• Asian-Australasian Journal of Animal Sciences
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• 제32권6호
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• pp.776-782
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• 2019

Objective: Fasting may lead to changes in the microbiota and activity in the rumen. In the present study, the effects of fasting on rumen microbiota and the impact of fasting on in vitro rumen fermentation were evaluated using molecular culture-independent methods. Methods: Three ruminally cannulated Holstein steers were fed rice straw and concentrates. The ruminal fluids were obtained from the same steers 2 h after the morning feeding (control) and 24 h after fasting (fasting). The ruminal fluid was filtrated through four layers of muslin, collected for a culture-independent microbial analysis, and used to determine the in vitro rumen fermentation characteristics. Total DNA was extracted from both control and fasting ruminal fluids. The rumen microbiota was assessed using denaturing gradient gel electrophoresis (DGGE) and quantitative polymerase chain reaction. Microbial activity was evaluated in control and fasting steers at various intervals using in vitro batch culture with rice straw and concentrate at a ratio of 60:40. Results: Fasting for 24 h slightly affected the microbiota structure in the rumen as determined by DGGE. Additionally, several microorganisms, including Anaerovibrio lipolytica, Eubacterium ruminantium, Prevotella albensis, Prevotella ruminicola, and Ruminobacter amylophilus, decreased in number after fasting. In addition, using the ruminal fluid as the inoculum after 24 h of fasting, the fermentation characteristics differed from those obtained using non-fasted ruminal fluid. Compared with the control, the fasting showed higher total gas production, ammonia, and microbial protein production (p<0.05). No significant differences, however, was observed in pH and dry matter digestibility. Conclusion: When in vitro techniques are used to evaluate feed, the use of the ruminal fluid from fasted animals should be used with caution.

• 환경생물
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• 제18권2호
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• pp.255-262
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• 2000

Synaptosome에 의한 [$^3$H]-serotonin의 일반적인 흡수특성과 이 과정에 납이 미치는 영향을 in vitro와 in vitro에서 관찰하였다. 흰쥐의 대뇌와 뇌간에서 각각 분리한 synaptosome의 흡수친화력은 대뇌가 Km=0.5$\mu$M, 뇌간이 Km=0.1$\mu$M로 모두 고친화성 흡수였고 뇌간에서 더 높았다. 또한 이 흡수과정에 sodium과 potassium이온이 영향을 미치는 것으로 나타났다. Synaptosome이 [$^3$H]-serotonin을 흡수하는 과정은 납에 의해 억제되었고 이러한 납의 독성영향은 in vitro와 in vitro에서 유사한 결과를 보였다.

• Asian-Australasian Journal of Animal Sciences
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• 제15권6호
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• pp.851-858
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• 2002

This study was conducted to determine effects of whole (intact), coarsely-ground (4 mm), finely-ground (1 mm), steam-flaked and steam-flaked-ground (1 mm) corns on in vitro and in situ DM digestibilities and also in vitro fermentation characteristics. After 48 h incubation, in vitro dry matter digestibilities of whole, steam-flaked, coarsely-ground, steam-flaked-ground, and finely-ground corns were 6.79, 61.68, 76.48, 85.72 and 90.31%, respectively. Steam-flaked-ground corn showed the highest digestibility until 24 h incubation (p<0.01). After 48 h incubation, pH of whole corn decreased with a small range. However the values of pH of other media significantly decreased (p<0.01). The gas productions of finely-ground and steam-flaked-ground corns were higher than those of the other corns (p<0.01). After 24 h incubation, $NH_3$-N concentration of finely-ground and steam-flaked-ground corns increased rapidly. Total VFA was the highest in finely-ground corn, followed by steam-flaked-ground, steam-flaked, coarsely-ground and whole corns. Incorporating steam-flaked corn resulted in the highest propionate concentration (p<0.01) and the lowest acetate : propionate value (p<0.05). Finely-ground corn showed the highest in situ DM digestibility throughout the incubation period (p<0.01), followed by coarsely-ground, steam-flaked and whole corns, respectively. Overall, DM of whole corn was merely digested in vitro as well as in situ.

• 한국동물생명공학회지
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• 제37권1호
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• pp.34-41
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• 2022

