Background: This study focused on reproductive traits in Hanwoo cattle, specifically the environmental factors affecting gestation length and birth weight. Methods: The records of 1,540 cows calved at the Hanwoo Research Institute from 2015 to 2023 were examined. This study analyzed two populations, linebreeding Hanwoo (LBH) and general Hanwoo (GH), with all cows undergoing estrus synchronization and artificial insemination. The R software was used to compare the differences between the two populations and analyze the environmental factors affecting each trait. Results: The results showed that the average gestation length for LBH was 283.28 ± 5.93 days, which was significantly shorter than that of the GH, which had an average of 285.63 ± 6.21 days (p < 0.001). The average birth weight of LBH calves was 25.10 ± 3.69 kg, significantly lighter than GH calves, which weighed 27.26 ± 4.11 kg on average (p < 0.001). Analysis of environmental factors revealed significant differences in the gestation length of LBH based on dam parity, year, and season of calving. However, no significant differences were observed based on calf sex. For LBH, birth weight showed significant differences based on dam parity, year of calving, and sex of the calf, but not the season of calving. In GH, gestation length varied with dam parity and calving season, but not with calving year or calf sex. The GH birth weight showed differences based on dam parity, year of calving, and calf sex, but not the season of calving. Conclusions: Reproductive traits in the Hanwoo cattle industry are economically vital but are heavily influenced by environmental factors due to their low heritability. An accurate evaluation of the genetic potential of these traits requires an analysis of the environmental factors affecting them. The results of this study serve as foundational data for predicting the potential for genetic improvement in the gestation length and birth weight of Hanwoo cattle.

Background: Sperm quality and the number of sperm introduced into the uterus during artificial insemination (AI) are pivotal factors influencing pregnancy outcomes. However, there have been no reports on the relationship between sperm concentration at AI and sperm quality in Hanwoo cattle. In this study, we examined sperm quality and pregnancy rates after AI using sperm inseminated at different concentrations. Methods: We evaluated the motility, viability, and acrosomal membrane integrity of sperm at different concentrations (10, 15, 18, and 20 million sperm/straw) in 0.5-mL straws. Subsequently, we compared the pregnancy rates after AI with different sperm concentrations. Results: After freeze-thawing, sperm at the assessed concentrations showed similar viability and acrosomal membrane integrity. After AI, cattle in the 10 million group had significantly lower pregnancy rates compared to those in the 18 and 20 million groups. Conversely, there were no statistically significant variances observed between cattle in the 10 and 15 million groups. Conclusions: Sperm at concentrations of 10, 15, 18 and 20 million per straw exhibited comparable motility, viability, and acrosomal membrane integrity. However, a concentration of at least 18 million sperm per straw is required to achieve a consistent rate of pregnancy rate in Hanwoo cattle after AI.

현재 한우에서는 영양대사물질 수준에 대한 연구가 미흡한 수준이다. 따라서 본 연구는 한우 번식우에 있어서 임신과 비임신 상태에 있어서 혈액내 주요 영양대사물질을 분석하였다. 한우 번식우 54두를 방목을 하였을 경우에는 혈액내 주요대사물질 6개 항목(AST, ALT, Cholesterol, Glucose, BUN, NEFA)을 분석하였다. 사육방법과 관련된 NEFA수준 조사에서 방목우 임신우 평균은 221±105 uEq/L이고, 사사 사육 임신우 평균 240±121 uEq/L로 나타났으나 유의적인 차이는 없었다. 콜레스테롤 수준은 사사 사육시 임신우 113.33±17.03 mg/dL이었고, 비임신우는 137.25±21.77 mg/dL로 비임신우에서 유의적으로(p<0.05) 높은 결과를 보였다. 사사 사육시 NEFA 수준(임신우 204±119 uEq/L과 비임신우 286±215 uEq/L)도 콜레스테롤 수준과 동일한 경향을 보였다. 분만 전ㆍ후 AST 수치는 각각 105.53±24 IU/L와 81.59±12.55 IU/L으로 분석되어 분만 후에서 유의적으로(p<0.05) 감소한 결과를 보였다. NEFA 수준에서는 분만 전 122±52 uEq/L, 분만 후 497±239 uEq/L 로서 분만 후에서 유의적으로(p<0.05) 높은 결과를 보였다. 이러한 NEFA 수준의 증가는 분만 후 번식우가 필요로 하는 영양대사물질이 증가하였기 때문인 것으로 사료된다. 이러한 결과를 종합하면 한우 번식우의 적정한 영양수준 관리는 생산성을 향상 시킬 수 있을 것으로 사료되며 한우 번식우에 대한 영양수준 연구는 추가적으로 연구가 이루어져야 할 것으로 사료된다.

