Drug-induced liver injury (DILI) is considered to be a significant cause of drug wastage. To mitigate clinical DILI risks, assessing drugs using human liver models is crucial since animal studies may fall short due to species-specific liver pathway variations. Cell-based preclinical hepatotoxicity testing is often pertinent. In the present study, cells from a human liver cancer line (HepG2 and HepaRG) were cultured in both formats of 2D and 3D spheroids to explore their responses to drugs. Liver-specific marker expressions across cell lines and culture formats were also examined to assess disparities in DILI marker expressions. After treating each cell with the drugs, cytotoxicity and liver injury markers aspartate aminotransferase and alanine aminotransferase were increased. In addition, liver specific markers albumin and urea decreased in a drug concentration-dependent manner. These findings were consistent with drug sensitivity. Additionally, mRNA expression levels of cytochrome P450 enzymes (CYPs) involved in hepatocellular drug metabolism were compared following treatment with enzyme inducers. CYP1A2 and CYP2C9 were not epxressed in HepG2 cells. HepaRG cells exhibited significantly increased expression of CYP1A2, 2C9, and 3A4 post-treatment. Notably, enzyme expression was notably higher in 3D cultures than in 2D cultures. Collectively, these findings suggest that HepaRG cells and 3D cultures hold promise for evaluating DILI during early-stage drug development.

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.

This study aimed to develop the in vitro method using domestic commercial diets to estimate nutrient digestibility in dogs. The existing in vitro method were tested and compared with literature data to develop new in vitro method. The development of in vitro method progressed as follows: modification of pepsin solution to an activated form and supplementation with 1% lipase. All the in vitro method progressed to 4 hours of stomach simulation and 2 hours of small intestine simulation. In vivo digestibility was measured using the same diets as beagle dogs. The supplementation of lipase methods showed significantly improved (p < 0.05) DM, OM, and EE than the existing and modified pepsin solution methods. The correlation between in vitro and in vivo data in DM, OM, and EE digestibility was high (r2 = 0.889, 0.907, and 0.721, respectively), and the correlation between in vitro and in vivo data in CP and GE digestibility was medium (r2 = 0.681 and 0.536, respectively). The current in vitro method is similar to in vivo digestibility and helps potentially predict digestibility for dogs. In conclusion, this developed in vitro method suggests that it can help estimate the nutrient digestibility of dogs' diets without in vivo experiments.

Water lettuce (Pistia stratiotes L.) is a free-floating perennial herbaceous plant with rosette leaves and a stem. Although this plant multiplies and has adverse effects on aquatic ecosystems, it can be used for biological purification of polluted water and production of valuable substances as a traditional medicine. In this study, we report a protocol to establish an in vitro micropropagation method based on direct shoot organogenesis from stem explants. In media comprising two types of basal medium and different growth regulators, multiple shoot organogenesis was observed on stems. The micropropagation method was most effective on Schenk and Hildebrandt (SH) medium with 1 mg/L indole-3-acetic acid and 2 mg/L 6-benzylaminopurine supplemented with 30 g/L sucrose, on which all explants produced multiple shoots. The shoots rooted spontaneously on solid half-strength Murashige and Skoog (MS) and SH media without growth regulators (1/2MSO and 1/2SHO). However, roots developed more vigorously in liquid media. Regenerated plants colonized and grew more rapidly in SH basal medium than in MS basal medium and produced 6–8 stolons within 2 weeks on 1/2SHO. In summary, we established a method for micropropagation in vitro through direct organogenesis of water lettuce, which shows the potential of water lettuce as a model aquatic plant for phytochemical and pharmacological research.

