• Journal of Animal Reproduction and Biotechnology
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• v.37 no.2
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• pp.80-86
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• 2022

The ability to determine the sex of bovine embryos before the transfer is advantageous in livestock management, especially in dairy production, where female calves are preferred in milk industry. The milk production of female and male cattle benefits both the dairy and beef industries. Pre-implantation sexing of embryos also helps with embryo transfer success. There are two approaches for sexing bovine embryos in farm animals: invasive and non-invasive. A non-invasive method of embryo sexing retains the embryo's autonomy and, as a result, is less likely to impair the embryo's ability to move and implant successfully. There are lists of non-invasive embryo sexing such as; Detection of H-Y antigens, X-linked enzymes, and sexing based on embryo cleavage and development. Since it protects the embryo's autonomy, the non-invasive procedure is considered to be the safest. Invasive methods affect an embryo's integrity and are likely to damage the embryo's chances of successful transformation. There are different types of invasive methods such as polymerase chain reaction, detection of male chromatin Y chromosome-specific DNA probes, Loop-mediated isothermal amplification (LAMP), cytological karyotyping, and immunofluorescence (FISH). The PCR approach is highly sensitive, precise, and effective as compared to invasive methods of farm animal embryonic sexing. Invasive procedures, such as cytological karyotyping, have high accuracy but are impractical in the field due to embryonic effectiveness concerns. This technology can be applicable especially in the dairy and beef industry by producing female and male animals respectively. Enhancing selection accuracy and decreasing the multiple ovulation embryo transfer costs.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.6
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• pp.693-699
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• 2010

Imprinted genes are essential for fetal development, growth regulation, and postnatal behavior. However, little is known about imprinted genes in livestock. We hypothesized that certain putatively imprinted genes affected normal peri-implantation development such as embryo elongation, initial placental development, and preparation of implantation. The objective of the present study was to investigate the mRNA expression patterns of several putatively imprinted genes during the porcine peri-implantation stages from day 6 to day 21 of gestation. Imprinted genes were selected both maternally (Dlk1, IGF2, Ndn, and Sgce) and paternally (IGF2r, H19, Gnas and Xist). Here, we report that the maternally imprinted gene IGF2 was expressed from day 6 (Blastocyst stage), but Dlk1, Ndn, and Sgce were not expressed in this stage. These genes were first expressed between days 12 and day 14. All the maternally imprinted genes studied showed significantly high expression patterns from day 18 of embryo development. In contrast, paternally imprinted genes IGF2r, H19, Gnas, and Xist were first expressed from day 6 of embryo development (BL). Our data demonstrated that the expression of H19 and Gnas genes was significantly increased from day 14 of the embryo developmental stage, while IGF2r and Xist only showed high expression after day 21. This study is the first to show that the putatively imprinted genes were stage-specific during porcine embryonic development. These results demonstrate that the genes studied may exert important effects on embryo implantation and fetal development.

• Journal of Embryo Transfer
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• v.9 no.3
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• pp.221-227
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• 1994

This study was conducted to examine the condition of in vitro culture system and the viability after embryo transfer of in vitro matured-in vitro fertilized (IVM-IVF) bovine embryos. The in vitro development to the blastocyst stage was enhanced by supplying bovine serum albumin(BSA) to co-culture medium with bovine oviduct epithelial tissue(BOET) compared with that in medium supplemented with fetal bovine serum(FBS) (41.2% vs. 26. 3%, P<0.05). After transfer of IVM-IVF blastocysts into the uterine horn of recipient females (Aberdeen Angus), one was pregnant to term and produced a head of male Korean native calf. These results confirm that the in vitro development of IVM-IVF bovine embryos is affected with different protein source in co-culture with BOET, and IVM-IVF embryos can develop to term after in vitro culture and embryo transfer.

• Journal of Sericultural and Entomological Science
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• v.8
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• pp.35-40
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• 1968

As a result of investigating the change of Ascorbic acid and Lipid which have a relation with metabolism of a silkworm egg in the process of the growth of embryo is silkworm eggs. The following facts have been found 1) Ascorbic acid has gradually increased before the period of the Byong B embryo and it has decreased after period of Byong B embryo. 2) Triglyceride and Total cholesterol has gradually increased before the period of the Byong B embryo and it has decreased after period of the Byong B embryo. 3) Phospholipid has gradually decreased before the period of the Byong A embryo and it increased during the Byong B embryo and decreased again at same stage. It has increased from the head pigment of embryo to hachting. 4) Free Fatty acid decreased during the Byong A embryo stage and increased from the Byong B embryo stage to the Ki A embryo stage and decreased again and increased shortly before the hachting.