In the present study, we examined the effect of different lecithin concentrations on spermatozoa characteristics after freeze-thawing. Hanwoo semen was collected from one bull and divided into five groups (tris-citric acid semen extender with 0, 0.1, 0.25, and 0.5% lecithin groups as well as a 20% egg yolk group). Semen extender with 20% egg yolk was used as control treatment. After the freeze-thawing of semen, spermatozoa motility, motility parameters, viability, acrosomal membrane integrity, mitochondrial membrane potential, and plasma membrane integrity were examined. In experiment 1, the effect of different lecithin concentrations on spermatozoa motility and associated parameters was examined. The 0.1% lecithin-treated spermatozoa showed greater fast progressive motility (%) in addition to higher VCL (μm/s), VSL (μm/s), and VAP (μm/s) when compared to other lecithin concentration groups and controls. In experiment 2, the effect of different lecithin concentrations on spermatozoa viability was examined. The 0.1% and 0.25% lecithin addition groups (55.4±7.3 and 51.7±11.2%) exhibited similar viability compared to the control group (54.1±12.6%). In experiment 3, the effects of different lecithin concentrations on viability, acrosomal membrane integrity, and mitochondrial membrane integrity of spermatozoa were examined. The percentage of live spermatozoa with an intact acrosome and high mitochondrial membrane potential in the 0.1% lecithin group was not significantly different compared to the control group (31.2±13.3 vs. 30.5±10.9%). In experiment 4, the effect of different lecithin concentrations on the plasma membrane integrity of spermatozoa was examined. The percentage of spermatozoa with a normal plasma membrane was similar between the 0.1% lecithin and control groups (31.2±13.3 vs. 30.5±10.9%). In conclusion, we suggest that semen extender supplemented with 0.1% lecithin can replace 20% egg yolk without reducing spermatozoa quality.

In the present study, we examined if deep uterine artificial insemination (DUAI) can improve the pregnancy rate of artificial insemination (AI) using epididymal spermatozoa (ES) in Hanwoo cattle. The estrus cycles of 88 Hanwoo cows were synchronized, and 17 cows were artificially inseminated using the DUAI method with ES, 20 cows were artificially inseminated via the uterine body (BUAI) method with ES, and as a control, 51 cows were inseminated by using the BUAI method with ejaculated spermatozoa from 1 proven bull after frozen thawing. The pregnancy rate of the DUAI method (58.8%) was higher than that of the BUAI method (25.0%, p = 0.0498). The motility of ES was examined immediately after thawing and after 3 and 6 h of incubation. The rapid progressive sperm motility of the control group was significantly higher than that of the ES group immediately after thawing and after 3 and 6 h of incubation (p < 0.05). The straight line velocity and average path velocity of the ES group after 6 h of incubation were significantly lower than those in the control group (p < 0.05). The linearity and amplitude of lateral head of ES were lower than those at 6 h (p < 0.05). The flagellar beat cross frequency and hyperactivation of ES were lower than the control spermatozoa immediately after thawing and at 3 h (p < 0.05). These motility parameters suggested that ES had a low motility and fertilization ability compared to the control spermatozoa. After frozen-thawing and 3 h of incubation, the percentage of live spermatozoa with intact acrosomes in the ES was significantly lower than that in ejaculated spermatozoa (p < 0.05). Our findings suggested that the DUAI method can overcome the low pregnancy rate of ES, despite the low motility, viability, and fertilization ability of ES.

