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). Cumulusoocyte 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.

본 연구는 조사료 원료인 마늘 껍질의 사료적 가치와 메탄 발생량을 조사하기 위해 in vitro 발효 실험을 수행하였다. Garlic husk를 제외하고 국내에서 널리 사용되는 4가지 조사료 원료(Oat hay, Annual ryegrass, Timothy, Tall fescue)를 실험의 처리구로 구성하였다. In vitro 48 시간 발효 후 건물 소화율, 섬유소 소화율, 암모니아태 질소, 휘발성 지방산, pH, 메탄 발생량을 평가하였다. 가스 발생량은 3, 6, 9, 12, 24, 36 및 48 시간에 각각 측정되었다. 최종 가스 발생량에서 Oat hay가 유의적으로 가장 높았다(p<0.01). Garlic husk는 처리구 중 가장 높은 메탄 발생량을 나타내었다(p<0.01). Garlic husk의 건물 소화율은 Oat hay보다 유의적으로 낮았지만 Annual ryegrass, Timothy, Tall fescue보다 높았다(p<0.01). 섬유소 소화율은 Annual ryegrass에서 가장 낮았다(p<0.01). 총 휘발성 지방산 생성량은 Oat hay에서 가장 높았고(p<0.01), Garlic husk, Timothy, Tall fescue는 유사하였다. 본 연구의 in vitro 발효 성상과 메탄 발생량을 고려할 때, Garlic husk는 국내 주요 조사료를 대체할 영양적 가치를 포함하고 있으나 메탄 저감 효과는 미미할 것으로 추정된다.

체외 환경에서 생산되는 배아 (Embryo)는 활성산소종 (Reaction oxygen species, ROS) 수준이 일정 수준을 초과함에 따라 산화적인 손상을 받게 된다. 선행연구에 따르면, 항산화제는 ROS를 감소시켜주는 효과를 가지기 때문에 ROS로부터 오는 배아의 단백질, DNA의 손상, 세포 자멸사를 방지하여 배아의 발달률을 향상시킨다. 이전연구에 따르면 항산화제로써 엘라그산 (Ellagic acid, EA)은 ROS를 효과적으로 제거하고, 난자의 산화스트레스를 방지하는 효과를 가지고 있다고 보고되었다. 그리하여, 본 연구를 통해 우리는 소의 수정란 배양체계 중 in vitro culture (IVC) 단계에서 EA의 농도 (0, 5, 10 μM) 별 첨가가 소의 수정란 발달률과, 질적 수준에 미치는 영향을 조사하고자 실험을 진행하였다. 결과적으로, 배반포의 단계별 발달 수준에서 cleavage 형성률은 EA첨가군과 대조군 간의 차이를 발견할 수 없었으나 배반포 형성률에서는 모든 EA 첨가군들이 대조군보다 높았고 EA 첨가군 중에 5 μM 첨가군이 가장 높았다 (p < 0.05). 생산된 배반포의 총 세포 수는 5 μM EA 첨가군이 대조군과 10 μM EA 첨가군 보다 유의적으로 높았으며, 대조군과 10 μM EA 첨가군 사이의 유의적 차이는 없었다 (Control vs. 5 μM vs. 10 μM; 137 ± 7.90 vs. 163.2 ± 7.42 vs. 138.8 ± 6.67, p < 0.05). 세포 자멸사 세포 수는 모든 EA 첨가군들이 대조군보다 유의적으로 낮았다 (Control vs. 5 μM vs. 10 μM; 22.65 ± 4.08, 9.61 ± 1.55, 6.14 ± 0.90, p < 0.05). ROS 수준에서 모든 EA 첨가군들과 대조군 간의 유의적 차이는 없었다 (Control vs. 5 μM vs. 10 μM; 6.81 ± 1.31, 3.86 ± 0.23, 4.11 ± 0.18, p < 0.05). qRT-PCR 실험 결과에서 Nrf2 gene expression은 대조군과, 5 μM 첨가군에서 유의적 차이가 없었으나, 10 μM 첨가군에서는 유의적으로 상향 조절된 것을 관찰하였다. Keap1 gene expression은 5 μM 첨가군에서 유의적으로 하향 조절된 것을 관찰하였다. 하지만 EA의 농도가 10 μM으로 높아짐에 따라 발현 수준이 증가한 것을 관찰할 수 있었다. CAT gene expression은 5 μM 첨가군에서 유의적으로 상향조절 되었으나 10 μM 첨가군에서는 유의적인 차이를 보이지 않았다. SOD1 gene expression은 대조군과 5 μM 첨가군은 유의적인 차이를 보이지 않았으나 10 μM 첨가군에서는 유의적으로 상향 조절된 것을 관찰하였다.

