Identification of matrix metalloproteinases secreted by human hepatocarcinoma HepG2 cells

Young Jae Lee,Keun Cheon Kim,Jeong Mook Lim,Seung Tae Lee 한국동물생명공학회(구 한국수정란이식학회) 2022 한국동물생명공학회지 Vol.37 No.1

To date, the development of anticancer drugs has been conducted using two-dimensional (2D) cell culture systems. However, since cancer cells in the body are generated and developed in three-dimensional (3D) microenvironments, the use of 2D anticancer drug screening can make it difficult to accurately evaluate the anticancer effects of drug candidates. Therefore, as a step towards developing a cancer cellfriendly 3D microenvironment based on a combination of vinylsulfone-functionalized polyethylene glycol (PEG-VS) with dicysteine-containing crosslinker peptides with an intervening matrix metalloproteinase (MMP)-specific cleavage site, the types of MMPs secreted from human hepatocarcinoma HepG2 cells, a representative cancer cell, were analyzed transcriptionally and translationally. MMP3 was confirmed to be the most highly expressed protease secreted by HepG2 cells. This knowledge will be important in the design of a crosslinker necessary for the construction of PEG-based hydrogels customized for the 3D culture of HepG2 cells.

A simple and rapid method for detection of single nucleotide variants using tailed primer and HRM analysis

Hyeonguk Baek,Inchul Choi 한국동물생명공학회(구 한국수정란이식학회) 2023 한국동물생명공학회지 Vol.38 No.4

Background: Single nucleotide polymorphisms (SNPs) are widely used genetic markers with applications in human disease diagnostics, animal breeding, and evolutionary studies, but existing genotyping methods can be labor-intensive and costly. The aim of this study is to develop a simple and rapid method for identification of a single nucleotide change. Methods: A modified Polymerase Chain Reaction Amplification of Multiple Specific Alleles (PAMSA) and high resolution melt (HRM) analysis was performed to discriminate a bovine polymorphism in the NCAPG gene (rs109570900, 1326T > G). Results: The inclusion of tails in the primers enabled allele discrimination based on PCR product lengths, detected through agarose gel electrophoresis, successfully determining various genotypes, albeit with some time and labor intensity due to the use of relatively costly high-resolution agarose gels. Additionally, high-resolution melt (HRM) analysis with tailed primers effectively distinguished the GG genotype from the TT genotype in bovine muscle cell lines, offering a reliable way to distinguish SNP polymorphisms without the need for time-consuming AS-PCR. Conclusions: Our experiments demonstrated the importance of incorporating unique mismatched bases in the allele-specific primers to prevent cross-amplification by fragmented primers. This efficient and cost-effective method, as presented here, enables genotyping laboratories to analyze SNPs using standard real-time PCR.

Reproductive management of dairy cows: an existing scenario from urban farming system in Bangladesh

Nayeema Khan Sima,Munni Akter,M. Nazmul Hoque,Md. Taimur Islam,Ziban Chandra Das,Anup Kumar Talukder 한국동물생명공학회(구 한국수정란이식학회) 2023 한국동물생명공학회지 Vol.38 No.4

Background: Reproductive management practices play crucial roles to maximize the reproductive performance of cows, and thus contribute to farm profitability. We aimed to assess the reproductive management of cows currently practiced in the dairy farms in an urban farming system. Methods: A total of 62 dairy farms were randomly selected considering all size of farms such as small (1-5 cattle), medium (6-20 cattle) and large farms (> 20 cattle) from selected areas of Dhaka city in Bangladesh. The reproductive managementrelated parameters viz. estrus detection, breeding method, pregnancy diagnosis, dry cow and parturition management, vaccination and treatment of reproductive problems etc. were obtained in a pre-defined questionnaire during the farm visit. Results: The visual observation method was only used (100.0%; 62/62) for estrus detection irrespective of size of the farms; while farmers observed cows for estrus 4-5 times a day, but only for 20-60 seconds each time. Regardless of farm size, 89.0% (55/62) farms used artificial insemination (AI) for breeding the cows. Intriguingly, all farms (100.0%) routinely checked the cows for pregnancy at 35-40 days post-breeding using rectal palpation technique by registered veterinarian. However, only 6.5% (4/62) farms practiced dry cow management. Notably, all farms (100.0%) provided nutritional supplements (Vit D, Ca and P) during late gestation. However, proper hygiene and cleanliness during parturition was not practiced in 77.4% (48/62) farms; even though 96.7% (60/62) farms treated cows by registered veterinarian for parturition-related problems. Conclusions: While farmers used AI service for breeding and timely check their cows for pregnancy; however, they need to increase observation time (30 minutes/ observation, twice in a day: early morning and early night) for estrus detection, consider dry cow management and ensure hygienic parturition for maximizing production.