In the present study, we examined the effect of straw size on spermatozoa motility, viability, acrosome integrity, mitochondrial membrane potential, and plasma membrane integrity after freezing-thawing. Hanwoo semen was collected from three bulls and diluted with an animal protein-free extender, divided into two groups, namely, 10 million spermatozoa in 0.25 mL and 20 million spermatozoa in 0.5 mL straw, and cryopreserved. In Experiment 1, the motility and motility parameters of the frozenthawed spermatozoa were evaluated. After freezing-thawing, the spermatozoa motility parameters fast progressive, straight line velocity, and average path velocity were compared between the 0.25 mL straw and 0.5 mL straw groups. They were 35.2 ± 1.0 and 32.3 ± 0.7%, 34.6 ± 0.7 and 31.8 ± 0.5 μm/s, 51.4 ± 1.3 and 47.1 ± 1.1 μm/s, 0.25 mL straw and 0.5 mL straw groups, respectively. In Experiment 2, the viability, acrosome membrane integrity, and mitochondrial membrane potential of the frozen-thawed spermatozoa were assessed. After freezing-thawing, the percentages of spermatozoa with live, intact acrosomes and high mitochondrial membrane potential were compared between the in 0.25 mL straw and 0.5 mL straw groups. They were 48.0 ± 2.6% and 35.6 ± 2.8% between the 0.25 mL straw and 0.5 mL straw groups. In Experiment 3, the plasma membrane integrity of frozen-thawed spermatozoa was compared. After freezingthawing, the plasma membrane integrity was higher for the in 0.25 mL straw group than the 0.5 mL straw group. They were 62.0 ± 2.2 and 54.1 ± 1.3% between the 0.25 mL straw and 0.5 mL straw groups. In conclusion, our results suggest that freezing semen in 0.25 mL straw improves the relative motility, viability, and acrosomal, mitochondrial membrane potential, and plasma membrane integrity of Hanwoo bull spermatozoa.

This study was conducted to examine the oocyte recovery efficiency through having an OPU session once and twice a week. Also, the oocyte recovery efficiency was examined by using OPU after two and three months of rest period. Six cows were used for oocytes collection and were randomly divided into two groups. In experiment 1, OPU sessions were conducted once and twice a week to collect oocytes. The collected oocytes between once and twice OPU groups were classified into four groups (grade 1, 2, 3 and 4) according to the quality of cumulus cells and ooplasm. Based on the result, the percentage of collected oocytes per aspirated follicle number was similar between once and twice OPU session groups (65.5 ± 1.9 and 68.7 ± 1.4 vs.). However, the percentage of grade 1 oocytes from the twice OPU session group was significantly high compared with that of the once a week OPU session group (25.3 ± 0.9 and 32.5 ± 1.2% vs. once and twice session group, respectively, p < 0.05). In experiment 2, the group with three months of rest period tended to have a high percentage of collected oocyte compared with the group with two months of rest period (64.6 and 70.9% vs. 2 and 3 months rest group, respectively, p = 0.62). The percentage of grade 4 in the group with three months of rest period was significantly low compared with the group with two months of rest period group (27.3 and 36.5% vs. two and three months rest group, respectively, p = 0.05). In conclusion, twice a week OPU session is suitable for collection of high quality oocytes by using OPU, and three months of rest period is needed for the recovery of oocyte quality of a donor cow.

본 연구는 한우 번식우에 있어서 영양대사물질 분석을 통하여 영양수준을 구명하여 번식우의 수태율 개선을 위한 기초 자료로 활용하기 위해서 실시하였다. 번식우의 정확한 영양수준 분석을 위해서 사료급여량을 80%, 100%, 120%로 구분하여 사양관리를 실시한 결과 Cholesterol과 BUN 농도가 120% 급여구에서 유의적으로 높은 결과를 나타내었다(p<0.05). 방목우 중에서 임신우와 비임신우의 영양대사물질 수준 분석 결과, Cholesterol, AST, NEFA 농도가 임신우에 비해 비임신우에서 유의적으로 높은 결과를 보였다(p<0.05). 이와 같이 임신과 관련한 영양수준 분석에 Cholesterol, AST, NEFA 의 3가지 항목을 설정하는 것이 필요할 것으로 사료된다. 방목과 사사 사육에 대한 결과 분석에서 Glucose 농도는 방목우 84.8, 비방목우 56.0 mg/dl 으로서 방목우에서 유의적으로 높은 결과를 보였고(p<0.05), Cholesterol 수준은 방목우에서 142.5 mg/dl로서 사사 사육 128.9 mg/dl 보다 유의적으로 높았으며(p<0.05), ALT(34.4 vs 27.1 IU/l)와 NEFA 농도(317.8 vs 160.2 ЧEq/l) 역시 방목우에서 유의적으로 높은 결과를 보였다(p<0.05). 결론적으로, 암소에 사료 급여시 Cholesterol, ALT, NEFA 수준을 낮출 수 있도록 하는 것이 한우 암소의 수태율을 높일 수 있을 것으로 사료된다.