Vitrification methods are commonly used for mammalian reproduction through the long-term storage of blastocyst produced in vitro. However, the survival and quality of embryos following vitrification are significantly low compared with blastocyst from in vitro production (IVP). This study evaluates that the survival of frozen-thawed bovine embryos was relevant to mitochondrial superoxide derived mitochondrial activity. Here we present supplementation of the cryopreservation medium with Mito- TEMPO (0.1 μM) induced a significant (p < 0.001; non-treated group: 56.8 ± 8.7%, reexpanded at 24 h vs Mito-TEMPO treated group: 77.5 ± 8.9%, re-expanded at 24 h) improvement in survival rate of cryopreserved-thawed bovine blastocyst. To confirm the quality of vitrified blastocyst after thawing, DNA fragmentation of survived embryos was examined by TUNEL assay. As a result, TUNEL positive cells rates of frozenthawed embryos were lower in the Mito-TEMPO treated group (4.2 ± 1.4%) than the non-treated group (7.1 ± 3.5%). In addition, we investigated the intracellular ROS and mitochondrial specific superoxide production using DCF-DA and Mito-SOX staining in survived bovine embryos following vitrification depending on Mito-TEMPO treatment. As expected, intracellular ROS levels and superoxide production of vitrified blastocysts after cryopreservation were significantly reduced (p < 0.05) according to Mito-TEMPO supplement in freezing medium. Also, mitochondrial activity measured by MitoTracker Orange staining increased in the frozen-thawed embryos with Mito-TEMPO compared with non-treated group. These results indicate that the treatment of Mito-TEMPO during cryopreservation might induce reduction in DNA fragmentation and apoptosis-related ROS production, consequently increasing mitochondrial activation for developmental capacity of frozen-thawed embryos.

We investigated the effect of a synthetic complement peptide C3a on the outcome of Brucella abortus 544 infection in a murine macrophage cell line RAW264.7 cell. First, we determined the highest non-cytotoxic concentration of the peptide in the cell line. We also found that the peptide significantly increased the growth of the bacteria at 8 and 24 h. Although the number of bacterial CFU was also elevated at 48 and 72 h, the increases were not significant as compared to controls. We further investigated the effect of C3a peptide on the growth of Brucella by pre-incubating the peptide at various temperatures and found that the effect was reversed at 24 h post-incubation suggesting that incubation of peptide at high temperatures including 65°C or 95°C could inactivate its action. This also could indicate the beneficial effect of high temperature during infection. Although several studies reported the inhibitory effect of different antimicrobial peptides including C3a, the present study preliminarily revealed that it had no positive contribution on the control of B. abortus 544 infection in vitro and indirectly to its receptor, CD88, which belongs to GPCR. Moreover, the encouraged further exploration of the effect of other similar peptides would be performed for the purpose of finding Brucella-host cell interaction for the control of disease progression.

In vitro maturation (IVM) of oocytes is the procedure where the immature oocytes are cultivated in a laboratory until they are mature. Since IVM oocytes generally have low developmental competence as compared to those matured in vivo, development of an optimal IVM culture system by fine-tuning culture conditions is crucial to maintain high quality. In-depth knowledge and a deep understanding of the in vivo physiology of oocyte maturation are pre-requisites to accomplish this. Within ovarian follicles, various signaling pathways that drive oocyte development and maturation regulate interaction between oocytes and surrounding somatic cells. This review discusses the sonic hedgehog (SHH) signaling pathway, which has been demonstrated to be intimately involved in folliculogenesis and oocyte maturation. Advances in elucidating the role of the SHH signaling pathway in oocyte maturation will aid attempts to improve the current inferior in vitro oocyte maturation system.

