The production of therapeutic protein from transgenic domestic animal is the major technology of biotechnology. Insulin-like growth factor-1 (IGF-1) is known to play an important role in the growth of the animal. The objective of this study is construction of knock-in vector that bovine IGF-1 gene is inserted into the exon 7 locus of β-casein gene and expressed using the gene regulatory DNA sequence of bovine β-casein gene. The knock-in vector consists of 5’ arm region (1.02 kb), bIGF-1 cDNA, CMV-EGFP, and 3’ arm region (1.81 kb). To express bIGF-1 gene as transgene, the F2A sequence was fused to the 5’ terminal of bIGF-1 gene and inserted into exon 7 of the β-casein gene. As a result, the knock-in vector is confirmed that the amino acids are synthesized without termination from the β-casein exon 7 region to the bIGF-1 gene by DNA sequence. These knock-in vectors may help to create transgenic dairy cattle expressing bovine bIGF-1 protein in the mammary gland via the expression system of the bovine β-casein gene.
Effectiveness of transgene transfer into genome is crucially concerned in mass production of the bio-pharmaceuticals using genetically modified transgenic animals as a bioreactor. Recently, the mammary gland has been considered as a potential bioreactor for the mass production of the bio-pharmaceuticals, which appears to be capable of appropriate post-translational modifications of recombinant proteins. The mammary gland tissue specific vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals. In this study, to minimize physiological disturbance caused by constitutive over-expression of the exogenous gene, we constructed new retrovirus vector system designed for mammary gland-specific expression of the hEPO gene. Using piggyBac vector system, we designed to express hEPO gene under the control of mammary gland tissue specific and lactogenic hormonal inducible goat β-casein or mouse Whey Acidic Protein (mWAP) promoter. Inducible expression of the hEPO gene was confirmed using RT-PCR and ELISA in the mouse mammary gland cells treated with lactogenic hormone. We expect the vector system may optimize production efficiency of transgenic animal and reduce the risk of global expression of transgene.
Sirtuin proteins are evolutionary conserved Sir2-related NAD+-dependent deacetylases and regulate many of cellular processes such as metabolism, inflammation, transcription, and aging. Sirtuin contains activity of either ADP-ribosyltransferase or deacetyltranfease and their activity is dependent on the localization in cells. However, the expression pattern of Sirtuins has not been well studied. To examine the expression levels of Sirtuins, RT-PCR was performed using total RNAs from various tissues including liver, small intestine, heart, brain, kidney, lung, spleen, stomach, uterus, ovary, and testis. Sirtuins were highly expressed in most of tissues including the testis. Immunostaining assay showed that Sirt1 and Sirt6 were mainly located in the nucleus of germ cells, spermatocytes, and spermatids in the seminiferous tubules, whereas Sirt2 and Sirt5 were exclusively present in the cytoplasm of germ cells and spermatocytes. Our results indicate that Sirtuins may function as regulators of spermatogenesis and their activities might be dependent on their location in the seminiferous tubules.
In order to achieve successful in vitro production of embryo, it is necessary to establish intrauterine environment during in vitro culture. Thus, this study was investigated to establish embryo culture system using co-incubated collagen matrix gel (CM) with endometrial epithelial cells (EC). Endometrial epithelial cells were isolated from porcine endometrium at follicular phase, the cells seeded in insert dish for co-incubation with CM-coated culture dish. Then, culture media treated with/without 2.0 IU/ml hCG or 10 ng/ml IL-1β. After incubation for 24 h, the co-incubated insert dishes were removed from CM-coated culture dish before embryo culture. Embryos at 48 h after in vitro fertilization (IVF) were cultured on the dish for 120 h with porcine zygote medium. We determined PTGS-2 expression in the ECs, VEGF protein in co-incubated CM with EC and observed cleavage rate and blastocyst development of embryos at 168 h after IVF. In result, expression of PTGS-2 was higher at co-incubated EC with hCG and IL-1β groups than EC without hCG and IL-1β. The VEGF protein was detected at co-incubated CM with EC, EC treated with hCG and IL-1β groups higher than CM group. Also, cleavage rate was no significantly difference among all group, however, blastocyst development was significantly higher in co-incubated CM with EC treated with hCG group than un-treated groups (p<0.05). Therefore, we suggest that novel embryo culture system using co-incubated collagen matrix gel with endometrial epithelial cells treated with IL-1β is beneficial and useful for enhancing the production of porcine blastocysts in vitro.