Effect of replacement feed ingredients of Micropterus salmoides in exotic species

Min-Gi Han,Ran Lee,Hyun Jung Park,Kyung Hoon Lee,Hyuk Song 한국동물생명공학회(구 한국수정란이식학회) 2023 한국동물생명공학회지 Vol.38 No.4

Background: Largemouth bass (Micropterus salmoides) is introduced species that has caused aquatic ecology activity both in vitro and in vivo were investigated for the possibility of application of the bass extract as an alternative feed ingredient. Methods: The bass oil was extracted using a 1-L supercritical extractor, while the protein was extracted from 250 g of bass dry matter, which was dissolved in 1 mL of H2O at 50℃. Both oil and protein extracts were evaluated antioxidant activities and the level of DPPH radical scavenging assay and nitric oxide (NO) production assay with lipopolysaccharide response. Oral administration of 6.6 μL/g bass protein and 5.38 μL/g bass oil conducted for investigating serological and physiological effect. Results: DPPH radical scavenging showed similar radical scavenging ability of 50 μM of ascorbic acid at 200 μg of protein and 10% of oil treatment. NO concentration was diminished by the treatment of bass oil. Oral administration of both bass oil and proteins to mice showed that the body weight increase rate of the bass oil treated group was significantly reduced by 1.55 g compared to the other groups. The number of white blood cells (WBC) was increased by 4.52 k/μL in the bass protein-treated group and 4.44 k/μL in the bass oil-treated group compared to the control group. However, the serum IgG level did not show a significant difference between the bass extract-treated groups and the control group. Conclusions: These studies demonstrate that both bass oil and proteins extracted from the bass not only provide excellent effects of antioxidant and immune activity but can also be used as functional food supplements.

Ovarian potential of Cameroonian Zebu cattle (Bos indicus ) slaughtered in Etoudi – Yaound?

Nsiyapnze Katte Yato Katte,Tchoffo Herve,Azafack Kana Dorice,Chongsi Margaret Mary Momo,Ngoula Ferdinand 한국동물생명공학회(구 한국수정란이식학회) 2023 한국동물생명공학회지 Vol.38 No.4

Background: This study helps to evaluate the Ovarian potential of Cameroonian Zebu cattle (Bos indicus) slaughtered in Etoudi – Yaound? for implementing Assisted Reproductive Techniques (ARTs). The aim was to enhance the productivity of the cattle sub-sector in Cameroon while conserving local genetic resources. Methods: A total of 144 cows, including 94 cycled cows and 50 pregnant cows, were included in the study. Live weights were determined by measuring the thoracic perimeter of each cow using a Rondo measuring tape. Age was determined postmortem through examination of dentition and horns, while the uterus and ovaries were removed, weighed, and classified based on physiological status (pregnant or nonpregnant). Follicles were counted, and their diameters were measured and categorized into small (Ø < 3 mm), medium (Ø 3-8 mm), and large (Ø > 8 mm). Results: The results revealed an average follicular population of 32.02 ± 9.31 per cow, with 22.43 ± 8.45 small follicles, 8.42 ± 3.87 medium follicles, and 0.76 ± 0.34 large follicles. The weight of the right ovary was significantly higher than that of the left ovary (p < 0.05), and cows aged 6 to 9 years exhibited a higher number of follicles compared to other age groups. Cows with medium (BCS = 3) and large (BCS = 4-5) body reserves had the heaviest ovaries. Additionally, pregnant cows had heavier uteri compared to non-pregnant cows, and cows with a body condition score of 3 or higher had higher uterine weights than lean cows (BCS = 1-2). Conclusions: This study demonstrates that age, body condition score, and pregnancy status influence ovarian weight. Body Condition Score serves as a reliable indicator of cow nutritional status, and cows with BCS of 3-5 are excellent candidates for in vitro production of embryos.