In this study, two epididymal spermatozoa recovery methods in relation to sperm number, motility, viability and acrosome reaction were examined. Seven bulls were castrated and 7 testicles with epididymides were transferred to the laboratoy. Epididymis in each bull was randomly used for flushing and mincing methods with semen extender (Optixcell, IMV, France). The recovered spermatozoa with adjusted sperm concentration to 40 × 106 cells/mL was diluted with optixcell and cryopreserved. In experiment 1, the difference in the total number of spermatozoa using flushing and mincing methods was insignificant (2570.0 and 2505.2 × 106 cells/mL, respectively). For experiment 2, the percentage of motile spermatozoa and motility parameters between flushing and mincing methods were studied through the use of sperm class analyzer after frozen-thawing. The percentage of total motile sperm between flushing and mincing methods was almost the same with 89.5±12.8 and 91.4±7.9%, respectively. The same is the case with experiment 3 wherein the viability and acrosomal integrity of frozen-thawed epididymal spermatozoa by flushing and mincing was insignificantly different. The results from the study showed that both flushing and mincing methods can be used for epididymal spermatozoa recovery in bull.

본 연구는 티모시 건초와 농후 사료 위주의 사료를 급여한 한우 씨수소 정소상체 정자 체외수정 효율 조사를 통해 정자의 활용 가능성을 조사하였다. 농후 사료는 체중의 1.8%를 급여하고 양질의 티모시 건초를 자유채식 시킨 14개월령 거세우의 정소에서 분리된 정소상체 미부의 정자를 회수하고 동결 흉해 후 체외수정을 실시한 결과는 다음과 같다. 웅성전핵과 자성전핵이 형성(2PN)된 난자는 정상수정으로, 1개의 전핵(1PN), Expanded Sperm Head (ESH), Polyspermy 형태는 비정상적인 수정의 형태로 평가하였다. 정상적으로 수정된 난자의 비율은 정소상체 정자의 경우 전체 침투율은 49.7% 그리고 정상적인 2PN을 가진 난자는 18.5%를 보였으며, 대조구 정자의 전체 침투율은 54.4%로서 정소상체 정자 보다 높은 결과를 보였으나 유의적인 차이를 보이지는 않았다. 정상적으로 2PN을 형성한 비율은 36.7%로서 정소상체 정자를 이용한 정자 보다 높았으나 유의적인 차이는 없었다. 체외수정 후 발달률 조사에서 정소 상체 정자의 분할률은 81.2%, 대조구 정자는 82.7%로 유사한 결과를 보였으나, 배반포 발달률은 정소상체 정자 24.4%와 대조구 정자 12.2%로 정소상체 정자를 사용한 난자의 발달에서는 유의적으로 높았다(p<0.05).

Serum metabolites were analyzed to investigate relationship of pregnancy and non-pregnancy Hanwoo cows. Totally, 251 Hanwoo cows were used in the present study. Grazing was carried out for 5 months in the pasture. In barn feeding, concentrate 3.0 Kg (TDN 68%, CP 14%) and rice straw 6 kg(TDN 50%, CP 6.5%) were fed. For artificial insemination (AI), progesterone-supplying device (CIDR) was introduced to vagina of Hanwoo cows and 2.0 mL of GnRH. One week after introduction, CIDR was removed and 5.0 mL of PGF2α was injected intramuscularly. After 2.5 day, AI was accompanied by a 2 mL of GnRH intramuscular injection and a second AI was carried out 3.5 day. The pregnancy diagnosis was confirmed by rectal palpation about 90 days after AI. Blood samples were collected from the jugular vein after 3 hours of feeding. Analysis of serum metabolites was performed on six types of metabolites: glucose(mg/dl), cholesterol(mg/mL), BUN(mg/dl), AST(U/L), ALT(U/l), and nonessterified fatty acids(NEFA, uEq/L). The metabolic profile test was analyzed by analyzer (Hitachi, 7020, Japan). Pregnant and non-pregnant groups showed serum metabolites as follows. In 60 pregnant group: Glucose 88.9 ± 2.5, Cholesterol 149.8 ± 4.9, BUN 16.9 ± 0.4, AST 99.1 ± 2.6, ALT 35.9 ± 0.9 and NEFA 326.7 ± 15.7. In 43 non-pregnant cow group: Glucose 89.2 ± 3.3, Cholesterol 165.9 ± 4.6, BUN 17.4 ± 0.6, AST 108.9 ± 0.6, ALT 37.8 ± 1.0 and NEFA 419.2 ± 32.8. Cholesterol, AST and NEFA levels in non-pregnant cows were significantly higher than those in pregnant cows (P<0.05). In sum of grazing and barn feeding group was totally 148 cows. Seventy nine pregnant cows showed high glucose and low NEFA levels compared to 69 non-pregnant cows (P<0.05). In conclusion, pregnant group showed high level of glucose and low level of cholesterol, NEFA. Further study needed to obtain more accurate level of metabolites in serum for pregnant and non-pregnant cows.

