Bos Taurus Coreanae Research Articles

Identification of Novel SNPs in Bovine Insulin-like Growth Factor Binding Protein-3 (IGFBP3) Gene

The insulin-like growth factors (IGFs), their receptors, and their binding proteins play key roles in regulating cell proliferation and apoptosis. Insulin-like growth factor binding protein-3 (IGFBP3, OMIM #146732) is one of the proteins that bind to the IGFs. IGFBP3 is a modulator of IGF bioactivity, and direct growth inhibitor in the extravascular tissue compartment. We identified twenty-two novel single nucleotide polymorphisms (SNPs) in IGFBP3 gene in Korean cattle (Hanwoo, Bos taurus coreanae) by direct sequencing of full gene including -1,500 bp promoter region. Among the identified SNPs, five common SNPs were screened in 650 Korean cattle; one SNP in promoter (IGFBP3 G-854C), one in 5'UTR region (IGFBP3 G-100A), two in intron 1 (IGFBP3 G+421T, IGFBP3 T+1636A), and one in intron 2 (IGFBP3 C+3863A). The frequencies of each SNP were 0.357 (IGFBP3 G−854C), 0.472 (IGFBP3 G-100A), 0.418 (IGFBP3 G+421T), 0.363 (IGFBP3 T+1636A) and 0.226 (IGFBP3 C+3863A), respectively. Haplotypes and their frequencies were estimated by EM algorithm. Six haplotypes were constructed with five SNPs and linkage disequilibrium coefficients (|D'|) between SNP pairs were also calculated. The information on SNPs and haplotypes in IGFBP3 gene could be useful for genetic studies of this gene. (Asian-Aust. J. Anim. Sci. 2005. Vol 18, No. 1 : 3-7)

햄스터와 소의 젖산탈수소효소에 대한 미토콘드리아 inhibitor의 영향

젖산탈수소효소(EC 1.1.1.27, lactate dehydrogenase, LDH) inhibitor는 햄스터와 소 골격근 미토콘드리아에서 분리하였다. 햄스터 골격근조직의 LDH inhibitor는 175 mM NaCl과 초음파로 분리하였다. 소 골격근조직의 미토콘드리아에서 분리된 inhibitor는 열에 강한 특성을 보였고, <TEX>$A_4$</TEX> 동위효소에 대한 저해정도가 높았으며, 분자량은 22,000 kDa으로 나타났다. Inhibitor는 심장조직을 제외한 골격근, 신장 및 간 조직의 미토콘드리아에서 LDH 결합 시 중요하게 관여하고 있었다. The lactate dehydrogenase (EC 1.1.1.27, LDH) inhibitors were isolated from the LDH-free crude mitochondrial fraction of skeletal muscle in Syrian hamster (Mesocricetus auratus) and Korean native cattle (Bos taurus coreanae). The LDH inhibitor in skeletal muscle of M. auratus was successfully isolated by the treatment with 175 mM NaCl and ultrasonic. The LDH inhibitor in skeletal muscle of B. taurus coreanae was highly stable to heat and LDH fu isozyme was largely inhibited by the LDH inhibitor. The molecular weight of inhibitor was 22 kDa. Inhibitor played an important role in the binding of LDH with the mitochondria in tissues of skeletal muscle, kidney and liver except heart.

Polymorphisms of the prion protein gene (PRNP) in Hanwoo (Bos taurus coreanae) and Holstein cattle

Polymorphisms in the prion protein gene (PRNP) in humans and sheep correlate with susceptibility to transmissible spongiform encephalopathies (TSEs). Bovine spongiform encephalopathy (BSE) has been reported in British and Japanese cattle; it has occurred thus far in Holstein cattle. BSE in Hanwoo (Bos taurus coreanae) cattle has not been diagnosed up to now. To characterize the bovine PRNP polymorphisms in Korean cattle, we analyzed the open reading frame (ORF) of PRNP in 120 Hanwoo (beef) cattle and 53 Holstein (dairy) cattle. Three polymorphisms were found, the third position of codon 78 (G-->A), the third position of codon 192 (C-->T), and the deletion of a single octa-repeat. An analysis of codon 78 revealed no difference in the genotype (P = 0.2026) or allele (P = 0.7180) frequencies between Hanwoo and Holstein animals. However, there were significant differences in the genotype (P < 0.0001) and allele (P < 0.0001) frequencies at PRNP codon 192 between Hanwoo and Holstein animals. The rate of Holstein animals with deletion of a single octa-repeat was 91.5% undeleted homozygotes, 8.5% heterozygotes (with R3 deletion), and 0% deleted homozygotes. However, none of the 120 Hanwoo animals had any octa-repeat deletions. The genotype (P < 0.0001) and allele (P < 0.0001) frequencies of a single octa-repeat-deletion were also significantly different between Hanwoo and Holstein animals.

