Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis (CLA), a chronic contagious disease in small ruminants. The prevalence of CLA has been reported to be >50% in Korean black goats. CLA is difficult to control due to a lack of efficient vaccines and treatment methods. Effective disinfection of the farm environment may be an alternative strategy for reducing the spread of C. pseudotuberculosis. The objective of this study was to evaluate the efficacy of commercial disinfectants against CLA. The six commercial disinfectants, largely composed of sodium dichloroisocyanurate, sodium hypochlorite, potassium monopersulfate triple salt, quaternary ammonium, citric acid, and copper sulfate, were tested against five different genotypes of C. pseudotuberculosis isolated from goat farms in Korea. Efficacy tests were performed in accordance with the disinfectant efficacy test guidelines recommended by the Animal and Plant Quarantine Agency of Korea with slight modifications. All disinfectants except for copper sulfate exhibited >99.99% killing efficacy under hard water conditions following 30 min of incubation, which is the recommended standard treatment time according to guidelines. The minimum bactericidal treatment time was evaluated by employing treatments for durations of 1, 5, and 15 min. The most effective compounds under hard water conditions were sodium dichloroisocyanurate, potassium monopersulfate triple salt, and sodium hypochlorite, exhibiting >99.99% killing efficacy after 1 min of treatment. In the aqueous solution forms, citric acid and the quaternary ammonium compound were the most effective, but required at least 5 min to kill >99.99% of the bacteria. The current study characterizes the killing efficacy of six commercial disinfectant active compounds against C. pseudotuberculosis. Thus, this study provides essential information regarding the efficacy of the disinfectants used to control CLA in goat farms.

The acrosome cap allows sperm to penetrate the egg membrane and produce male pronuclei within female chicken eggs, facilitating successful fertilization. Given this, it is important to establish practical methods for evaluating the integrity of the acrosome cap and thus the quality of the rooster’s sperm. There are several established methods for evaluating the acrosomes of mammalian sperm, but none of these methods are suitable for evaluating the acrosome status of rooster spermatozoa. Therefore, a simplified method for evaluating the rooster acrosome is needed. Here we evaluated the usefulness of CBB (coomassie brilliant blue) staining of the acrosome at concentrations of 0.04%, 0.08%, and 0.3% CBB solutions. Our data revealed a clear staining pattern for intact acrosome caps at 0.04% and 0.08% CBB but not at 0.3% CBB. This protocol revealed differences in acrosome integrity between fresh and frozen rooster sperm smears suggesting that CBB staining may facilitate easier semen evaluation in roosters. This protocol allows for the accurate differential staining of acrosome cap in rooster spermatozoa.

난자의 성숙과정과 노화에 관한 이해는 인공수정과 체외수정 최적기를 판단하기 위하여 가장 중요한 연구내용으로 알려져 있다. 이러한 기작은 번식 호르몬들에 의하여 조절되는 것으로 알려져 있으나 난자 세포질 변화에 관한 내용은 잘 알려져 있지 않다. 본 연구에서는 산화질소물(nitric oxide, NO)이 난자 성숙과정에서 증가하는 것을 밝혔으며 난자의 미성숙단계(germinal vesicle stage, GV)와 난자핵막붕괴단계(germinal vesicle breakdown, GVBD) 및 성숙완료단계(metaphase II, MII)단계에서 생산되는 NO의 양을 비교하였다. 또한, 난자를 체외에서 배양할 때, MII단계로 성숙되지 않는 성장 단계의 난자에서는 NO의 증가 현상을 관찰할 수 없었고, 세포질이 불균일한 노화된 난자에서는 NO가 증가된 상태로 유지되는 특성이 있음을 밝혔다. 이러한 결과는 NO의 작용이 난자의 성숙과정과 난자 노화과정에서 중요한 기능을 담당하고 있음을 보여주고 있다.

