Current developments in IVF and animal cloning have resulted in increasing demand for large quantities of oocytes and ovarian follicles at specific stages of development. These medical and scientific needs may be met by developing an optimal culture system for preantral follicles. In this study, we investigated the growth of porcine preantral follicle cultures in different media and in the presence and absence of serum. Follicles were manually dissected from ovaries obtained from prepubertal gilts at a local slaughterhouse, and cultured for 3 days in M199 or NCSU23 medium supplemented with porcine FSH, transferrin, L-ascorbic acid and insulin. Follicle diameters were measured on day 1 and 3 of culture. In Experiment 1, the effect of supplementing culture medium with fetal calf serum (FCS) on porcine preantral follicle growth was examined. In the group of cultures supplemented with FCS, follicle diameter after 3 days of culture, survival rate and antrum formation rate in the FCS group were significantly higher than those of the control group. In Experiment 2, the effects of culture medium (M199 and NCSU23) on follicle growth were compared. Follicle diameters were increased in the M199 group, compared with those in NCSU23 (p<0.05), but we observed no significant differences in survival and antrum formation rates between cultures grown in the two media. In conclusion, supplementation of the culture medium with serum enhances preantral follicle growth and antrum formation, and M199 is superior to NUSU23 for porcine preantral follicle culture in vitro.

The 34 potently pig pheromonal odorants (1-32, 5755 & 7113) through structure-based virtual screening and ligand-based virtual screening method were selected and their ADMET and pharmacokinetics characters were evaluated and discussed quantitatively. The pheromonal odorants were projected on the following pre-calculated models, Caco-2 cell permeability, blood-brain barrier permeation, hERG inhibition and volume-distribution. From the results of in silico study, it is found that an optimal compound (31) either penetrating or have a little (Pcaco2=－8.143) for Caco-2 cell permeability, moderate penetrating ability (PBBB=0.082) for blood-brain barrier permeation, the low QT prolongation (PhERG=1.137) for the hERG K+ channel inhibition, and low distribution into tissues (PVD=－5.468) for volume-distribution. Therefore, it is predicted that the compound (31) a topical application may be preferable from these based foundings.

To search the potent pig pheromonal odorants through receptor-based approach methods, molecular dockings between 680 Flavornets as substrate molecule and pig odorants binding proteins OBP (1HQP) and PBP (1GM6) as receptor, and QSPR (quantitative structure-property relationship) analyses from physico-chemical parameters of Flavornets and their docking scores (DS) were performed and discussed quantitatively. From the basis on the findings, the optimal value (MSA)opt.=407.595 Å2 of MSA (molecular surface area; Å), and RB (number of rotational bond) had the Flavornets will be able to increase DS. Therefore, it is expected that the stearyl alcohol from DS and H-bond type between substrate and receptor would be shows the character as potent pig pheromonal odorant.

The binding affinity constants (p(Od)50) and molecular docking scores (DS) between porcine odorant binding proteins pOBP (1HQP) and pPBP (1GM6) as receptor and a series of tetrahydrofuran-2-yl (A & B) analogues as substrate, and their interactions were discussed quantitatively using three-dimensional quantitative structure-activity relationship (3D-QSAR) models. The statistical qualities of the optimized CoMFA models for pOBP were better than those of the CoMSIA models. The binding affinity constants and DS between substrate and receptor molecules were dependent upon steric and hydrophobic interaction. The DS constants of the substrates into the binding site of OBP (1HQP) were bigger than those of PBP (1GM6). The resulting contour maps produced by the optimized CoMFA model were used to identify the structural features relevant to the binding affinity in binding site of pOBP.