Black soldier fly (BSF, Hermetia illucens) has been noted as an excellent feed ingredient. However, there is limited information on rearing and processing technology. Thus, this study was conducted to evaluate the substrates for rearing BSF and the optimal processing method for BSF performance. Study separated as 2 experiment, BSF rearing and drying method(Exp 1.) and EP-processing method(exp 2.). During the study, 30 clutches were reared, with 10 per substrate. Three substrates, namely food waste (FW), tofu by-product (TF), and vegetable waste with two drying methods, namely hot air dry (AD) and microwave dry (MW) at expanding (EP) ratios of 5:5 and 7:3, were examined by evaluating their rearing performance, nutrient contents, in vitro digestibility and lipid oxidation stability during storage (0, 14 and 28 days). In experiment 1, the rearing substrates and drying methods were evaluated. Compared with that of conventional methods (FW, AD), the TF substrates and MW method showed higher dry matter contents (3.43%) and in vitro digestibility (1.62%) but lower ether extract contents(3.53%; p<0.05). However, the malondialdehyde (MDA) concentration under MW treatments decreased during storage (5.77%, 4.69% and 3.24%; p<0.05). In experiment 2, compared with that of the 7:3 EP-BSF ratio, the 5:5 EP-BSF ratio showed higher in vitro digestibility (2.70%) and lower MDA concentration during storage (19.19%, 7.96% and 6.42%; p<0.05). In conclusion, the optimal conditions for BSF rearing and ensuring product quality were TF substrates, MW methods and a 5:5 corn:BSF ratio. Therefore, the optimal conditions for producing EP-BSF can present an excellent feed ingredient alternative for swine feed.

A semi-natural composite of κ-carrageenan and bentonite, two natural biopolymers, was synthesized through free radical polymerization. This synthesis aimed to obtain a biodegradable, biocompatible, and swellable composite that is environmentally friendly. The components used in this synthesis are readily available, making it economically feasible and promising for potential biomedical applications. The composite is pH-responsive and intended for oral delivery of metformin hydrochloride and aminophylline, which have low bioavailability and undesirable side effects, respectively. The organic composite exhibits the advantage of reducing drug release in the acidic gastric medium. This composite is a stimuli-responsive polymeric material that has garnered significant attention in recent years for its application in oral drug delivery systems. These materials enable site-specific and controlled drug release while minimizing toxicity. The carrageenan-g-poly(acrylamide-co-acrylic acid)/bentonite composite was characterized using Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), and field emission scanning electron microscopy (FE-SEM), which confirmed the successful synthesis of the composite. The swelling behaviour and point of zero charge of the composite were studied at different pH values, which showed a strong influence on the swelling properties of the composite. The drug loading capacity of the composite was measured at pH 5.3, and it was 70.60 mg/g for metformin and 95.66 mg/g for aminophylline at pH(3). The in vitro release profile of both drugs from the composite was also affected by the ionic strength, and it exhibited a lower release rate with higher salt concentration. The maximum release percentage of the drugs from carrageenan-g-poly(acrylic acid-acrylamide)/bentonite in simulated gastric, intestinal, and colon fluids was achieved within 40 h. The maximum release was 80% for metformin in simulated intestinal fluid (SIF) and 75% for aminophylline after 40 h.

본 실험에서는 외인성 효소 첨가제 및 혼합 세균 배양을 통한 고상발효(Solid-state fermentation, SSF)가 채종박(Rapeseed meal, RSM)의 체외건물소화율(In vitro dry matter digestibility, IVDMD) 및 단쇄지방산(Short chain fatty acid) 생성에 미치는 영향을 조사하기 위해 수행되었다. 외인성 효소 칵테일(첨가 및 미첨가) 및 RSM에 대한 SSF(발효 및 비발효)를 나타내는 2 x 2 요인 설계가 적용되었다. 3-step 돼지 소화율 모델을 적용하여 채종박의 건물 소화율을 분석하였으며, 72시간 대장발효 후 상층액을 수집하여 단쇄지방산 생성량을 분석한 후 칼로리 단위로 변환하여 가소화에너지 소화율을 분석하였다. 소장 (IVDMDh) 및 전장 (IVDMDt) 건물소화율에서는 고상발효된 채종박이 더 높게 나타났다 (각각 p < 0.01). 마찬가지로, 외인성 효소 첨가제 처리구에서 채종박의 소장 소화율(IVDMDh)이 증가하는 경향을 나타냈다(p = 0.06). Acetic acid 및 butyric acid의 생산은 대조구에 비해 고상발효 처리 시 유의하게 더 생산되었으며 (각각 p < 0.01), 이는 총 단쇄지방산의 생산 증가 경향을 나타냈다(p = 0.09). 에너지 소화율에서는 채종박의 고상발효 및 외인성 효소제 첨가가 유의적으로 높게 나타났다 ( p < 0.01). 그러므로 채종박의 고상발효 처리는 단백질 이용성을 비롯한 영양적 가치를 향상시키는데 효과적이라고 사료된다.