본 연구는 엘크 암사슴(Cervus canadensis)의 사육방식에 따른 사료섭취량, 체중변화 및 자록의 성장에 미치는 영향을 확인하고 적정 방목강도를 구명하기 위하여 수행되었다. 본 실험에 사용 된 공시가축은 3~4년생 엘크 암사슴 16두(평균체중 : 236.2 ± 15.7 kg)를 이용하였으며, 방목초지는 3년 이상된 톨 페스큐 위주의 기성 혼파초지로서 초종구성은 톨 페스큐(약 50%), 오차드 그라스(약 10%), 켄터기 블루그라스(약 5%)와 피, 바랭이 등으로 구성되었다. 방목초지의 수분함량은 19.51∼22.61%로 방목기간 동안 유사하였으며, 조단백질 함량은 6~7월에 유의적으로 높게 나타났다. 조지방과 조회분 함량은 가을로 갈수록 점차 증가하였으며, NDF와 ADF함량은 각각 53.65∼60.18%과 26.08∼29.10% 로 조사되었다. 실험기간동안 보충사료 섭취량은 월별로 처리구 사이에 유의적인 차이가 나타나지 않았으나, 조사료와 총 건물섭 취량은 5월을 제외하고 처리구 사이에 유의적인 차이가 나타났다 (p<0.05). 특히 8월에는 GR처리구의 조사료 섭취량이 급격히 감소하였는데, 여름철 환경변화에 따른 것으로 사료된다. 반면, 9월 포유를 위해 부족한 영양소를 섭취하기 위해 사초섭취량이 급격히 증가하였다. 우리 연구에서 암사슴의 채식패턴은 수사슴과 차이를 보였으며, 이는 실험기간 자록의 이유 및 포유기간에 따른 영향을 받은 것으로 사료된다. 본 실험에서 사양방식에 따른 엘크 암사슴의 체중은 유의적 차이가 나타나지 않았으나, GR처리구에서 자록의 성장률이 빠른 것으로 나타났다. 그러나, GR처리구에서 BF처리구에 비해 낮은 이유율이 나타나 분만 전후 사사사육을 통해 이유율을 높여주는 것이 필요할 것으로 판단되며, 목지 내에서 분만 시 포유경험이 있는 산차가 높은 암사슴을 이용하고 방목강도를 높여주는 것이 이유율을 향상시키는 데 도움을 줄 수 있을 것으로 사료된다. 엘 크 암사슴의 방목강도는 연평균 15두/ha로 나타났는데, 이는 다마 암사슴(Dama dama)에 비해서는 체중대비 높게 나타났으나, 엘크 수사슴에 비해서는 낮은 수치를 보여주었다. 이러한 결과는 기성초지 이용으로 인해 사초의 생산성이 감소한 결과로 사료되며, 앞으로 방목초지의 혼파방법과 기성초지의 비배관리 등을 통해 초지 활용성을 높일 수 있는 연구가 추가적으로 필요할 것으로 사료된다.

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.

Mesenchymal stem cells (MSCs) are capable of differentiating into mesenchymal tissue such as bone, cartilage, muscle, and adipose, and have been isolated and characterized from various species. Deer adipose tissue-derived MSCs (dAD-MSCs) have not been studied and deer bone marrow-derived MSCs (dBM-MSCs) have not been fully characterized. In this study, we firstly isolated MSCs from deer tissues and then compared characteristics of dAD-MSCs and dBM-MSCs. dAD-MSCs and dBM-MSCs exhibited significant increase in proliferation under low-glucose DMEM culture condition during 20 and 10 passages consecutive passages, respectively. Both cells expressed cell surface markers such as CD73, CD90, and CD105, but did not express CD34 and CD45. Two types of cells expressed stemness markers (Oct4, Sox2, and Nanog) and exhibited differentiation potential into mesodermal lineages. Both cells exhibited osteogenic and chondrogenic differentiation potential, but poor adipogenic differentiation potential. Specifically, dAD-MSCs have a greater capacity for chondrogenic differentiation potential compared to dBM-MSCs. Collectively, we successfully isolated dAD-MSCs from deer for the first time. This study suggests that adipose tissue of deer could be used as a source of deer MSCs.

