• Journal of The Korean Society of Grassland and Forage Science
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• v.44 no.2
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• pp.99-105
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• 2024

This study was conducted to estimate the effects of the forage process on rumen fermentation characteristics and greenhouse gas emissions of rye. Rye was grown at the Taeyoung Livestock farm and harvested at the heading stage. The harvested rye (5 kg) was sub-sampled for fresh forage, hay, and silage in triplicates. The sub-sampled rye was freeze-dried or air-dried for fresh forage or rye hay, respectively. For rye silage, the sub-sampled rye forage was ensiled into a 10 L mini bucket silo and stored for 90 days. For 72 h rumen incubation, each forage (0.3 g) was placed into the incubation bottle with the rumen mixture (30 mL) in quadruplicates. After the incubation, total gas was measured and sub-sampled for CO₂ and CH₄ analyses, and the bottle content was centrifuged for in vitro digestibilities of dry matter (IVDMD) and neutral detergent fiber (IVNDFD), and rumen fermentation characteristics. Silage had higher crude protein, crude ash, and acid detergent fiber concentrations than fresh forage and hay but lower non-fiber carbohydrates and relative feed value (p<0.05). And, silage had higher lactic acid bacteria than the other forages but lower pH (p<0.05). After 72 h incubation in the rumen, fresh forage had higher IVDMD and butyrate content than the other forages (p<0.05). However, silage had higher rumen pH and propionate content than the other forages but lower A:P ratio (p<0.05). Regarding greenhouse gases, silage had lowest total gas (mL/g DMD and NDFD) and CH₄ (mL/g DMD and NDFD) emissions, while fresh forage had lowest CO₂ (mL/g DMD) emission (p<0.05). Therefore, this study concluded that the ensiling process of rye can effectively mitigate greenhouse gas emissions of Hanwoo.

• Journal of Veterinary Clinics
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• v.24 no.4
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• pp.486-492
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• 2007

Seminal plasma(SP) is usually removed from semen that is to be cryopreserved. However, some reports indicate that SP has beneficial effects on spermatozoa during chilling and freezing. The purpose of this study was to determine the effect of SP on sperm survival by adding SP to the extender before cooling and freezing canine spermatozoa. In replicate experiments, ejaculates obtained from four healthy dogs(1-4 years old) of various breeds were pooled, centrifuged at $300{\times}g$ for 10 min at $25^{\circ}C$, and the supernatant of seminal plasma was decanted. Spermatozoa were suspended in egg yolk-Tris(EYT) buffer. The study comprised two experiments: [Exp 1] Sperm were suspended in EYT extender containing either 0, 20, 40, 80 or 100% SP and were slowly cooled to $4^{\circ}C$ for 2h or held at $25^{\circ}C$ as controls. Sperm concentration was adjusted to $2{\times}10^8/ml$. [Exp II] Sperm samples, each of which contained $1{\times}10^8/ml$, were assigned to nine groups to be frozen. In the first four groups, sperm in EYT containing either 20, 40, 80 or 100% SP were cooled to $4^{\circ}C$, then diluted to contain final concentrations of EYT+0.6M glycerol and then were frozen. The final concentrations of SP were 10, 20, 40 or 50%. In the other four groups, sperm in EYT alone were first cooled slowly to $4^{\circ}C$, then diluted to contain final concentrations of EYT+0.6M glycerol plus 10, 20, 40 or 50% SP and then were frozen. Spermatozoa, which chilled in EYT alone and diluted to contain final concentrations of EYT+0.6M glycerol without seminal plasma, and then frozen, was regarded as control. Spermatozoa were frozen at $25^{\circ}C/min$ of cooling rate in plastic straws that were suspended above liquid nitrogen and thawed in water at $38^{\circ}C$ for 1 min. Sperm survival was assayed by determining progressive motility and integrity of plasma and acrosome membranes. Progressive motility was determined by microscopic examination at $200{\times}$ magnification. Membrane integrity was assessed by use of a double fluorescent dye, and acrosome integrity by staining sperm with Pisum sativum agglutinin. The results of the first experiment showed that adding SP did not improve motility of spermatozoa compared to those incubated without SP regardless of temperature. The results of the second experiment showed that spermatozoa suspended in EYT+0.6M glycerol containing SP exhibited the higher progressive motility before being frozen(P<0.05). However, frozen-thawed spermatozoa that had suspended in EYT+0.6M glycerol containing SP showed the similar or lower viability(P<0.05). In summary, although seminal plasma did not affect spermatozoa that were chilled in EYT without cryoprotectant(CPA), addition of seminal plasma to EYT containing CPA did significantly improved progressive motility of canine spermatozoa that were chilled.

