The in vitro experiment was conducted to ensure the supplemental level of spent Flammulina velutipes mushroomsubstrates (SMS) as an energy source in manufacturing of whole crop sorghum silage. Sorghum harvested at heading stage wasensiled with spent mushroom substrates of 20% (S-20), 40% (S-40) and 60% (S-60) as fresh matter basis for 6 week. Theexperiment was conducted by 3, 6, 9, 12, 24, 48 hrs of incubation time with 3 replications. The silages were evaluatedfermentation characteristics and dry matter digestibility (DMD) in vitro. The pH of in vitro solution was inclined to decrease withelapsing the incubation time, and that of the S-20 was significantly (P<0.05) lower than the other treatment at 48 hr ofincubation. Gas production was greater (P<0.05) in the S-20 than the other treatments at 6 and 12 hrs of incubation. Themicrobial growth in vitro was inclined to decrease following 24 hr of incubation, and thereafter sustained the similar levels. Invitro dry matter digestibility (IVDMD) was lowered by increasing the supplemental level of spent mushroom substrate, and was alow level in the S-60 throughout whole incubation time. Althoughthe IVDMD for S-40 was steadily increased from 9 hr ofincubation and reached to similar level with the S-20 at 48 hourof incubation, however SMS for whole crop sorghum silagefermentation might as well add about 20 to 30% in fresh matterbasis when considering DMD.

In this study, we investigated and analyzed the registration, sales and regulatory management system of in vitro diagnostic veterinary medical reagents (IVDVMRs) in Korea. The registration of IVDVMRs has gradually increased since 2000, and total of 233 products from 58 companies were registered from 1975 to 2014. The market size of IVDVMRs is estimated to be approximately 12 billion Won per year from 2011 to 2013: the export sales and proportion was estimated to be 36.8% as 4.4 billion Won in 2013. Of these products, the ranking of the sales were canine heartworm, bovine tuberculosis, swine fever, porcine reproductive and respiratory syndrome, canine distemper+adenovirus+parvovirus disease, foot and mouth disease, etc. In vitro diagnostic human medical reagents were diverted biological medicine from the medical devices by the revision of the Pharmaceutical Affairs Law Enforcement Regulations in 2014 in Korea. In contrast, in vitro diagnostic devices for animal were still managed as medical devices and biological medicines, respectively. The diagnostic reagents for infectious diseases have neither classification nor grade systems. Good manufacturing practices (GMP) requirements on IVDVMRs were also exempted from the current system. This study suggested that the registration of the IVDVMRs has increased since 2005, and regulations of these devices should be improved for the effective operating system.

Many pronuclear stage eggs were used to generate transgenic mice (Tg) by microinjection. In this study, we used in vitro fertilized mouse eggs, followed by ultrarapid freezing to establish a simple procedure for production of Tg mice. We produced in vitro fertilized mouse eggs and cryopreserved them by ultrarapid freezing method. A total of 139 cryopreserved-thawed pronuclear eggs, of which 101 (72.6%) were survived following microinjection of chicken b-actin promoter-driven firefly improved luciferase cDNA (β-act/luc+) and were transferred into 5 recipients. All recipients became pregnant and gave birth to a total of 15 (14.8%) pups. As a control, same DNA construction (β- act/luc+) was also injected into 450 in vitro fertilized eggs, of which 338 (75.1%) were survived and then were transferred into 14 recipients. Eleven (78%) mice became pregnant and littered a total of 54 (19.1%) pups. Southern blotting analysis of Tg mice indicated that one (1/15, 6.6%) and three (3/54, 5.5%) transgenic mice were production from cryopreserved and in vitro fertilized eggs, respectively. All Tg mice produced from both eggs showed the expression of improved luciferase gene. These results indicated that efficiency of produced of Tg mice from cryopreserved eggs was comparable to that from in vitro fertilized eggs. Furthermore, it is suggested that microinjection of transgene into in vitro fertilized eggs cryopreserved by ultrarapid freezing is an easy and conveniently method for production of Tg mice.