The addition of growth factors and cytokines to in vitro culture (IVC) media could affect embryo development and the quality of the resulting blastocysts. The present study was performed to investigate the effect of porcine induced pluripotent stem cell (piPSC)-culture conditioned medium (CM) on the in vitro maturation (IVM) and development of parthenogentic embryos (parthenotes) in pigs. Cumulus-oocyte complexes (COCs) or activated oocytes were cultured in IVM or IVC medium supplemented with 0 (control), 25, or 50% of stem cell medium (SM) or CM, respectively. The maturation rate of CM-25% group was significantly improved when compared with control group (p<0.05), but that was not different among SM or CM groups. Blastocyst formation rate was significantly higher in CM-25% group (29.2%) than that of control (20.7%), SM-50% (19.6%) and CM-50% (23.66%, p<0.05). Cell number and the apoptotic cell index in blastocysts was significantly lower in SM-25% than in CM-25% group (p<0.05). The embryo quality related genes, OCT4, KLF4, TERT and ZFP42, were significantly increased in CM-25% group compared with control (p<0.05). In conclusion, the addition of 25% of CM to IVM and IVC medium positively influences not only the developmental potential also quality of parthenotes in pig.
The objective of this study was to monitor health conditions of genetically identical somatic cells cloned Korean white cattle, endangered indigenous cattle (EIC) and indigenous cattle (IC) by analysis of hematologic characteristics. Naturally ovulated oocytes and donor cells were used for somatic cell nuclear transfer (SCNT). Donor cells and enucleated oocytes were followed by electric fusion, chemical activation and surgical embryo transfer into the oviducts of surrogate females. Two recipients became pregnant; two maintained pregnancy to term, and one live cattle were delivered by caesarean section. The cloned Korean white cattle were genetically identical to the nuclear donor cattle. As a result, the mean values of RBC and platelet of cloned cattle and white cattle were significantly decreased by age (p<0.05). The mean values of RBC, HCT, MCV and MCHC between cloned cattle and IC of the same age (1 ~ 2 years) showed the statistical significance (p<0.05). Also, in the WBC of Korean white cattle, the estimated values were decreased according to the age from 12.0×103/μl under 1 year to 11.0×103/μl over 1 years respectively. Although clone-cattle had lower numbers of RBC than reference range, the most of RBC and WBC related heamatologic results of cloned cattle were not different when compared to reference range. This study suggests that cloned Korean white cattle derived from SCNT did not have remarkable health problems, at least in the growth pattern and hematological parameters. In addition, this study provides a valuable resource for further investigations of the preservation of rare genetic stocks underlying traits of interest in cattle.
This study was conducted to determine the relationship between elapsed time after semen preservation on the changes of bacteria and semen quality. Semen was diluted with BTS(Beltsville Thawing Solution) extender without antibiotic for 7 days and sperm parameter and fertility were measured. Sperm motility was measured by CASA and total bacteria number was counted after 22～24 hr incubation from counting agar plate in which sperm dilute to 10 ～106 in 0.9% saline solution and inoculate to agar. Acrosomal integrity was measured by Chlortetracycline (CTC) staining. CTC patterns were uniform fluorescence over the whole head (pattern F), characteristic of incapacitated acrosome- intact spermatozoa; fluorescence-free band in the post-acrosomal region (pattern B), characteristic of capacitated acrosome-intact spermatozoa; and almost no fluorescence over the whole head except for a thin band in the equatorial segment (pattern AR), characteristic of acrosome reacted spermatozoa. Total number of bacteria was significantly increased (p<0.0001) 3 days after preservation. Sperm motility, viability, and morphological abnormality on elapsed time after preservation were lower from 5 (77.24±6.47, p<0.001) and 7 days (77.24±6.47, p<0.001) after preservation compared to 1 (15.71±7.18) and 3 days(18.39±7.22) after preservation, respectively. Sperm viability was significantly lower (53.25± 35.03, p<0.0001) at 7 days after preservation. Morphological abnormality of sperm was lower (p<0.001) at 1 (15.71±7.18) and 3 (18.39±7.22) days compared to 5 (21.84±7.91) and 7 (22.59±9.93) days after preservation. Acrosomal integrity and capacitation rate (pattern F) were significantly lower (p<0.001) from 5 days after preservation. Based on the data we obtained from this study suggested that semen preserved more than 5 days without antibiotic would not recommend use for artificial insemination.