In this study, we examined number, motility and plasma membrane integrity of spermatozoa from six regions of epididymis in bull. Six testicles with epididymides were castrated from six bulls (mean±standard error, age of days = 441.3±9.6, body weight (kg) = 367±8.4, scrotal circumference (cm) = 30.7±0.4) at Hanwoo Research Institute, NIAS and transported to laboratory within 1 hour. Testicular weight, length, width and circumference were recorded. Epididymis in each bull was randomly used for recovery of spermatozoa. Epididymis was divided into six regions: efferent duct (ED), caput, corpus, proximal cauda (Pcauda), distal cauda (Dcauda) and vas deferens (VD). In experiment 1, we examined sperm number of each region of epididymis. Each region of epididymis contained different number of spermatozoa: ED (37.8±15.7 × 106cells/ml, 8.2%), caput (93.6±18.8 × 106cells/ml, 20.2%), corpus (33.0±8.5 × 106cells/ml, 7.1%), Pcauda (104.2±23.5 × 106cells/ml, 22.5%), Dcauda (180.5±32.5 × 106cells/ml, 39.0%) and VD (14.0±5.0 × 106cells/ml, 3.0%). In experiment 2, sperm motility of each epididymal region was examined by computer assisted sperm analysis (SCA, MicroOptic) system. Sperm motility was divided into 4 groups (fast progressive, slow progressive, non-progressive and immotile) based on WHO guideline. Percentages of fast progressive of Pcauda and Dcauda (11.0±2.3 and 15.4±3.6%) were significantly higher than that of ED, Caput, Corpus and VD which is 0.1±0.1, 1.5±0.6, 1.9±0.7 and 0.3±0.2%, respectively (p<0.05). In experiment 3, percentage of intact plasma membrane spermatozoa of each regions were examined by hypoosmotic swelling test. Percentages of intact plasma spermatozoa were not significantly different among six regions of epididymis: ED, caput, corpus, Pcauda, Dcauda and VD which is 68.0±8.6, 74.0±5.3, 68.5±6.2, 70.8±5.5, 71.0±5.8 and 64.6±10.8%, respectively. In conclusion, in the present study, we found out distribution, motility and plasma membrane integrity of spermatozoa from six regions of epididymis in Hanwoo bull. These results will be contributed to basic research about spermatozoa transportation and characters in epididymis of bull.