국내 육성 프리지아(Freesia)가 농가에 보급되고 시간이 지나면서 바이러스에 감염되거나 품질이 저하되는 문제가 발생하고 있다. 고품질을 유지하기 위해서는 생장점 배양을 통해 무병종구를 만들고 기내에서 급속 대량증식하여 농가에 보급하는 일이 시급하다. 본 연구에서는국내에서 육성된 프리지아 ‘Shiny Gold’와 ‘Gold Rich’의자구를 이용하여 효과적인 급속 대량증식 기술을 찾고자실험을 수행하였다. 배지는 MS기본배지에 호르몬을 첨가하지 않은 배지와 5.0mg•L−1 NAA+5.0mg•L−1 Kinetin를 넣은 호르몬배지 2종류를 이용하였다. 치상방법은 자구를 횡으로 절단한 절편체를 정상으로 치상(정상치상)한경우와 위아래를 거꾸로 치상(역위치상)하는 2가지 방법을 이용하였다. 치상한 절편체는 23oC의 암배양실에서 5주간 배양한 후, 광도 60µmol•m−2s−1 (16/8, 주/야)의 광환경으로 옮겨 주었다. 치상 10주째에 두 품종 모두 역위치상하였을 때, 정상치상에 비해 부정아 발생이 2~6배 정도 증가하였다. 이러한 결과는 호르몬 유무에 상관없이 비슷한 결과를 보였다. 역위치상하고 19주 후 ‘ShinyGold’의 신초수는 기본배지에서 절편체당 42.7개로 정상치상에 비해 약 2.5배 증가되었고, 호르몬 첨가배지에서는 17.7개로 정상치상에 비해 약 7.1배 증가되었다. ‘GoldRich’를 역위치상 하였을 때, 기본배지에서는 신초수가 절편체당 55.4개로 대조구에 비해 약 1.7배 증가되었고, 호르몬 첨가배지에서는 61.0개로 대조구에 비해 약 2.4배증가되었다. 결론적으로 프리지아 자구를 기내에서 급속대량증식 하고자 할 때, 호르몬 유무와 상관없이 자구를횡으로 절단하여 역위치상 하는 방법이 효율적이라는 것을 알 수 있었다.

These studies were conducted to establish the practical Hanwoo (Korean native cattle) improvement system through the combining of embryo transfer technology and confirming individual Hanwoo oocyte culture system and to investigate that correlation of Hanwoo carcass classification (intramuscular marbling) and oocyte donor for blastocyst production in vitro. In case of Hanwoo, the carcass meat quality grades were divided to grade 1⁺⁺, 1⁺, 1, 2, and 3 depends on fat distribution of longest muscle cross-sectional surface. As results, the numbers of follicular oocytes collected from individual fundamentally-registered Hanwoo yielded 1⁺⁺, 1⁺, 1, 2 and 3 meat quality were 9.5, 9.4, 8.5, 8.8 and 8.8 per ovary, respectively. The numbers of retrieval oocyte from follicles were significantly higher in the cattle yield 1⁺⁺, 1⁺ meat quality than in the cattle yield 1, 2 and 3 meat quality (p<0.05). The rates of blastocyst formation were 18.2, 21.3, 29.4, 30.9, and 31.5% in the cattle yield 1⁺⁺, 1⁺, 1, 2 and 3 meat quality of after in vitro maturation, respectively. It was significantly lower in the cattle yield 1⁺⁺ and 1⁺ meat quality than in the cattle yield 1, 2 and 3 meat quality (p<0.05). In order to evaluate embryos quality, TUNNEL assay was conducted for each meat quality grade using blastocyst stage embryo on day 8. The results showed that apoptosis cell number was higher tendency in the cattle yield 1⁺⁺and 1⁺ meat quality (81 and 79, respectively) than in the cattle yield 1, 2 and 3 meat quality (51, 48 and 50, respectively) but there was no statistical significance in each group. After embryo transfer, the conception rate of recipient was 53.5 (23 out of 43), 52.1 (38 out of 73) and 58.0% (58 out of 100) in the meat quality of 1, 1+ and 1++, respectively. These results showed that the conception rate was significantly higher in the cattle yield 1 meat quality than in the cattle yield 1⁺⁺, 1⁺ , 2, and 3 meat quality (p<0.05). In summary, these results indicate that the application of confirming Hanwoo individual oocyte culture system and embryo transfer technology can make good use of the genetic resources conservation and improvement of Hanwoo. Relevance of the meat quality grade and reproductive ability of carcasses of Hanwoo will be considered to be one of the effective means for the associated research with obesity and reproduction.

