• Korean Journal of Environmental Agriculture
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• v.41 no.3
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• pp.206-216
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• 2022

BACKGROUND: Dithiocarbamate fungicide propineb can be analyzed quantitatively by derivatization reaction followed by HPLC/UVD, which has high reproducibility and stability. However, the presence of high protein in soybeans and peas affects the derivatization process resulting in extremely low recoveries. Therefore, this study was conducted to improve the analytical method for analysis of propineb in soybeans and peas by applying a deproteinization process using chloroform-gel method. METHODS AND RESULTS: The deproteinization process was carried out up to 6 times for soybeans and 5 times for peas using 50 mL chloroform. After 4 times of deproteinization process followed by a derivatization reaction with methyl iodide, the recovery yields of propineb in both pulses were >90%. However, the recovery yield tended to decrease when the deproteinization process was performed more than 5 times. The method limit of quantification (LOQ) was 0.04 mg/L. The recovery conducted in triplicate at 10 times and 50 times of the LOQ ranged from 87.2 to 95.0 % with a coefficient of variation <10%. CONCLUSION(S): This study confirmed that 4 times of deproteinization process using the chloroform-gel method was effective when derivatizing and analyzing dithiocarbamate fungicides in pulses with high protein content. However, depending on the initial protein content present in the pulses, there was a difference in the recovery: the lower the protein content, the higher the recovery rate of propineb. It is expected that the method proposed in this study could be applied to remove high content of protein as analytical interference substance from agricultural samples.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.2
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• pp.130-135
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• 2022

Epididymal sperm cryopreservation provides a potential method for preserving genetic material from males of endangered species. This pilot study was conducted to develop a freezing method for tiger epididymal sperm. We evaluated post-thaw sperm condition using testes with intact epididymides obtained from a Siberian tiger (Panthera tigris altaica) after castration. The epididymis was chopped in Tyrode's albumin-lactate-pyruvate 1x and incubated at 5% CO₂, 95% air for 10 min. The Percoll separation density gradient method was used for selective recovery of motile spermatozoa after sperm collection using a cell strainer. The spermatozoa were diluted with modified Norwegian extender supplemented with 20 mM trehalose (extender 1) and subsequent extender 2 (extender 1 with 10% glycerol) and frozen using LN₂ vapor. After thawing at 37℃ for 25 s, Isolate^® solution was used for more effective recovery of live sperm. Sperm motility (computerized assisted sperm analysis, CASA), viability (SYBR-14 and Propidium Iodide) and acrosome integrity (Pisum sativum agglutinin with FITC) were evaluated. The motility of tiger epididymal spermatozoa was 40.1 ± 2.0%, and progressively motile sperm comprised 32.7 ± 2.3%. Viability was 56.3 ± 1.6% and acrosome integrity was 62.3 ± 4.4%. Cryopreservation of tiger epididymal sperm using a modified Norwegian extender and density gradient method could be effective to obtain functional spermatozoa for future assisted reproductive practices in endangered species.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.37 no.3
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• pp.250-256
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• 1992

This study was carried out to obtain basic genetic informations in pea(Pisum sativum L.). A series of field experiments were carried out with seven pea cultivars and F$_1$ hybrids for three years. Based on the information collected from a set of diallel crosses, the behaviours of inheritance of seed yield and ten major agronomical characters were observed by the estimation of heterosis and combining ability. Heterosis for seed yield per plant was 79% and those for yield components such as number of seeds per plant, number of pods per plant, stem length, stem diameter, number of branches per plant, also showed relatively high values. General combining ability(GCA) and specific combining ability(SCA) were significantly different among major agronomic characters, and the variances of GCA were higher than those of SCA latter. Among the tested cultivars, Alderman was found to be the most promising parent based upon the GCA effects on the seed yield and yield components. The crosses of Sugar Ann$\times$Alderman, Wando$\times$Alderman and JI 1198$\times$Chinju ＃6, showed the highest SCA effects on seed yield and yield components.

