This study was conducted to investigate the optimal artificial insemination (AI) time with diagnostic kit at ovulation time. We already applied the patent about the protein in the cow heat mucose in external reproductive tract. And we would examine the accuracy for detection of cow heat by the kit produced with the protein. Evaluation of optimal heat detection was tried two time at 12 hrs and 24 hrs after the heat. And then, AI service also performed two times with no relation to the results of heat diagnosis by heat detection kit and pregnancy rates were checked with rectal palpation on 60th day after AI. Heat diagnostic results by kit in natural heat after 12 hrs in Hanwoo cows were showed 31.3~75.0% on positive in first heat detection and 33.3~100.0% on positve in second heat detection. In the 1st positive results were significant different (p<0.05), but 2nd positive were not. The results of heat detection showed different result on regional influence and individual cow effects. The pregnancy rates of first trial of heat detection were showed 34.4~78.7% on positive and 21.3~68.8% on negative after the diagnosis by heat detection kit. And the pregnancy rates of next trial of heat detection were showed 33.3~85.7% on positive and 14.3~66.6% on negative after the heat diagnosis. Both positive results of first trial and next trial also were showed significant different (p<0.05), but negative results were not. In positive result, first trial of total pregnancy rates was higher than the next trial of pregnancy, but there showed opposite results on negative results. In conclusion, the optimal heat detection kit is suitable to ordinary Hanwoo cows and it suggested that we have to improve the kit’s accuracy by detecting the materials like proteins related optimal AI time.

The coat color of mammals is determined by the melanogenesis pathway, which is responsible for maintaining the balance between black-brown eumelanin and yellow-reddish pheomelanin. The melanogenesis-associated genes controlling pigmentation act as a complex and interact with each other to cause phenotypic and genotypic variations in cattle. That the MC1R genotype of Korean native cattle with dark muzzle was e/e or E+/e, while the genotype of Korean native cattle with light muzzle was E+/E+, which is a variant of the MC1R genotype in the Korean native cattle. Especially, the MC1R expression type is shows how much pigmentation, important factor in deciding its status in the coat and nose colours. However, information regarding the coat or nose colours-associated gene regulation of korean cattle is not yet unknown. Therefore, in this study was to investigate the expression patterns of melanogenesis-associated genes in black dot nose(korea brindle cattle) and normal nose(korea native cattle). Using microarray clustering and real-time polymerase chain reaction techniques, we analysed that the expression of genes involved in the mitogen-activated protein kinase (MAPK) and Wnt signaling pathways is distinctively regulated in the dark and light muzzle tissues. Differential expression of tyrosinase was also noticed, although the difference was not as distinct as those of MAPK and Wnt. We hypothesize that emphasis on the MAPK pathway in the Korea brindle cattle induces eumelanin synthesis through the activation of cAMP response elementbinding protein and tyrosinase, while activation of Wnt signaling counteracts this process and raises the amount of pheomelanin in the native cattle. Regarding the increasing interest in the genetic diversity of cattle stocks, genes we identified for differential expression in the brindle cattle vs. native cattle may serve as novel markers for genetic diversity among cows based on the coat and muzzle color phenotype.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis. This study was conducted to clarify the seroprevalence of BLV in the Republic of Korea. Blood samples were obtained from Korean native cattle farms in all provinces of South Korea except Jeju. A commercial indirect enzyme-linked immunosorbent assay with 4,498 samples to detect antibodies against BLV was conducted. The results revealed that the prevalence of BLV was dependent upon age, with increasing prevalence among cattle occurring until they were 5 years old. The highest seroprevalence in cattle was observed in Chungnam (29.6%) and the lowest was observed in Jeonnam (2.6%). The mean overall prevalence for BLV antibodies in the survey was 10.2%, indicating that BLV is widespread nationwide.

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.