손 위생 제품이 다양화됨과 동시에 각 활용 방법에 따 라 그 효능을 평가하는 여러 시험 방법들이 보고되고 있 다. 하지만 평가 방법에 따라 각 제품의 항균 효능은 다 르게 나타나며, 이로 인해 제품의 실제적인 효능을 확인 하는 데에 어려움이 있을 수 있다. 손 위생 제품의 효능 평가방법 비교에 초점을 둔 연구는 매우 제한적이며, 특 히 돼지피부를 이용한 ex vivo에 대한 연구는 극히 드물 다. 이에 본 연구는 손 위생 제품 중 리브온 소독제와 워 시오프 세정제에 대해 각각의 항균 평가 방법을 종합적으 로 비교했고, ex vivo 시험에 영향을 미칠 수 있는 요인을 파악하여 연구 단계에서 효율적인 ex vivo 시험의 신뢰성 을 향상시키고자 하였다. in vitro 시험으로써 액체 현탁을 기반으로 하는 time-kill 시험을 진행했고, in vivo 시험은 최소 20명의 참여자를 대상으로 진행되었다. ex vivo 시험 은 규격화된 돼지 피부를 이용하여 in vivo 시험과 동일한 방법으로 진행하면서 소독제의 최적 처리량과 세정제 사 용 시 첨가되는 물의 양을 제안했다. 시험에 사용된 손 소 독제는 in vitro 시험에서 모두 5 log 이상의 세균 감소율 을 보인 반면, ex vivo와 in vivo에서는 훨씬 낮은 살균 활 성을 보였으며, 특히 알코올 함량이 낮은 손 소독제에서 는 1 log 미만의 살균 활성을 나타냈다. 반면에 손 세정 제의 in vitro 시험 결과, 대장균에 대해서는 1 log 이하의 낮은 항균력을 보였으나, ex vivo 와 in vivo 시험 결과에 서는 이보다 높은 항균력을 유사하게 나타냈다. 본 연구 에서는 ex vivo 와 in vivo 시험 방법이 리브온과 워시오 프 타입 제품의 두가지 다른 항균 메커니즘을 반영할 수 있음을 확인했다. 이로 인해 최적의 조건으로 설정된 ex vivo 시험은 빠르고 정확한 항균 평가법이 될 수 있음을 제시한다.

This study is concerned with the optimization of the manufacturing process of a hot water extract containing antioxidant activity from Lycium barbarum, traditionally known to have various physiological activities. For the establishment of the optimization process, the central composite design of response surface methodology(RSM) was used. Thirteen extraction processes were performed by encoding the independent variables, extraction temperature (65.9oC–94.1oC) and extraction time (2.59 hr–5.41 hr). As a result of the experiment, the optimal manufacturing conditions for the extract were 340.0 mg/100 g of GAE at an extraction temperature of 94.1oC and an extraction time of 5 hr. The maximum yield of flavonoids was 22.44 mg/100 g of HES at an extraction temperature of 94.1oC and an extraction time of 4 hr. The conditions for producing the extract with the maximum antioxidant capacity (DPPH 92.12%) were 90oC and 4.5 hr extraction time. Therefore, the optimal manufacturing process conditions for extracts containing total phenol content, flavonoid content, and DPPH radical scavenging activity, which are dependent variables, were extraction temperature of 90-95oC and extraction time of 4 hr, which were not significantly different from the actual values. Therefore, Lycium barbarum extract rich in total phenol and flavonoid content related to antioxidant function is expected to be used as a functional food and cosmetic material.