In this study, we examined sperm penetration and blastocyst developmental rate of oocytes to determine fertilizability of cauda epididymal spermatozoa in Hanwoo bull. One testicle with epididymides were castrated from one Hanwoo bull (14 months of age) and transported to laboratory. Spermatozoa recovered from cauda epididymis by mincing with semen extender (Optixcell, IMV, France) and cryporeserved in liquid nitrogen tank until use. As control, frozen Hanwoo semen was used. Cumulus oocyte complexes (COCs) were collected from follicles (2-8 mm) of slaughtered ovaries and 10 to15 COCs were matured in 50μl droplet with M-199 media supplemented with 10% fetal bovine serum, 10μg/ml FSH, 10μg/ml LH, 10μg/ml EGF for 22 to 24 hours in a humidified atmosphere of 5% CO2 in air. After maturation of COCs, matured COCs were co-incubated with cauda epididymal spermatozoa in 100μl droplet in modified Brackett and Oliphant media supplemented with 2.5 mM theophylline for 12 or 18 hours under 5% CO2 in air. Sperm concentration was adjusted to 5 × 106cells/ml. After IVF for 18 hours, presumptive zygotes were cultured in modified synthetic oviductal fluid with 1mM glutamine, 12 essential amino acids, 10 μg/ml insulin under 5% CO2, 5% O2 in air. In experiment 1, we examined sperm penetration rate at 12 hours of IVF of frozen-thawed epididymal sperm. Total penetration rate among cauda epididymis and control were not significantly different (mean±standard error, cauda epididymis and control vs. 49.7±11.3 and 54.4±12.8%) In experiment 2, cleavage and blastocyst development rate were evaluated at day 2 and day 8 after IVF for 18 hours. Cleavage rate among cauda epididymis and control was similar different (cauda epididymis and control vs. 81.2±3.4 and 82.7±2.5%). However, blastocyst developmental rate of cauda epididymis group was significantly higher than that of control group (cauda epididymis and control vs. 24.4±1.6 and 12.2±2.8%, p<0.05). In conclusion, cauda epididymal spermatozoa in Hanwoo bull has high fertilizability and embryo development. Cauda epididymal sperm can be used as an alternative to ejaculated frozen sperm in vitro.

In this study, we examined total number, motility and plasma membrane integrity of epididymal spermatozoa from cauda epididymis of bull after preservation at 4ºC. Totally, 23 testicles were castrated from 23 bulls (mean±standard error, age of days = 426.0±7.3, body weight (kg) = 379.7±8.4, scrotal circumference (cm) = 31.0±0.4) at Hanwoo Research Institute, NIAS, and transported to laboratory and preserved on 1, 4 and 6 days at 4 ºC. As control, epididymal spermatozoa recovery from 7 testicles was conducted after transportation to laboratory immediately. In experiment 1, we compared total number of spermatozoa among groups. Total number of spermatozoa from epididymis was not significantly on different preservation day of 0, 1, 4 and 6 which is 1778.0±304.7, 1824.8±343.9, 1228.4±91.7, 1201.8±178.6×106 cells/ml, respectively). In experiment 2, we examined spermatozoa motility and motility parameters (VCL (μm/s), VSL (μm/s), VAP (μm/s), LIN (%)) by computer assisted sperm analysis (SCA, MicroOptic) system. Percentage of motile on 0 and 1 day (88.9±5.2 and 85.8±6.1) was significantly higher than that on 4 and 6 days (32.6±6.5 and 34.3±8.25). Percentage of VCL (μm/s) on 0 and 1 day (93.5±7.6 and 83.0±14.9) was significantly higher than that on 4 and 6 days (36.6±5.1 and 39.5±5.5) (p<0.05). Percentage of VSL (μm/s) on 0 day (28.0±2.1) was significantly higher than that on 1, 4 and 6 days (20.2±3.0, 9.0±2.0 and 8.5±1.6, p<0.05). Percentage of VAP (μm/s) on 0 and 1 days (49.4±3.8 and 41.3±6.6) was significantly higher than that on 4 and 6 days (18.2±3.0 and 19.3±2.8, p<0.05). Percentage of LIN (%) on 0 day (30.7±2.6) was significantly higher than that on 4 and 6 days (23.4±2.7 and 21.1±1.0, p<0.05). Motility of spermatozoa was divided into 4 groups (fast progresive, slow progressive, non-progressive and immotile) based on WHO guideline. Percentage of fast progressive on day at 0 was significantly higher than that on 1, 4 and 6 days (0, 1, 4 and 6 days vs. 19.8±1.9, 10.2±1.1, 2.6±1.0 and 2.3±1.2%, respectively). In conclusion, cauda epididymal spermatozoa should be recovered within one day after preservation at 4 ºC to recover high quality of epididymal spermatozoa in Hanwoo bull

We observed the external genitalia and uterus of a 24-month-old freemartin Hanwoo. The vulva was smaller than observed in a normal female Hanwoo, while the clitoris was larger in the freemartin. The angle between the external genitalia and the perineum also varied. Upon internal genital examination, the uterus of the freemartin was a thin tube approximately 18 cm in size and had not differentiated into a normal uterus and uterine horns.