The two dimensional quantitative structure-activity relationships (2D-QSARs) models concerning the binding affinity constants (p[Od.]50) between 2-cyclohexyltetrahydropyrane and 2-cyclohexyltetrahydrofurane analogues as substrates, and bovine odorant binding protein (bOBP) as receptor were derived by multiple regression analyses method and discussed. The statistical quality of the optimized 2D-QSAR model (5) was good (r=0.907). From the model, the binding affinity constants (p[Od.]50) were dependent upon the optimal value ((TL)opt.=2.737) of total lipole (TL) of substrate molecules. Based on these findings, the high active compounds predicted by optimized 2D-QSAR model (5) were 2-(dimethylcyclohexyl)tetrahydropyrane molecule and their isomer molecules. The binding affinity constants regarding bOBP of the tetrahydrofuryl-2-yl family compounds were dependent upon the hydrophobicity (logP) of whole substrate molecules. In any case of porcine odorant-binding proteins (pOBP), the constants were dependent upon the hydrophobicity (πx=logPx－logPH) of substituents (R) in substrate molecules. Also, from the optimal values of hydrophobic constant, the hydrophobicity for bOBP influenced ca. twice time bigger (bOBP>pOBP) than that for pOBP.

To search a new pig pheromonal odorant, the N1-allyl-N2-(tetrahydrofuran-2-ylmethyl)oxalamide molecule predicted by ligand based approach and molecular docking method was synthesized by nucleophilic addition-elimination reaction (AdNU-E) between N-allyloxalamic acid ethylester and tetrahydrofurylmethylamine. According to the evaluation results for efficiency of pig estrus control, the synthesized pig pheromonal N1-allyl-N2-(tetrahydrofuran-2- ylmethyl)oxalamide molecule advanced the estrus by 11.3 days (p<0.05) compared with the non-pheromone group. And from these results, it is predicted that the synthesized pig pheromonal compound will be able to increase the reproduction efficiency of pig.

돼지 페르몬성 냄새 물질을 탐색하기 위하여 tetrahydrofuran-2-yl 계 화합물들과 관측된 결합 친화력상수(Obs.p[Od]50) 사이의 정량적인 구조-활성관계(QSAR)로부터 4개 형태의 모델(2D-QSAR, HQSR, CoMFA 및 CoMSIA)들이 유도되었다. Ligand based approache로부터 최적화된 CoMFA 모델(예측성; r2cv.(q2)=0.886 및 상관성 r2RCV 0.984)이 가장 좋은 모델이었다. CoMFA 모델로부터 돼지 페르몬성 냄새 물질로 예측된 N1-allyl-N2 -(tetrahydrofuran-2-yl)methyl) oxalamide (P1), 2- (4-trimethylammoniummethylcyclohexyloxy)tetrahydrofurane (P5) 및 2- (3-trimethylammonium-methylcyclohexyloxy)tetrahydrofurane (P6) 분자들은 비교적 높은 결합 친화력 상수값(Pred.p[Od]50) =8~10)과 몇 가지 독성에 대하여 낮은 독성간을 나타내었다.

This study was carried out to investigate the effects of cryoprotectants, warming solution and removal of lipid on open pulled straw vitrification (OPS) method of porcine embryos produced by nuclear transfer (NT) of fetal fibroblasts. All solutions used during vitrification were prepared with holding medium consisting of 25 mM Hepes buffered TCM199 medium containing 20% fetal bovine serum (FBS) at 38.5℃. The blastocysts derived from NT with or without lipid were vitrified in each medium of different concentrations of dimethyl sulfoxide (DMSO) and ethylene glycol (EG). Also, blastocysts after cryopreservation were warmed into different concentrations of sucrose in warming solution. The optimal concentrations of cryoprotectants in vitrification solution were 10% DMSO + 10% EG in vitrification solution 1 (VS1) and 20% DMSO + 20% EG in vitrification solution 2 (VS2). The optimal concentrations of sucrose were 0.3 M sucrose in warming solution 1 (WS1) and 0.15 M sucrose in warming solution 2 (WS2). Lipid removal from oocytes before NT enhanced the viability of NT embryos after vitrification. Our results show that use of the OPS method in conjunction with lipid removal provides effective cryopreservation of porcine nuclear transfer embryos.