Prolactin is an anterior pituitary hormone involved in various physiological phenomenon including reproduction. The prolactin receptor (PRLR) is detected in diverse tissues such as brain, ovary, placenta and uterus in several mammalian species. A total of 227 pigs [Korean native pigs (KNP) 27; Landrace pigs 29; Korean native pigs x Landrace F1 91; Nanchuckmacdon pigs 80] were used to investigate the allele frequency difference of the prolactin receptor (PRLR) gene among the four pig lines. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with Alu I restriction enzyme was used to determine the genotypes of PRLR. Frequencies of PRLR alleles among the four different pig lines were not significantly different (Chi-square=3.94, DF=3, P=0.27). A total of 40 Nanchuckmacdon pigs were used to investigate the effect of the prolactin receptor (PRLR) gene on total number of piglets born (TNB), number of piglets of alive (NBA) using general linear model implemented in MINITAB software. For TNB, the AB genotype had higher genotypic value (10.61) than the values of AA (9.83) and BB (10.30). Likewise, the AB genotype had higher genotypic value (8.96) than the values of AA (8.18) and BB (8.90) for NBA. However, these associations of the PRLR gene with TNB and NBA were not statistically significant. In conclusion, it is necessary to increase the sample size for investigating the effect of the PRLR gene on TNB and NBA in pigs.

Blood types in pigs are divided into two types, A and O type. It is important to select and breed O-type pigs that have no hematologic rejection, because the blood type of the donor is an important factor causing immune rejection in xenotransplantation. Therefore, the gene that determines the blood type is GGTA1(glycoprotein, alpha-1,3-galactosyltransferase 1), which generally belongs to the ABO blood group. This study was carried out to develop a simple and accurate method by analyzing the structure of GGTA1 gene which determines blood type. A primer was designed to allow easy identification of the blood type of the target in the first and second deletion regions of intron 7 to distinguish between the A and O genotypes. That is, for the purpose of identifying the blood type using length difference after the PCR, the forward and reverse primers were designed in the highly conservative region. As a result, a pair of primers were prepared and PCR amplification was performed to distinguish three types of genotypes, AA, AO, and OO, using the length difference by electrophoresis. Using the above primers, the parents and their offspring were compared with each other to confirm the correct genetic pattern. And, in four pig breeds, the genes were amplified and the genotype could be correctly identified. In this study, we could diagnose the blood type of AA, AO, OO genotype of pigs by using primer of INDEL region. Especially, since it is possible to diagnose the genotype by the length difference, it is possible to diagnose it quickly and accurately from the gene amplification to the genotype reading

Spermatozoa viability can be assessed by microscopy, flow cytometry, and other methods using fluorescent stain. Flow cytometry can be used to examine the morphological and functional characteristics of spermatozoa in a short time. The purpose of this study was to compare the viability of cryopreserved spermatozoa in Jeju black cattle by two dual fluorescent stain methods. Semen of Jeju black cattle raised in Subtropical Livestock Research Institute, National Institute of Animal Science, RDA were collected with artificial vaginal technique. Sperm was diluted with Triladyl®-egg yolk diluent and then was performed cryopreservation.There was no significant difference in viability of spermatozoa according to the two dual fluorescent stain methods. However, when the distribution of spermatozoa according to the staining method was compared, the spermatozoa group stained with 6-CFDA/PI was more clearly distinguished than the spermatozoa group stained with calcein AM/PI.

An intensive analysis was conducted of the maternal lineages of the Jeju native pigs (JNPs). A total of 100 mtDNA sequences from Asian wild boars (AWB), European wild boars (EWB), Asian domestic pigs (ADP), European domestic pigs (EDP), and JNPs were used for the phylogeny and network analyses. Two distinct JNP groups were found JNPA and JNPE in the Asian and European cluster. The maternal lineage of JNPE was the closest to that of EWB and a clear haplogroup sharing an identical haplotype (hap16) among 15 individuals of JNPE and 2 individuals of EWB was identified. However, except for hap18, no EDP shared any identical haplotypes with JNPE, suggesting that no obvious maternal contribution of EDP has occurred in JNPE in recent years. The possible existence of an additional and unknown path of maternal lineage from EDP into JNPE could therefore be postulated, in addition to those from AWB and ADP into the JNPA groups. Thus, JNPE appeared to have a pure maternal lineage that had no recent contact with EDP, and both the JNPA groups and JNPE are pure Jeju native pigs.