The objective of this study was to investigate the comparison of transferable embryos and pregnancy rate between Hanwoo and Chickso. The results obtained were as follows: No differences were observed in the efficiency of superovulation rates on Hanwoo 78%, and Chickso 85%, respectively. The mean number of total embryos are each 14.76± 2.16 and 6.23±1.07. So the mean number of transferable embryos are each 10.94±1.91 and 4.58±1.05. In addition, the mean number of total Hanwoo embryo from <10 and 10≤ of corpora luteum was 0.50±0.50, 11.56±1.92, respectively. In case of Chickso, The mean number of transferable embryo from <10 and 10≤ of CL was 2.75±1.39, 6.00±1.00, respectively. The pregnancy rates were Hanwoo 40%, and Chickso 37% following transfer of fresh embryos produced in vivo. Also, the pregnancy rates of Chickso 60% were significantly greater (p<0.05) than the Hanwoo 42.48% following transfer of following transfer of frozen embryos, respectively. In conclusion, these results suggest that Chickso may be effectively used for transferable embryos production in Hanwoo. Although the transferable embryos number was not enough, it seems the Chickso greatly affect pregnancy rate. The results indicated that the possibility of transferable embryos from Chickso for embryo transfer could be confirmed in this study. Based on the present findings, it was suggested that it is very important to evaluate in vivo embryo production and pregnancy rate of embryo transfer following superovulation for effective Hanwoo and Chickso production.
DNA methyltransferase 1 (Dnmt1) gene contains three different isoform transcripts, Dnmt1s, Dnmt1o, and Dnmt1p, are produced by alternative usage of multiple first exons. Dnmt1o is specific to oocytes and preimplantation embryos, whereas Dnmt1s is expressed in somatic cells. Here we determined that porcine Dnmt1o gene had differentially methylated regions (DMRs) in 5’-flanking region, while those were not found in the Dnmt1s promoter region. The methylation patterns of the porcine Dnmt1o/Dnmt1s DMRs were investigated using bisulfite sequencing and pyrosequencing analysis through all preimplantation stages from one cell to blastocyst stage in in vivo or somatic cell nuclear transfer (SCNT). The Dnmt1o DMRs contained 8 CpG sites, which located in —640 bp to —30 bp upstream region from transcription start site of the Dnmt1o gene. The methylation status of 5 CpGs within the Dnmt1o DMRs were distinctively different at each stage from one-cell to blastocyst stage in the in vivo or SCNT, respectively. 55.62% methylation degree of the Dnmt1o DMRs in the in vivo was increased up to 84.38% in the SCNT embryo, moreover, de novo methylation and demethylation occurred during development of porcine embryos from the one-cell stage to the blastocyst stage. However, the DNA methylation states at CpG sites in the Dnmt1s promoter regions were hypomethylated, and dramatically not changed through one-cell to blastocyst stage in the in vivo or SCNT embryos. In the present study, we demonstrated that the DMRs in the promoter region of the porcine Dnmt1o was well conserved, contributing to establishment and maintenance of genome-wide patterns of DNA methylation in early embryonic development.