체외 배양액에 성장호르몬 및 사이토카인의 첨가는 초기배 발육 및 생산된 배반포의 질에 영향을 미칠 수 있다. 본 연구는 돼지 유도만능줄기세포(porcine induced pluripotent stem cell, piPSC)의 조정배지(conditioned medium, CM)가 돼지 난자의 체외성숙 및 단위발생 후 초기배 발육에 미치는 영향을 검토하기 위하여 수행하였다. 난자-난구세포 복합체(cumulus-oocyte complex, COC)는 0(control), 25, or 50%의 줄기세포 배양액(stem cell medium, SM) 또는 CM이 첨가된 체외성숙 배양액으로 배양하였으며, 성숙된 난자는 활성화 유도 후 같은 농도의 SM 또는 CM을 첨가한 체외배양액에서 배양하였다. 체외 성숙율은 CM-25% 그룹에서 대조구보다 유의적으로 높았으나(p<0.05), 다른 SM 또는 CM 처리구와는 차이가 없었다. 배반포 형성율은 CM-25% 그룹(29.2%)에서 대조구(20.7%), SM-50%(19.6%) 및 CM-50%(23.66%) 처리구보다 유의적으로 높았다(p<0.05). 배반포에서의 세포수 및 세포사 비율은 SM-25% 그룹이 대조구에 비하여 유의적인 차이가 나타났다(p<0.05). 난자의 질과 연관되어 있는 유전자들(Oct4, Klf4, Tert 및 Zfp42)의 발현은 CM-25% 그룹에서 대조구보다 유의적으로 증가되었다(p<0.05). 따라서 본 실험의 결과 체외성숙(IVM) 및 체외발달(IVC) 배양액에 25% 수준의 CM의 첨가는 돼지 단위발생 난자의 배발달과 난자의 질적 향상에 기여하는 것으로 사료된다.

This study was conducted to verify the effects of Erigeron annuus powder on serum lipid levels of high-fat diet-induced mice from a nutritional viewpoint. Erigeron annuus powder has been used as a folk remedy since ancient times in Korea. There was no significant difference in the weight of the kidneys and spleens of the mice. The high-fat diet group had a significantly higher kidney weight compared to other groups (p<0.05). In the group of mice fed 20% Erigeron annuus powder with a high-fat diet, the concentration of serum LDL-cholesterol was high (p<0.05), whereas the concentration of triglyceride was remarkably lower compared to other groups (p<0.05). The group fed 10% Erigeron annuus with a high-fat diet had the lowest concentration of blood phospholipids (p<0.05) as well as the highest alkaline phosphatase and alanine aminotransferase levels in blood (p<0.05). There was no difference in blood insulin concentration. However, blood leptin concentration was significantly higher (5.88±3.53 ng/dL) in mice fed a high-fat diet compared to other groups (p<0.05). Measurements of Erigeron annuus revealed that TPC, ABTS+ radical scavenging activity of trolox, DPPH radical scavenging activity, and the measured value of FRAP were higher in the ethanol extract than in the water extract. Especially, the antioxidant activity effects were excellent for the ABTS+ radical scavenging activities of trolox and FRAP values of Erigeron annuus. Therefore, Erigeron annuus powder showed antioxidant activity. Hence, Erigeron annuus powder drastically lessened triglyceride concentration in blood in high-fat diet-induced mice. Thus, the powder is considered to have utility in the food processing industry. Additional related experiments are ongoing.