Alterations affecting the status of robustness and health can bring about physiological changes including hematological parameters in pigs. To identify quantitative trait loci (QTL) associated with 8 hematological phenotypes (one leukocyte trait, six erythrocyte traits, and one platelet trait), we performed a genome-wide association study using the Porcine SNP 60K BeadChip in an intercross population between Landrace and Korean native pigs. A total of 36,740 SNPs from 816 F2 offspring were analysed for each blood related traits after filtering by quality control. Data were analysed genome-wide rapid association using the mixed model and regression (GRAMMAR) approach. A total of 257 significant SNPs (P<1.36x10-6) on SSC3, 6, 8, 13, and 17 were detected for blood related traits in this study. Interestingly, the genomic region between 17.9 and 130 Mb on SSC8 was found to be significantly associated with RBC, MCV, and MCH. Our results include 5 significant SNPs within five candidate genes (KIT, IL15, TXK, ARAP2, and ERG) for hematopoiesis. Further validation of these identified SNPs could give valuable information for understanding the variation of hematological traits in swine.

Previously, we reported a quantitative trait locus (QTL) that affect total teat number (TTN) on pig chromosome 7 (SSC7) in a large F2 intercross population between Landrace and Korean native pigs. The aim of this study was to refine the QTL associated with TTN and to identify positional candidate gene(s) within the refined genomic region. TTN was recorded in 1,105 F2 progeny. All experimental animals were genotyped using 998 informative single nucleotide polymorphism (SNP) markers located on SSC7. A haplotype-based linkage and association analysis using the PHASEBOOK programme was applied to perform high-resolution QTL analysis. Additionally, linear mixed-effect models were used to assess the effect of a positional candidate gene on TTN and other economically important traits [i.e., thoracic vertebrae number (THO), carcass body length (CBL) and weight (CW), back fat thickness (BFT) and intramuscular fat content (IMF) in loin muscles]. Joint linkage and association analysis refined the critical region to a 1.07 Mb region that included a novel positional candidate gene, BRMS1L, that encodes the breast cancer metastasis-suppressor 1-like protein, which could possibly be implicated in normal mammary gland development. Significant association of an SNP marker (g.-1087 G>A) in the 5’-flanking region of BRMS1L with TTN (P=1.10x10-8), THO (P=5.80x10-4), and CBL (P=0.038) was observed. Based on these data, we propose BRMS1L as a positional candidate gene for TTN in pigs. After validation of the association in other independent populations and further functional studies, these results could be useful in optimizing breeding programmes that improve TTN and other economically important traits in swine

In the swine industry, growth related traits are important economic traits directly linked to profitability. Representative growth traits include daily gain, back fat thickness, and carcass weight. This study was conducted to search for positional candidate genes associated with the carcass weight through a genome-wide association study(GWAS) using suggestive levels of statistical thresholds in pigs. As a result of the genome-wide analysis of the associations with carcass weight, the single nucleotide polymorphism(SNP) markers with suggestive significance were identified in 1 SNP marker on chromosome 2(ALGA0015365) and 1 SNP marker on chromosome 4(ALGA0023678). We could select positional 2 candidate genes, located close to the SNP markers with suggestive significance levels. The SNP markers in adjacent to the 2 genes(LOC100519538, LOC100737583) may provide basic data regarding the marker-assisted selection for the carcass weight trait in pigs.

