한우(韓牛, Korean Cattle, Bos taurus coreanae)는 모색에 따라 황소, 칡소, 흑소로 나 누어지고 있다. 칡소는 현재 1,700여두가 전국에 사육되고 있는 것으로 추정하고 있다. 그러나 종모우로 활용 가능한 수소의 숫자가 매우 적어 근친의 위험도가 높다. 본 연구 에서는 우량 칡소를 선발하기 위하여 성장단계별 체형을 측정하고, 12개월 및 30개월 령 에 신선정액과 동결융해 정액의 활력을 비교하였다. 칡소의 발육단계별 체형은 체고, 흉위, 십자부고, 체장, 흉심, 흉폭, 고장, 요각폭, 곤폭, 좌골폭을 각각 6개월, 12개월, 18개월, 24개월 및 36개월 이상으로 구분하여 측정하였다. 정액 채취는 인공질을 이용하여 채취하였고, 정액동결용 완충액은 Triladyl를 이용하였다. 칡소 수소의 성장단계별 체형을 조사한 결과, 12개월령 흉위, 체고, 십자부고, 체장, 흉 심, 흉폭, 고장, 요각폭, 곤폭 및 좌골폭이 각각 평균 156.0 cm, 113.2 cm, 118.6 cm, 122.6 cm, 59.5 cm, 31.7 cm, 43.0 cm, 34.7 cm, 36.6 cm 및 19.8 cm였고, 30개월령에 는 각각 평균 214.8 cm, 140.5 cm, 140.5 cm, 179.3 cm, 83.2 cm, 48.6 cm, 62.3 cm, 53.9 cm, 51.0 cm 및 34.7 cm로 조사되었다. 칡소 육성우의 모색을 조사한 결과, 전체 호반 무늬를 가진 개체는 9.6%, 부분 호반 무늬는 59.0%, 호반 무늬 없이 갈색인 개체는 20.5% 그리고 흑색인 개체는 10.8%였다. 15개월령 칡소와 30개월령 칡소의 신선 및 동 결융해 정액의 활력을 조사한 결과, 15개월령 수소의 정액량이 평균 2.3ml로서, 30개월 수소의 정액량의 평균 5.0ml로서 유의차가 인정되었다(p<0.05). 채정 직후의 신선정자의 생존율은 15개월 및 30개월 수소가 각각 평균 93.7% 및 88.3%, 운동성은 각각 97.0% 및 88.3%로서 운동성은 15개월령이 유의하게 높았다(p<0.05). 한편 동결융해 정자의 생 존율은 각각 평균 56.0% 및 58.0%였고, 운동성은 각각 평균 64.0% 및 70.7%로서 차이 가 없었다. 본 연구를 통하여 칡소의 동결정액 생산을 위한 체형이 우수한 개체의 선발이 가능하 였으나, 대량 증식을 위한 추가적인 연구가 필요할 것으로 생각된다.

The purpose of this study was undertaken to evaluate of cryopreservation efficiency in α 1,3-galactosyltransferase knock-out(GalT KO) cloned miniature pig sperm. To compare ability of frozen-thawed sperm characteristics, three different pig strains (GalT KO) cloned miniature pig, PWG miniature pig and Duroc were used. The ejaculated semen from the three pig species was diluted with same volume extender and added to LEY solution for freezing. The diluted semen was placed in 0.5 ml straws, and freezing was initiated by exposing the straws to liquid nitrogen (LN2) vapours for 10 min before placing them into LN2 for cryopreservation. A fter thawing, the sperm ability were assessed for viability (SYBR-14/PI staining), abnormality (Rose Bengal staining), and acrosome status (intactness, intensity and capacitation) (chlorotetracycline, CTC staining). The viability of frozen-thawed GalT KO pig sperm had no significant difference as compared with Duroc and PWG miniature pig sperm. However, The CTC pattern of frozen-thawed GalT KO cloned miniature pig spermatozoa showed significantly lower rates in F pattern and AR pattern (p<0.05) and significantly higher rates in B pattern than Duroc and PWG miniature pig (p<0.05). The abnormality of GalT KO cloned miniature pig sperm was significantly lower as compared to Duroc and PWG miniature pig sperm (p<0.05). In conclusion, GalT KO cloned miniature pig semen can be cryopreserved successfully and used for artificial insemination reasonably.

