• Journal of Animal Reproduction and Biotechnology
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• v.36 no.3
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• pp.121-128
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• 2021

Increasing the efficiency of HR (homologous recombination) is important for a successful knock-in. Rad51 is mainly involved in homologous recombination and is associated with strand invasion. The HR-related mismatch repair system maintains HR fidelity by heteroduplex rejection and repair. Therefore, the purpose of this study is to control Rad51, which plays a critical role in HR, through UV-induced DNA damage. It is also to confirm the effect on the expression of MMR related genes (Msh2, Msh3, Msh6, Mlh1, Pms2) and HR-related genes closely related to HR through treatment with the MMR inhibitor CdCl₂. The mRNA expression of Rad51 gene was confirmed in both HC11 cells and mouse testes, but the mRNA expression of Dmc1 gene was confirmed only in mouse testes. The protein expression of Rad51 and Dmc1 gene increased in UV-irradiated HC11 cells. After 72 hours of treatment with 1 ㎛ of CdCl₂, the mRNA expression level of Msh3, Pms2, and Rad51 decreased, but the mRNA expression level of Msh6 and Mlh1 increased in HC11 cells. There was no significant difference in Msh2 mRNA expression between CdCl₂ untreated-group and the 72 hours treated group. In conclusion, HR-related gene (Rad51) was increased by UV-induced DNA damage. Treatment of the MMR inhibitor CdCl₂ in HC11 cells decreased the mRNA expression of Rad51.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.2
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• pp.113-120
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• 2022

Sperm cryopreservation is a fundamental process for the long-term conservation of livestock genetic resources. Yet, the packaging method has been shown, among other factors, to affect the frozen-thawed (FT) sperm quality. This study aimed to develop a new mini-straw for sperm cryopreservation. In addition, the kinematic patterns, viability, acrosome integrity, and mitochondrial membrane potential (MMP) of boar spermatozoa frozen in the developed 0.25 mL straw, 0.25 mL (minitube, Germany), or 0.5 mL (IMV technologies, France) straws were assessed. Post-thaw kinematic parameters were not different (experiment 1: total motility (33.89%, 32.42%), progressive motility (19.13%, 19.09%), curvilinear velocity (42.32, 42.86), and average path velocity (33.40, 33.62) for minitube and the developed straws, respectively. Further, the viability (38.56%, 34.03%), acrosome integrity (53.38%, 48.88%), MMP (42.32%, 36.71%) of spermatozoa frozen using both straw were not differ statistically (p > 0.05). In experiment two, the quality parameters for semen frozen in the developed straw were compared with the 0.5 mL IMV straw. The total motility (41.26%, 39.1%), progressive motility (24.62%, 23.25%), curvilinear velocity (46.44, 48.25), and average path velocity (37.98, 39.12), respectively, for IMV and the developed straw, did not differ statistically. Additionally, there was no significant difference in the viability (39.60%, 33.17%), acrosome integrity (46.23%, 43.23%), and MMP (39.66, 32.51) for IMV and the developed straw, respectively. These results validate the safety and efficiency of the developed straw and highlight its great potential for clinical application. Moreover, both 0.25 mL and 0.5 mL straws fit the present protocol for cryopreservation of boar spermatozoa.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.2
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• pp.136-143
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• 2022

Recent progress has been made to establish intestinal organoids for an in vitro model as a potential alternative to an in vivo system in animals. We previously reported a reliable method for the isolation of intestinal crypts from the small intestine and robust three-dimensional (3D) expansion of intestinal organoids (basal-out) in adult bovines. The present study aimed to establish next-generation intestinal organoids for practical applications in disease modeling-based host-pathogen interactions and feed efficiency measurements. In this study, we developed a rapid and convenient method for the efficient generation of intestinal organoids through the modulation of the Wnt signaling pathway and continuous apical-out intestinal organoids. Remarkably, the intestinal epithelium only takes 3-4 days to undergo CHIR (1 µM) treatment as a Wnt activator, which is much shorter than that required for spontaneous differentiation (7 days). Subsequently, we successfully established an apical-out bovine intestinal organoid culture system through suspension culture without Matrigel matrix, indicating an apical-out membrane on the surface. Collectively, these results demonstrate the efficient generation and next-generation of bovine intestinal organoids and will facilitate their potential use for various purposes, such as disease modeling, in the field of animal biotechnology.