The aim of this study was to identify quantitative trait loci (QTLs) influencing teat number traits in an F2 intercross between Landrace and Korean native pigs (KNP). Three teat number traits (left, right, and total) were measured in 1105 F2 progeny. All experimental animals were genotyped with 173 informative microsatellite markers located throughout the pig genome. We detect that seven chromosomes harbored QTLs for teat number traits: genome regions on SSC1, 3, 7, 8, 10, 11, and 13. Six of fourteen identified QTL reached genome-wide significance. In SSC7, we identified a major QTL affecting total teat number that accounted for 5.6% of the phenotypic variance, which was the highest test statistic (F-ratio = 61.1 under the additive model, nominal P = 1.3×10-14) observed in this study. In this region, QTL for left and right teat number were also detected with genome-wide significance. With exception of the QTL in SSC10, the allele from KNP in all 6 identified QTLs was associated with decreased phenotypic values. In conclusion, our study identified both previously reported and novel QTL affecting teat number traits. These results can play an important role in determining the genetic structure underlying the variation of teat number in pigs.

Using a partial D-loop sequence of mtDNA, a comprehensive molecular evolutionary analysis was performed of the maternal lineages of the Jeju native pigs(JNPs) that presented in Jeju Island. A total of 100 DNA sequences from Asian domestic pigs(ADP), European domestic pigs(EDP), Asian wild boars(AWB), European wild boars(EWB), and JNPs were used for the molecular evolutionary analyses including phylogeny and network analyses. The most fitted model for the phylogenetic analysis was determined using hierarchical likelihood-ratio tests conducted by MrModeltest implemented in the PAUP package. A consensus tree was established from 5,000,000 iterations using the MR BAYS program. Three recognizable JNP groups–one(JNPE) in the European cluster and two(JNPA1 and JNPA2) in the Asian cluster–were detected in the estimated phylogenetic tree and network. The maternal lineage of JNPE was the most adjacent to that of EWB and a clear haplogroup sharing an identical haplotype(hap16) among 15 individuals of JNPE and 2 individuals of EWB was detected.

Growth traits, such as body weight, directly influence productivity and economic efficiency in the swine industry. In this study, we estimate heritability for body weight traits usinginformation from pedigree and genome-wide single nucleotide polymorphism (SNP) chip data. Four body weight phenotypes were measured in 1,105 F2 progeny from an intercross between Landrace and Jeju native black pigs. All experimental animals were subjected to genotypic analysis using PorcineSNP60K BeadChip platform, and 39,992 autosomal SNP markers filtered by quality control criteria were used to construct genomic relationship matrix for heritability estimation. Restricted maximum likelihood estimates of heritability were obtained using both genomic- and pedigree- relationship matrix in a linear mixed model. The heritability estimates using SNP information were smaller (0.36-0.55) than those which were estimated using pedigree information (0.62-0.97). To investigate effect of common environment, such as maternal effect, on heritability estimation, we included maternal effect as an additional random effect term in the linear mixed model analysis. We detected substantial proportions of phenotypic variance components were explained by maternal effect. And the heritability estimates using both pedigree and SNP information were decreased. Therefore, heritability estimates must be interpreted cautiously when there are obvious common environmental variance components.

This study tested the association between genotypes of the single nucleotide polymorphism (SNP) marker, rs81437607 and capric acid (FA_C10_0) compositions in longissimus dorsi muscle in pigs. Eighteen fatty acid (FA) compositions were measured in a total of 974 F2 animals among 1,106 F2 progeny produced between Landrace and Jeju Black Pig (JBP). Among FA compositions tested, we identified a cluster of highly significant SNPs for capric acid compositions on 58 Mb position of Sus scrofa chromosome 12 (SSC12) using genome-wide association study (GWAS) with F2 genotypes from SNP panel analysis. GWAS results showed that the rs81437607 was the highest trait-related SNP marker with capric acid levels. Three genotypes (C/C, C/T and T/T) of rs81437607 marker were found in F2 population by further pyrosequencing. Association analysis results showed the significant differences between rs81437607 genotypes and capric acid compositions (P<0.05). The F2 pigs harboring rs81437607 C/C (0.119±0.002%) and C/T (0.116±0.002%) genotypes showed additively higher levels of capric acid content than those of T/T homozygotes (0.109±0.002%) (P=1.30×10-12). These results suggested that the genetic variations of rs81437607 may be helpful to find causative variants and assist as molecular genetic markers for improving the capric acid contents in longissimus dorsi muscle in pigs.