한우 어미소와 송아지의 행동 특성

한우 어미소(24개월령)와 송아지(1 개월령)를 대상으로 6일 동안 07:00시부터 18:00시까지 42가지의 세부 행동을 관찰 분석하였다. 42가지의 세부 행동 중에서 서 있는 자세(ST), 걸어다니는 동작(WA), 가슴을 대고 누워 있는 자세(LD), 옆으로 누워있는 자세(LF), 놓여있는 여물 또는 사료를 먹는 동작(EA), 냄새를 맡는 동작(SF), 서서 자신을 핥는 동작(EA), 냄새를 맡는 동작(SF), 서서 자신을 핥는 동작(SG), 새끼나 상대방을 핥아주는 동작(PG), 몸을 비비는 동작(RB), 물을 마시는 동작(DR), 서서 반추행동을 하고 있는 동작(SR), 가슴을 대고 누어서 반추 행동을 하고 있는 동작(LR), 서서 꼬리를 흔드는 동작(STW), 서서 반추 행동을 하며 동시에 꼬리를 흔드는 동작(SRW), 젖을 빠는 동작(SUK), 그리고 새끼나 다른 소에 볼을 대는 동작(CR)과 같은 16가지의 행동을 주로 나타내었다. 즉, 유지행동, 사회행동, 섭식행동을 주로 나타내었다. 어미소는 LD가 25%로 가장 높은 빈도를 보였고, 다음으로 ST가 23%, EA가 13%, LR이 10%, SR이 7%, SG와 RB가 4%, 그리고 PG와 SF가 3%의 빈도를 보였다. 송아지는 LD가 52%로 가장 높은 빈도를 보였다. 송아지는 LD가 52%로 가장 높은 빈도를 보였고, 다음으로 ST가 28%, LF가 4%, EA와 WA가 3%, 그리고 SF와 SUK가 2%의 빈도를 보였으며, 그 외의 행동들은 1% 미만이었다. 그리고 주요 시간대인 07:00시, 12:00시, 18:00시에 어미소와 송아지의 행동 양상을 살펴보았는데, 어미소와 송아지 모두가 시간대별로 다른 행동 양상을 나타내었다. 향후 계절 및 우사 등에 따른 행동 양식 차이에 대한 심도 깊은 연구가 이루어져야 할 것으로 판단된다. This study was performed to investigate the behavioral characteristics of Hanwoo(Bos taurus coreanae) cows and their calves. Four cows and their calves were placed in pens. The behaviors of each cow and calf were recorded using a time lapse VCR from 07:00 h to 18:00 h for 6 days and were analyzed using the scan point sampling method. The cow's behaviors in order of frequency were LD(lying down, 25%), ST(standing, 23%), EA(eating, 13%), LR(lying down rumination, 10%) and SR(standing rumination, 7%). The cows spent less than 3% of their time PG(pairwise grooming) and SF(snifting). The calves mainly showed LD(52%), ST(28%), LF (lying flat, 4%), EA(3%) and WA(walking, 3%). The calves spent less than 2% of their time SF(sniffing) and SUK(suckling), The behavioral patterns of cows and their calves at 07:00 h, 12:00 h and 18:00 h were different, especially in ST and LD.