헤스페리딘(Hesperidin, HD)은 다양한 식물체에 존재하는, 강한 항산화 기능을 가진 대표적인 flavonoid의 일종이다. 본 연구에서는 수용성 HD인 Hesperidin glucoside(HDG)가 가지는 세포손상 회복, 항염증 인자억제 및 melanin 생성억제 활성을 세포수준에서 비교하였다. HDG는 HD에 당전이 효소반응 으로 제조되었으며, HD에 비해 20,000배 이상 수용해도가 증가되었다. HaCaT 세포주에 대한 세포독성은 HDG가 HD에 비해 월등히 낮았다. HD와 HDG는 모두 자외선 조사된 HaCaT 세포에서 세포생존율 회 복효과를 나타내었다. 또한 HD와 HDG는 세포내 산화질소(NO), 종양괴사인자-α(TNF-α) 및 인터루킨 -6(IL-6)과 같은 염증 매개체 및 cytokine을 감소시켰으며, HD 보다는 HDG의 효과가 다소 우수하였다. Melanoma B16F10 세포주를 이용한 melanin 형성능과 tyrosinase 저해활성을 측정한 결과, HD와 HDG 모두 효과를 나타내었으며 HDG가 약간 우수한 결과를 보였다. 결론적으로, HD의 당전이체인 HDG는 HD에 비해 동등이상의 세포손상 회복, 염증성 매개체 및 cytokine 억제능과 melanin 형성억제능을 나타내 었으며, HDG의 높은 수용성과 낮은 세포독성 등의 특성은 다양한 분야에서의 용도를 확대시킬 수 있을 것으로 보인다.

Radiation workers, especially those dealing with Uranium isotopes, can potentially intake Uranium -containing materials through their respiratory and digestive systems. According to the “Regulations on the Measurement and Calculation of Internal Exposure” from Nuclear Safety and Security Commission (NSSC), those who intend to work in or enter the nuclear facilities with a risk of exceeding 2 mSv exposure per year should be examined the internal exposure. However, when it comes to in-vitro bioassay, Uranium intake through drinking water can affect the quantitative analysis. The International Commission on Radiological Protection (ICRP) reported in ICRP Publication 23 (Report on the Task Group on Reference Man) that the reference man excretes Uranium in the urine (0.05-0.5 μg/day) and feces (1.4-1.8 μg/day). Korea Atomic Energy Research Institute (KAERI) set the 90.5 ng/day as the 238U background of workers handing Uranium based on the daily Uranium intake of Koreans. In this research, we examined the possible effects of Uranium in drinking water on internal exposure by analyzing the concentration of Uranium in bottled waters from various water sources sold in the domestic market and a water from the water purifier. The 238U concentration results of analyzing 11 bottled waters and 1 purified water, were ranged from 0 to 10.2 μg/L. All the results were satisfied the standard of 30 μg/L according to “Regulations for Drinking Water Quality Standards and Inspection” enacted by the Ministry of Environment. However, various concentrations were shown depending on the water sources. Assuming that these concentrations of water are consumed by drinking 1 L per day, the internal dose assessment result is 0 to 0.94 mSv. On the other hand, if it is assumed to be inhaled, it can be an overestimated because the dose coefficient of inhalation, Type M is higher than that of ingestion, f1=0.02 which are the values recommended by ICRP Publication 78 (Individual Monitoring for Internal Exposure of Workers) when the Uranium compound is unspecified. In case of two workers at KAERI, the daily excretion of urine was 151 and 120 ng/day respectively in the first quarter monitoring. However after changing the kind of drinking water in the second quarter monitoring, it dropped to 17.4 and 15.4 ng/day respectively. Through this study, it is confirmed that the Uranium background in urine can be analyzed differently depending on the kind of drinking water consumed by each worker. Depending on the Uranium concentration of drinking water, the internal exposure dose assessment can be overestimated or underestimated. Therefore, the Uranium concentration and intake amount according to the kind of drinking water should be considered for in-vitro bioassays of Uranium handlers. Furthermore, if necessary, the Uranium isotope ratio analysis in urine and the handling information should be comprehensively considered. In addition, in order to exclude the effect of intake through the digestive system, replacing the kind of drinking water can be considered. The additional analysis such as in-vivo bioassay and 24 hours urine analysis rather than spot samples can be also recommended.