This study was conducted to investigate the breeding status of farms to improve the production efficiency of Hanwoo calf. The study was conducted on 45 farms divided into two groups. This study was conducted to investigate the breeding size and breeding area of Hanwoo cows. The average age at first delivery of Hanwoo was 28.7 months. The number of artificial insemination per pregnancy was 1.45±0.32, and the number of artificial insemination days after birth was 119.8 days. Conception rates were 75.2±16.93% for small farms and 70.6±17.46% for medium sized farms and 71.4±11.03% for large farms. When we looked at farming methods, ‘the farmers using estrus observation aids’ had 10.42% higher calf production rate than the ‘unused farmers’. The farms vaccinated with IBR and BVDV for breeding cattle showed a 4.41% decrease in abortion, stillbirth and mortality. According to farming conditions, conception rate and delivery rate improved by 3.47% and 18.29%, respectively, when grazing and exercising were performed. Observation, immunization and grazing were found to be important indicators for improving calf production efficiency in Hanwoo farm. This study can be used as a research data to improve the reproductive rate of farmhouse sites through the survey on the breeding status of Hanwoo farmers.

Commercial applications of OPU/IVP were to produce embryos and calves from high genetic cows.The aim of this present study was to compare the number of recovered oocytes and cultured in vitro produced embryos from Ovum Pick-up (OPU). OPU derived embryo production was carried out of oocytes by ultrasonographic guided follicular aspiration and then produced in vitro produced blastocysts by IVP culture system. In result, the rate of recovered oocytes was obtained 612 (57.2%) and 451(73.7) G1+G2 grade oocytes. No difference of recovered rate (51.1~62.1%) was seen in six donor. The rate of cleavage and blastocyst development were obtained 320 (70.9%) and 78 (24.4%) that was 3.3±0.4 cleaved embryo and 0.9±0.2 blastocysts per session. Cleavage rate of OPU oocytes in No. 6 donor was 90.6%, significantly (P<0.05) higher than that in the other donors, However, blastocysts was similar (25.8~30.0%). In conclusion, limited numbers of OPU oocytes had competent development when cultured in SOF culture medium

The present study was conducted to investigate the effect of BHT supplementation on sperm motility, viability, acrosomal integrity and plasma membrane integrity after frozen-thawing. One ejaculate was collected from one fertile Hanwoo bull by using artificial vagina at Hanwoo Research Institute. The ejaculate was transferred to laboratory immediately and diluted with pre-warmed semen extender (Optixcell, France) (1:1). Sperm dilutions were extended to a final concentration of 40 x 106 sperm/ml, and divided into 5 groups according to BHT concentration (0, 0.5, 1.0, 2.0 and 4.0 mM) and cryopreserved in LN2 tank until evaluation. Frozen-thawed semen was transferred to 1.5 ml tube and incubated for 0, 2 and 4h. Sperm motility and motility parameters (total motility, VSL with 25μm≥, VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were evaluated by sperm class analysis (SCA, IVOS, Spain). There were not significant effects of BHT supplementation (0, 0.5, 1.0 and 2.0 mM) on total motile and VSL with 25μm≥ at 0, 2 and 4h. However, 4.0 mM of BHT supplementation showed negative effect on total motile (26.3%), VSL with 25μm≥ (1.3%) at 0 h (p<0.001). The viability and acrosomal integrity of spermatozoa were evaluated by Trypanblue/Giemsa staining method and divided into 4 groups; live and intact acrosome (LIA), live and damaged acrosome (LDA), dead intact acrosome integrity (DIA), dead damaged acrosome (DDA). There were no significant differences of LIA, LDA, DIA and DDA on various BHT concentrations at 0 and 2 h. However, 4.0 mM BHT supplementation showed decreased LIA compared with 0, 0.5, 1.0 and 2.0 mM BHT at 4 h (34.6, 37.1, 43.6, 45.4 and 14.7% vs. 0, 0.5, 1.0, 2.0 and 4.0 mM, irrespectively; p<0.01). Addition of 4.0 mM of BHT showed negative effect on plasma membrane integrity compared with that of 0, 0.5, 1.0 and 2.0 BHT at 2 h (71.9, 64.2, 64.6, 67.5 and 31.7 % vs. 0, 0.5, 1.0, 2.0 and 4.0 mM, irrespectively; p<0.05). In conclusion, various BHT concentrations on optixcell extender showed no improvement on sperm motility, viability and plasma membrane integrity.