In vivo oocytes grow and mature in ovarian follicles whereas oocytes are matured in vitro in plastic culture dishes with a hard surface. In vivo oocytes show a superior developmental ability to in vitro counterparts, indicating suboptimal environments of in vitro culture. This study aimed to evaluate the influence of an agarose matrix as a culture substrate during in vitro maturation (IVM) on the development of pig oocytes derived from small antral follicles (SAFs). Cumulus-oocyte complexes (COCs) retrieved from SAFs were grown in a plastic culture dish without an agarose matrix and then cultured for maturation in a plastic dish coated without (control) or with a 1% or 2% (w/v) agarose hydrogel. Then, the effect of the soft agarose matrix on oocyte maturation and embryonic development was assessed by analyzing intra-oocyte contents of glutathione (GSH) and reactive oxygen species (ROS), expression of VEGFA, HIF1A, and PFKP genes, and blastocyst formation after parthenogenesis. IVM of pig COCs on a 1% (w/v) agarose matrix showed a significantly higher blastocyst formation, intra-oocyte GSH contents, and transcript abundance of VEGFA. Moreover, a significantly lower intra-oocyte ROS content was detected in oocytes matured on the 1% and 2% (w/v) agarose matrices than in control. Our results demonstrated that IVM of SAFs-derived pig oocytes on a soft agarose matrix enhanced developmental ability by improving the cytoplasmic maturation of oocytes through redox balancing and regulation of gene expression.

• 농업과학연구
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• 제48권1호
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• pp.73-81
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• 2021

The current study investigated how Saccharomyces cerevisiae ameliorates the adverse effects of aflatoxin on in vitro rumen fermentation. In this study, five groups (T₁: Control [basal feed]; T₂: T₁ + 300 ppb aflatoxin B1 [AFB₁] and T₃, T₄, and T₅: T₂ with 0.05, 0.1, and 0.2% of S. cerevisiae, respectively) were prepared and incubated in vitro. The results revealed that truly degradable dry matter (TDDM), gas production (GP), microbial biomass production (MBP), truly degradable organic matter (TDOM), partitioning factor (PF), total volatile fatty acids (TVFA), acetate (A), propionate (P) and butyrate (B) values in the control group (T₁) were higher (p < 0.05) than those of the AFB₁ fed group (T₂). The A : P ratio in the control group (T₁) was reduced (p < 0.05) when compared to that of the T₂ group. The TDDM, TDOM, GP, TVFA, A, P, and B values of T₃, T₄, and T₅ improved with the increasing levels of S. cerevisiae; however, the values of group T₅ were lower (p < 0.05) than that of the control. The values of MBP, A : P ratio and PF in group T₅ were statistically similar to that of the control. It was concluded that the inclusion of S. cerevisiae (0.05 to 0.20%) to the AFB₁ (300 ppb) contaminated feed partially to completely ameliorated the adverse effects of AFB₁ on the in vitro rumen fermentation parameters.

• Reproductive and Developmental Biology
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• 제35권1호
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• pp.85-91
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• 2011

The objective of this study was to evaluated the efficiency on sperm cryosurvival and ability of in vitro fertilization using Triladyl and Lactose Egg-Yolk(LEY) as extenders for cryopreservation of separated sperm by 65% percoll in miniature pig. Sperm viability was measured with SYBR-14/PI double stained sperm by flow cytometry. Ability on embryo cleavage rate and blastocyst development were observed by in vitro fertilization after frozen-thawing of sperm separated by 65% percoll. The experimental groups were designed that separated sperm by 65% percoll with Triladyl (ST) or LEY(SL) and unseparated sperm with Triladyl(UT) or LEY(UL) for cryopreservation. As a results, the viability was significantly(p<0.05) higher in ST(55.1%), SL(63.1%), UL(58.8%) than UT(38.2%) group. Sperm viability in SL(63.1%) group was significantly(p<0.05) higher than other experimental groups. On the other hand, embryo cleavage rate was significantly(p<0.05) higher in ST(79.1%), SL(83.2) than UT(74.1) and UL(75.7%) groups at 96h after in vitro fertilization. Blastocyst development was also significantly(p<0.05) higher in ST(21.5%), SL(20.9%) than UT(17.0%) and UL(18.8%) groups. In conclusion, cryopreservation of miniature boar sperm separated by 65% percoll were beneficial to viability and capacity on in vitro fertilization.

• Asian-Australasian Journal of Animal Sciences
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• 제14권6호
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• pp.754-758
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• 2001

Bovine oocytes with compact and complete cumulus cells were cultured for up to 24h in TCM199 buffered with 25mmol/l hepes and supplemented with 10% FBS (fetal bovine serum), 1mg/ml $17{\beta}$-estradiol, 20IU/ml hCG(human chorionic gonadotropin). All of the oocytes were divided into at 6 groups depending upon incubation times (control, 0 hour, 6 hours, 12 hours, 16 hours, 18 hours). To all experimental media, $200{\mu}g/ml$ puromycin was added at different incubation times mentioned above. Following these culture times, in vitro insemination was conducted with frozen-thawed bovine spermatozoa in medium BO (Brackett and Oliphant medium for in vitro insemination) with $10{\mu}g/ml$ BSA(bovine serum albumin) and 10 mg/ml heparin added. After 22h culture, the oocytes were fixed with acetic alcohol solution and stained with orcein acetic solution to evaluate sperm nuclear progression. Addition of puromycin after 0, 6 and 12 h of culture resulted in near of oocyte maturation at the M1 stage. Contrariwise, puromycin addition after 12 h of culture led to restoration of nuclear progression to M2 stage. On the one hand, puromycin affected the synthesis of Cyclin B protein that may be involved in the oocyte maturation and sperm capacitation for in vitro fertilization. The present study suggests the participation of protein synthesis, cyclin B, in the oocyte development from M1 to M2 stages in vitro.