대반하[大半夏: Pinellia tripartita(Blume) Schott]는 천남성과에 속하는 초본성 식물로써, 중요 한약재인 반하[半夏: Pinellia ternata(Thunb.) Makino]의 국내 자생 동속 종으로 활용가치가 높은 식물이나 반하에 비해 생리학 및 유전학 등 관련 연구가 거의 진행되지 않았다. 본 연구는 대반하의 소괴경 형성 및 분화에 적합한 최적 배지와 식물생장조절물질의 농도를 규명하고, 유전적인 안전성 및 토양환경 순화조건을 검증하여 향후 반하의 대체 약재로 활용시 이용가능한 배양묘 대량생산조건을 확립하기 위하여 수행되었다. 자생지에서 채집한 대반하는 소독 후 MS 기본배지에서 배양하여 기내식물체를 확보하였고, 소괴경 분화와 비대 최적배지 규명을 위해서 10종류의 다양한 식물배지에서 6주간 배양 한 결과, Nitsch & Nitsch(NN) 배지에서 가장 효과적이었다. 기내 배양 시 발생한 캘러스를 이용하여 식물체 최적 재분화 조건을 규명하기 위하여, 명/암 반응과 BA 호르몬의 다양한 농도별(0.5ppm-3.0ppm) 처리를 통해서 4주 동안 배양한 결과, 명상태에서 BA 1.0 ppm이 첨가된 배지에서 가장 효과가 높았다. 최적 기내배양 및 재분화 개체는 2주 후 토양으로 이식하여 환경적응을 유도한 후 배양묘 대량 생산조건을 설정하였다. 기내배양 및 재분화를 통해 생산된 배양묘의 유전적 안전성을 검증하기 위해서 각 증식 단계별 식물조직 및 캘러스 시료의 RAPD genomic profile을 분석한 결과, 원 식물체와 각 단계별 개체의 유전적 안전성이 증명되었다.

Pigs are considered an ideal source of human disease model due to their physiological similarities to humans. However, the low efficiency of in vitro embryo production (IVP) is still a major barrier in the production of pig offspring with gene manipulation. Despite ongoing advances in the associated technologies, the developmental capacity of IVP pig embryos is still lower than that of their in vivo counterparts, as well as IVP embryos of other species (e.g., cattle and mice). The efficiency of IVP can be influenced by many factors that affect various critical steps in the process. The previous relevant reviews have focused on the in vitro maturation system, in vitro culture conditions, in vitro fertilization medium, issues with polyspermy, the utilized technologies, etc. In this review, we concentrate on factors that have not been fully detailed in prior reviews, such as the oocyte morphology, oocyte recovery methods, denuding procedures, first polar body morphology and embryo quality.

Terfenadine (TFN) was a second generation histamine receptor antagonist. Although several studies have reported the regulatory effect of H1-histamine receptor antagonists in human cancer cell lines, its effect in oral cancer remains unclear. In this study, we focused on addressing the anti-cancer activity of TFN in human oral cancer cell lines. The anti-cancer activities of TFN were performed by tryphan blue exclusion assay, 4'-6-diamidino-2-phenylindole (DAPI) staining, live/dead assay and Western blot analysis. TFN induced a significant reduction of the growth in three different human oral cancer cell lines (MC3, HSC4 and Ca9.22). TFN markedly induced apoptosis through DNA damage and increase in cytotoxicity. It also accumulated cleaved PARP and caspase 3. This process was due to cleavage of caspase 8 and Bid protein. The results from this study strongly demonstrated that the cleavages of caspase 8 and Bid are required for the apoptotic activity of TFN in human oral cancer cells. Taken together, these findings suggest TFN as a potent anticancer drug candidate for the treatment of oral cancer.