The bovine fatty acid binding protein 4 and 5 (FABP4 and 5) is a major positional and physiological candidate gene for the bovine marbling and carcass weight. The aim of this study was to evaluate the association between economic traits of Korean cattle (Hanwoo) and genetic variation in fatty acid binding protein 4 and 5 (FABP4 and 5) genes within carcass/meat quality traits and the before/after of fatting in breed Hanwoo. Here, we characterized the nucleotide polymorphism of FABP4 and 5 in 86 cattle. We were detected the variability of three types (GG, AG, and AA) by PCR, and economic traits were analyzed by the mixed regression model implemented in the ASReml program. As the result of statistical and supersonic analysis, FABP4 gene was highly showed significant effect (p<0.006) on marbling score (MS), in contrast FABP5 gene was lowed (p<0.084) on MS before fatting. But, FABP4 gene was highly showed significant effect (p<0.0054) on MS, in contrast FABP5 gene lowest (p<0.0899) on MS in the after of fatting. Compare to supersonic result before fatting in FABP4 gene, it was detected type GG: (p<7.18), AG: (p<8.50), and AA: (p<10.50) (n=50), showed type GG: (p<4.88), AG: (p<2.33), and AA: (p<0.00) after weed out (n=20). Futhermore, it was detected type GG: (p<9.30), AG: (p<7.95), and AA: (p<7.40) (n=50) before fatting in the FABP5 gene. It was shown type GG: (p<2.67), AG: (p<3.50), and AA: (p<5.00) after weed out (n=50). Our results indicate that FABP4 and 5 gene transcription is regulated by the environment of feeding and management, and suggest that feeding and management could be potential key in determining FABP4 and 5 genes transcription for carcass/meat quality traits in breed Hanwoo.
Sphingosine-1-phosphate (S1P) has a many function involved proliferation, differentiation and survival of many cells. In this study, to investigate whether S1P improve the developmental competence of porcine embryos, 50 nM of S1P were supplemented during in vitro maturation (with EGF or without EGF) medium and/or in vitro culture (IVC) medium. Addition of S1P was significantly increased the rate of oocytes reaching metaphase II (MII) compared to the control (83.5 vs. 64.1%) in without EGF medium, but not with EGF medium (89.5 vs. 84.6%). When treated with 1 μM of N1N-dimethylsphingosine (DMS), a sphingosine kinase inhibitor which is blocked endogenous generation of S1P, the meiotic progression rates to MII stage (without EGF: 45.2 and with EGF: 66.7%) were significantly decreased and degeneration rates (without EGF: 51.2 and with EGF: 30.1%) were increased in both medium compared to control group during IVM periods. Also, the rates of blastocyst formation was significantly increased in the S1P treated group compared to control group (29.0 vs. 19.2%) of EGF supplemented medium, whereas there were no effect in the EGF free medium (9.0 vs. 10.5%). After 12 h IVM, the phosphorylation of ERK1 and ERK2, which is major signaling pathway of MAP kinase, were increased in the S1P group than that of control or DMS group. When supplemented of S1P during IVC, the rates of blastocyst formation and total cell number (30.2% and 40.6) were significantly increased in S1P-treated group compared with control (20.1% and 32.5), DMS (12.3% and 25.1), and S1P plus DMS group (24.7% and 33.6). The percentage of apoptosis nuclei in the S1P group was significantly decreased than other groups. Also, the rates of blastocyst formation (26.7 vs. 14%) and total cell number (42.8 vs. 32.5) were significantly increased in the S1P group than those of control group when S1P added during the entire IVM and IVC periods. Taken together, our results indicate that S1P supplementation in IVM and/or IVC medium affects beneficial effect of meiotic maturation and subsequent developmental competence of porcine embryos.
This study was carried out to investigate artificial insemination (AI) failure status and frozen semen characteristics in Korean proven bulls‘ number (KPN) semen used for AI of Hanwoo cows in Gangwon East region (Gangneung, Donghae, Taebaek, Samcheok, Sokcho, Yangyang, Goseong). Among semen used for AI, AI failure rate showed lowest at KPN506 (27.6%), whereas highest at KPN593 (77.2%). Correlations of AI failure in between Korean proven bulls semen and cows was 0.2941, which means that AI failure rate of Korean proven bulls semen may have respectable effect on reproduction of Hanwoo cow. In addition, present study was conducted to investigate spermatozoal viability rate, ruptured acrosome rate and active mitochondria in frozen Korean proven bulls semen with flow cytometry. The semen of KPN593 showed significantly (p<0.05) higher viability rate in KPN593 (30.49%) than that in KPN637 (37.34%). Furthermore, percentage of ruptured acrosome was lower in KPN637 as 21.37% than in KPN637 (21.37%), but it was not statistically significant. In conclusion, these results indicate that choice of Korean proven bulls semen may correlate positively with conception rate in Hanwoo cow. Therefore, KPN with high AI failure rate might be avoid to increase conception rate and characteristics of frozen semen might be evaluated before its use for AI.