The present study was conducted to investigate the effect of different heights from liquid nitrogen (LN2) vapor on sperm motility and morphology after frozen-thawing. Two ejaculates were collected from 2 fertile Hanwoo bulls (A and B) by using artificial vagina at Hanwoo Research Institute. After collection, ejaculates were transferred to laboratory immediately and diluted with semen extender (Optixcell, France). Sperm dilutions were extended to a final concentration of 40 x 106 sperm/ml, and cooled at 4°C for 4 h and loaded to 0.5 ml straws. The straws were divided into 2 groups. Straws were placed in 3 or 9 cm of LN2 vapor for 14 min and then plunged into LN2 tank and cryopreserved until evaluation. Sperm motility and motility parameters (total motility, VSL with 25μm≥, VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were evaluated by sperm class analysis (SCA, IVOS, Spain) after frozen-thawed. In bull A, 3cm group showed higher percentages of total motility, VSL with 25μm and VAP compared those with 9cm group (98.0 vs. 93.4%, 62.4 vs. 54.0% and 98.6 vs. 93.2%, 3 vs. 9 cm, irrespectively; p<0.001). In bull B, frozen-thawed sperm of 3cm group showed higher percentages of VSL with 25μm, VCL, VSL, VAP and BCF compared with those of 9cm group (43.5 vs. 26.0%, 123.8 vs. 111.6 μm, 62.9 vs. 57.3 μm and 81.5 vs. 72.5 μm; 3 vs. 9 cm, irrespectively; p<0.001). The viability and acrosomal integrity of spermatozoa were evaluated by Trypanblue/Giemsa staining method divided into 4 groups; live and intact acrosome (LIA), live and damaged acrosome (LDA), dead intact acrosome integrity (DIA), dead damaged acrosome (DDA). In bull A, frozen-thawed sperm of 3 and 9cm groups showed no significant difference in LIA, LDA, DIA and DDA. In bull B, 3 cm group showed higher LIA and lower DIA compared with those of 9 cm group (73.2 vs. 23.7% and 23.7 vs. 32.2%, 3 vs. 9 cm, irrespectively; p<0.001). We suspected that 3 cm vapor on LN2 vapor might be affected positively spermatozoa viability and acrosomal integrity compared with 9 cm group. In conclusion, semen freezing procedure in the present study will improve sperm quality after frozen thawing.

The study aims to assess viability, acrosomal menbrane, integrity and mitochondria membrane potential of sperm separated using a percoll density gradient(45% and 90%) and swim-up methods using Hanwoo epididymal sperm frozen semen. Briefy, motile sperm separated using a percoll gradient and swim-up. 25 μl of sperm dilution from droplets were transferred to 1.5 mL tube and incubated with fluorescent probes at 39°C in dark as follows. After incubation, 75 μl of 5,5',6,6'-tetrachloro-1,1',3,3'- -JC-1; Mitochondrial Membrane Potential Detection kit, Cell Technology Inc., USA) working solution was mixed with sperm dilution and incubated for 30 min. One μl of Hoechst 33342 (H1339; Molecular Probe, Eugene, OR, USA) stock solution was mixed with sperm dilution and incubated for 10 min. And then, 0.5 μl of propidium iodide (PI) stock solution and 0.5 μl of fluorescein peanut agglutinin FITC conjugate (FITC-PNA; Vector Laboratories, FL-1071) were mixed with sperm dilution and incubated for 8 min. After mixing with fluorescent probes and sperm dilution, 5 μl of stained sperm dilution was mounted on a slide glass and covered with cover glass. More than 200 sperms in a slide glass were counted with × 400 magnification by fluorescent microscope (Eclipse Ci_L, Nikon, JAPAN) and evaluated viability, acrosomal membrane integrity, and mitochondrial membrane potential of sperm with triple band filter (DAPI/FITC/TRITC; Nikon, Tokyo, Japan). Live sperm were stained with Hoechst 33342(blue) and dead sperm were stained with PI (red). Damaged acrosomal membrane of sperm was stained with FITC-PNA (green) and intact acrosomal membrane of sperm was not stained. Both of sperm swim-up method with or without BSA separated to Live intact mitochondria (15.39±4.31 vs, 12.58±3.74, and) without significant difference. and percoll density gradient method also similar (7.29±6.54), swim-up method of sperm preparation appeared to be more efficacious in percentage recovery of motile sperm concentration compared to Density gradient method.