• Reproductive and Developmental Biology
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• 제39권2호
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• pp.29-36
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• 2015

Pigs are considered an ideal source of human disease model due to their physiological similarities to humans. However, the low efficiency of in vitro embryo production (IVP) is still a major barrier in the production of pig offspring with gene manipulation. Despite ongoing advances in the associated technologies, the developmental capacity of IVP pig embryos is still lower than that of their in vivo counterparts, as well as IVP embryos of other species (e.g., cattle and mice). The efficiency of IVP can be influenced by many factors that affect various critical steps in the process. The previous relevant reviews have focused on the in vitro maturation system, in vitro culture conditions, in vitro fertilization medium, issues with polyspermy, the utilized technologies, etc. In this review, we concentrate on factors that have not been fully detailed in prior reviews, such as the oocyte morphology, oocyte recovery methods, denuding procedures, first polar body morphology and embryo quality.

• Asian-Australasian Journal of Animal Sciences
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• 제17권10호
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• pp.1369-1373
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• 2004

The percentages of sperm motility and normal acrosome on the liquid boar semen diluted and preserved at $4^{\circ}C$ with lactose hydrate, egg yolk and N-acetyl-D-glucosamine (LEN) diluent were significant differences according to preservation day and incubation time, respectively. The sperm motility steadily declined from 96.9% at 0.5 h incubation to 78.8% at 6 h incubation at 1 day of preservation. However, the sperm motility rapidly declined after 4 day of preservation during incubation. The normal acrosome steadily declined from 93.3% at 0.5 h incubation to 73.8% at 6 h incubation at 1 day of preservation. However, the normal acrosome rapidly declined after 3 day of preservation during incubation. The rates of sperm penetration and polyspermy were higher in 5 and $10{\times}10^6$ sperm/ml than in 0.2 and $1{\times}10^6$ sperm/ml. Mean numbers of sperm in penetrated oocyte were highest in $10{\times}10^6$ sperm/ml compared with other sperm concentrations. The rates of blastocysts from the cleaved oocytes (2-4 cell stage) were highest in $1{\times}10^6$sperm/ml compared with other sperm concentrations. In conclusion, we found out that liquid boar sperm stored at $4^{\circ}C$ could be used for in vitro fertilization of pig oocytes matured in vitro. Also, we recommend $1{\times}10^6$sperm/ml concentration for in vitro fertilization of pig oocytes.

• Asian-Australasian Journal of Animal Sciences
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• 제20권4호
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• pp.461-465
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• 2007

Porcine Endogenous Retroviruses (PERVs) are a major concern in relation to xenotransplantation. Previous research indicated that PERVs are present at about 50 copies in the pig genome and their chromosomal insertion sites are different among pig breeds. We examined nine Korean native pigs and seven Asian Wild Boars for the presence of a PERV-A at SSC 1q2.4 and a PERV-B at SSC 7p1.1-2 previously reported in a Large White pig. The PERV-B at locus 7p1.1-2 displayed insertional variability in Korean native pigs and Asian Wild Boars. Using the primers for the PERV-A at 1q2.4 from Large White pig, we only can amplify an unclassified 798 bp sequence, which showed insertional variability only in Korean native pigs. This study indicates that there are differences within and between Asian and European pigs in PERV insertions and suggests that selection could generate PERV-free lines of pigs more suitable for xenotransplantation.

