The ovum pick up(OPU) technique can be used to produce embryos after in vitro culture of ovarian oocytes, can be used for early securement for effective herd early proliferation and excellent Hanwoo genetic resources, It is attracting attention as a very important technique for establishing technology. In addition to in vitro culture techniques, the number of oocytes retrieved depends on the environment and timing of the OPU. This study was conducted to compare and examine seasonal effect to the differences in the number of recovered oocytes, recovery rate and embryo development rate using Korean cattle kept in animal genetic resource research center by OPU technique. The grade of COCs was evaluated by microscopic examination. Grade A had 3 or more layers of cumulus cell and compact cytoplasm. Grade B had 1~3 layers of cumulus cell and compact cytoplasm. Grade C had 1 layers cumulus cell and compact cytoplasm. Grade D was denuded oocyte and poor cytoplasm. The recovery rate was 47.8±3.4% in summer (June to August) and 51.6±3.8% in autumn (September to October). The number of oocytes was 5.7±0.6 in summer and 5.2±0.7 in autumn. Oocyte grade A and B was 46.2%±6.3% in summer and 51.1±5.0% in autumn. The cleavage rate was 46.1±7.1% in summer and 65.0±11.3% in autumn. Blastocyst development rate was 19.9±9.4% in summer and 29.0±8.7% in autumn. There was no difference the recovery rate of oocytes and the number of embryos between summer and autumn. Cleavage rate and blastocyst rate of autumn was higher than summer. we will investigate to study the appropriate method for the production of Hanwoo embryos and the systematic comparison.

Cryopreservation of boar semen is continually researched in reproductive technologies and genetic resource banking in breed conservation. For evaluating the boar semen quality, sperm motility (MOT) is an important parameter because the movement of spermatozoa indicates active metabolism, membrane integrity and fertilizing capacity. Recently, polymorphisms reported to be significant association with sperm MOT. This study was conducted to evaluate the SNP in the coding region of ESR1 (g.672C>T inexon 1) as a positional controlling for motility and kinematic characteristics of post-thawed boar semen. To results,The g.672C>T was significantly associated with frozen semen motility and kinematic characteristics. g.158 T>C SNP was high significantly associated with MOT, VCL, VSL and VAP Also, the SNP was low significantly associated with ALH.Therefore, we suggest that theSNP in the coding region of ESR1 (g.672C>T in exon 1) may be used as a molecular marker for Duroc boar Post-thawed semen quality.

Estrogen receptor 2 (ESR2) is involved in oestrogen-related apoptosis in cell cycle spermatogenesis but their effects have not yet confirmed in pig. Therefore, this study was performed to investigatetheir association with semen motility and kinematic characteristics. DNA samples from 126 Duroc pigs with records of sperm motility and kinematic characteristics [Total motile spermatozoa (MOT), Curvilinear velocity(VCL), Straight-line velocity(VSL), the ratio between VSL and VCL(LIN), Amplitude of Lateral Head displacement(ALH)] were used in present study. A single nucleotide polymorphism (g.35547A>G) was associated with MOT, VCL, VAP and ALH in Duroc population (p < 0.05). Therefore, we suggest that the porcine ESR2 may be used as a molecular marker for Duroc boar semen quality, although its functional effect was not clear yet. These results will improve the understanding of the functions of the ESR2 in spermatogenesis within the reproductive tracts, and will shed light on ESR2 as a candidate gene in the selection of good sperm quality boars.