본 연구는 long-chain fatty acids 첨가 시 발효 48 h 에 in vitro 반추위 발효와 메탄 생성에 미치는 영향을 구명하고자 수행하였다. 반추위액은 외과적 방법으로 누관이 장착된 홀스타인에서 채취하였으며, 공시축의 사양관리는 조사료와 농후사료 6:4 비율로 하였다. 60 mL serum bottle 에 buffer 10 mL, 티모시 0.3 g, 위액 5 mL 를 각각 넣고 fatty acids(stearic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid ; 0.001%/15 mL)를 첨가한 뒤 발효시간대별(3, 6, 12, 24, 36 및 48 h)로 5 처리 3 반복 수행하였다. 발효종료시간인 48 h 에서 pH 는 oleic acid 와 linoleic acid 는 대조구에 비해 유의적(p<0.05)으로 높게 측정되었다. 48 h 에 총가스 발생량은 stearic acid 가 유의적(p<0.05)으로 높게 측정되었고 메탄 발생량은 arachidonic acid 에서 유의적(p<0.05)으로 높게 측정되었다. 건물 소화율은 발효 36 h 에서 linoleic acid 가 다른 처리구에 비해 높았으나, 발효 48 h 에서는 처리구간 유의차가 없었다. 휘발성 지방산 농도와 butyric acid 는 발효 3 h, 12 h 에서 long-chain fatty acid 처리구가 대조구에 비해서 유의적(p<0.05)으로 높게 나타났다. 3 h, 12 h 에서 acetic acid는 linoleic acid, linolenic acid 가 다른 처리구보다 유의적(p<0.05)으로 높게 나타났고, propionic acid 는 stearic acid, oleic acid 에서 다른 처리구에 비해 유의적(p<0.05)으로 높게 측정되었다. Protozoa 의 생존 수와 사멸 수는 처리구간의 차이를 나타내지 않았다. Bacteria 는 linolenic acid 와 arachidonic acid 가 다른 처리구에 비해 유의적(p<0.05)으로 높았다. Fungi 는 oleic acid 가 linoleic acid 를 제외한 나머지 처리구에서 유의적(p<0.05)으로 높게 측정되었다. 본 연구의 결과에 의해 발효 48 h 에 0.001%의 oleic acid 와 linoleic acid 의 첨가는 메탄 발생량을 감소시키는데 효과적이라는 것을 알 수 있었다.

The PHBV nonofibrous membrane fabricated by electron-spinning method for tissue engineered bone regeneration scaffold was evaulated in terms of cellular prolieration and cryopreservation efficiency. The rat calvarial periosteum derived primary cells were cultured with PHBV nanofibrous membranes and analyzed the cellular proliferation and differention fashion and cryopreservation potential by in vitro MTT assay as well as ALP staining and Alizarine red staining with or without cryopreservation for 2 weeks. The rat calvarial periosteum derived primary cells cultured with PHBV nonofibrous membrane showed favorable proliferation and alkaline phosphatase activity with numerous mineral nodule formation regardless of cryopreservation, even though its efficiency was slightly decreased in cryopreserved condition. These findings suggest that PHBV nanofibrous membrane can be applicable as an efficient cell engineered membrane for guided bone regeneration or scaffold for tissue engineered bone regeneration.

The objective of this study was to investigate the efficiency of nicotinic acid on sperm cryosurvival and fertilization ability in frozen-thawed boar semen. Boar semen was collected by glove-hand method and was frozen using freezing solution treated to 0, 5, 10 and 20 mM of nicotinic acid. The frozen sperm for sperm characteristic analysis was thawed such as viability, acrosome reaction, and mitochondrial integrity. The frozen-thawed sperm was estimated by SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction and Rhodamine123/PI double staining for mitochondrial integrity using a flow cytometry. The embryo was estimated in vitro development and DCFDA staining for reactive oxygen species assessment. As results, frozen-thawed sperm viability was significantly higher in 5 and 10 mM (61.1 ± 1.5%, 64.7 ± 2.0%) of nicotinic acid than other groups (0 mM, 52.1 ± 2.3%; 20 mM, 47.8 ± 5.1%, P<0.05). The live sperm with acrosome reaction was significantly higher in 5 and 10 mM of nicotinic acid (26.1 ± 1.8%, 24.9 ± 1.5%) than other groups (0 mM, 35.3 ± 0.8%; 20 mM, 36.5 ± 1.9%, P<0.05). The live sperm with mitochondrial integrity was significantly higher in 5 and 10 mM (84.2 ± 3.6%, 88.4 ± 2.3%) of nicotinic acid than other groups (0 mM, 77.3 ± 4.4%; 20 mM, 73.3 ± 3.6%, P<0.05). Blastocyst rate of in vitro development was significantly higher in 10 mM (17.0 ± 1.3%) of nicotinic acid than other groups (0 mM, 9.4 ± 0.5%; 5mM, 12.6 ± 0.8%; 20 mM, 5.0 ± 1.0%, P<0.05). Moreover, total cell number was higher in 5 and 10 mM (53.6 ± 2.9%, 57.9 ± 2.8%) of nicotinic acid than other groups (0 mM, 41.0 ± 1.4%; 20 mM, 23.2 ± 2.8%, P<0.05). Hydrogen peroxide in embryos was lower in 5 mM nicotinic acid (0.7 ± 0.1%) than other groups (0 mM, 1.0 ± 0.1%; 10mM, 0.9 ± 0.0%; 20 mM, 1.4 ± 1.0%, P<0.05). In conclusion, nicotinic acid-treated semen improves cryosurvival and quality of spermatozoa. Also, the fertilized oocytes with nicotinic acid improve quality of embryo and blastocyst formation.