• 한국수정란이식학회지
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• 제25권4호
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• pp.215-219
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• 2010

The objective of this study was to examine the reproductive characteristics of cloned miniature piglets produced from surrogate domestic pigs. Somatic cell nuclear transfer (SCNT) miniature pig embryos were transferred into domestic pigs. As controls, domestic pigs of the same breed with surrogates for SCNT embryos and miniature pigs of the same breed with the somatic cell donor were bred by artificial insemination and natural mating, respectively. Surrogate domestic pigs that farrowed cloned miniature piglets had a significantly longer gestation length (118.1 days) than conventionally bred domestic (115.4 days) and miniature (115.5 days) pigs. Furthermore, the birth weight of cloned miniature piglets produced from domestic pigs (743 g) was significantly greater than that of miniature piglets produced by natural breeding (623 g). Also, cloned miniature piglets had a significantly lower weaning rate (49.7%) than conventionally produced domestic (91.5%) and miniature (100%) piglets. No differences were observed between female and male cloned piglets in gestation length, litter size, birth weight, or weaning rate. Our results demonstrate that gestation length is extended in domestic pigs that are transferred with SCNT miniature pig embryos and that cloned miniature piglets have increased birth weight and high pre-weaning mortality.

• Asian-Australasian Journal of Animal Sciences
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• 제13권2호
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• pp.258-264
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• 2000

Production systems for cloned pigs are very important not only for an increase in production of superior animals but also for the production of knockout animals with organs that do not contain antigens for xenotransplantation or to analyze functions of isolated human genes. At present, however, effective systems have not been developed. We have tried to produce cloned pigs by transfering cultured cells into enucleated oocytes and obtained some cloned embryos. To develop a production system for cloned pigs, the basic technologies needed to support such an effort must be improved.

• 한국동물생명공학회지
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• 제35권3호
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• pp.258-264
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• 2020

This study investigated the effect of variation in the number of somatic-cell-cloned embryos and their developmental stage at transfer on pregnancy, as well as the influence of the estrus status of recipient pigs on in vivo development of cloned porcine embryos after embryo transfer. For somatic cell nuclear transfer (SCNT), fibroblast cells were obtained from a male porcine fetus. Recipient oocytes were collected from prepubertal gilts at a local abattoir and then cultured. After SCNT, reconstructed embryos of different numbers and developmental stages were transferred into recipient pigs. The developmental stage of the cloned embryos and the number of transferred embryos per surrogate showed no significant differences in terms of the resulting cloning efficiency. However, the pregnancy rate improved gradually as the number of transferred cloned embryos was increased from 100-150 or 151-200 to 201-300 per recipient. In pre-, peri-, and post-ovulation stages, pregnancy rates of 28.6%, 41.8%, and 67.6% and 16, 52, and 74 offspring were recorded, respectively. The number of cloned embryos and estrus status of the recipient pig at the time of transfer of the cloned embryo affect the efficiency of pig production; therefore, these variables should be particularly considered in order to increase the efficiency of somatic cell pig cloning.

• Reproductive and Developmental Biology
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• 제31권4호
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• pp.227-233
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• 2007

This study was conducted to examine the mRNA expression of apoptosis-related and imprinted genes and methylation pattern of the differentially methylated region (DMR) of H19 gene in day 35 of SCNT pig fetuses. The day 35 of natural mating (control) or cloned (clone) pig fetuses were recovered from uterus. Endometrium from dam and liver from fetus were obtained, respectively. mRNA expression was evaluated by real-time PCR and methylation pattern was analyzed by bisulfite sequencing method. The Bcl-2 mRNA expression in clone was significantly lower than that of control (p<0.05). The mRNA expression of H19 gene in both endometrium and liver was significantly higher in clone than that of control, respectively (p<0.05). The level of IGF-2 mRNA in liver of clone was significantly lower than that of control (p<0.05), whereas the mRNA expression of IGF2-R gene in liver of clone was significantly higher than that of control (p<0.05). The DMR of H19 was lower methylation pattern in clone than that of control. These results suggest that the aberrant mRNA expression of apoptosis-related and imprinted genes and the lower DMR methylation pattern of imprinted gene may be closely related to the inadequate fetal development of cloned fetus.