Changes in contents of free sugars, amino acids, and fatty acids of legumes were analyzed for each phase of in vitro digestion. In addition, contents of resistant starch in raw and digested pulses were compared. Soybeans, kidney beans, cowpeas, and chickpeas were analyzed. An in vitro digestion model was used to analyze contents of nutrients using LC-MS and GC-MS. Stachyose in kidneybean, cowpea, and chickpea increased as the digestion phase progressed. In four types of legumes, raffinose slightly decreased or showed no significant difference between the Oral phase and the BBMV phase. Content of glucose, a monosaccharide, increased during the BBMV phase. During the digestion phase, levels of free amino acids and free fatty acids also increased. Content of resistant starch was reduced compared to that in the raw material. It was 0.01g/100 g food in soybean, 1.06 g/100 g food in red kidney bean, 0.77g/ 100g food in cowpea, and 0.76 g/100 g food in chickpea. It was confirmed that nutrients in the in vitro digestion model were liberated at each digestion phase with changes in the content of resistant starch. These results are expected to be used as fundamental data for obtaining bioavailability of nutrients.

Leaf-spray in vitro bioassays appraise new aphicidal formulations for managing deleterious plant-feeding aphids. The formulation may utilize alternative and integrated strategies. However, leaf spraying even under controlled conditions may affect aphid reproduction and mortality. This study examines leaf spray applications for optimum and reproducible aphicidal results using tobacco leaves overlaid on cotton fabric or water agar surfaces. Infestation of the undersides of tobacco leaves with nymphs of green peach aphids was used in the assays. Spray distance and volume were optimized using water-sensitive paper to ascertain the best surface coverage. Overlays of the leaves on water agar caused less mortality and greater reproduction than the use of cotton fabric. The relative humidity of the insect-rearing chambers changed with the watering regime for the insect - rearing chambers with cotton fabric; 60% relative humidity was optimal. Relative humidity was not affected by the concentration of agar in the water agar chambers. Applications of the chemical aphicidal standard, Sulfoxaflor, under the optimized conditions exhibited similar times for lethality although the rate was faster with leaves on the cotton fabric than on water agar. These studies establish reproducible and sensitive techniques for assessing the lethality and effects on reproduction of potential aphicidal products.

Background: The successful production of superior or transgenic offspring from in vitro produced embryos in cattle relies heavily on the quality of blastocyst stage embryos. In order to enhance the developmental competency of these embryos, a novel culture method was devised. Methods: This study utilized stem cell culture medium (SCM) from hESCs as a supplement within the culture medium for bovine in vitro produced embryos. To gauge the efficacy of this approach, in vitro fertilized embryos were subjected to culture in CR1aa medium enriched with one of three supplements: 0.3% BSA, 10% FBS, or 10% SCM. Results: The blastocyst development and hatching rates of one-cell zygotes cultured in CR1aa medium supplemented with SCM (23.9% and 10.2%) surpassed those cultured in CR1aa medium supplemented with BSA (9.3% and 0.0%) or FBS (3.1% and 0.0%) (p < 0.05). Furthermore, post-zygotic gene activation, cleaved embryos cultured in CR1aa medium supplemented with SCM (57.8% and 34.5%) exhibited notably higher rates (p < 0.05) compared to those cultured with BSA (12.9% and 0.0%) or FBS (45.7% and 22.5%) supplementation. Furthermore, the microinjection of SCM into the cytoplasm or pronucleus of fertilized zygotes resulted in elevated blastocyst development and hatching rates, particularly when the microinjected embryos were subsequently cultured in CR1aa medium supplemented with SCM from the 8-cell embryo stage onwards (p < 0.05), in contrast to those cultured with FBS supplementation. Conclusions: In conclusion, this study conclusively demonstrated that the incorporation of SCM into the culture medium significantly enhances the developmental progress of preimplantation embryos.