• Korean Journal of Animal Reproduction
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• v.27 no.3
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• pp.215-223
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• 2003

This study was to investigate the effects of fertilization time and culture medium of pig oocytes matured in-vitro by liquid boar sperm. The sperm rich fraction (30∼60 ml) was slowly cooled to room temperature (20∼23$^{\circ}C$) by 2 h after collection. Semen was transferred into 15 ml tubes, centrifuged at room temperature for 10 min 800 ${\times}$ g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5 ml of the LEN diluent to provide 1.0${\times}$10$^{9}$ sperm/ml at room temperature. The resuspended semen was cooled in a refrigerator to 4$^{\circ}C$. The medium used for oocyte maturation was TCM-199 supplemented with 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 $\mu\textrm{g}$/ml insulin, 2 $\mu\textrm{g}$/ml vitamin B$_{12}$ , 25 mM HEPES, 10 $\mu\textrm{g}$/ml bovine apotransferrin, 150 $\mu$M cysteamine, 10 IU/ml PMSG, 10 IU/ml hCG, 10 ng/ml EGF, 0.4% BSA, 75 $\mu\textrm{g}$/ml sodium penicillin G, 50 $\mu\textrm{g}$/ml streptomycin sulfate and 10% pFF. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. Oocytes were inseminated with liquid boar sperm stored at 4$^{\circ}C$ for 2 days after collection. Oocytes were coincubated for 1, 3, 6 and 9 h in 500 ${mu}ell$ mTBM fertilization media with 1.0${\times}$10$^{6}$ sperm/ml concentration, respectively. Thereafter, oocytes were transferred into 500 ${mu}ell$ NCSU-23, HEPES buffered NCSU-23, PZM-3 and PZM-4 culture media, respectively, for further culture of 6, 48 and 144 h. The rates of sperm penetration and male pronuclear formation were higher in the fertilization times for 6 and 9 h than in those for 1 and 3 h. The rates of cleaved oocytes were higher in the fertilization times for 6 and 9 h (85.0 and 84.6%) than in those for 1 and 3 h (61.1 and 76.8%). The percentage of blastocyst formation from the cleaved oocytes was highest in the fertilization time for 6 h (33.6%) than in that for 1, 3 and 9 h (11.4, 23.0 and 29.6%). Mean cell numbers per blastocyst were 32.9, 27.6, 26.3 and 24.4 in the fertilization times for 6, 9, 3 and 1 h, respectively. The rate of blastocyst from the cleaved oocytes and the number of cells per blastocyst were higher in HEPES buffered NCSU-23 culture medium than in NCSU-23, PZM-3 and PZM-4 culture media. In conclusion, we found out that liquid boar sperm stored at 4$^{\circ}C$ could be used for in-vitro fertilization of pig oocytes matured in-vitro. Also, we recommend the coincubation time of 6 h in 500 ${mu}ell$ TBM fertilization medium with 1${\times}$10$^{6}$ sperm/ml concentration and the HEPES buffered NCSU-23 culture medium for in-vitro fertilization of pig oocytes matured in-vitro.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.167-176
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• 1997