This study was conducted to establish a freezing method of miniature pig spermatozoa. The semen ejaculated from PWG M-type miniature pig was collected by gloved-hand method. The semen was diluted with same volume extender (m-Modena B). The frozen solution used frozen solution of four different (LEY, TCG, BF-5 and m-Modena+egg yolk) for find optimal frozen solution in miniature pig sperm. The diluted semen for frozen rate assay was added to LEY solution (solution Ⅰ: 11% lactose+egg yolk; solution Ⅱ: solutionⅠ+glycerol+OEP), and frozen depending on freezing rate by the three different freezing methods (A: until 5℃ for 1 hrs, holding at —102℃ for 10 min; B: until 5℃ for 2 hrs, holding at —102℃ for 10 min; C: until 5℃ for 3 hrs, holding at —80 and —102℃ for 10 min). Semen cooled until 5℃ was added with glycerol 1, 3 and 5%, and take a equilibrium time for 0, 10 and 30min. Frozen-thawed sperm were evaluated for viability, acrosomal status and morphological abnormality. The results of frozen-thawed sperm ability by frozen solution, viability was higher in LEY solution compared to other three different frozen solution. AR pattern of LEY solution were lower than other three different frozen solution. The results of freezing rate, viability was higher in B method compared to other methods (p<0.05). Acrosomal statute was intacted in A and B methods than C method. The experiment for glycerol condition was showed that sperm viability was higher in extender with 1% and 3% glycerol and equilibrium time of 0 min. The acrosome damage was lower in extender with 1% glycerol and equilibrium time of 10 min than other conditions. In conclusion, the optimal conditions for cryopreservation of miniature pig spermatozoa obtained in LEY frozen solution, cooling rate of 1～2 hrs, 1～3% glycerol concentrations and glycerol equilibrium time of 0～10 min.

One-step dilution and direct transfer would be a practical technique for the field application of frozen embryo. This study was to examine whether Jeju Black Cattle (JBC, Korean Cattle) can be successfully cloned from vitrified and one-tep diluted somatic cell nuclear transfer (SCNT) blastocyst after direct transfer. For vitrification, JBC-SCNT blastocysts were serially exposed in glycerol (G) and ethylene glycol (EG) mixtures〔10% (v/v) G for 5 min., 10% G plus 20% EG (v/v) for 5 min., and 25% G plus 25% EG (v/v) for 30 sec.〕which is diluted in 10% FBS added D-PBS. And then SCNT blastocysts were loaded in 0.25 ml mini straw, placed in cold nitrogen vapor for 3 min. and then plunged into LN2. One-step dilution in straw was done in 25℃ water for 1 min, by placing vertically in the state of plugged- end up and down for 0.5 min, respectively. When in vitro developmental capacity of vitrified SCNT blastocyst was examined at 48 h after one-step dilution, hatched rate (56.4%) was slightly lower than that of control group (62.5%). In field trial, when the vitrified-thawed SCNT blastocysts were transferred into uterus of synchronized 5 recipients, a cloned female JBC was delivered by natural birth on day 299 and healthy at present. In addition, when the short tandem repeat marker analysis of the cloned JBC was evaluated, microsatellite loci of 11 numbers was perfectly matched genotype with donor cell (BK94-14). This study suggested that our developed vitrification and one-step dilution technique can be applied effectively on field trial for cloned animal production, which is even no longer in existence.

The objective of this study was to examine the effect of various discontinuous Percoll washing conditions on motile sperm recovery rate and motion kinematics. Frozen semen samples from 3 bulls (0.5 ml plastic straws, 6% glycerol in egg yolk-Tris-glycerol extender) were thawed in 37℃ water bath for 1 min. After thawing, the mixed semen samples were randomly allocated to 12 treatment groups. Briefly, the spermatozoa were centrifuged for three different time lengths (10, 20, and 30 min) at two gravities (300×g and 700×g) through two concentrations of discontinuous Percoll density gradient of 1 ml 90%: 1 ml 45% Percoll and 2 ml 90%: 2 ml 45% Percoll to remove extender, debris, and dead spermatozoa. Motile sperm recovery rate and motion kinematics were evaluated by computer assisted sperm analyzer using Makler counting chamber. Sperm motility (%) and motile sperm recovery rate showed similar pattern in all treatment groups. However, sperm motility (%) and motile sperm recovery rate were highest at 700×g for 30 min through a discontionous Percoll density gradient of 1 ml 90%: 1 ml 45% Percoll. There were no significant differences in motion kinematics after various Percoll washings. These results suggest that force of centrifugation, centrifugation time, and Percoll volume significantly affect motile sperm recovery rate.

노상토의 동상 가능성이 없는 경우 동상방지층을 생략한다. 이러한 조건에서 노상토의 동결 융해가 발생하면, 동상이 없는 상태에서 탄성계수 변화를 수반하고, 포장설계에서 이를 합당하게 고려해야 한다. 동결 융해 과정의 연속적인 탄성계수 변화를 효과적으로 규명하고자 비파괴시험인 충격공진시험(IR, Impact Resonance)을 도입하였다. 동결 융해 과정에서 수분의 공급이 차단된 폐쇄형 동결조건에서 노상토의 탄성계수는 동결 융해 반복횟수에 무관하게 일정한 값으로 나타났다. 또한 시험에 적용한 모든 함수비 및 다짐도 조건에서 동결전의 탄성계수와 동결 융해 후의 탄성계수는 변화가 거의 없이 일정한 값으로 나타났다. 즉 폐쇄형 동결 융해 조건에서는 노상토의 융해 강성도 감소(thaw-weakening)가 발생하지 않았다. 동결시 탄성계수는 다짐도 및 함수비에 따라서 변화하였으나, 포장설계 관점에서는 무시 가능한 수준으로 나타났다.