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

Cryopreservation of bovine embryos is used to efficiently implant surrogate mothers. It has been widely accepted that high lipid content in the oocyte interrupts its survival during freeze-thaw cycles. Serum component in the culture medium is thought to increase the embryo`s lipid contents. Conversely, L-carnitine stimulates lipid metabolism by transporting long chain fatty acids into the mitochondria. Objective of this study was to analyze the effect of L-carnitine supplementation in IVM medium and defined IVC medium on the development, lipid contents and the cryosurvival of bovine IVF embryos. 0.0, 1.5, 3.0 and 6.0 mM L-carnitine was supplemented in IVM medium, respectively (IVM-LC 0.0, LC 1.5, LC 3.0 and LC 6.0). Development rate from the 2cell to the morula stages was higher in IVM-LC 3.0 groups than those of IVM-LC 6.0 (p<0.05). But there were no significant differences among the other groups in the blastocyst rates and lipid content results. When 0.0, 1.5, 3.0 and 6.0 mM L-carnitine were supplemented in IVC medium (IVC-LC 0.0, LC 1.5, LC 3.0 and LC 6.0), development competence was not significantly different between those embryos. Lipid contents of embryos treated L-carnitine (IVC-LC 1.5, 3.0 and 6.0) were significantly lower than embryos of non-treated group. L-carnitine was supplemented 0.0, 1.5, 3.0, 6.0 mM during IVM and 3.0 mM during IVC (LC 0.0 - 3.0, LC 1.5 – 3.0, LC 3.0 – 3.0, LC 6.0 – 3.0) and cryosurvival of blastocysts confirmed after freezing-thawing. There were no significant differences on development, but LC 3.0 – 3.0 was significantly lower lipid contents than other groups. And LC 3.0 – 3.0 had better survival rates and hatched rates of blastocysts than LC 0.0 – 0.0. In conclusion, supplementation of L-carnitine in defined IVC medium decreases lipid contents. And L-carnitine supplementation improves cryosurvival and developmental ability of bovine IVF embryos.

To improve survival rates of vitrified pig oocytes, the treatment of cytoskeletal stabilizer on an appropriate time is one of the possible approaches. However, the exact treatment timing and effect of cytoskeletal stabilizer such as cytochalasin B (CB) is not well known during oocyte vitrification procedures. Thus, the present study was conducted to determine optimal treatment timing of CB during vitrification and warming procedures. In experiment 1, the survival rates of the post-warming pig oocytes were analyzed by fluorescein diacetate (FDA) assay with 4 classifications. In results, post-warming oocytes showed significantly (p<0.05) decreased number of alive oocytes (31.8% vs. 86.4%) compared to fresh control. In detail, the significant difference (p<0.05) was found only in strong fluorescence (18.2% vs. 70.5%) not in intermediate fluorescence groups (13.6% vs. 15.9%). In experiment 2, CB was treated before (CB-Vitri) and after (Vitri-CB) vitrification. In results, group of Vitri-CB showed significantly (p<0.05) higher (91.6%) survival rates compared to group of CB-Vitri (83.7%), significantly (p<0.05) and comparable with group of Vitri Control (88.7%) by morphological inspection. In FDA assay results, group of Vitri-CB showed significantly (p<0.05) higher (44.2%) survival rates compared to groups of CB-Vitri (36.7%) and Vitri Control (35.1%). In conclusion, the increased survival rates of post-warming pig oocyte treated with Vitri-CB method are firstly described here. The main finding of present study is that the CB treatment during recovery could be helpful to refresh the post-warming pig oocyte resulting its improved survival rates.

The early-onset familial Alzheimer's disease (EOFAD/ FAD), the less common type of Alzheimer's disease (AD) currently affects a vast number of individuals worldwide. This type is being inherited as an autosomal dominant fashion. Missense mutations on Amyloid precursor protein (APP) and Presenilins 1 and 2 (PSEN1 & PSEN2) are known as major genetic factors in FAD. Conversely, missense mutations on microtubule-associated protein tau (MAPT) are also thought to involve. Up to date, several triple-transgenic animal models with muted forms of the human APP, PSENs and MAPT have been reported. Compared to other animals, canines are more emotional and their disease signs can be easily diagnosed. This attempt was to develop a triple transgenic canine model for the AD. We have obtained the coding sequences of APP, PSEN1 and MAPT from Dana-Farber/Harvard Cancer Center DNA resource core at HMS and incorporated several common AD mutations. The transgenic construct is composed of hNSE (ENO2) promoter-driven three AD genes fused together with modified 2A sequences. It was transfected into the canine fetal fibroblasts which were then used to perform somatic cell nuclear transfer (SCNT). The viable transgenic embryos were obtained after in vitro culture and the GFP was detected. In this study, we have successfully produced viable triple transgenic canine cloned embryos using SCNT technique. These transgenic canine embryos will be further developed into canines with FAD. The transgenic canines will be a good candidate in the AD research field.