본 조사연구는 충남 예산지역에 위치하고 있는 젖소 사 육농가 중 10개 전업 검정농가를 대상으로 2015년 1월부터 2015년 12월까지 농협중앙회 젖소개량사업소의 검정성적 을 통하여 유량 및 유조성분을 조사·분석하여 젖소농가의 경영지표 설정과 농가의 경영기술 개선을 위한 기초적 자 료를 얻고자 실시한 결과를 요약하면 다음과 같다. 평균 산유량은 33.62kg로 나타났다. 산차별로는 유의한 차이 (p<0.05)를 보였으며 1산차는 29.60kg로 가장 낮은 산유량 을 나타났으나, 2산차 이상부터 높은 산유량을 보였다. 산 유량별로도 유의한 차이를 보였으며(p<0.05), 계절별 평균 산유량은 봄에 34.51kg으로 다른 계절보다 높은 유량을 보 였다(p<0.05). 산차별의 유지율은 유의성 없이 1산에서 6산 차까지 비슷한 결과를 보였다. 산유량별 간에 유지율은 유 의한 차이(p<0.05)가 있었고, 계절별 간에는 유의한 차이 (p<0.05)를 보였는데, 겨울에 유지율 4.15%로 가장 높은 성 적을 보였으나, 여름에 3.61%로 가장 낮은 함량을 보였다. 산차별 간의 단백율은 유의한 차이 없이 평균치와 비슷한 수준을 보였다. 산유량별 간의 유단백율은 유의한 차이 (p<0.05)로 1일 30kg미만 군에서는 3.51% 높은 유단백율을 보였고, 40kg이상 군은 3.14%로 가장 낮은 함량으로 나타났다. 계절별 간의 유단백율은 가을과 겨울은 각각 3.40% 와 3.41%으로 높았으나, 봄과 여름은 각각 3.28%와 3.27% 로 낮은 수준를 보였다(p<0.05). 총고형분은 산차별 간에 1 산차가 8.92%로 가장 높은 함량를 보였으나(p<0.05), 산차 가 커질수록 감소현상을 보였다. 산유량별에 따른 총고형 분율은 1일 30 kg미만 군에서 8.92%로 높았으나, 40kg이상 군은 8.62%로 낮은 총고형분율을 나타났다(p<0.05). 계절 별 간의 총고형분율은 가을과 겨울에 각각 8.87%와 8.84% 으로 높은 함량이 었으나, 봄과 여름에는 각각 8.77%와 8.75%로 낮았다(p<0.05). 산차별간의 체세포수는 1산차가 100.75천개/ml로 가장 낮은 수를 나타냈으나, 산차가 커질 수록 체세포수는 증가현상을 보였다(p<0.05). 산유량별로 는 1일 30kg미만 군은 157.00천개/ml의 높은 체세포수로 유의한 차이를 보였으나(p<0.05), 40kg이상 군의 123.64천 개/ml로 낮은 수로 나타났다. 산유량은 유지율, 유단백율 및 총고형분율과 각각 -0.378, -0.534 및 –0.370으로 음(-)의 상관계수를 나타냈다. 유지율은 유단백율 및 총고형분율과 의 상관계수는 각각 0.600과 0.493으로 매우 높은 양(+)의 관계를 보였다. 유단백율과 총고형분율 간에는 0.833으로 양(+)의 매우 높은 상관관계를 보였다. 산유량과 유지율의 단순회귀모형은 y=-0.0297x+4.907(R2=0.1426), 유지율과 유 단백율은 y=0.2824x+2.2482(R2=0.3606), 유단백율과 총고형 분율은 y=0.8564x+5.9404(R2=0.6942)를 나타냈다. 따라서 젖소의 산유량과 유성분은 계절적 요인, 그리고 체세포수 는 산차와 산유능력에 영향을 받는 것으로 나타나 적절한 젖소의 산유량과 유질개선에 각별한 관리가 요구된다.

This study investigated the changes in appetite and behavior of cattle and pigs after foot-and-mouth disease (FMD) vaccination. This study involved ten calves and ten pigs, each divided into two groups of five animals. One group of each animal was vaccinated with an FMD vaccine (FMD-V), and the other group was used as a non-treated control (CON). Each animal’s appetite and behavior were observed before vaccination and for seven days post-vaccination. In the FMD-V groups, appetite and behavior scores during the seven days post-vaccination were significantly decreased compared to those in the CON groups. The only exception was the seventh day post-vaccination in the swine behavior scores.