This study was carried out to evaluate the potential for preselection of transgenic embryos prior to transfer into recipient animals. In these experiments, I used a 3.2 kb transgene which contained the neomycin resistance gene (neo) and lac Z gene driven by the $\beta$ actin promoter (iacZ Ineo). Oocytes were aspirated from abattoir ovaries, matured in TCM-199 supplemented with 10% fetal bovine serum (FBS), 5 ${\mu}\textrm{g}$/ml LH, 0.5 ${\mu}\textrm{g}$/ml FSH, 100 unit/ml penicillin, and 100 ${\mu}\textrm{g}$/ml streptomycin for 22 to 24 hrs then inseminated with a sperm suspension of 1 X 10$^6$ sperm/ml containing 5 ${\mu}\textrm{g}$/ml of heparin. At 18-20 hrs after insemination, cumulus cells were removed by vortexing and pronuclei of centrifuged zygotes microinjected with the lacZ/neo construct (3 ng/$\mu$l). All cultures were carried out in CR1aa with transfected BRL monolayers containing 3 mg/ml BSA, 20 $\mu$/ml NEM amino acids and 40 $\mu$I/ml BME amino acids. To identify the appropriate concentration of G418 for selection, non-microinjected zygotes were cultured in the presence of 0, 50, 100 and 200 $\mu$g/ml of G418. After 8 days of culture in these treatments, 44/145 (30.3%), 13/150 (8. 7%), 1/151 (0.7%) and 0/134 (0.0%) devel-oped to the blastocyst stage in 0, 50, 100 and 200 $\mu$g/ml of G418, respectively. A total of 1,127 zygotes were microinjected and placed into culture (without G418) and subsequently 710 (63.0%) cleaved. At 48 hrs post-injection, embryos ($\geq$2-cell) were randomly assigned to control (medium alone) or G418 (100 ${\mu}\textrm{g}$/ml) treatments. A control culture of 740 non-microinjected embryos from the same replicates of embryos were also placed into control medium. After 8 days in culture, 54/343 (15.7%) and 22/367 (6.0 %) of the microinjected embryos developed to the blastocyst stage in control and G418 media, respectively. A total of 151/740 (27.2%) of the non-microinjected embryos placed in the control medium developed to the blastocyst stage. The blastocysts in the control treatment had a mean of 70.7 ${\pm}$ 4.7 cells of which 23.1 ${\pm}$ 2.6 (32.7%) stained for $\beta$-Gal activity. B1astocysts in the G418 treatment had a mean of 48.8${\pm}$7.5 cells of which 40.3 ${\pm}$ 4.1 (82.6%) stained for $\beta$-Gal ac tivity (P<0.05). In the control treatment 26 of 30 (87.0%) blastocysts had some cells with $\beta$-Gal activity while all of the blastocysts in the G418 treatment had some cell with $\beta$-Gal ac tivity (P<0.05). However, ICM colonies in either control or G418 treatments were not expressed any epiblast cell except of trophetoderm celIs. The $\beta$-actin promoter/lacZ gene may not be e expression or silence expression in epiblast cells These results clearly show an enrichment of blastocysts expressing the transgene in the majority of their cells after culture in the presence of G418. The exogeneous gene was not express a and silence in ICM colonies especiallly epiblast cells except of trophectederm cells. Even though the higher rate cell number expressed of exogeneous gene in the G418 treatments, a total cell number was decrease significantly (P<0.05) of which might be also drop of the establishment of ES like-cell colonies and production of transgenic animals. However, futher studies need to determine the viability of these selected embryos and the avability of production of transgenic offspring.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.18 no.3
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• pp.195-205
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• 1985

As a part of investigation to use the Anatrctic krill, Euphausia superba, more effectively as a food source, processing conditions, utilizations and storage stability of krill paste (intermediate product of krill) were examined and also chemical compositions of krill paste were analyzed. Frozen raw krill was chopped, agitated with $25\%$ of water to the minced krill and then centrifuged to separate the liquid fraction from the residue. This liquid fraction was heated at $98^{\circ}C$ for 20 min. to coagulate the proteins of krill, and it was filtered to separate the protein fraction. Krill paste was prepared with grinding the protein fraction, adding $0.2\%$ of polyphosphate and $0.3\%$ of sodium erythorbate to the krill paste for enhancing of functional properties and quality stability. The krill paste was packed in a carton box, and then stored at $-30^{\circ}C$. Chemical compositions of krill paste were as follows : moisture $78\%$, crude protein $12.9\%$, crude lipid $5.9\%$, and the contents of hazardous elements of krill paste as Hg 0.001 ppm, Cd 1.15 ppm, Zn 9.1 ppm, Pb 0.63 ppm and Cu 11.38ppm were safe for food. The amino acid compositions of krill paste showed relatively high amount of taurine, glutamic acid, aspartic acid, leucine, lysine and arginine, which occupied $55\%$ of total amino acid and also taurine, lysine, glycine, arginine and proline were occupied $65\%$ of total free amino acid. Fatty acid compositions of krill paste consist of $32.4\%$ of saturated fatty acid, $29.6\%$ of monoenoic acid and $38.0\%$ of polyenoic acid, and major fatty acids of product were eicosapentaenoic acid ($17.8\%$), oleic acid ($16.9\%$), palmitic acid ($15.3\%$), myristic acid ($8.7\%$) and docosahexaenoic acid ($8.4\%$). In case of procssing of fish sausage as one of experiment for krill paste use, Alaska pollack fish meat paste could be substituted with the krill paste up to $30\%$ without any significant defect in taste and texture of fish sausage, and the color of fish sausage could be maintained by the color of krill paste. Judging from the results of chemical and microbial experiments during frozen storage, the quality of krill paste could be preserved in good condition for 100 days at $-39^{\circ}C$.

