantly increased diameter growth and $CO_2$ exchange rate, but decreased leaf dry weight. Our results suggest that the higher N- or P-fertilization affect nitrogenase activity and mycorrhizal development but the effects are changed by their interactions.

• Asian-Australasian Journal of Animal Sciences
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• v.9 no.6
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• pp.737-741
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• 1996

Dry matter yield of reed canarygrass was investigated in uncultivated rice paddy in Korea, and an attempt was made to estimate the most economic and efficient cutting frequency and rates of nitrogen (N) fertilization, for increased production of reed canarygrass. Total dry matter yields of reed canarygrass per year were 7.4-15.7, 8.5-16.1, and 7.5-13.4 tons/ha in 3, 4, and 5 cutting frequencies, respectively, and over the N treatments of 0-120 kg N/ha/cut. When cut 3 or 5 times annually, the 2nd cut produced the highest proportion of total yield at 38.4 and 33.0%, respectively, when cut 4 times the 3rd cut was highest (38.3%). The ranges of economic N level, limiting N level and efficiency of dry matter production were 243.3-293.0, 387.2, and 14.6 kg DM/kg N, respectively. These indicators were particularly low for the 5 cutting frequency possibly because of the unusually high temperature in the summer season. The best cutting frequency for the dry matter production of reed canarygrass in 1994 was 3 per year because of the higher efficiency of dry matter production.

• Journal of Embryo Transfer
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• v.22 no.1
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• pp.1-7
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• 2007

This study was conducted to examine the role of intact cumulus cells during in vitro fertilization (IVF) on sperm penetration, male pronuclear (MPN) formation and subsequent embryo development of oocytes matured and fertilized in vitro. Cumulus-oocyte complexes obtained from the slaughtered gilt ovaries were matured for 44 h in TCM199 containing 10% porcine follicular fluid, epidermal growth factor and hormones. After maturation culture, denuded oocytes or oocytes with intact cumulus cells were coincubated with frozen-thawed boar semen for 8h in a modified tris-buffered medium containing 5mM caffeine and 10mM calcium chloride. Putative zygotes were fixed and examined for sperm penetration and MPN formation (Experiments $1{\sim}3$), or cultured in North Carolina State University-23 medium fo. 156 h (Experiment 3). In Experiment 1, sperm penetration was examined after insemination of denuded oocytes and oocytes with intact cumulus cells at the concentration of $7.5{\times}10^5$ sperm/ml. Optimal sperm concentration for IVF of cumulus-intact oocytes was determined in Experiment 2 by inseminating intact oocytes with $2{\sim}5{\times}10^6$ sperm/ml. In Experiment 3, denuded or intact oocytes were inseminated at the concentrations of $7.5{\times}10^5$ and $4.0{\times}10^6$ sperm/ml, respectively, and in vitro embryo development was compared. Sperm penetration was significantly (p<0.01) decreased in cumulus-intact oocytes compared to denuded oocytes (35.2% vs. 77.4%). Based on the rates of sperm penetration and normal fertilization, the concentration of $4.0{\times}10^6$ sperm/ml was optimal for the IVF of intact oocytes compared to other sperm concentrations. The presence of intact cumulus cells during IVF significantly (p<0.05) improved embryo cleavage (48.8% vs. 58.9%), blastocyst (BL) formation (11.0% vs. 22.8%) and embryo cell number $(22{\pm}2\;vs.\;29{\pm}2\;cells)$ compared to denuded oocytes. In conclusion, these results suggest that intact cumulus cells during IVF inhibit sperm penetration but improve embryo cleavage, BL formation and embryo cell number of porcine embryos produced in vitro.

• Korean Journal of Ichthyology
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• v.31 no.1
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• pp.23-29
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• 2019

The embryonic development and hatchability of the artificially fertilized eggs of convict grouper (Hyporthodus septemfasciatus) ♀${\times}$giant grouper (Epinephelus lanceolatus) ♂ (CGGG) were compared to those of the maternal pure species (convict grouper ♀${\times}$♂, CG) to establish a novel grouper hybrid for aquaculture industry. The fertilized eggs were divided into nine 5-L beakers (3,000~5,000 eggs/beaker) filled with UV sterilized seawater and incubated at a temperature range of $23.5{\sim}24.8^{\circ}C$ (32.1~32.8 psu). The percentages of fertilization and hatching of CGGG were $69.4{\pm}1.5%$ and $59.0{\pm}5.1%$, respectively and were significantly lower than those of CG (p<0.05). The CGGG proceeded normal embryonic development similar to that of CG, but showed an irregular cleavage, immature embryonic body and spinal deformity in hatched larvae. The incubation time from fertilization to hatching of CGGG was 31 hrs, which was approximately 2 hrs slower than that of CG. Our study provided the possibility of mass production of grouper hybrid CGGG larvae.

• Applied Biological Chemistry
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• v.22 no.3
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• pp.181-189
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• 1979

In order to establish fertilizing technique for the increase of grain yield and its stability, the total absorbed amount of nutrients and nutritional status of rice plants at different growth stages were studied with respect to the nutrient contents and its relation to grain yield and yield components. This experiment was carried at three different level of nitrogen fertilization; two different seeding and transplanting times, and eight cultivars including both Japonica-and Tongil-type. The results of a part of these experiments are summarized as fallows: 1. The culm length tended to Increase with increasing fertilizer amount, but no significant difference between ordinary and heavy fertilization was observed in Tongil-type cultivars. 2. The panicle length was some what longer in ordinary and heavy fertilization than non-fertilization. 3. As the N-fertilizer level increases, the number of panicles per plant increased with higher response in Japonica-type cultivars than in Tongil-type cultivars. 4. The number of grains per panicle increased significantly in ordinary and heavy fertilization, compared to non-fertilization, but the difference in grain number per panicle among fertilizer treatment was smaller in Japonica-type cultivars than Tongil-type. 5. The 1,000 grains weight showed no significant difference among fertilization levels in ordinary transplanting, but indicated gradual decreasing tendency as the transplanting delayed. 6. In percentage of ripeness, there was no significant difference in ordinary transplanting, but hightly significant in late transplanting among N-fertilization levels and cultivars. 7. In yield, highly significant difference was shown among N-fertilizer levels and cultivars In Tongil-type variety, higher yield was obtained in ordinary fertilization than heavy fertilization. 8. The straw weight showed the tendency to increase in higher nitrogen level, but no significant difference between ordinaly and heavy fertilization was observed in Tonsil-type cultivars.