In this study, swine and cattle farms located in Jeollanam-do were selected to analyze and evaluate the components of odorants in livestock facilities. In addition, a preliminary survey of the literature was conducted to establish a sampling and analysis method for phenol and indoles which are major components of odor emissions from livestock facilities, yet are not regulated by the laws. To establish a sampling and analysis method for phenol and indoles, Tedlar bag and Tenax-TA sorbent tube was used as background concentration of blank sample and samples according to the elapsed time. The results obtained indicate the GC/MS analysis with Tenax-TA sorbent tube sampling was an effective method for measuring the compounds of phenol and indoles. In the swine facility, the rankings of the odorants in order, from highest to lowest, were ammonia, sulfuric compounds, phenol/indoles, volatile fatty acids. The main odorants were hydrogen sulfide (41.3%) and 4-methylphenol (p-cresol, 13.9%). In the swine slurry storage, hydrogen sulfide (33.7%), ammonia (18.8%), and 3-methylindole (skatole, 15.7%) were the main odorants, and hydrogen sulfide (31%) and i-valeric acid (32.4%) were the main odorants in the cattle farms.

This study aimed to indentify single nucleotide polymorphisms (SNPs) in exon region of the glycerol-3-phosphate dehydrogenase 1 (GPD1) gene and to evaluate their associations with meat yield and quality traits in Hanwoo (Korean cattle). Two SNP markers, g.2766C>T and g.5105A>G were identified in the exons 5 and 8, respectively. Genotyping of the two SNPs was carried out using PCR-RFLP analysis in 309 Hanwoo steers to evaluate their associations with meat yield and quality traits. As a result, g.2766C>T in exon 5 was significantly associated with carcass weight (CW) and marbling score (MS). Animals with the CC genotype of g.2766C>T had heavier CW than those with the CA or AA genotype. Animals with the CC genotype of g.2766C>T also had higher MS than those with the CA or AA genotype. Additive effect was also observed with CW and MS traits. We constructed haplotypes by linkage disequilibrium analysis and analyzed association between haplotypes and meat yield and quality traits. Haplotype of GPD1 gene was associated with CW. As a result, animals with CA haplotype had heavier CW than TG haplotype. These findings suggest that the SNPs of bovine GPD1 gene may be a useful molecular marker for selection of meat yield and quality traits in Hanwoo.

CRISPR/Cas9-induced knock-out/-in can be occurred at specific locus in the genome by non-homologous end joining (NHEJ) or homology directed repair (HDR). Here, we demonstrate the targeted insertion into the specific loci of embryo fertilized by semen from transgenic cattle via CRISPR/Cas9 system. Recently, we published on the efficient generation of transgenic cattle using the DNA transposon system (Yum et al. Sci Rep. 2016 Jun 21;6:27185). In the study, eight transgenic cattle were born following transposon-mediated gene delivery system (Sleeping Beauty and Piggybac transposon system) via microinjection. In the analysis of their genome stability using next-generation sequencing, there was no significant difference in the number of genetic variants between transgenic and non-transgenic cattle. All the transgenic cattle have grown up to date (the oldest age: 33 months old, the youngest age: 15 months old) without any health issue. One of transgenic male cattle expressing GFP reached puberty and semen was collected. Over 200 frozen semen straws were produced and some were used for in vitro fertilization (IVF). On seven days after IVF, expression of GFP was observed at blastocyst stage and was seen in 80% of the embryos. Another application is to edit the GFP locus of the transgenic cattle because long-term and ubiquitous expression of transgene didn’t affect their health. In one cell stage embryos produced using GFP frozen-thawed semen, microinjection of sgRNA for GFP, Cas9, together with donor DNA that included RFP and homology arms to link the double-strand break of sgRNA target site into fertilized eggs resulted in expression of RFP. This indicated that the GFP locus of transgenic cattle shows potential candidates for stable insertion of the functional transgene. Knock-out/-in for editing GFP locus using CRISPR-Cas9 might be a valuable approach for the next generation of transgenic models by microinjection. In conclusion, we demonstrated P-112 that transgenic cattle via transposon system are healthy to date and germ-line competence was confirmed. The GFP locus will be used as the potential target site for future gene engineering via genome-editing technology. Finally, all those animals could be a valuable agricultural and veterinary science resource for studying the effects of gene manipulation on biomedical research and medicine. This work was supported by BK21 PLUS Program for Creative Veterinary Science and Seoul Milk Coop (SNU 550-20160004).