쌀가루의 활용도를 높이기 위해서 제과 품목 중에 쌀파이를 제조하였고, 쌀파이에 볶음땅콩가루, 새싹땅콩가루를 첨가하여 쌀파이의 품질 특성을 분석하였다. 수분함량은 밀 파이에 비해 쌀파이가 낮았고, 볶음땅콩가루, 새싹 땅콩가루 등 부재료를 첨가했을 때 수분함량이 낮게 나타났다. pH는 밀파이에 비해 쌀로 만든 파이에서 pH가 낮게 나타났으며, 볶음땅콩가루나 새싹 땅콩가루를 첨가하였을 때 pH가 높게 나타났다. 반죽의 밀도는 밀파이에 비해 쌀로 만든 파이에서 밀도가 낮게 나타났으며, 볶음땅콩가루나 새싹 땅콩가루를 첨가했을 때 쌀파이보다는 밀도가 높게 나타났다. 손실률은 밀파이에 비해 쌀로 만든 파이에서 높게 나타났으며, 볶음땅콩가루나 새싹 땅콩가루를 첨가했을 때 쌀파이보다는 손실률이 낮게 나타났다. 쌀로 제조한 파이에서 볶음땅콩가루와 새싹땅콩가루를 첨가했을 때 명도(L)와 황색도(b)는 감소하고, 적색도(a)는 증가한 경향을 보였다. 쌀파이의 경도는 밀파이보다는 쌀가루로 만든 파이에서 경도가 증가하였으며, 볶음땅콩가루, 새싹땅콩가루를 첨가할수록 경도는 낮아졌다. 쌀파이의 지방산 조성은 포화 지방산은 myristic acid, palmitic acid, stearic acid로 나타났다. 단일불포화지방산은 주로 oleic acid로 구성된 것으로 확인되었고, 다가불포화지방산의 조성은 linoleic acid로 구성된 것으로 확인되었으며, 밀에 비해서 쌀가루로 제조한 파이에서 높게 나타났다.

Epizootic HPAIV, H5N6, and H5N8 infections produced severe loss in poultry and wild birds in the Republic of Korea from 2016 to 2017. But pathological lesions and antigen distribution of the novel HPAIV H5N6 clade 2.3.4.4 in natural cases have been rarely reported. Herein, we describe the pathological lesions and antigen localization in chickens (layer and Korean native), ducks, and Japanese quail naturally infected by HPAIV H5N6. Grossly, severe reddening, swelling, and some necrotic foci, which were similar to septicemia or viremia, were observed in skin and many visceral organs including trachea, lung, liver, spleen, and pancreas. Histopathologically, pulmonary congestion and edema, as well as necrotizing hepatitis, splenitis, pancreatitis, myocarditis, and encephalitis were observed. Immunohistochemically, numerous HPAIV antigens were detected in necrotic parenchymal cells and in blood vessels of the respiratory, lymphoid, digestive, urinary, nervous, and cardiovascular systems. The results indicate that HPAIV H5N6 spread to the entire body via blood and caused severe damage throughout the entire body. The HPAI H5N6 clade 2.3.4.4 virus was isolated from samples of all four cases.

To control an external parasitic mite, a honey bee line possessing high hygienic behavior (HHB) against an external parasitic mite, Varroa destructor, has been bred in South Korea and an assessment method has been necessitated to diagnose HHB line from the low hygienic behavior (LHB) line. Thus, in this study, we developed single nucleotide polymorphism (SNP) markers from whole genome sequencing of each 20 worker bees from HHB and LHB lines of A. mellifera ligustica (Hymenoptera: Apidae). An average of 319,445,977 sequence reads was mapped to the known A. mellifera reference genome (an average of 87.46%). In 2,316,128 and 3,266,756 SNPs from each HHB and LHB line, an average of 93.6% and was located in the intergenic spacers and introns, whereas, the remaining 6.4% was located in the genic region, respectively. Among them 20 SNPs that were fixed at each line possessing within-individual homozygosity were selected and each four SNPs were used to diagnose the two honey bee lines either by typical PCR-restriction fragment length polymorphism method or allele-specific PCR. The remaining six SNPs had the size difference, enabling relatively easy differentiation between the two honey bee lines on typical agarose gel and another remaining six SNPs only has sequence difference including SNP sites. Thus, these SNP markers can be used to diagnose the honey bee line with HHB from LHB line against V. destructor.