The present study was to assess the in vivo embryo production efficiency using the semen separated according to sex during superovulation in Hanwoo. Seventy Hanwoo donor cows were flushed on day 7 of estrus cycle with same FSH and artificial insemination by the same technicians. Embryos were recovered on 7 days after the third insemination by flushing the uterus with embryo collection medium. KPN semen straws used artificial insemination contained 20 million sperm (total number 60 million per donor). Sex-sorted semen straws contained 4 million sperm (total number 12 million per donor). The results obtained were as follows: No differences were observed in the efficiency of superovulation rates on KPN semen 87%, and sexed semen 100%, respectively. The mean numbers of total embryos are each 12.58 ± 8.31 and 13.25 ± 7.86. The mean numbers of transferable embryos, sexed semen were significantly lower than KPN semen (3.75 ± 1.98 vs. 8.23 ± 6.07, P<0.05). The rates of unfertilized embryos from superovulation using sexed semen were significantly higher than KPN semen (50% vs. 15%, P<0.05). The rate of degenerated 2-cell embryos from sexed and KPN semen was 60.87% and 11.11%, respectively (p<0.05). In conclusion, these results indicate that superovulation using sexed semen was useful, but efficient embryo production was important to reducing the damage caused by the Flowcytometer-based sperm sorting procedure.

To preserve genetic materials, cryopreservation of the semen from live animals is the main technique to establish cryo-banking system which could be used for artificial insemination and embryo transfer. However, the population of Korean black goat (KBG) becomes to dwindle in number and is now faced genetic erosion by crossbreeding with non-native breeds in small KBG farms. In this study, simple freezing method was used to preserve frozen semen from KBG using spermatozoa of cauda epididymis (CE) and electro-stimulated semen (ES). The negative effects of seminal plasma on fresh sperm was confirmed using precipitation test of Triladyl egg yolk diluent and sperm viability after thawing was compared between CE and ES spermatozoa. When seminal plasma of fresh ES semen was washed with semen washing media (SWM), the rates of live sperm shown no significant difference between CE and ES spermatozoa before freezing. However, the survival rate of frozen/thawed CE sperm was higher than ES (74.6±10.6% vs 53.8±5.2%) with significant difference (p < 0.05). The results of longevity test on frozen/thawed sperm from CE showed healthier sperm than ES. Therefore, spermatozoa from CE could be used for cryo-banking system in KBG lines. The more studies are needed to increase survival rate of ES semen.

To increase the productivity of in vitro development, the antioxidants have been used for culture system of bovine oocytes and embryos. However, comparative studies on these molecules are rare and direct beneficial effects on blastocyst production cannot be discriminated for best results. The study was conducted to determine the influence of N-acetyl-L-cysteine (NAC), N-acetyl-L-cysteine amide (NACA), glutathione (GSH) and cysteamime (CYS) on maturation competence of COCs from GV to MII stage and productivity of blastocyst formation during in vitro fertilization and culture. There was no difference among maturation rates of oocytes to metaphase II with polar body with antioxidants for any of the treatment groups (p>0.05). However, the significant improvement on the rate of blastocysts (32.3±5.0%) was found in 0.1 mM CYS treatment than 0.3 mM NAC, 0.2 mM NACA or 0.5mM GSH (p<0.05). The addition of NAC (18.8±3.7%) or NACA (21.2±3.9%) did not improve development competence to morula and blastocysts than control (24.4±4.1%) and GSH (26.5±5.0%) (p>0.05). Our study showed that medium supplementation with CYS during IVM and IVC improved the rate of bovine embryo development but not with NAC, NACA and GSH addition.

The elevated temperature and high humidity has been known as main reason for heat stress on animals and cause detrimental effects on productivity of organisms and physiological conditions of normal bioactivities. The aims of this study were to evaluate the relationship between time of heat shock simulation during in vitro maturation and developmental competence of subsequent embryo after in vitro fertilization. Heat shocked cumulus-oocyte complexes (COCs) of Korean native cattle were subjected to normal conditions for 22, 21, 18 and 12 h respectively and transferred to heat stress inducing condition at 40.5 °C in other incubator for 0 (control), 1 and 4 h. After maturation for 22 h, the oocytes were fertilized and cultured in mSOF media for 8 d and examined the developmental capacity of embryos. There were no differences in maturation and cleavage rates between 0, 1 and 4 h heat socked oocytes, but blastocysts formation were lower in the 4 h heat stressed oocytes. The apoptotic cells of developed blastocysts were also increased in at day 8 with 4 h heat shocked oocytes. These results indicate that heat shock on oocytes during maturation could cause negative effects on the developmental competence of embryos.