This in vitro study investigated the enhancement of rumen bacterial adhesion on a substrate to increase the digestibility of rice straw in Hanwoo cattle. The rice straw was pretreated with enzymes, probiotics, or ammonia, and the effects on the enhancement of bacterial adhesion and fiber degradation were analyzed using in vitro rumen fermentation. Enzyme treatment included spraying of cellulase and xylanase at 40 and 120 U per g of rice straw, respectively; Saccharomyces cerevisiae culture of 1.0×107 CFU was sprayed as a probiotic treatment per gram of rice straw; ammonia was sprayed at 0.3% per gram of rice straw. Following enzyme treatment, Fibrobacter succinogenes formed a higher adhesion colony than the control group (7.26±0.03 and 8.43±0.20) after 6h and 12h of in vitro culture (p<0.05), respectively. Attachment of Ruminococcus flavefaciens also increased following enzyme treatment (p<0.05) after 6 and 12 h compared to that of the control (5.18±0.06 and 6.60±0.15); and R. albus attachment showed a significant increase compared to that of the control (5.94±0.15) after 6 h of incubation (p<0.05). Probiotic treatment increased attachment of F. succinogenes in comparison with untreated rice straw after 6 h and 12 h of fermentation (p<0.05); R. flavefaciens attachment showed an increase only after 6 h of culture (p<0.05); R. albus was not affected. Attachment of F. succinogenes, R. flavefaciens, and R. albus increased with ammonia treatment after 6 h and 12 h (p<0.05). Dry matter digestibility was higher after the enzyme treatment (3.45±0.21 and 7.04±0.09) than in the control group(1.85±0.08 and 3.94±0.04) after 6 and 12 h of in vitro culture (p<0.05), respectively. It was also higher than that of untreated rice straw after probiotic and ammonia treatments (p<0.05). There was an increase in the enhancement levels of bacterial adhesion depending on the type of fibrolytic bacteria following enzyme, probiotic, and ammonia treatment. These treatments improved digestibility. This enhancement is considered to be greater following enzyme and ammonia treatments than with probiotics.

The objective of this study was to evaluate effects of feeding methods on in vitro ruminal fermentation, total gas and methane production in Hanwoo steers. Six Hanwoo steers fitted with rumen cannula (430 ± 21 kg of body weight) were randomly assigned to one of three feeding systems: 1) feeding forage 1 hour after concentrate, 2) feeding concentrate 1 hour after forage, 3) feeding mixed ration. Rumen fluid sampled from each animals was incubated 24 hours with maize or timothy substrates in in vitro. Ruminal pH was increased in feeding method 2 or maize substrate than that of other methods or timothy substrate (P < 0.001). The production of total volatile fatty acid, acetate, propionate, butyrate, and valerate were increased when steers fed diets using feeding method 1 or rumen fluid was incubated with maize substrate (P < 0.001). Increased production of total gas and methane was observed in feeding method 1 and maize substrate compared to those of other methods or timothy substrate (P < 0.001). Due to the inconsistent results between ruminal fermentation and gas production in this study, further research is required to estimate effects of feeding method on enteric fermentation and gas production in in vivo.

In this study, the effect of forage sources in the total mixed ration (TMR) on in vitro goat rumen fermentation was investigated. Rice straw (RS), Italian ryegrass (IRG), timothy (TIM), and alfalfa (ALF) were used as forage sources. Each forage source was mixed with a commercial goat concentrate diet in the ratio of 1:1. Total 4 TMR were prepared. Rumen simulated in vitro fermentation using goat rumen fluid collected from the slaughterhouse was conducted until 72 th . For fermentation parameters, gas production (GP), volatile fatty acids (VFAs), and ammonia nitrogen (NH3-N) were examined. All assays were performed at 24 th , 48 th , and 72 th h of incubation individually. Contents of crude protein and non-fibrous carbohydrate were greater in the order of RS < IRG < TIM < ALF. Significant treatment effects were found in valerate and NH3-N at 24 th h of incubation (p<0.05). ALF showed the greatest contents of them and RS was the lowest. At 48 th incubation, a significant effect was detected at GP (p<0.05) and RS was greater than others. However, GP of RS was lower than others at 72 th . Significant effects on Total VFA, butyrate, and valerate productions were found at 72 th h of incubation (p<0.05). ALF showed the greatest production. Methane production from all treatments was not significantly different for each incubation time (p>0.05). The present study provided primary information on how goat rumen fermentation responds to different nutrient contents and forage sources of TMR. And the information could be used for the design or optimizing economical diet formulation for goats.