A separate procedure of fusion and activation in an ear fibroblast nuclear transfer program improves preimplantation development of bovine reconstituted oocytes

This study was conducted to examine whether preimplantation development of bovine (“Han Woo,” Bos Taurus coreanae ) oocytes reconstituted with ear fibroblasts could be improved by a modified procedure of fusion and activation. In Experiment l, enucleated oocytes were reconstituted with ear fibroblast by a combined procedure of electric fusion and activation at either 24 or 28 hours after IVM. The 28 hours reconstitution yielded more blastocysts (4% vs. 21%, P = 0.0025) and higher ratio of blastocysts per 2-cell embryos (0.05 vs. 0.25, P = 0.003) than the 24 hours. In Experiment 2, enucleated oocytes were reconstituted by one of the three fusion and activation protocols; 1) a combined procedure of electric fusion and activation at 28 hours after IVM, 2) a combined procedure of electric fusion and chemical activation at 28 hours, and 3) a separate procedure of electric fusion at 24 hours and chemical activation at 28 hours. When compared two combined procedures, chemical activation with 5 μM ionomycin for 4 minutes did not promote embryo development and significantly reduced the fusion rate (42% vs. 53%, P = 0.0395). However, significant (P < 0.0113) increases in the development to 2-cell (90% vs. 70 to 74%) and blastocyst (47% vs. 7 to 13%) stages and in the ratio of blastocysts per 2-cell embryos (0.52 vs. 0.11 to 0.18) were obtained by a separate procedure of electric fusion and chemical activation than by the combined procedures. This separate protocol did not reduce the fusion rate compared with the combined procedures (58%). In conclusion, improved development of oocytes reconstituted with ear fibroblasts was achieved by undergoing a separate procedure of electric fusion and chemical activation 4 hours apart.

Production of HanWoo (Bos taurus coreanae) fetuses following interbreed somatic cell nuclear transfer.

The possibility of producing HanWoo (Bos taurus coreanae) calves from transferable bovine embryos, obtained by interbreed nuclear transfer using Holstein cytoplasts and surrogates, was investigated. As donor nuclei, HanWoo fetal fibroblasts were used. Cells were induced into quiescence by serum deprivation for 4-7 days before nuclear transfer. In vitro matured Holstein oocytes were enucleated, and single donor cell was placed into the perivitelline space of enucleated oocyte. After reconstruction, the embryos were fused. activated and cultured. On day 7, the embryos that developed to the blastocyst stages were transferred into Holstein recipient cows on day 6 to 7 of estrous cycle (estrus=0). The reconstructed embryos were successfully fused (58.8%; 47/80), cleaved (91.5%; 43/47), and developed to blastocysts (29.8%; 14/47). Eleven blastocysts were transferred into 5 Holstein recipient cows. Two recipients were pregnant, confirmed by ultrasonography at day 60 of gestation. But, one of them was opened between on day 80 to 100 of pregnancy, and the other had a stillbirth on day 255. The stillborn calf was physically normal, and we couldn't find any evidence of anomaly. These results show that cells derived from HanWoo somatic cell lines can be reprogrammed by interbreed nuclear transfer and develop subsequently in vivo as well as in vitro.

Purification and embryotropic roles of tissue inhibitor of metalloproteinase-1 in development of "HanWoo" (Bos taurus coreanae) oocytes co-cultured with bovine oviduct epithelial cells.

This study was conducted to purify a tissue inhibitor of metalloproteinase (TIMP)-1 in a serum-free medium conditioned with bovine oviduct epithelial cells (BOEC) and to evaluate its effect on development of "HanWoo" (Bos taurus coreanae) embryos to the blastocyst stage. In the first study using SDS-PAGE electrophoresis, the presence of 32 kDa proteins, which contains TIMP-1, was detected in the medium conditioned with BOEC, and TIMP-1 was then purified from the medium by gel filtration and HPLC techniques. When examined TIMP-1 secretion, fluorescent foci indicating the secretion of TIMP-1 were found after stained BOEC with fluorescein isothiocyanate. In the next experiment, two-cell embryos derived from in vitro-fertilization were cultured in a serum-free medium, to which 0, 1.25, 2.5 or 5 microg/ml of purified TIMP-1 was supplemented. More (P<0.05) embryos developed to the morula and blastocyst stages after the addition of 2.5 microg/ml to culture medium than after no addition. In conclusion, our data indicate that BOEC secrete TIMP-1 and this glycoprotein promotes the prehatched development of "HanWoo" embryos derived from in vitro-fertilization.