We developed a polymerase chain reaction (PCR)-based molecular method for sexing and identification using sexual dimorphism between the Zinc Finger-X and -Y (ZFX-ZFY) gene and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for mitochondrial DNA (mtDNA) cytochrome B (CYTB) gene in meat pieces and commercial sausages from animals of different origins. Sexual dimorphism based on the presence or absence of SINE-like sequence between ZFX and ZFY genes showed distinguishable band patterns between male and female DNA samples and were easily detected by PCR analyses. Male DNA had two PCR products appearing as distinct two bands (ZFX and ZFY), and female DNA had a single band (ZFX). Molecular identification was carried out using PCR-RFLP of CYTB gene, and showed clear species classification results. The results yielded identical information on the sexes and the species of the meat samples collected from providers without any records. The analyses for DNA isolated from commercial sausage showed that pig was the major source but several sausages originated from chicken and Atlantic cod. Applying this PCR-based molecular method was useful and yielded clear sex information and identified the species of various tissue samples originating from livestock.

본 연구는 교잡돈의 출하일령은 약 200일 이고, 175두의 돼지를 도축하여 3개의 그룹(59 ~ 95kg, 96 ~ 110kg, 111 ~ 148kg)으로 나누어, 등심, 상완세갈래근, 대퇴두갈래근, 반막모양근의 육질 특성을 조사하였다. 콜라겐과 지방함량은 반막모양근, 대퇴두갈래근이 111 ~ 148kg 그룹에서 다름 그룹에 비 해 낮은 함량을 나타내었으며(p<0.05), 단백질함량은 반막모양근, 대퇴두갈래근과 상완세갈래근이 111 ~ 148kg 그룹에서 다른 그룹에 비해 높은 함량을 나타내었다(p<0.05). 등심과 반막모양근의 명도는 111 ~ 148kg 그룹에서 높게 나타내었으며(p<0.05), 적색도와 황색도는 유의적인 차이가 나타나지 않았 다(p>0.05). pH는 등심에서 체중이 증가함에 따라 낮은 값을 나타냈었다(p<0.05). 가열감량은 등심, 반 막모양근, 대퇴두갈래근, 상완세갈래근이 111 ~ 148kg 그룹에서 보다 59 ~ 95kg 그룹에서 낮게 나타 났으며(p<0.05), 보수력은 반막모양근과 대퇴두갈래근에서 체중이 증가할수록 높게 나타났다(p<0.05). 전단력은 등심과 상완세갈래근에서 체중이 증가할수록 낮게 나타났다(p<0.05). 등심의 지방산 분석 결 과는 96 ~ 110kg 그룹에서 oleic acid, MUFA, MUFA/SFA가 높았고(p<0.05), 111 ~ 148kg 그룹에서 trans-vaccenic acid와 SFA가 높았지만(p<0.05), UFA는 낮았다(p<0.05).등심의 관능평가는 연도와 다 즙성이 96 ~ 110kg 그룹에서 높은 점수를 받았다(p<0.05).

The aim of this study was to develop the production of crossbred pigs suitable to the Korean consumers’demand. Crossbred (Jeju native pig × Landrace) pigs (n=175) at the age of 200 days, approximately classified into three slaughter weight groups (59~95 kg, 96~110 kg and 111~148 kg) were used to investigate the carcass traits and retail cuts characteristics. We have chosen longissimus dorsi, triceps brachii, biceps femoris, semimembranosus muscles, shoulder butt, thoracic vertebra (5-11th) and last thoracic vertebra for investigations of aforementioned parameters. The results showed that the 111~148 kg group had larger loin area, higher scores for meat color, quality and carcass grade than the other remaining groups (p<0.05). Weights of shoulder butt, rib, picnic shoulder, loin, belly, tenderloin, ham retail cuts, lean meat, fat and bone increased with increasing carcass weight (p<0.05). Ratios of shoulder butt, picnic shoulder, tenderloin, ham, lean meat, skin and bone in the 59~95 kg group were higher than that of the other groups (p<0.05). Ratios of belly in the 96~110 kg group were higher than that in the 59~95 kg group (p<0.05) but similar to the ratios of the 111~148 kg group. Total length, longest length and shortest length of belly increased with increasing carcass weight (p<0.05). Additionally, the longest width and shortest width of belly in 59~95 kg group were lower (p<0.05) than that in the other remaining groups. Length of shoulder butt increased with increasing carcass weight (p<0.05) whereas, the width of shoulder butt showed no differences between the groups. Weights of triceps brachii of picnic shoulder, biceps femoris and semimembranosus muscles of ham increased with increasing carcass weight (p<0.05). Overall, the crossbred pigs of 111~148 kg group possessed better quality and carcass grade however, the yield of belly in 96~110 kg group was higher than the other groups. Based on the consumption preference of Korean consumers therefore, the slaughter weight of crossbred pigs at ≥96 kg could produce higher economic benefit.