Russula rosacea, a mycorrhizal fungus, has been used for edible and medicinal purposes. This study was conducted to evaluate the in vitro antioxidant, anti-hyperglycemic, anti-cholinesterase, and nitric oxide inhibitory effects of the fruiting bodies from R. rosacea extracted with methanol, and hot water. The 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activities of the methanol and hot water extracts (2.0 mg/ml) of R. rosacea were comparable with BHT, the positive control. The chelating effects of the mushroom and hot water extracts were significantly higher than that of BHT. The reducing power of methanol and hot water extract (6 mg/ml) were significantly lower than that of BHT. Seven phenolic compounds were detected from acetonitrile and hydrochloric acid solvent extract of the mushroom. alpha-amylase and alpha-glucosidase inhibitory activities of methanol and hot water extracts were lower than that of acarbose, the positive control. The acetylcholinesterase and butyrylcholinesterase inhibitory effects were moderate compared with galanthamine, the standard drug. Nitric oxide (NO) production in lipopolysaccharide (LPS) induced RAW 264.7 cells were inhibited significantly by the mushroom extracts in a concentration dependent manner. Therefore, we demonstrated that fruiting bodies of R. rosacea possess in vitro antioxidant, anti-hyperglycemic, anti-cholinesterase, and NO production inhibitory activities. The experimental results suggest that the fruiting bodies of R. rosacea are good natural antioxidant, anti-hyperglycemic, anti-cholinesterase, and anti-inflammatory sources.

To investigate the antioxidative potential of ramie (Boehmeria nivea L.) leaves, alcoholic extracts with different ethanol concentration were prepared. The extract obtained by using 70% ethanol possessed the highest total phenolic content. The extraction yield and total phenolic content of the ethanolic extract were 15.46% and 105.0 μg chlorogenic acid equivalent (CAE)/mg, respectively. Therefore, the antioxidant activities of the extract were evaluated as 2,2diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, reducing power and nitrite scavenging ability. EC 50 value for radical scavenging and nitrite scavenging activities, which is the effective concentration at which 50% of DPPH radicals and nitrites are scavenged, were 34.72 μg/mL and 52.99 μg/mL, respectively. EC 50 value for reducing power, which is the effective concentration at which the absorbance is 0.5, is 44.39 μg/mL. All antioxidant activities increased as extract concentration increased. These results imply that the ethanolic extract of ramie leaves has the potential to be utilized as an effective antioxidant source.

Extracts from Aloe vera leaves, Aloe arborescens leaves, Aloe vera callus, Portulaca oleracea and cacao (Theobroma cacao L.) bean husk (CBH) were prepared using acetone, chloroform, ethanol, hexane, and water. Solvent extracts of Aloe vera leaf had very high antioxidant activities showing IC50 values in the ranges of 0.02-0.17 mg/ mL, and had the highest total phenolic and flavonoid content among the tested samples. We hypothesized that Aloe vera leaf and CBH extracts might possess considerable in vitro anti-glycation activities. Indeed, these extracts strongly inhibited the formation of advanced glycation end-products from RNase in the presence of ribose. The chloroform extract of Aloe vera leaf showed the strongest inhibition of AGE formation (99.9%), followed by the 95% acetone extract (92.8%) at a concentration of 1 mg/mL, exhibiting higher anti-glycation activities than those of AG and rutin (73.4% and 96.1% at 1 mg/mL, respectively). The anti-glycation activity of all extracts was correlated positively with their total contents of phenolics and flavonoids. We conclude that Aloe vera leaf extracts and their constituents may be used as anti-glycation agents.