The CRISPR/Cas9 system is proved to be a powerful tool for knock-out and knock-in in various species. By introducing genetic materials of two components (Cas9 and small guide (sg) RNA) into cells or pronuclear of the fertilized embryo, gene editing occurs. Some studies reported that efficiency of gene editing would be increased as Cas9 was integrated into cells or animals since Cas9 is indispensable in the CRISPR/Cas9 system. Accordingly, the production of Cas9 expressing cattle may provide the broadly used gene editing platform in cattle. For this study, Cas9 and RFP genes were cloned into PiggyBac (PB) transposon system. PB-Cas9-RFP and transposase were microinjected into 1436 in vitro fertilized embryos and 241 blastocysts were formed. Blastocysts with RFP expression accounting for 14.1% of total formed blastocysts were selected and transferred into 5 recipient cow. After gestation periods, four transgenic cattle were delivered without any veterinary assistance. From a transgenic cattle, ear skin tissue was collected for primary culture. On those primary cells, sgRNAs in DNA form for various genes such as PRNP, RB1 and BLG were transfected as 2ug of sgRNA per 5x105 cells using Nucleo factor system (Neon®, invitrogen, program#16). As expected, every group of each sgRNA delivered was confirmed to be mutated by T7E1assay. Those data demonstrated that for the first time, transgenic cattle with Cas9 expression were born, grown up to date and will be avaluable resource for genome-editing in cattle. This work was supported by BK21PLUS Program for Creative Veterinary Science and Seoul Milk Coop (SNU550-20160004).

Jeju Black Cattle (JBC) is an indigenous species of Korea and their mass production and industrialization are required for this high quality indigenous species. For production of elite JBC zygotes, selection of high quality sperm is necessary for in vitro fertilizatioin. In this study, we compared the sperm fertility and developmental capacity of IVF embryos using various JBC sperm (Bull A, B and C). The frozen semen was thawed and confirmed sperm viability and motility. In addition, frozen-thawed sperm was used for a chlorotetracycline(CTC) staining assay and in vitro fertilization. Sperm were classified into three staining patterns. The F pattern is indicative of uncapacitated sperm, the B pattern is indicative of capacitating and capacitated sperm and the AR pattern is indicative of acrosome-reacting sperm or acrosome-reacted sperm, respectively. Several kinds of JBC sperm was inseminated in 44 ㎕ IVF drop contained 10 oocytes with sperm concentration of 1 × 106 cells/ml, and then 2 ㎕ heparin and 2 ㎕ PHE (20 μM penicillamine, 10 μM hypotaurine, 2 μM epinephrine) were added. The sperm viability and motility were higher in sperm 3 species (n=8). When we confirmed sperm capacitation, F pattern and B pattern rate were higher than AR pattern in sperm A group. After IVF, the rates of cleavage and blastocyst development were higher in sperm C group compared to other sperm group. However, the cell number of blastocyst was higher in sperm E group. These results demonstrate that the use of sperm C was effective in production of elite JBC IVF embryos. Additional experimental data are required for more accurate analysis.

High reproductive efficiency is a prerequisite for dairy animals in order to optimize dairy production. An accurate and early pregnancy diagnosis is a crucial aspect for better reproductive management in livestock. The indigenous, visual and clinical methods have various limitations including accuracy, sensitivity, specificity, later stages of applicability and requires highly skilled manpower. This hurdles the researchers to ensue further research on developing novel early pregnancy diagnostics for dairy animals. However, the advantage of molecular techniques like proteomics has given a new hope to look for pregnancy biomarkers in animal research. In this study, we assayed the pregnancy-associated glycoprotein (PAG) levels using anti-bPAG antibody. Serum plasma samples were collected 30, 60, 90 and 120 days after insemination. Cattle diagnosed pregnant with singleton are used for this experiment in which blood samples were collected. The plasma PAG level is gradually increased after insemination to until 60 days and drastically decreased after that. The PAG level was approximately 2-fold greater during 30 days compared with 60 days. These results concluded to validate our other pregnancy diagnosis methods including circulating microRNA and cell free DNA concentration (data not shown). Despite this proteomic approach, validating all other molecular technique results may give the exact time for early pregnancy diagnosis.