Precise, rapid and simple methods for species identification in animals are among the most important techniques in the livestock industry and research fields including meat classification. In this study, polymerase chain reaction (PCR) based molecular identification using inter species polymorphisms were examined by PCR-restriction fragment length polymorphism (RFLP) analysis for mitochondrial DNA (mtDNA) cytochrome b (CYTB) gene sequences among four mammalian livestock animals (cattle, horse, goat and pig). The results from PCR-RFLP analysis using the AluI restriction enzyme were also provided for the species-specific band patterns among CYTB gene sequences in these four species. The AluI-digestion for CYTB genes provided interesting migration patterns differentially displayed according to each species. Cattle and horse had one AluI-recognition site at different nucleotide positions and their AluI-digested fragments showed different band patterns on the gels. Pig had two AluI-recognition sites within the amplified CYTB sequences and produced three bands on the gels. Goat had no AluI-recognition site and was located at the same position as the uncut PCR product. The results showed the species-specific band patterns on a single gel among the four livestock animal species by AluI-RFLP. In addition, the results from blind tests for the meat samples collected from providers without any records showed the identical information on the species recorded by observing their phenotypes before slaughter. The application of this PCR-RFLP method can be useful and provide rapid, simple, and clear information regarding species identification for various tissue samples originating from tested livestock species.

This study was carried out to examine a molecular marker system for parentage test in Jeju Black cattle (JBC). Based on the preliminarily studies, we finally selected for construction of a novel genetic marker system for molecular traceability, identity test, breed certification, and parentage test in JBC and its related industrial populations. The genetic marker system had eight MS markers, five indel markers, and two single nucleotide polymorphisms (SNPs; g.G299T and g.del310G) within MC1R gene which is critical to verify the breed specific genotypes for coat color of JBC differing from those of exotic black cattle breeds such as Holstein and Angus. The results showed lower level of a combined non-exclusion probability for second parent (NE-P2) of 4.1202×10-4 than those previously recommended by International Society of Animal Genetics (ISAG) of 5.000×10-4 for parentage, and a combined non-exclusion probability for sib identity (NE-SI) of 2.679×10-5. Parentage analysis has been successfully identified the JBC offspring in the indigenous population and cattle farms used the certified AI semens for production using the JBC-derived offspring for commercial beef. This combined molecular marker system will be helpful to supply genetic information for parentage test and traceability and to develop the molecular breeding system for improvement of animal productivity in JBC population.

The present study was to assess the in vitro viability and sexing rate of bovine embryos. Blastocysts were harvested on day 7～9 day after insemination(in vitro and in vivo), and the sex of the embryos was examined using the LAMP method. Embryo cell biopsy was carried out in a 80 μl drop Ca2+, Mg2+ free D-PBS and, biopsied embryos viability were evaluated after more 12 h culture in IVMD culture medium. The formation of recovered embryo to expanded and hatching stages had ensued in higher of sexed embryo in vivo than in vitro (100% vs. 89%, p<0.05), and in vitro, the rates of degeneration after sexing were significantly (p<0.05) higher in vitro than in vivo(11% vs. 0.0%). The rates of the predicted sex were female 61% vs. 56%, and male 39% vs. 44% in vivo and in vitro, respectively. The rates of survival following different biopsy methods were seen between punching and bisection method in vivo and in vitro (100% vs. 89% and 100% vs, 78% respectively). Biopsy method by punching was significantly (p<0.05) higher than bisection between produced embryos in vivo and in vitro. The present data indicate that with microblade after punching for embryo sexing results in high incidence of survivability on development after embryo biopsy. It is also suggested that LAMP-based embryo sexing suitable for field applications.