Phasianus colchicus is not only a beautiful bird but also a great value in science and under the threat of endanger. Hence, the aim of this study was to isolate the pheasant male germ cells (mGCs) and then induce them into elongated sperm-like cells in vitro. The mGCs were purified and enriched by a two-step plating method based on the different adherence velocities of mGCs and somatic cells. The percentage of the c-kit positive cells and c-kit negative cells examined by flow cytometry analysis (FCA) was 92.87% and 2.57%, respectively. Subsequently, the mGCs were induced for 48h in DMEM/F12 medium supplemented factors such as retinol acid, testosterone and bovine FSH, followed by 5 weeks in culture. We found that some elongated sperm-like cells appeared initially in vitro under inducement of stimulated factors. The elongated sperm-like cells showed in the expression of changed morphology and post-transcriptional marker such as spermatid associated (SPERT), spermatid perinuclear RNA binding protein (STRBP), round spermatid basic protein 1 (RSBN1) and SPER1L. Moreover, in DNA content identified assay, induced cells showed that the 1C DNA population markedly increased in differentiated group but it was not change in undifferentiated group. Successful in vitro differentiation of pheasant testicular germline cells into spermatids appears to offer extremely attractive potential for the conservation of endangered birds and treatment of male infertility.

Nutritive value analysis of feed is very important for the growth of livestock, and ensures the efficiency of feeds as well as economic status. However, general laboratory analyses require considerable time and high cost. Near-infrared reflectance spectroscopy (NIRS) is a spectroscopic technique used to analyze the nutritive values of seeds. It is very effective and less costly than the conventional method. The sample used in this study was a corn kernel and the partial least square regression method was used for evaluating nutrient composition, digestibility, and energy value based on the calibration equation. The evaluation methods employed were the coefficient of determination (R2) and the root mean squared error of prediction (RMSEP). The results showed the moisture content (R2val=0.97, RMSEP=0.109), crude protein content (R2val=0.94, RMSEP=0.212), neutral detergent fiber content (R2val=0.96, RMSEP=0.763), acid detergent fiber content (R2val=0.96, RMSEP=0.142), gross energy (R2val=0.82, RMSEP=23.249), in vitro dry matter digestibility (R2val=0.68, RMSEP=1.69), and metabolizable energy (approximately R2val >0.80). This study confirmed that the nutritive components of corn kernels can be predicted using near-infrared reflectance spectroscopy.

Differentiated nuclei can experimentally be returned to an undifferentiated embryonic status after nuclear transfer (NT) to unfertilized metaphase II (MII) oocytes. Nuclear reprogramming is triggered immediately after somatic cell nucleus transfer (SCNT) into recipient cytoplasm and this period is regarded as a key stage for optimizing reprogramming. In a recent study (Dai et al., 2010), use of m-carboxycinnamic acid bishydroxamide (CBHA) as a histone deacetylase inhibitor during the in vitro early culture of murine cloned embryos modifies the acetylation status of somatic nuclei and increases the developmental competence of SCNT embryos. Thus, we examined the effects of CBHA treatment on the in vitro preimplantation development of porcine SCNT embryos and on the acetylated status of histone H3K9 on cloned embryos at the zygote stage. We performed the three groups SCNT: SCNT (NT), CBHA treatment at the porcine fetus fibroblast cells (PFFs) used as donor cells prior to SCNT (CBHA-C) and CBHA treatment at the porcine SCNT embryos during the in vitro early culture after oocyte activation (CBHA-Z). The PFFs were treated with a 15 μM of CBHA (8 h) for the early culture and the porcine cloned embryos were treated with a 100 μM concentration of CBHA during the in vitro early culture (10 h). Cleavage rates and development to the blastocyst stage were assessed. No significant difference was observed the cleavage rate among the groups (82.6%, 76.4% and 82.2%, respectively). However, the development competence to the blastocyst stage was significantly increased in CBHA-Z embryos (22.7%) as compared to SCNT and CBHA-C embryos (8.6% and 4.1%)(p<0.05). Total cell numbers and viable cell numbers at the blastocyst stage of porcine SCNT embryos were increased in CBHA-Z embryos as compared to those in CBHA-C embryos (p<0.05). Signal level of histone acetylation (H3K9ac) at the zygote stage of SCNT was increased in CBHA-Z embryos as compared to SCNT and CBHA-C embryos. The results of the present study suggested that treatment with CBHA during the in vitro early culture (10 h) had significantly increased the developmental competence and histone acetylation level at the zygote stage.