Background: Pluripotent stem cells (PSCs) including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer the immense therapeutic potential in stem cell-based therapy of degenerative disorders. However, clinical trials of human ESCs cause heavy ethical concerns. With the derivation of iPSCs established by reprogramming from adult somatic cells through the transgenic expression of transcription factors, this problems would be able to overcome. In the present study, we tried to differentiate porcine iPSCs (piPSCs) into endothelial cells (ECs) for stem cell-based therapy of vascular diseases. Methods: piPSCs (OSKMNL) were induced to differentiation into ECs in four differentiation media (APEL-2, APEL-2 + 50 ng/mL of VEGF, EBM-2, EBM-2 + 50 ng/ mL of VEGF) on cultured plates coated with matrigel® (1:40 dilution with DMEM/F-12 medium) for 8 days. Differentiation efficiency of these cells were exanimated using qRT-PCR, Immunocytochemistry, Western blotting and FACS. Results: As results, expressions of pluripotency-associated markers (OCT-3/4, SOX2 and NANOG) were higher observed in all porcine differentiated cells derived from piPSCs (OSKMNL) cultured in four differentiation media than piPSCs as the control, whereas endothelial-associated marker (CD-31) in the differentiated cells was not expressed. Conclusions: It can be seen that piPSCs (OSKMNL) were not suitable to differentiate into ECs in the four differentiation media unlike porcine epiblast stem cells (pEpiSCs). Therefore, it would be required to establish a suitable PSCs for differentiating into ECs for the treatment of cardiovascular diseases.

진딧물 방제제 개발을 위해 In vitro 경엽살포 검정방법이 널리 사용되고 있다. 이러한 신소재 진딧물 방제 제형은 종합방제와 화학농약의 대안 으로 많은 연구가 진행되고 있다. 하지만, 경엽살포 검정방법은 환경이 조절되는 실내에서도 진딧물의 증식과 살충에 영향을 받는다. 본 연구에서는 담배를 기주로 하여 솜과 한천방법을 이용하여 진딧물 방제제 검정을 위한 최적 경엽살포 확립하고자 하였다. 진딧물 검정 챔버에 솜과 한천을 넣은 후 담배 잎과 진딧물 3-4령 약충을 접종하였다. Water-sensitive paper를 이용하여 경엽살포 시에 가장 표면 피복이 높은 최적 경엽살포 거리와 살 포량을 확립하였다. 대조구로 물을 처리한 구에서 한천 방법이 솜 방법에 비해 살충율이 낮고, 증식율이 높았다. 솜 검정 방법에는 곤충 검정 챔버의 상대습도를 60% 이상 유지시켰을 때 가장 최적 조건이었지만, 한천 검정 방법에서는 한천의 농도에 상대습도 차이가 없었다. 최적화된 조건하에서 대조화학 농약, Sulfoxaflor, 경엽살포 시 솜 방법에서 살충율이 한천방법보다 빨랐지만, 최종 살충율은 통계적으로 유의하지 않았다. 본 연구는 살 진딧물 물질을 검정 시 재현성과 활용성이 가능한 최적화된 증식율과 살충율 검정 조건을 제시하였다.

Background: Despite considerable technological advancements, polyspermy remains a significant challenge in in vitro fertilization (IVF) procedures in pigs, disrupting normal embryonic development. Here, we aimed to determine whether optimal fertilization conditions reduce the polyspermy incidence in pigs. Methods: In vitro -matured oocytes were co-incubated with sperm according to a modified two-step culture system. Results: In the first experiment, oocytes were briefly co-incubated with sperm, washed in IVF medium, and then moved to fresh IVF medium for 5 or 6 h. Although the 6 h sperm-free cultured group had a higher penetration rate than the 5 h cultured group, the polyspermy rate significantly increased in the 6 h sperm-free cultured group. The gamete co-incubation period was either 20 or 40 min. The 40 min cultured group had a higher rate of blastocyst formation and number of total cells in blastocysts than the 20 min cultured group. In experiment 2, oocytes were inseminated with sperm separated by Pecroll treatment. Percoll treatment increased the rate of oocyte penetration and blastocyst formation compared to the control. In experiment 3, fertilized oocytes were cultured in 25 μL microdroplets (10 gametes/drop) or 500 μL (100 gametes/well) of culture medium in 4-well plates. The large volume of medium significantly reduced the number of dead oocytes and increased the rate of blastocyst formation compared to the small volume. Conclusions: Collectively, these results demonstrate that various fertilization conditions, including modified co-culture period, active sperm separation, and culture medium volume, enhance fertilization efficiency and subsequent embryonic development by decreasing polyspermy occurrence.