Morphological differences according to uterine tissue remodeling during estrus between sika deer and water deer

Min-Gee Oh,Yong-Su Park,Sang-Hwan Kim 한국동물생명공학회(구 한국수정란이식학회) 2023 한국동물생명공학회지 Vol.38 No.4

Background: Water deer and sika deer, which breed in the wild environment, are known to have similar reproductive physiology mechanisms. Therefore, this study aimed to analyze the differences in uterine development between water deer and sika deer during estrus. Methods: MMPs and uterine development-related factors were analyzed and morphological differences were compared in the uterus of sika deer captured near Russia near Korea and water deer captured in the wild in Korea. Results: In terms of morphological differences in the uterus, the glands that form villus within the endometrium of the water deer were newly developed, and the formation of small glands was high, but the villus and glands of the sika deer were expanded, and the stroma zone in the myometrium was higher than that of the water deer. Development has increased. Additionally, the expression of PAPP-A and VEGF factors was increased in the endometrium of water deer than in sika deer, but the actions of MMPs were increased in sika deer. Conclusions: As a result of this study, there is a significant difference in the development of glands in the endometrium of water deer and sika deer during estrus, and it is believed that there is a significant difference in the development of the uterus due to the physiological effects of estrus between water deer and sika deer. Additionally, it is believed that there will be differences in the timing at which pregnancy can be decided.

A study on the correlation between vaginal conductivity and estrus in Hanwoo (Bos taurus coreanae ) cows

Junkoo Yi,Jinyeon Park,Jaejung Ha,Daejung Yu,Woo-Sung Kwon,Daehyun Kim 한국동물생명공학회(구 한국수정란이식학회) 2023 한국동물생명공학회지 Vol.38 No.4

Background: Estrus in cows can be detected through vaginal electrical resistance or conductivity. However, there are no studies measuring vaginal electrical resistance in Hanwoo cows. This study aims to measure the vaginal electrical resistance value in Hanwoo cows and compare it with estrus and ovulation. Methods: Vaginal electrical resistance values of 73 Hanwoo cows were measured before and after estrus at the Gyeongsangbuk-do Livestock Research Institute. Measurements were taken on days -6, -3, -2, -1, 0, 1, 2, 3, and 6 of artificial insemination. Large follicles and ovulation were confirmed using transvaginal ultrasonography. Results: The vaginal electrical resistance averaged 225.6 ± 6.3 Ω days before the artificial insemination date, decreasing until the day of artificial insemination. The average vaginal electrical resistance was 163.7 ± 4.6 Ω on the date of artificial insemination, and 188.8 ± 4.3 Ω one day after artificial insemination, when large follicles were observed. In addition, on the 6th day after artificial insemination, the vaginal electrical resistance averaged 231.4 ± 5.5, which was similar to the 6th day before artificial insemination (222.5 ± 6.3). Transvaginal ultrasonography showed that most of the cows ovulated one day after artificial insemination. Conclusions: The accuracy of estrus is high if the vaginal electrical resistance is measured for cows with confirmed estrus, making is a potentially useful for determining the timing of artificial insemination.

Genetic evaluation for economic traits of commercial Hanwoo population using single-step GBLUP

Gwang Hyeon Lee,Khaliunaa Tseveen,Yoon Seok Lee,Hong Sik Kong 한국동물생명공학회(구 한국수정란이식학회) 2023 한국동물생명공학회지 Vol.38 No.4

Background: Recently, the single-step genomic best linear unbiased prediction (ssGBLUP) method, which incorporates not only genomic information but also phenotypic information of pedigree, is under study. In this study, we performed a ssGBLUP analysis on a commercial Hanwoo population using phenotypic, genotypic, and pedigree data. Methods: The test population comprised Hanwoo 1,740 heads raised in four regions of Korea, while the reference population used Hanwoo 18,499 heads raised across the country and two-generation pedigree data. Analysis was performed using genotype data generated by the Hanwoo 50 K SNP beadchip. Results: The mean Genome estimated breeding values (GEBVs) estimated using the ssGBLUP methods for carcass weight (CWT), eye muscle area (EMA), back fat thickness (BFT), and marbling score (MS) were 7.348, 1.515, -0.355, and 0.040, respectively, while the accuracy of each trait was 0.749, 0.733, 0.769, and 0.768, respectively. When the correlation analysis between the GEBVs as a result of this study and the actual slaughter performance was confirmed, CWT, EMA, BFT, and MS were reported to be 0.519, 0.435, 0.444, and 0.543, respectively. Conclusions: Our results suggest that the ssGBLUP method enables a more accurate evaluation because it conducts a genetic evaluation of an individual using not only genotype information but also phenotypic information of the pedigree. Individual evaluation using the ssGBLUP method is considered effective for enhancing the genetic ability of farms and enabling accurate and rapid improvements. It is considered that if more pedigree information of reference population is collected for analysis, genetic ability can be evaluated more accurately.