• Journal of Veterinary Clinics
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• v.30 no.6
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• pp.442-448
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• 2013

The aim of the present study was to develop glycerol-free TRIS extender using glucose for dog sperm cryopreservation. We determined the appropriate concentration of glucose in glycerol-free TRIS and the exposure time in glycerol-free TRIS containing 0.3 M glucose at $4^{\circ}C$. Ejaculates of six dog sperm were cooled in glycerol-free TRIS through $4^{\circ}C$ for 100 min, cooled at $4^{\circ}C$ in TRIS with different glucose concentrations 0 M, 0.04 M, 0.1 M, 0.2 M and 0.3 M, respectively for 30 min followed by cryopreservation. After thawing at $37^{\circ}C$ for 25 sec, membrane and acrosome integrities of dog sperm were evaluated. In addition, the effect of exposure time (10, 30, 50 and 70 min) of sperm to glycerol-free TRIS containing 0.3 M glucose at $4^{\circ}C$ on progressive motility, viability, and DNA integrity following sperm cryopreservation was studied. Membrane integrity and acrosome integrity were assessed by 6-carboxyfluoresceindiacetate (6-CFDA)/propidium iodide (PI) fluorescent staining and Pisum sativum agglutinin conjugated to fluorescein isothiocyanate, respectively. DNA integrity was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling, using flow cytometry. Sperm frozen in glycerol-free TRIS supplemented with 0.2 M or 0.3 M glucose have an intact plasma membrane (CFDA+/PI-) after cryopreservation than sperm frozen in the extenders with lower glucose concentrations (p<0.05). Acrosome integrity was significantly higher in the 0.3 M group than less than 0.1 M groups (p<0.05). The sperm DNA fragmentation index did not differ according to exposure time, although progressive motility was significantly higher in the 50 min exposure group than the other groups (p<0.05). These results indicate that cryopreservation of dog sperm is feasible and yields more motile sperm following freezing and thawing in glycerol-free TRIS containing 0.3 M glucose with the exposure time for 50 min at $4^{\circ}C$.

• 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 Plant Tissue Culture
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• v.26 no.1
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• pp.41-47
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• 1999

Phenylalanine ammonia-lyase (PAL; EC 4, 3, 1, 5) genomic clones were isolated from tomato(Lycopersicon esculentum L.) genomic DNA libraries using tomato PAL5 cDNA sequences as probes. The nucleotide sequences of tPAL1, tPAL4 and tPAL5 were compared. tPAL5 contains an open reading frame encoding a polypeptide of 722 amino acids, interrupted by a 710 bp intron in the codon for the amino acid 139. tPAL1 encodes a polypeptide of 249 amino acids which is much shorter than tPAL5 gene due to a premature stop codon and does not contain an intron. tPAL4 encodes a polypeptide of 357 amino acids, interrupted by a 305 bp intron in the codon for the amino acid 138. Premature stop codons observed in tPAL1 and tPAL4 gene produce a short polypeptide rather than a normal polypeptide (722 aa). tPALl shows 87.2% homology with tPAL4 and 85.3% homology with tPAL5 gene whereas tPAL4 showes 91.4% homology with tPAL5 at nucleotide level. In general, phylogenetic analysis showed that genes isolated from tomato, potato, and sweet potato were belong to the same group and another dicot plants such as parsley, bean, soybean, pea and alfalfa formed another group. PAL genes isolated from rice and yeast showed very low homology with other PAL genes and formed the other group.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.10
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• pp.1417-1425
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• 2014