• Journal of Animal Reproduction and Biotechnology
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• v.37 no.4
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• pp.298-302
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• 2022

South Korea has a temperate climate with four distinct seasons. However, summers are extremely hot and humid, which negatively affects industrial animal production. Hanwoo are native cattle that have traditionally been raised in the natural environment of Korea. The present study investigated the effects of birth and lactation season on the birth and weaning weights of Hanwoo calves. Data were collected from 100 local breeding farms between 2016 and 2021. A total of 56,970 (males, 29,530; females, 27,440) Hanwoo calves were classified according to sex or birth and weaning season (March-May, spring; June-August, summer; September-November, fall; and December-February, winter). The birth weight of Hanwoo calves differed according to the birth season. As such, birth weight of the summer-born calves was the lowest. Additionally, the 90-day weaning weight was positively correlated with birth weight. Interestingly, however, the 90-day weaning weight was not related to the birth season but was related to the 2-month seasonal effect during the lactation period. Furthermore, the 90-day weaning weight was the lowest during the summer lactation period. In the beef cattle industry, daily weight gain is an important economic characteristic related to feed efficiency and growth. Our findings will contribute the management of Hanwoo cattle and analysis of changes in economic characteristics due to high temperatures.

• Journal of Animal Reproduction and Biotechnology
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• v.38 no.2
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• pp.70-76
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• 2023

Background: Despite considerable technological advancements, polyspermy remains a significant challenge in in vitro fertilization (IVF) procedures in pigs, disrupting normal embryonic development. Here, we aimed to determine whether optimal fertilization conditions reduce the polyspermy incidence in pigs. Methods: In vitro-matured oocytes were co-incubated with sperm according to a modified two-step culture system. Results: In the first experiment, oocytes were briefly co-incubated with sperm, washed in IVF medium, and then moved to fresh IVF medium for 5 or 6 h. Although the 6 h sperm-free cultured group had a higher penetration rate than the 5 h cultured group, the polyspermy rate significantly increased in the 6 h sperm-free cultured group. The gamete co-incubation period was either 20 or 40 min. The 40 min cultured group had a higher rate of blastocyst formation and number of total cells in blastocysts than the 20 min cultured group. In experiment 2, oocytes were inseminated with sperm separated by Pecroll treatment. Percoll treatment increased the rate of oocyte penetration and blastocyst formation compared to the control. In experiment 3, fertilized oocytes were cultured in 25 µL microdroplets (10 gametes/drop) or 500 µL (100 gametes/well) of culture medium in 4-well plates. The large volume of medium significantly reduced the number of dead oocytes and increased the rate of blastocyst formation compared to the small volume. Conclusions: Collectively, these results demonstrate that various fertilization conditions, including modified co-culture period, active sperm separation, and culture medium volume, enhance fertilization efficiency and subsequent embryonic development by decreasing polyspermy occurrence.

• Journal of Animal Reproduction and Biotechnology
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• v.38 no.3
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• pp.109-120
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• 2023

Background: Pluripotent stem cells (PSCs) including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer the immense therapeutic potential in stem cell-based therapy of degenerative disorders. However, clinical trials of human ESCs cause heavy ethical concerns. With the derivation of iPSCs established by reprogramming from adult somatic cells through the transgenic expression of transcription factors, this problems would be able to overcome. In the present study, we tried to differentiate porcine iPSCs (piPSCs) into endothelial cells (ECs) for stem cell-based therapy of vascular diseases. Methods: piPSCs (OSKMNL) were induced to differentiation into ECs in four differentiation media (APEL-2, APEL-2 + 50 ng/mL of VEGF, EBM-2, EBM-2 + 50 ng/mL of VEGF) on cultured plates coated with matrigel^® (1:40 dilution with DMEM/F-12 medium) for 8 days. Differentiation efficiency of these cells were exanimated using qRT-PCR, Immunocytochemistry, Western blotting and FACS. Results: As results, expressions of pluripotency-associated markers (OCT-3/4, SOX2 and NANOG) were higher observed in all porcine differentiated cells derived from piPSCs (OSKMNL) cultured in four differentiation media than piPSCs as the control, whereas endothelial-associated marker (CD-31) in the differentiated cells was not expressed. Conclusions: It can be seen that piPSCs (OSKMNL) were not suitable to differentiate into ECs in the four differentiation media unlike porcine epiblast stem cells (pEpiSCs). Therefore, it would be required to establish a suitable PSCs for differentiating into ECs for the treatment of cardiovascular diseases.