Cryopreservation of germ cells from genetically proven animals could be a source of restoration tools from the risk of extinction or disappearance of wanted characteristics. Using frozen semen, the genetic gains of Korean native cattle have been increased greatly for 70 years. The preservation of genetic resources as a form of frozen semen straw has limited availability due to the numbers. To circumvent this weakness of frozen semen, we tested two re-freezing methods with different initial thawing temperatures using frozen Korean proven semen and rare breed semen from albino, black and chikso breeders. It has been known that human sperm could resist to cryo-damages by repeated freeze-thaw cycles, but not for Korean proven bulls number (KPN) or for rare breeds. Total 7 frozen semem from brindled(2), black(1), Korean Albino(2) and KPN(1) bulls were used for our research. After thawing straws under 5°C/2min or 37°C/40sec with low temperature water bath and thermo jug, spermatozoa were re-diluted with triladyl diluents after first thawing and re-frozen. Sperm motilities were compared between animals and treated groups after re-thawing. Mean values of motility and viability of refrozen/thawed sperm for expansion of the number of straws were significantly higher in 5°C than in 37°C (P< < 0.05). However, the activity of viable sperm thawed at 5°C was significantly decreased after first and second thawing. It is estimated that re-freezing of frozen semen from rare Korean native cattle is possible with resistant properties of survived spermatozoa.

Multiple interferon tau (IFNT) genes exist in bovine. An antiluteolytic substance secreted by the bovine conceptus and primarily responsible for maternal recognition of pregnancy is bovine trophoblast protein 1 (bIFNT1), a new type I interferon tau (IFNT) genes. The objectives of this research were to investigate whether multiple, distinct gene encode bIFNT1 and other type I bIFNT gene in the bovine genome and to examine expression of bIFNT1 and other bIFNTc1 mRNAs during conceptus development. These transcrips could be regulated through caudalrelatedhomeobox-2 (CDX2) and ETS2 and/or AP1 (JUN) expression, a transcription factor implicated in the control of cell differentiation in the trophectoderm. The presence of mRNAs encoded by bIFNT1 and type I bIFNTc1 genes were examined quantitatively via reverse transcription-polymerase chain reaction (RT-PCR) analysis of total cellular RNA (tcRNA) extracted from on day 17, 20 and 22 bovine conceptuses. The expression level of bIFNT1 was higher on day 17 transcripts were gradually weakly detectable on day 20 and 22. However, the other bIFNTc1 gene examined transcripts was highly expressed on day 20 and transcripts were weakly detectable on day 17 and 22 bovine conceptuses. Furthermore, human choriocarcinoma JEG3 was co-transfected with an -1kb-bIFNT1/c1-Luc constructs and several transcription factor expression plasmids. Compared to each -1kb-bIFNT1/c1-Luc increased when this constructs were co-transfected with, ETS2, AP1(JUN), CREBBP and/or CDX2. Also, bIFNTc1 gene was had very effect on activity by alone ETS2, and AP1 (JUN) expression factors in choriocarcinoma JEG3 cell. However, bIFNT1 gene expression of the upstream region was not identified. We demonstrated that the activities of bIFN genes are regulated by differential, tissue-specific and developmental competence during pregnancy.

The periods of elevated temperature and high humidity has been longer since last ten years and cause problems in program of artificial insemination or at the efficiency of in vitro production of transferable embryos. The aims of this study were to evaluate the relationship between time of heat shock (0, 1, 2 and 4), during in vitro maturation and developmental competence of subsequent embryo after in vitro fertilization. The develpmentat rate and percetage of apoptotic cells were evaluated on matured oocyte and day 8. 41℃ Heat treatment after IVM culture significantly decreased the developmental capacity of IVF embryos. Also the number of apoptotic cell in COCs, morula and blatostcysts was started to increase at 2 hr heat treatment but did not affect on the rate of maturation. These results indicate that heat treatment for 2 to 4 hr at 41℃ have negative effects on maturation rate of COCs and lower the developmental competence of heat shocked oocyte derived embryos.