The selection of morphologically normal spermatozoa is critical to obtain high breeding performances in boar breeding farms and artificial insemination (AI) centers. Parameters for the selection of semen mainly include total sperm motility, concentration, and morphology. However, these primary parameters are often not reliable for discriminating between normal and abnormal, non-fertilizable spermatozoa. The present study was designed to compare the motion characteristics, fertilization ability using in vitro fertilization (IVF), and acrosome formation of the semen from boars having low (boar number 2012) and normal (boar number 2004 and 2023) breeding performances. The ultimate goal was to identify additional simple and easy criteria for the selection of normal sperm. There was no significant difference between boar 2004 and boar 2023 sperm total motility in computer assisted sperm analysis. However, boar number 2012 semen presented a significantly reduced population of rapid moving spermatozoa and an increased population of slow moving spermatozoa compared to boar numbers 2004 and 2023. Analysis of detailed motion characteristics revealed that sperm from boar number 2012 had significantly reduced motility in progressiveness, average path velocity, straight-line velocity (VSL), curvilinear velocity (VCL), straightness, and linearity. The assessment of the fertilizing ability by IVF also showed that sperm from boar number 2012 showed a fertility rate of 3.4%, whereas sperm from boar number 2023 had a fertility rate of 75.45%. Interestingly, most of the sperm nuclei were found on the peripheral area of the oocytes, suggesting that the sperm from boar number 2012 lacked penetration ability into the oocyte zonapellucida. The acrosome formation analysis using Pisum sativum agglutinin staining demonstrated that the sperm from boar number 2012 had a defect in acrosome formation. Consequently, primary parameters for selecting semen before AI such as motility are not sufficient to select normal and fertilizable spermatozoa. In conclusion, the present study suggests that the acrosome staining and detailed motion characteristics such as progressiveness, VCL, and VSL should be included in determining semen quality together with primary parameters for successful AI and high breeding performance in the swine industry.

• Korean Journal of Agricultural Science
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• v.12 no.1
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• pp.22-30
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• 1985

Protoplasts of Pisum sativum L. were isolated and cultured from leaf mesophyll tissue. The successful yield of protoplast was obtained in an enzyme solution of 2% 'Onozuka R-10' and 2 % 'Macerozyme R-10' contained 6mM $CaCl_2{\cdot}2H_2O$ within 4 hours. They were divided in B5 culture medium supplemented with 2mg/l kinetin, 1mg/l 2, 4-D and 0.2% Difcobacto agar. Divisions of the protoplasts were continued and led to colony formation for 1 months. The colony from protoplasts of pea mesophyll tissue was formed to callus after subculture in a medium contained macronutrients and amino acids of BII medium and micronutrients and vitamins of B5 medium, and also supplemented with 2mg/l kinetin 2mg/l NAA.

• Korean Journal of Breeding Science
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• v.41 no.3
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• pp.261-270
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• 2009

Oxidative stress is a major damaging factor for plants exposed to environmental stresses. In order to develop transgenic rice plants with enhanced tolerance to various environmental stresses, PsAPX1, the gene of ascorbate peroxidase isolated from Pisum sativum was expressed in chloroplast under the control of an oxidative stress inducible sweet potato peroxidase2 (SWPA2) promoter (referred to as PsAPX1 plants). PsAPX1 transgenic plants showed enhanced tolerance to various environmental stresses, such as 170 mM NaCl, UV-B, ozone, 20% PEG, and drought in compared with non-transgenic (NT) plants. These results suggest that chloroplast-targeted over-expression of PsAPX1 gene could be very useful strategy for developing transgenic rice plants with increased tolerance to environmental stresses.

• Korean Journal of Food Science and Technology
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• v.52 no.6
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• pp.641-648
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• 2020

The objective of this study was to optimize the mixing ratio of mixed bean protein concentrate (MBPC) and to improve the quality of sausage analogues. Soybean (Glycine max MERR), mung bean (Phaseolus radiatus L.), red bean [Vigna angularis (Wild.)], and pea (Pisum sativum L.) were mixed and processed to produce a MBPC, which was used to make a sausage analogue. The protein, moisture, and carbohydrate content were significantly (p<0.05) different among the samples. A significant (p<0.05) improvement was observed in textural properties (hardness, gumminess, and chewiness), cooking loss, frying loss, and emulsion stability of the sausage analogue. This study suggested the possibility of attaining high-quality sausage analogues and partial sausage analogues using MBPC, which could serve as a potential ingredient in meat analogues.