콘크리트의 박리(scaling)는 수분의 존재하에 동결융해 싸이클에 따른 콘크리트의 점진적인 표면열화이다. 특히, 이것은 제설제에 염화물의 존재가 콘크리트 표면박리(스켈링)와 더불어 심한 경우, 굵은골재의 노출 및 탈리로 이어질 수 있다. 본 연구에서는 콘크리트의 스켈링에 대한 저염화물계 제설제(low chloride deicier, LCD)와 염화칼슘 및 염화나트륨 제설제의 상대적인 영향을 ASTM C672에 준하여 실시하였다. 시험 제설제의 농도는 1, 4, 10% 이고, 수돗물은 기준으로 사용하였다. 박리량은 중량으로 평가하였다. 연구결과 4% 농도를 적용하였을 때, 동결융해 56 싸이클 후 콘크리트의 박리는 수돗물에 비해 LCD 용액에서 약 9배, 염화칼슘 용액에서 약 18배, 염화나트륨 용액에서 약 33배 정도 크게 발생하였다. 용액의 농도에 따라서는 고농도인 10%에 비해 4% 농도에서 표면 박리가 가장 현저하게 발생하였는데, 이는 스켈링 발생이 염농도가 3~4%일 때 가장 현저해진다는 기존의 연구결과와 일치함을 알 수 있었다(일본콘크리트공학회, 1999). 또한 콘크리트가 경화된 후, 현장에서 염화나트륨 및 저염화물계 제설제(LCD, 염소이온 중량비 50%)가 살포되고 동결융해 싸이클에 노출된 경우, 제설제에 노출되지 않은 경우의 콘크리트 동해열화에 대해, 콘크리트의 공기량에 따른 영향을 실험적으로 연구하였다. 연구 결과 동결융해 싸이클에 따른 콘크리트 시편은 제설제에 노출되지 않은 것 보다 염화물 제설제 노출에서 스켈링이 더 심한 것으로 나타났고, 염화물 제설제에 노출된 시편이 노출되지 않은 시편 보다 중량 손실이 2배나 되었다. 콘크리트 시편의 상대 동탄성계수는 염화물 제설제에 노출되지 않은 것과 비교하여 염화물 제설제에 노출된 것에서 더 빠르게 감소하였다. 또한 염화나트륨 제설제에 노출된 콘크리트 시편의 상대 동탄성계수는 저염화물계 제설제에 노출된 것 보다 더 빠르게 감소하였다. AE 콘크리트는 염화물과 동결융해 싸이클에 노출되었을 때, Non-AE 콘크리트 보다 성능저하가 크게 지연되었다.

The objective of this study was to evaluated the efficiency of sperm cryosurvival using each extenders Triladyl and LEY containing egg yolk to the cryopreservation of separated sperm by percoll in miniature pig. The ejaculated semen from miniature pig was separated by 65% percoll and un-separated sperm as a control before freezing. The freezing of diluted semen added with Triladyl containing egg yolk and LEY solution (solution Ⅰ: 11% Lactose or Triladyl + egg yolk; solution Ⅱ: solutionⅠ + glycerol + OEP). Analysis of sperm ability was estimated by viability, capacitation acrosome reaction using chlortetracycline (CTC) the morphologic abnormality and hypoosmotic swelling test(HOST). The groups were designed that as separated sperm by Percoll with Triladyl(ST) or LEY(SL) for cryopreservation. And unseparated sperm with Triladyl(UT) or LEY(UL). As a results, the viability was higher significantly(p<0.05) in ST, SL, UT than UL extender. The morphologic abnormality was measured significantly (p< 0.05) lower in ST than other extenders. The AR-patterned in CTC analysis was measured significantly(p<0.05) lower in SL and UL than other extenders. In conclusion, using Triladyl extender resulted in viability and morphology of separated sperm by percoll that were effective than using LEY extender, but it resulted in capacitation acrosome reaction was lower than using LEY extender.