Cymbidium flower is mainly grown for exportation to China and Japan, but detection of a few pests including the cotton aphid, Aphis gossypii (Hemiptera: Aphidae), necessitated post-harvest treatment for casual exportation. Thus, we irradiated electron beam to cotton aphids occurring in cymbidium to establish post-harvest method for casual exportation of the flowers. For cymbidium, six categories of product quality were examined after eight different doses were irradiated (100, 200, 300, 400, 500, 600, 800 and 1,000 Gy). One thousand Gy to cymbidium caused an extreme deterioration only in vase life in both colors compared to control (0 Gy). In the case of cotton aphids, adult longevity decreased from 11.23 (100 Gy) to 4.70 (400 Gy) when four different doses were irradiated (100, 200, 300, and 400 Gy), with control being an average of 20.89 days. The numbers of total first instar nymph produced per adult was not significantly differed among five doses (2.21 ~ 2.74 individuals), but was substantially lower compared to control (an average of 51.46; P < 0.0001). Live F1 nymphs did not develope to adults at all five doses, except for a single nymph at 100 Gy, which was dead right after emergence. The results of probit analysis indicated that majority of adults required 3.33 ~ 7.55 days for 90% mortality at 200 Gy and higher, but at 100 Gy it required 41.56 days. Therefore, higher than 100 Gy might be required for complete control of adult cotton aphids and their F1.

In order to accumulate the ecological information of Drosophila suzukii for export negotiations of domestic strawberry, we weekly monitored occurrence of D. suzukii at three strawberry fields located in Gok-seong, Dam-yang and Hwa-sun using the best combination of trap and attractant from November 2017 to May 2018, the period of strawberry production. Monitoring was carried out at three sites (a hill located 150 meter distant to greenhouse, outside greenhouse and inside greenhouse) in Gok-seong. Drosophil suzukii was trapped at three short periods from November to the beginning of December, from the end of December to the beginning of January, and from April in a hill. At outside greenhouse, D. suzukii was trapped at two short periods from November to the early December and the beginning of May. In inside greenhouse, it occurred once after May 24th. Gok-seong data can be summarized as more occurrence for longer period as the distance to greenhouse increases. Dam-yang occurred once in a short period inside greenhouse and hill, respectively on May. In case of Hwa-sun, D. suzukii did not trapped in whole trapping sites during trapping period, except for the surrounding grape and peach cultivation areas at the end of November. Summarized, no occurrence periods are from November 20th, 2017 to May 14th, 2018 inside greenhouse; from December 11th, 2017 to May 3rd, 2018 outside greenhouse; and from January 8th, 2018 to April 12th, 2018 on a hill. In connection with strawberry culture, the D. suzukii tends to occur at the beginning and the end of the strawberry production period. We expect this result can serve as a helpful data for export negotiation for quarantine.

The nerippe fritillary butterfly, Argynnis nerippe , is listed as an endangered species in Korea. Establishment of effective conservation strategies can be aided by the development and application of molecular markers that can be used to investigate the population genetics of the butterfly. Therefore, in this study, we identified ten microsatellite markers specific to A. nerippe using the Next-Seq 500 platform, and applied these markers to investigate the characteristics of five South Korean butterfly populations. Genotyping of 48 A. nerippe individuals from five localities showed that at each locus the number of alleles ranged from 4 to 14, and that the observed and expected heterozygosities were 0.324–0.863 and 0.138–0.985, respectively. Significant deviation from the Hardy–Weinberg equilibrium was not observed at any locus. Population structure analysis indicated that there are two genetic groups in Korea, but no population-based gene pool assignments were found. Analysis of FST, RST, and a principal coordinates analysis suggested that the Gureopdo and Yaecheon populations were isolated from other populations. Genetic isolation of the Gureopdo population may be a consequence of unequal population change between Gureopdo and inland populations and to the offshore habitat of Gureopdo. Genetic isolation of the Yaecheon population may be a consequence either of the southernmost location of the population or of the limited sample size available. Further studies with increased sample sizes will be necessary to draw robust conclusions on population isolation and to devise conservation strategies.