This study was conducted to report the census survey results of south Korean Chikso (Korean Brindle Cattle) distribution and to diagnose the current coat color expression patterns. Two years Chikso census showed that there were 2,413 Chikso in 2013 and 2,754 in 2014 in south Korea. Number of animals between 0 to 35 months of age was 1,632 heads in 2014 which comprised 59% of total Chikso population size. The percentages of animals between 36 ~ 71 months of age and over 6 years of age were 31% and 10% of total. Out of 2,416 animals with class numbers assessed by their coat colors, 1,249 heads (52%) were in group I (with black stripes, class no. 1 ~ 3) and the others (class no. 4 ~ 7) were in group II (1,167 heads, 48%). Among the 1,551 animals that were photographed twice in 2013 and in 2014, 226 animals were assessed different coat color class numbers, and round 90% of which were within the ages less than 48 months. The number of animals switched in coat color pattern groups over a year was 56, which was 3.6% of total number of animals on survey. And around 88% of the animals switched in group category was of the animals younger than 24 months of age. Therefore, we conclude that the coat color pattern becomes rather stable at around 24 months of age in Chikso populaion in south Korea.

The Korean native cattle, Hanwoo, is the most popular breed of beef cattle in Korea. However, the reproductive performance data are limited although reproduction is one of the most economically and biologically important in beef production. Therefore, this study was undertaken to investigate reproductive performance parameters including calving interval, parity for life time production. Data collected from 206,827 calvings were analyzed. There were no significant differences in calving interval and gestation days as parity increased from 2nd and 13rd parity cow, from spring to winter. However, we found a dramatic increase in calving interval after year 2000. About 1 month were increased per year ( y = 30.578x + 344.45 R² = 0.9157). Interestingly, we observed that parities for life time can be affected by birth weight. Calves with 23 kg at birth showed highest parities, 3.4±2.0 times. In summary, this study provides valuable data on reproductive performance of Hanwoo and the data presented here can be used as a standard target for optimising and enhancing reproductive performance.

Wholesale beef price is the critical factor for determining Korean native cattle, Hanwoo, farm’s income in short-term. Wholesale beef price has seasonality due to high demand in Korean traditional holidays such as Korean thanksgiving day and lunar new year’s day. Therefore, it is important to make reproduction and marketing plans for Korean Hanwoo farmers, in order to increase their farm income. However, there is no study available on changes in the expected farm income depending on reproduction and marketing schedules. This study analyzed the expected farm income per head depending on the monthly-based marketing schedules. The analysis was conducted based on the seasonality of wholesale beef price, reproduction efficiency, operating costs, relationship between carcass grade and slaughter age. The result shows that slaughter Hanwoo at the age of 29 months-old in August and January generating the highest expected farm income per head.

The aim of the present study was to evaluate and estimate timing of artificial insemination (AI) in Hanwoo heifer (Korean native cattle) that is the most popular breed of beef cattle in Korea. To determine changes in body weight of heifers around AI, body weight were measured at different stages either before or after AI. We found that daily body weight gain was higher in the pregnant cows after AI. We also investigate correlation between body mass measured by shoulder height and body length, and conception rates, used (body length+ height)2 instead of height2 for body mass index (BMI), and found that relatively more BMI heifers (>55) showed higher conception rates. Finally, we estimated body weight by measuring should height (SH), heart girth (HG), and body length (BL); BW=3.93372*HG-2.90985*SH-0.021*BL. In addition, we observed that HG is most closely correlated with BW; y(BW) = 1.77355*x(HG), R² = 0.98881. In summary, we can determine the best timing of AI using body measurement and its application including BMI.