Vitamin C promotes the early reprogramming of fetal canine fibroblasts into induced pluripotent stem cells

Sang Eun Kim,Jun Sung Lee,Keon Bong Oh,Jeong Ho Hwang 한국동물생명공학회(구 한국수정란이식학회) 2023 한국동물생명공학회지 Vol.38 No.4

Background: Canine induced pluripotent stem cells (iPSCs) are an attractive source for veterinary regenerative medicine, disease modeling, and drug development. Here we used vitamin C (Vc) to improve the reprogramming efficiency of canine iPSCs, and its functions in the reprogramming process were elucidated. Methods: Retroviral transduction of Oct4, Sox2, Klf4, c-Myc (OSKM), and GFP was employed to induce reprogramming in canine fetal fibroblasts. Following transduction, the culture medium was subsequently replaced with ESC medium containing Vc to determine the effect on reprogramming activity. Results: The number of AP-positive iPSC colonies dramatically increased in culture conditions supplemented with Vc. Vc enhanced the efficacy of retrovirus transduction, which appears to be correlated with enhanced cell proliferation capacity. To confirm the characteristics of the Vc-treated iPSCs, the cells were cultured to passage 5, and pluripotency markers including Oct4, Sox2, Nanog, and Tra-1-60 were observed by immunocytochemistry. The expression of endogenous pluripotent genes (Oct4, Nanog, Rex1, and telomerase) were also verified by PCR. The complete silencing of exogenously transduced human OSKM factors was observed exclusively in canine iPSCs treated with Vc. Canine iPSCs treated with Vc are capable of forming embryoid bodies in vitro and have spontaneously differentiated into three germ layers. Conclusions: Our findings emphasize a straightforward method for enhancing the efficiency of canine iPSC generation and provide insight into the Vc effect on the reprogramming process.

Anti-oxidative effects of broccoli (Brassica oleracea var. italica ) sprout extract in RAW 264.7 cell and cisplatin-induced testicular damage

Won-Young Lee,Hyun-Woo Shim,Hyun-Jung Park 한국동물생명공학회(구 한국수정란이식학회) 2023 한국동물생명공학회지 Vol.38 No.4

Background: Brassica oleracea var. italica (broccoli), a rich source of antioxidants, can prevent various diseases and improve human health. In this study, we investigated the antioxidative effects of broccoli sprout extract on oxidative stress induced by lipopolysaccharide and cisplatin in cell and organ tissue models. Methods: Antioxidative effect of BSE was evaluated using DPPH and ABTS in RAW 364.7 cells, and effects of BSE on testes were investigated using Cisplatin-induced testicular damage model with an in vitro organ culture system. Results: The DPPH assay showed that the antioxidant activity of the alcoholic broccoli sprout extract was higher than that of the water extract. Additionally, the expression levels of antioxidation-related genes, Nrf2 , Gsr , HO-1, and catalase , were significantly increased in broccoli sprout extract-treated RAW 264.7 cells, and the extract suppressed lipopolysaccharide-induced mitochondrial dysfunction. Based on the results in the RAW 264.7 cell culture, the antioxidative effects of the extracts were investigated in a mouse testis fragment culture. The expression of Nrf2 , HO-1 , and Ddx4 was clearly decreased in cisplatin-treated mouse testis fragments and not in both broccoli sprout extract- and cisplatin-treated mouse testis fragments. In addition, the oxidative marker O-HdG was strongly detected in cisplatin-treated mouse testis fragments, and these signals were reduced by broccoli sprout extract treatment. Conclusions: The results of this study show that broccoli sprout extracts could serve as potential nutraceutical agents as they possess antioxidant effects in the testes.