Bumblebee, Bombus ardens ardens (Apidae: Hymenopera), is an important resource for pollination that is most widely distributed in Korea. This study utilized microsatellite markers for investigation of genetic diversity and geographic relevance of the B. a. ardens populations in Korea. Through Next Generation Sequencing analysis, we identified 10 microsatellite markers and genotyped for 107 individuals of B. ardens collected from 10 populations. At each locus the number of alleles ranged from 10 to 23; the observed and expected heterozygosities ranged from 0.8909 to 1.0000 and 0.6641 to 0.8422, respectively; and inbreeding coefficient(FIS) ranged from –0.5053 to –0.0891. Significant deviation from the Hardy–Weinberg equilibrium was not observed at any locus. Population structure analysis indicated that there are three genetic groups in Korea with each Jeongseon and Ulleung-do composed of different gene pool from the remaining other populations. Similarly, Principal coordinates analysis also showed the same pattern. FST and RST analyses showed that each Jeongseon and Ulleung-do population had a significant genetic distance from other populations. Considering these results, genetic isolation of Ulleung-do may be explained by “Oceanic island” status and Jeongseon, which showed the positive FIS (0.069) and genetic isolation may be caused by its location on the east side of Baekdudaegan and by on-going inbreeding with a small population size.

Bombyx mandarina (Lepidoptera: Bombycidae) is generally regarded as the ancestor of the domesticated B. mori. Recently, over 40 mitochondrial genomes (mitogenomes) mainly from B. mori strains preserved in China and wild individuals of B. mandarina were sequenced to verify the progenitor of B. mori. At this point, we also were curious about the origin and the relationships of Korean silkworms to foreign B. mandarina and B. mori. As a first step, we sequenced the complete mitogenome of the B. mandarina collected in Korea and compared it to pre-exsiting data (37 strains of B. mori and 14 individuals of B. mandarina). The complete mitogenome of B. mandarina was 15,694-bp long, consisted of 13 protein-coding genes, two rRNA genes, 22 tRNA genes and one non-coding region. The 494-bp long A+T-rich region possessed the highest A/T content (95.3%) than any other region of the genome. Overall, the general mitogenome characteristics of the genus Bombyx species have an identical gene arrangement, similar A/ T content (average 82.3%) and so on. Phylogenetic analysis, however, showed that B. mori and B. mandarina formed a distant group each with the highest nodal support. For more findings of mitogenome characteristics of Bombyx including the Korean B. mandarina and those preserved in Korea more mitogenomes, particularly from Korea, might be needed.

The dung beetle, Copris tripartitus, has been listed as an endangered insect in Korea. In contrast to typical genomic characteristics, the 15,457-bp long, the complete mitochondrial genome (mitogenome) of C. tripartitus has a few unusual characteristics. The genome has the trnP-trnT arrangement at the ND4L-ND6 junction, which is found only in two species of Scarabaeoidea, including C. tripartitus, in 53 available species. Twelve protein coding genes (PCGs) have typical ATT and ATG start codons, whereas COII had the start codon GTA, which is unprecedented in Scarabaeidae. The 297-bp A + T-rich region, which is the shortest available in Scarabaeidae, harbors three repeat units (26, 29, and 21 bp), each consisting of duplicated identical sequences interspersed in the A + T-rich region. Phylogenetic analyses were performed using the concatenated sequences of the 13 PCGs and two rRNA genes using both the Bayesian inference (BI) and maximum likelihood (ML) methods for the families Scarabaeidae and Lucanidae that are available for their mitogenomes in Scarabaeoidea. Our consensus phylogeny yielded monophylies of each family and the subfamily Scarabaeinae in Scarabaeidae with the high nodal supports (0.99-1 by BI and 79-100 by ML). Among the three tribes, represented by multiple genera in Scarabaeinae each Oniticellini and Onitini was supported as monophyletic groups with the highest nodal supports (1 by BI and 100 by ML), whereas Onthophagini was non-monophyletic group, presenting the tribe relationships: ((((Oniticellini + Onthophagini [Caccobius, Milichus, Onthophagus]) + Onthophagini [Digitonthophagus, Phalops]) + Onitini) + (Phanaeini + Eurysternini) + Coprini).