• 한방안이비인후피부과학회지
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• 제19권1호
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• pp.55-64
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• 2006

Objective : The purpose of this study was to investigate effect of Gungguitakli-San(GTS) on the anti-tumor, immunocytes. Methods : This study estimated the proliferation of L1210 and S-180 cell lines, mouse splenocytes and thymocytes in vitro, and estimated the proliferation of L1210 cell, S-180 cell, thymocytes and splenocytes and body weight in S-180 cells-transplanted mice. The cytotoxicity and proliferation of cells were tested using a colorimetric tetrazoliun assay(M1T assay). Results : The results of this study were obtained as follow ; 1. GTS was significantly increased in the proliferation of thymocytes and splenocytes In vitro. 2. GTS was significantly showed cytotoxicity on the L1210 cell lines and 8-180 cell lines in vitro. 3. GTS was significantly showed cytotoxicity on the L1210 cell lines in vivo. 4. GTS was significantly increased in the weight of mice and decreased weight of sarcoma, in S-180 cells transplanted mice. 5. GTS was significantly increased in the period of survive, in S-180 cells transplanted mice. Conclusions : The author thought that GTS had action of anti-cancer by becoming immunocytes activity and by cytotoxicity of cancer cells.

• Reproductive and Developmental Biology
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• 제29권2호
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• pp.133-139
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• 2005

Oxidative stress is one of the major causes of failure in in vitro storage of boar semen. Reactive oxygen species (ROS) are known to be important mediators of such stress. The present study examined the effects of pyruvate and taurine on sperm motility and expression of BAD, Cytochrome c, Caspase-3 and Cox-2 protein in in vitro storage of boar semen, and tested the effect of semen treated with antioxidant with or without hydrogen peroxide on the development of IVM/IVF porcine embryos. Semen samples were transported to the laboratory at $17^{\circ}C$ within 2 hr after collection and were treated with different concentration of pyruvate $(1\~10mM)$ and taurine $(25\~100mM)$ with or without 250uM $H_2O_2$ respectively. The supplementation of pyruvate and taurine increased sperm motility in boar semen during in vitro incubation at $37^{\circ}C$. Expression of apoptosis protein (BAD, cytochrome c, caspase-3 and cox-2) were reduced in the group of boar semen treated with pyruvate and taurine when compared to the other groups. The developmental rates of IVM/IVF porcine embryos fertilized by semen treated with pyruvate and taurine were significantly increased when compared to control (P<0.005). These results indicate that supplementation of pyruvate and taurine as antioxidants in boar semen extender can improve the semen quality and increase in vitro development of porcine IVM/IVF embryos when boar semen treated with antioxidants was used for in vitro fertilization.

• Reproductive and Developmental Biology
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• 제37권1호
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• pp.51-55
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• 2013

The present study was conducted to examine the effect of antioxidant treatment during parthenogenetic activation procedure on the reactive oxygen species (ROS) levels and in vitro development of porcine parthenogenetic embryos. Porcine in vitro matured oocytes were activated by a combination of electric stimulus and 2 mM 6-dimethylaminopurine (6-DAMP) before in vitro culture. During the activation period, oocytes were treated with $50{\mu}M$ ${\beta}$-mercaptoethanol (${\beta}$-ME), $100{\mu}M$ L-ascorbic acid (Vit. C) or $100{\mu}M$ L-glutathione (GSH). To examine the ROS level, porcine parthenogenetic embryos were stained in $10{\mu}M$ dichlorohydrofluorescein diacetate ($H_2DCFDA$) dye 20 h after culture, examined under a fluorescence microscope, and the fluorescence intensity (pixels) were analyzed in each embryo. The parthenogenetic embryos were cultured for 6 days to evaluate the in vitro development. The apoptosis was measured by TUNEL assay. The $H_2O_2$ levels of parthenogenetic embryos were significantly lower in antioxidant treatment groups ($26.9{\pm}1.6{\sim}29.1{\pm}1.3$ pixels/embryo, p<0.05) compared to control ($33.2{\pm}1.7$ pixels/embryo). The development rate to the blastocyst stage was increased in antioxidant treatment groups (32.0~32.5%) compared to control (26.9%, p<0.05), although, there was no difference in apoptosis among groups. The result suggests that antioxidant treatment during parthenogenetic activation procedure can inhibit the ROS generation and enhance the in vitro development of porcine parthenogenetic embryos.