The aim of this study was to evaluate the effect of α-tocopherol on blastocysts development and subsequent cryosurvival of the vitrification. The α-tocopherol(0, 100, 200, 400 μM) was added in to culture medium for the bovine embryos. The blasocysts from the α-tocopherol and untreated control groups were then frozen-thawed, and their cryosurvival was assessed by in vitro culture for 48 h. There were no differences in the overall cleavage rate(56.14±4.66, 58.18±4.70, 62.97±6.86 and 51.17±7.28) among four treatment groups. However, in blastocyst development and total cell number were significantly higher in α-tocopherol 200 μM(38.60±7.12; 106.33±3.50) to culture medium than other treatment groups(29.30±5.24, 31.60±7.12 and 26.37±4.18; 101.36±5.12, 97.27±2.87, and 91.23±7.52 respectively). Before and after vitrification, the total cell number and blastocyst development of embryo were significantly higher in July to August than September to October. In conclusion, addition of α-tocopherol 200 μM to in vitro bovine embryo culture medium was beneficial for improving embryo quality by decreasing the embryo damage blsstocysts cell number and improving the tolerance of the embryos to cryopreservation.

한우 이력추적제에 적용되는 11개의 MS marker (TGLA227, BM2113, TGLA53, ETH10, SPS115, TGLA126, TGLA122, ETH3, ETH225, BM1824 and INRA23)와 성감별을 위한 2개의 sexing primer로 조합된 하나의 Multiplex PCR set를 이용하여 모근에서 추출한 genomic DNA를 이용해 3510두의 대량 시료를 분석한 결과 3.93%의 genotyping 실패율로 성공적인 분석결과를 얻었다. 무작위교배집단으로 가 정 시 동일개체출현확률 (PI)은 1.31×10-23, 반형매교배집단으로 가정 시 동일개체출현확률 (PIhalf-sibs)은 2.52×10-16 그리고 전형매교배집단으로 가정 시 동일개체출현확률 (PIsibs)은 1.09×10-6으로 나타나 현재 사용 중인 11종의 MS marker는 범용적으로 사용하여도 무방할 것으로 재확인 되었다. 또한 생산 및 사 육단계의 생우의 경우 모근을 이용하여 DNA를 추출하는 것은 시료 채취 시 소에게 주어지는 스트레스를 최소화 시킬 수 있을 뿐만 아니라 대립유전자형 분석에 있어서 시간적, 경제적인 효율성을 높일 수 있었 다. 또한 모근 채취 부위 중 등, 배, 꼬리상부와 꼬리하부를 이용하여 검정한 결과 꼬리하부의 모근을 이 용하여 5~13가닥을 사용했을 때 최적의 분석결과를 보였다. 최종적으로 한우의 사육단계 대량 유전자형 분석에 적용 가능한 96 well 단위를 기본으로 하는 모근 DNA분리 체계를 확립하였다.

The Korean native pig (KNP) have been considered as animal models for animal biotechnology research because of their relatively small body size and their presumably highly inbred status due to the closed breeding program. However, little is reported about the use of KNP for animal biotechnology researches. This study was performed to establish the somatic cell nuclear transfer (SCNT) protocol for the production of swine leukocyte antigens (SLA) homotype-defined SCNT KNP. The ear fibroblast cells originated from KNP were cultured and used as donor cell. After thawing, the donor cells were cultured for 1 hour with 15 μM roscovitine prior to the nuclear transfer. The numbers of reconstructed and parthenogenetic embryos transferred were 98 ± 35.2 and 145 ± 11.2, respectively. The pregnancy and delivery rate were 3/5 (60%) and 2/5 (40%). One healthy SLA homotype-defined SCNT KNP was successfully generated. The recipient-based individual cloning efficiency ranged from 0.65 to 1.08%. Taken together, it can be postulated that the methodological establishment of the production of SLA homotype-defined cloned KNP can be applied to the generation of transgenic cloned KNP as model animals for human disease and xenotransplantation researches.