The purpose of this study was to introduce a new in vitro method for evaluating the antimicrobial activity of toothpaste, reflecting the actual toothbrushing time and the dilution of toothpaste by salivation. We designed three experimental groups and one negative control group. The experimental groups were (1) 90 μL of toothpaste + 10 μL 1X phosphate-buffered saline (PBS, 9/10 dilution group), (2) 50 μL of toothpaste + 40 μL 1X PBS (1/2 dilution group), and (3) 25 μL of toothpaste + 65 μL 1X PBS (1/4 dilution group). During toothbrushing, saliva is continuously secreted into the oral cavity and the toothpaste concentration is diluted over time during toothbrushing. Therefore, the 1/2 and 1/4 dilution experimental groups were added. The negative control group was toothpaste diluted 20,000-fold with 1X PBS. Miracle Fresh Doctor toothpaste and Streptococcus mitis KCOM 1350, Prevotella intermedia KCOM 1107, Fusobacterium nucleatum subsp. polymorphum KCOM 1322, and Aggregatibacter actinomycetemcomitans KCOM 1306 were used as the toothpaste and target bacterial strains, respectively. The number of bacterial cells plated on agar plates in the negative control group was 1,000 CFU. If the number of colonies on the experimental group plate was less than one, the treatment was considered to have > 99.9% bactericidal activity. These results suggest that this new in vitro method for antimicrobial evaluation could be used as the standard method for testing the antimicrobial activity of toothpaste.

The present study evaluated the influence of rumen inocula of different breeds on in vitro fermentation with forage and concentrate substrates. An in vitro was conducted under a 2×2 factorial arrangement with two breeds (Jersey and Holstein steers) and two feed substrates (forage and concentrate) as factors. Three Jersey and 3 Holstein steers were used for the source of in vitro inocula. Metataxonomic analysis of donor rumen fluids showed that Firmicutes was more abundant in Jersey, while Bacteroidetes in Holstein steers. In vitro ㏗ was lower in the fermented inocula of Jersey steers and in the concentrate substrate (p<0.05). After 24h, higher gas production, dry matter, and neutral detergent fiber degradability, and total volatile fatty acids concentration were noted in concentrate substrate (p<0.05). After 24h, inocula of Jersey steers had higher methane and ammonia-nitrogen (p<0.05). After 24h, fermented inocula of Holstein steers produced higher propionate (p<0.05). Conversely, in vitro butyrate production was higher in the fermented inocula from Jersey steers (p=0.072) and in those with concentrate substrate (p<0.05). After 24h, the total bacterial population (log10 c opies) was h igher in t he fermented inocula received from Jersey steers and in the concentrate substrate whereas, Fibrobacter succinogenes and Ruminococcus flavefaciens population were higher (p<0.05) only in the concentrate substrate. Overall results suggest that rumen inocula of different donors influence in vitro fermentation either with forage or concentrate substrates.

This study was conducted to evaluate the effect of rumen origin lactate-utilizing bacteria (LUB) as one of the potential treatments on subacute ruminal acidosis (SARA) during in vitro challenge compared to buffering agents (NaHCO3, sea minerals, MgO) and direct-fed microorganism (yeast). We hypothesized that rumen LUB (RLUB) could be a potential treatment to treat ruminal acidosis. The supplementation level of other treatments was determined by referring to previous studies in the literature. The 108 CFU/g freeze-dried RLUB isolated from Hanwoo cattle were compared with 0.1% NaHCO3, 0.8% of MgO, 0.5% sea mineral and 0.4% yeast during in vitro SARA challenge. Rumen fluid collected from one cannulated Holstein and one Hanwoo steer fed by maize silage was mixed with 0.5g feed consisted of 0.05g forage and 0.45g concentrate. These mixtures were incubated in triplication for 3, 6, 12 and 24h. After 6h of incubation, along with MgO and sea minerals, RLUB treatment showed higher (p<0.05) ㏗ values than control with no significant differences in total volatile fatty acid concentration. However, in the same period, the propionate concentration and A:P ratio were higher in RLUB than in the other treatment (p<0.05), which might alter the fermentation pattern. On the other hand, the RLUB treatment produced a higher (p<0.05) ammonia-N concentration. Based on these results, we can conclude that RLUB might have the potential to alleviate SARA. Nonetheless, further study on its mechanism in SARA is required, especially with live animals.