• Journal of Animal Reproduction and Biotechnology
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• v.39 no.3
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• pp.201-211
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• 2024

Background: Hanwoo cattle farmers aim to improve calf production and reproductive efficiency. Recovery of the reproductive tract postpartum is a critical factor influencing the postpartum period and conception of breeding cows. This study aimed to precisely analyze the recovery process of the reproductive tract in primiparous Hanwoo postpartum and to establish recovery criteria. Methods: Ten primiparous Hanwoo cows were used in this study. After parturition, estrus was examined daily using visual observations and estrus detection patches. Ovarian recovery, cervical diameter, and uterine horn diameter were examined using ultrasonography four times per week. Results: The analysis revealed that the first estrus occurred at 19.1 ± 6.5 days postpartum, the first ovulation at 27.1 ± 4.5 days, and the first normal estrus cycle at 39.2 ± 6.4 days. The ovulation rate during the first estrus was 40%. A normal estrus cycle occurred in 11.1% of patients at the first ovulation. The cervix diameter recovered to 42.0 ± 3.5 mm and the uterine horn diameter to 34.4 ± 7.1 mm by 24 days postpartum, with the difference in uterine horn diameter recovering to 2.6 ± 1.2 mm by 31 days postpartum. Conclusions: This study can aid in determining the optimal breeding time for postpartum primiparous Hanwoo cow and provide foundational data for Hanwoo breeding studies.

• Journal of Animal Reproduction and Biotechnology
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• v.39 no.3
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• pp.153-163
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• 2024

Background: The clinical application of canine mesenchymal stem cells (MSCs) necessitates efficient and safe culture methods to produce large quantities of cells. Traditionally, fetal bovine serum (FBS) has been used for MSC expansion, but it carries risks such as contamination and adverse immune responses. Methods: In this study, we investigate the efficacy and efficiency of canine allogeneic serum as an effective alternative to FBS for the in vitro culture of canine MSCs. We measured the population doubling time of canine MSCs in allogeneic serum conditions and utilized qRT-PCR, flowcytometric analysis, and cellular staining/color-metric assay for investigating its effects on cellular senescence during long-term culture and the expression of key pluripotency-related transcriptomes. Results: Our findings demonstrate that canine MSCs cultured with allogeneic serum exhibited enhanced proliferation rates, reduced cellular senescence, and lower apoptosis levels compared to those cultured with FBS. Additionally, the expression of key pluripotency-related transcription factors, including Oct4, Sox2, and Nanog, was increased in canine MSCs cultured with allogeneic serum. Conclusions: These results highlight the potential of canine allogeneic serum to provide a safer and more effective culture environment, supporting the large-scale expansion and maintenance of canine MSCs for clinical applications.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.6
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• pp.794-798
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• 2004

An attempt was made to improve the efficiency of growth and lactation performance of dairy heifers subjected to a stair-step growth scheme using rice straw as the sole roughage source. Twenty-four young Holstein heifers were randomly assigned to either control or test group. The control diet met the National Research Council (NRC) requirement, with heifers calving at 24 to 26 mo of age. The test group was individually fed according to a schedule of 3, 2, 4, 2, 5 and 2 mo in which feed intake was alternately 20% below or 25% above the NRC requirements. Heifers on the stair-step growth pattern gained more body weight and consumed less dry matter (10.80 and 11.22%, respectively), resulting an increasing growth efficiency compared with the control. Body condition, first estrus, first conception, services per conception and calving difficulty (data not shown) were not affected. Milk yield of the test group was 8.5% higher than that of the control group. During the early lactation period, the milk yield was significantly higher in the stair-step group than in that of the control group (p<0.05). Milk composition was not affected by compensatory growth induced by the stair-step scheme. Also, weight at calving and calf growth performance was not affected by stair-step growth. The results indicate that using rice straw as a sole roughage source in a stair-step compensatory growth scheme can contribute to the improvement of growth efficiency and early lactation performance.

• Reproductive and Developmental Biology
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• v.33 no.1
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• pp.63-69
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• 2009

In this study we tried to construct a more efficient tetracycline-inducible gene expression system by replacing previous key elements with more advance ones. At the beginning, we substituted PGK (phophoglycerate kinase) promoter for CMV (cytomegalovirus) promoter to control "$rtTA2^sM2$" which has been known for high induction efficiency in response to tetracycline. With this modification, expression of the EGFP marker gene under the induction condition was significantly increased. Next, we replaced "TRE" fragment with a modified version named "TRE-tighf" which has been reported to have higher affinity and specificity to the transactivator by minor base change of the "TRE" DNA fragment sequence. Use of "TRE-tighf" instead of "TRE" resulted in more than 10 fold increment in terms of induction efficiency and significant decrement of background expression in non-inducible condition. By combining PGK promoter and "TRE-tight" fragment, we could upgrade previous tetracycline-inducible system to show more stringent turn on/off gene switch ability and stronger expression of the gene of our interest. Use of this newly developed system must be very helpful to the studies of gene expression, especially to the transgenic animal study in which non-controllable constitutive expression of the transgene has been one of the urgent problems to be solved.