The present study was conducted to develop a simple method for porcine oocyte maturation without CO2 regulation. In experiment 1, we evaluated that the effect of CO2 non-supplement on porcine oocyte maturation. Cumulusoocyte complexes (COCs) were collected from 2～6 mm follicles and divided into three groups (Control, tube-CO2, and tube-non-CO2). For control, COCs were cultured in 4-well multidish in a CO2 incubator. For tube-CO2, COCs were cultured in a round-bottom tube in a CO2 incubator, and for tube-non-CO2, COCs were cultured in a round-bottom tube sealed tightly without CO2 supplement in a dry incubator. The proportion of oocytes reached to metaphase II (M-II) was not significantly different among three groups (87.9% to 91.4%). In experiment 2, we evaluated the effect of CO2 non-supplement during oocyte maturation on development of embryos. Oocytes with a polar body were divided into two groups (Control and tube-non-CO2) and applied 1.1 kV/cm or 1.2 kV/cm voltages for parthenogenetic activation. After activation, embryos were cultured for 6 days and examined the development. The proportion of embryos cleaved was not significantly different among treatment (86.3% to 91.5%). The proportion of embryo reached to blastocyst stage was not significantly different among treatment (13.9% to 25.2%). The cell number of blastocysts was not significantly different among treatment (29.0 to 32.4). In conclusion, oocytes cultured in a dry incubator without CO2 supplement have enough competence to development after parthenogenetic activation. These results would be useful for transporting oocytes or embryos a long distance.
Male factor infertility or sub-fertility contributed half of all cases of infertility while the semen abnormality is the current topic of argument. Conventional analysis of semen showed poor correlation with fertility. Therefore, evaluation of current semen analysis method is necessary to improve standards of semen assessment. The goal of this study was to investigate that correlation between motion kinematic before and after capacitation and litter size in porcine. Sperm motility and kinematics were measure by computer-assisted sperm analysis (CASA). The motility of spermatozoa was positively correlated with curvilinear velocity (VCL), average path velocity (VAP), and mean amplitude of head lateral displacement (ALH) (p<0.05). Where as VCL positively correlated with VSL, VAP and ALH (p<0.01). Straight-line velocity (VSL) was positively correlated with VAP and ALH (p<0.01). VAP was significantly positively correlated with ALH (p<0.01). Also, we found significant positive correlation among variation of VSL, VAP and ALH (p<0.05). No motility and kinematic parameter are correlated with litter size. However, litter size was significantly correlated with breed (p<0.05). Our results suggested that analysis of sperm motility and kinematics using CASA is questionable for prediction of litter size. However, it has some practical importance to evaluate semen commercially.
The objective of this study was to examine the effect of thymidine treatment during in vitro maturation (IVM) of porcine follicular oocytes on blastocyst development. Porcine oocytes were treated with thymidine (10 mM, 20 mM and 30 mM) for 2 or 6 hr in the preiods of IVM I and/or II. The survival rates of the blastocysts in the 6 hr treatment groups of 10 mM and 20 mM during IVM I period were significantly higher than those of control group (p<0.05). However, the survival rate of the blastocysts in the 2 hr treatment group of 20 mM during IVM II period was significantly higher than control group (p<0.05). Furthermore, the survival rate of the blastocysts in the 6 hr treatment group of 30 mM during IVM II period was significantly lower than control group (p<0.05). Consistent with the previous result, blastocyst development of both IVM I and II treatment group was also showed as similar pattern. Total and apoptotic cell numbers of blastocysts derived from thymidine treated porcine oocytes were examined by using Tunel assay. The results showed that there was no significant differences in total cell number of blastocysts between thymidine treated and untreated groups. However, apoptosis-positive cells in the thymidine treated group (6 hr IVM I) were significantly lower than those of other groups (p<0.05). Taken together, these results indicate that high quality oocytes were selected by DNA synthesis mechanism according to high concentration thymidine treatment during porcine oocyte maturation. Therefore, we concluded that presumptive selected oocytes by thymidine treatment during maturation periods improved the further embryo development and embryonic quality of IVF embryos by decreasing the incidence of apoptosis in preimplantation porcine embryos.