The techniques for the collection, cooling and freezing of semen and artificial insemination of horses are not fully understood in Korea. We investigated the percentages of total motile (TM) and progressively motile (PM) sperms after the collection, cooling and freezing of stallion semen. The average volume of semen was 167 ml in Thoroughbred and 68 ml in Arab. The average numbers of spermatozoa in Thoroughbred and Arab were and respectively. The average percentages of TM and PM were 82.3% and 88.6% in Thoroughbred, and 61.4% and 82.6% in Arab, respectively. The average percentage of TM at 4 hr after cooling at was significantly lower than that at 0 hr (<), but the percentage of PM was similar between 66.5 and 73.2% at 0, 1, and 4hr. The average percentage of frozen-thawed Thoroughbred semen frozen in MFR5 extender was 56.2%, which was significantly higher than that of the semen frozen in LE extender (average 32.9%, p<0.05). The percentage of TM in Arab was similar for semen frozen in MFR5 extender and LE extender (18.2% and 21.2%, respectively), but the percentage of PM was significantly higher in sperm frozen in MFR5 extender than in sperm frozen in LE extender (69.0% vs. 36.4%, p<0.05). Four mares were artificially inseminated by Thoroughbred frozen-thawed semen and one of them fertilized at 11 day after artificial insemination. In this study, the collection, cooling and freezing of equine semen were possible under domestic conditions.

This study was performed to investigate the effect of laser-assisted hole in the zona pellucida (ZP) of frozen-thawed ICR mouse embryos on the process of hatching that is critical for expanded blastocysts to implant into endometrium, Vitrification medium, composed of ethylene glycol and sucrose supplemented with 7.5% (w/v) PVP, was used to freeze cell stage embryos recovered from oviducts of superovulated and mated female mice before storing them in . Right after thawing them, a laser beam was shot to make a hole in ZP followed by culturing in KSOM for and examining development to blastocyst and hatching every 12 hr. Laser-treated embryos showed significantly higher hatching rate compared to control (92.9% vs. 22.1%, p<0.05). From around Day 4, blastocysts developed from laser-treated embryos started hatching while the blastocysts of control group failed to hatch showing a lot of shrinkage. This study shows that a laser-assisted hole in ZP improves the hatching rate of blastocysts developed from frozen-thawed, in vitro cultured ICR mouse embryos.

The aim of this study was to evaluate the effects of orvus es paste(OEP) on the sperm characteristics during freezing in boar semen. Semen quality was evaluated the motility, membrane integrity, mitochondria function, acrosome status, viability and abnormality. Boar semen were frozen until 5℃ for 2 hours using cell freezer and making the straws, and then freezing by lowing the straws into styrofoam box on the 8cm above the LN2 and plunged into LN2 for cryopreservation. In different concentration of OEP (0, 0.25, 0.5 and 1.0%) into cryo-extender, sperm motility, membrane integrity, acrosome status, viability and mitochondria function were significantly higher in 0.5% OEP than those of any other groups, but sperm abnormality were highest in 1.0% OEP group among all treatment groups (P<0.05). In the relationships of the evaluation methods for sperm viability, CBB vs membrane integrity, CBB vs HO/PI and CBB vs mitocondria function were positively correlated (0.67~0.92). Among the evaluation methods, CBB vs membrane integrity, CBB vs HO/PI and CBB vs mitocondria function were significantly correlated (P<0.001). These results of this study indicate that supplementation of 0.5% OEP in boar semen cryo-extender can improve the semen quality.

본 연구는 0.5 ml straw를 이용한 정자 동결융해 시 융해 온도가 동결정자의 성상에 미치는 영향을 파악하고 미니 돼지의 동결정액에 최적화된 적정 융해 조건을 찾기 위하여 실시하였다. 정액동결 straw를 37, 50 및 70℃에서 각각 5초, 10초 및 45초간 융해하여 생존율(SYBR-14/PI staining), 정자원형질막기능검사 (Hypoosmotic Swelling Test) 및 첨체반응율 (CTC : chlorotetracycline staining)을 검사 한 결과 70℃에서 5초간 융해한 정자의 생존율과 CTC 결과가 37℃와 50℃에서 10초 및 45초간 융해한 처리구보다 유의적(p<0.05)으로 높은 생존율과 낮은 비율의 첨체 반응율을 얻었다. 따라서 미니 돼지의 동결 정액은 고온에서 단시간 융해를 하는 것이 정자의 성상에 유리한 것으로 사료된다.

본 연구는 mSOF(modified synthetic oviduct fluid medium) 배양액을 이용하여 와 배양 소적에서 일본 흑우의 수정란 생산 효율을 개선하기 위하여 수행하였다. 난구세포가 부착된 미성숙 난자는 각각 단독 배양조건( 소적) 및 그룹 배양 조건 ( 소적)에서 실시하였고 배양액은 TCM-199의 기본 배지에 10% FCS, 0.02IU/ml FSH와 를 첨가하여 사용하였다. 배반포 단계로 발육한 수정란은 1.5M ethylene