• Journal of Preventive Medicine and Public Health
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• v.25 no.2 s.38
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• pp.127-140
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• 1992

This study was done to identify the association between serum copper and zinc levels and the cirrhosis and hepatocellular carcinoma(HCC), and to evaluate its diagnostic value on liver diseases. Sixty-three healthy persons, 60 patients with cirrhosis and 33 patients with hepatocellular carcinoma were rendomly selected and investigated for their general characteristics from October 1990 to August 1991. For analysis of the biochemical markers in liver function test and the serum copper and zinc levels, their fasting venous blood were sampled at 9:00 to 11:00 in the morning and centrifuged to separate the serum within one hour. All the samples were immediately analysed for biochemical markers and stored at $-20^{\circ}C$ in polypropylene tubes further copper and zinc analysis. Mean of serum coppper levels was $91.97{\pm}4.76{\mu}g/dl$ in control, $106.21{\pm}2.73{\mu}g/dl$ in cirrhosis and $127.05{\pm}0.77{\mu}g/dl$ in HCC. The value of HCC was statistically significantly higher than that of the control and cirrhosis(p<0.05). Serum zinc levels were $110.82{\pm}7.24{\mu}g/dl$ in control, $68.10{\pm}5.43{\mu}g/dl$ in cirrhosis and $63.78{\pm}2.20{\mu}g/dl$ in HCC. The values of cirrhosis and HCC were statistically significantly lower than that of control(p<0.05). The Cu/Zn ratio was statiatically significantly different among three groups(p<0.05). Test total protein, albumin, ALP and total bilirubin of biochemical markers of liver function were statistically significantly different among three groups(p<0.05). Differences between cirrhosis and HCC for ALT and AST, and between the control and HCC for direct bilirubin were not statistically significant. Biochemical markers statistically significantly correlated with serum copper and zinc levels and Cu/Zn ratio(p<0.05), were variable in three groups. In multiple logistic regression, odds ratio of serum copper level and Cu/Zn ratio had no statistical significance on the cirrhosis and the HCC, but that of serum sinc was statistically significant as 0.951 and 0.952(p<0.05). Serum copper and zinc levels and Cu/Zn ratio were not statistically significantly different between the cirrhosis and HCC. H\Albumin, ALP, zinc, total bilirubin and age among all variables were selected as main variables for three-group discriminant analysis. Percentage of 'grouped' cases correctly classified by these five variables was 98.4 for control, 73.4 for cirrhosis, 75.7 for HCC and 84.0 for all subjects. This study suggests that zinc level is considered to play a role as diagnostic marker on the hepatic disorders and be more useful than serum copper level and Cu/Zn ratio in diagnosis of the liver diseases.

• The Korean Journal of Nuclear Medicine
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• v.38 no.4
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• pp.300-305
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• 2004

Purpose: We underwent this study to evaluate the factors which influence labeling efficiency when modified in vivo erythrocyte labeling technique was used. Materials and methods: Thirty healthy volunteers (M:F=19:11, age:$25{\pm}2$ yrs) were enrolled in this study. Totally, two hundred ten samples were obtained from them. The 1 mg of stannous pyrophosphate was injected intravenously at the beginning of labeling. After suitable tinning time (5 min, 20 min, 35 min) passed by, blood (5 mL, 3 mL or 1 mL) was withdrawn into 10 mL syringe previously containing Tc-99m (740 MBq) and anticoagulant (heparin, ACD or CPDA) through 19-gauged scalp needle. The generator ingrowth time of Tc-99m was within 24 hrs in each case. The blood samples were placed on rotating invertor during incubation (10 min, 25 min, 40 min) but some of them were not. Immediately after the conclusion of incubation, the labeled blood specimens to analyze were centrifuged. and then %Unbound Tc-99m was calculated. Statical analysis was used paired T-test and one way ANOVA with SPSS 10.0. Results: The binding efficiency at 1 mL of blood volume was $73{\pm}32%,\;91{\pm}10%$ at 3 mL and $96{\pm}7%$ at 5 mL (p<0.01). The binding efficiency at 5 min of tinning time was $45{\pm}23%,\;98{\pm}6%$, at 20 min and $97{\pm}8%$ at 35 min (p<0.001). The binding efficiency at 10 min of incubation time was $96{\pm}7%,\;95{\pm}12%$ at 25 min and $98{\pm}3%$ at 40 min (p>0.05). The binding efficiency in case of using rotating invertor was $96{\pm}7%$ and the binding efficiency in case of not using it was $87{\pm}18%$ (p>0.05). There was no significant difference between them. In binding efficiency according to kinds of anticoagulants, ACD was $98{\pm}4%$, CPDA was $97{\pm}6%$ and heparin was $89{\pm}20%$ (p<0.001). Conclusion: When modified in vivo erythrocyte labeling technique is used with Tc-99m, the methods to obtain the highest labeling efficiency are as follow. The withdrawing blood volume should be over 3 mL, tinning time should be kept between 20 min and 35 min, and incubation time should be kept between 10 min and 40 min. ACD or CPDA have to be used as a anticoagulant except heparin and the blood samples should be placed on rotating invertor during incubation.