• Reproductive and Developmental Biology
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• 제33권3호
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• pp.157-162
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• 2009

This study was performed to comprehend the developmental characteristics of cloned embryos knocked out (KO) of $\alpha$-1,3-galactosyltransferase (GalT) gene. Immature oocytes were collected and cultured for 40 hrs (1-step) or 20hrs (with hormone) + 20hrs (without hormone) (2-step). The embryos transferred with miniature pig ear fibroblast cell were used as control. The reconstructed embryos were cultured in PZM-3 with 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. To determine the quality of the blstocysts, TUNEL and quantitative realtime RT-PCR were performed. The embryos were transferred to a surrogate (Landrace) at an earlier stage of the estrus cycle. The maturation rate was significantly higher in 2-step method than that of 1-step (p<0.05). The blastocyst development of GalT KO embryos was significantly lower than that of normal cloned embryos (p<0.05). The total and apoptotic cell number of GalT KO blastocysts was not different statistically from control. The relative abundance of Bax-$\alpha$/Bcl-xl ratio was significantly higher in both cloned blastocysts than that of in vivo blastocysts (p<0.05). Taken together, it can be postulated that the lower developmental potential and higher expression of apoptosis related genes in GalT KO SCNT embryos might be a cause of a low efficiency of GalT KO cloned miniature pig production.

• 농업과학연구
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• 제44권3호
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• pp.309-317
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• 2017

Melatonin (N-acetyl-5-methoxytryptamine) is an indole synthesized from tryptophan by the pineal gland in animal. The major function of melatonin is to modulate circadian and circannual rhythms in photoperiodic mammals. Importantly, however, melatonin is also a free radical scavenger, anti-oxidant, and anti-apoptotic agent. Recently, the beneficial effects of melatonin on oocyte maturation and embryonic development in vitro have been reported in many species such as pig, cattle, sheep, mouse, and human. In this review, we will discuss recent studies about the role of melatonin in the production of porcine and bovine oocytes and embryos in vitro in order to provide useful information of melatonin in oocyte maturation and embryo culture in vitro.

• 미생물학회지
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• 제42권2호
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• pp.89-94
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• 2006

본 연구는 치주질환 병인론 연구에 빈번히 사용되는 Prevotella intermedia ATCC 49046 균주를 특이적으로 검출 및 동정할 수 있는 PCR primer를 개발하기 위하여 시행하였다. P. intermedia ATCC 49046 유전체 DNA를 추출하고, Hind III 제한효소를 이용하여 무작위 클로닝법으로 유전체 DNA 절편을 얻었다. Southern blot 분석법을 이용하여 DNA 절편의 특이성을 조사하였고, chain termination 법을 이용하여 핵산염기서열을 결정하였다. 이를 바탕으로 PCR primer를 설계하고, P. intermedia ATCC 49046에 대한 균주 특이성 및 검출 한계(민감도)를 조사하였다. Southern blot 분석 결과 Pig6 DNA probe는 서양인에서 분리 동정된 P. intermedia 균주와만 hybridization하였고, 한국인에서 분리 동정된 P. intermedia균주들과는 반응이 없었다. Pig6 DNA probe는 813 bp의 핵산염기로 구성되어 있었으며, 이를 바탕으로 설계된 Pig6-F3와 Pig6-R3 primer 쌍에 의해서는 서양 균주에 특이적인 PCR산물이 증폭되었다. Pig6-60F와 Pig6-770R primer 쌍에 의해서는 P. intermedia ATCC 49046 유전체 DNA에서만 특이적인 PCR 산물이 증폭되었다. 두 가지 primer 쌍들 각각에 대한 P. intermedia 유전체 DNA량의 검출 한계를 알아보기 위한 민감도실험 결과 두가지 primer 쌍들 모두 4 pg (약2000마리)까지 검출 가능하였다. 이상의 연구결과를 종합하면, Pig6-60F와 Pig6-770R primer쌍은 P. intermedia ATCC 49046을 균주 특이적으로 동정할 수 있어, 이 균주의 보존적 측면에서 유용하게 이용될 수 있을 것으로 사료된다.