During the oocyte maturation, antioxidants may be beneficial for futher developmental competence against reactive oxygen species (ROS) because the media for oocytes lack boiomolecules that serve as scavengers. In this study, N-acetyl-L-cysteine (NAC), N-acetyl-L-cysteine amide (NACA), glutathione (GSH) and cysteamime were compared to determine the effects of protection for ROS from GV to MII stage when supplemented during in vitro maturation (IVM) and in vitro culture (IVC) of bovine oocytes. NAC is one of well known ROS scavanger and NACA is modified form of NAC to help permeation into cytosolic area of oocytes. Significant improvement on the development undergoing blastocysts (32%, vs 18%, 22%) were found when cysteamine (0.1mM) was added to the maturation medium than NAC (0.3 mM), NACA (0.2mM) or GSH (0.5 mM) as compared to control medium with antioxydents. However, the addition of NAC(18%) or NACA(21%) to media did not improve the proportion of oocytes undergoing development to morula and blastocysts than control (24%) and GSH (26%). Our study showed that medium supplementation with cysteame during IVM and in vitro culture (IVC) improved the rate of bovine embryo development, in contrast to extracellular antioxidants like NAC, NACA and GSH that caused no improvement.

Historically, Korea old cattle had been consisted with various lines of coat color brindle, black and white-brown breeds or more. The two rare lines of black and white coat color are maintained for animal resources and preserved critically. The present study was carried out to evaluate potential usage of cysteamine supplementation during in vitro matration (IVM) and in vitro culture/production of embryo (IVP) by transvaginal ultrasound-guided follicle aspiration (Ovum Pick-Up: OPU) for the establishment of cryo-banking system. Immature slaughterhouse-derived cumulus-oocyte complexes (SL-COCs) were matured in IVM medium supplemented with 0, 0.1, 0.3 or 0.9 mM cysteamine, and then cultured in mSOF-BAS for 8 days after in vitro fertilization. The treatment of 0.1 mM cysteamine on SL-COCs showed higher rate of blastocyst, so OPU-derived COCs from rare breeds were matured in TCM media supplemented with or without 0.1 mM cysteamine, FSH and 5% FBS. The embryos were evaluated their developmental stages on day 8. During IVM, cysteamine treatment significantly increased the embryo production rate of slaughterhouse-derived COCs (19.6% vs. 30.5%). The presence of cysteamine during IVM of OPU-derived COCs from rare Korean cattle breeds (albino white and black line) also increased embryo production rates than those from SL-COCs (27.4% vs. 41.9% and 36.4%). With these results, cysteamine treatment during IVM is one of key factors IVP of blastocysts to establish banking system of endangered rare Koarean cattle with OPU derived transferable blastocysts.

During the ovary preservation in low temperature, the cumulus oocyte complexes(COCs) lose their developmental competences after in vitro fertilization. We used phosphate-buffered saline (PBS) as a basic solutions of at various temperatures (25, 15 or 5 ℃) and supplemented them with 1mM glucose and 0.5mM glutamine as a source of carbohydrate metabolites. After recovery of COCs and in vitro fertilization, a significantly higher number of oocytes developed into blastocysts. The developmental competence of embryos that were originated from ovaries preserved at 15 ℃ was increased compared to those of 25 or 5 ℃. The maturation rate of oocytes was not differed between 24 and 36 h at 15 ℃ but showed lower than control group (71% versus 78%). In vitro-fertilized oocytes from ovaries stored at 25 ℃ for 24 h or at 5 ℃ for 24 h had a significantly decreased developmental potentials, but at 15 ℃ did not (27% versus 29% of blastocysts to develop into day 8). With these results, bovine ovaries can be preserved at 15 ℃ for 36 h without decreasing developmental capacity of in vitro-fertilized oocyte at least to the blastocyst stage. This information provides valuable information of preserving ovaries for embryo transfer or in vitro embryo production.

In the field of reproductive medicine, assessment of sperm motility is a key factor for achieving successful artificial insemination, in vitro fertilization, or intracellular sperm injection. In this study, the motility of boar sperms was estimated using real-time imaging via confocal microscopy. To confirm this confocal imaging method, flagellar beats and whiplash- like movement angles were compared between fresh and low-temperature-preserved (17℃ for 24 h) porcine sperms. Low-temperature preservation reduced the number of flagellar beats from 11.0±2.3 beats/s (fresh sperm) to 5.7±1.8 beats/s and increased the flagellar bending angle from 19.8°±13.8° (fresh) to 30.6°±15.6°. These data suggest that sperm activity can be assessed using confocal microscopy. The observed motility patterns could be used to develop a sperm evaluation index and automated confocal microscopic sperm motility analysis techniques.