• Molecules and Cells
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• v.37 no.2
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• pp.126-132
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• 2014

As a tumor suppressor homologue during mitosis, Chk2 is involved in replication checkpoints, DNA repair, and cell cycle arrest, although its functions during mouse oocyte meiosis and early embryo development remain uncertain. We investigated the functions of Chk2 during mouse oocyte maturation and early embryo development. Chk2 exhibited a dynamic localization pattern; Chk2 expression was restricted to germinal vesicles at the germinal vesicle (GV) stage, was associated with centromeres at pro-metaphase I (Pro-MI), and localized to spindle poles at metaphase I (MI). Disrupting Chk2 activity resulted in cell cycle progression defects. First, inhibitor-treated oocytes were arrested at the GV stage and failed to undergo germinal vesicle breakdown (GVBD); this could be rescued after Chk2 inhibition release. Second, Chk2 inhibition after oocyte GVBD caused MI arrest. Third, the first cleavage of early embryo development was disrupted by Chk2 inhibition. Additionally, in inhibitor-treated oocytes, checkpoint protein Bub3 expression was consistently localized at centromeres at the MI stage, which indicated that the spindle assembly checkpoint (SAC) was activated. Moreover, disrupting Chk2 activity in oocytes caused severe chromosome misalignments and spindle disruption. In inhibitor-treated oocytes, centrosome protein ${\gamma}$-tubulin and Polo-like kinase 1 (Plk1) were dissociated from spindle poles. These results indicated that Chk2 regulated cell cycle progression and spindle assembly during mouse oocyte maturation and early embryo development.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.1
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• pp.94-101
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• 2020

In the early development of parthenogenetic embryo, cytoplasm and nucleic acid fragmentation may be a cause of lower embryo development. The purpose of this study was to evaluate whether embryonic development and apoptosis factors can be reduced by controlling the in-vitro culture environment by the addition of hormones, pregnancy serum and uterine milk. Our study showed that the activity of Casp-3 increased within the cytoplasm when artificially used hormones to induce the incubation environment, and PCNA's manifestation was low. However, the addition of pregnant serum appeared to lower the Casp-3 activity compared to the other groups. In addition, MMP-9 activity was increased and early embryo development and cytoplasmic fidelity were also increased. Therefore, the results of the present study showed that the use of gestational serum in the development of parthenogenetic embryo inhibit apoptosis and increases cytoplasmic reorganization by natural environmental control in in vitro culture.

• Development and Reproduction
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• v.18 no.3
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• pp.139-143
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• 2014

Different with other fishes, the guppies (Poecilia reticulata) is ovoviviparity, which retain their fertilized eggs within the follicle throughout gestation. The synchronously growing diplotene oocytes store nutrients in droplets and yolk, before their maturation and fertilization. The lecithotrophic strategy of development entails the provisioning of embryos with resources from the maternal yolk deposit rather than from a placenta, it allows the extracorporeal culture of guppy embryo. Studies on their early development of live bearers like the guppy including lineage tracing and genetic manipulations, have been limited. Therefore, to optimize conditions of embryo in vitro culture, explanted embryos from pregnant females were incubated in embryo medium (L-15 medium, supplemented with 5, 10, 15, 20% fetal bovine serum, respectively). We investigated whether the contents of FBS in vitro culture medium impact the development of embryos, and whether they would hatch in vitro. Our study found that in 5% of FBS of the medium, although embryos developed significantly slower in vitro than in the ovary, it was impossible to exactly quantify the developmental delay in culture, due to the obvious spread in developmental stage within each batch of eggs, and embryos can only be maintained until the early-eyed. And although in culture with 20% FBS the embryos can sustain rapid development of early stage, but cannot be cultured for the entire period of their embryonic development and ultimately died. In the medium with 10% and 15% FBS, the embryos seems well developed, even some can continue to grow after follicle ruptures until it can be fed. We also observed that embryonic in these two culture conditions were significantly different in development speed, in 15% it is faster than 10%. But 10% FBS appears to be more optimizing condition than 15% one on development process of embryos and survival rate to larvae stage.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.73-73
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• 2002

Nuclear transfer (NT) techniques have advanced in the last years, and cloned animals have been produced by using somatic cells in several species including pig. However, it is difficult that the nuclear transfer porcine embryos development to blastocyst stage overcoming the cell block in vitro. Abnormal segregation of chromosomes in nuclear transferred embryos on genome activation stage bring about embryo degeneration, abnormal blastocyst, delayed and low embryo development. Thus, we are evaluated that the correlations of the frequency of embryo developmental rates and chromosome aberration in NT and In viかo fertilization (IVF) derived embryo. We are used for ear-skin-fibroblast cell in NT. If only karyotyping of embryonic cells are chromosomally abnormal, they may difficultly remain undetected. Then, we evaluate the chromosome aberrations, fluorescent in situ hybridization (FISH) with porcine chromosome 1 submetacentric specific DNA probe were excuted. In normal diploid cell nucleus, two hybridization signal was detected. In contrast, abnormal cell figured one or three over signals. The developmental rates of NT and IVF embryos were 55% vs 63%, 32% vs 33% and 13% vs 17% in 2 cell, 8 cell and blastocyst, respectively. When looking at the types of chromosome aberration, the detection of aneuploidy at Day 3 on the embryo culture. The percentage of chromosome aneuploidy of NT and IVF at 4-cell stage 40.0%, 31.3%, respectively. This result indicate that chromosomal abnormalities are associated with low developmental rate in porcine NT embryo. It is also suggest that abnormal porcine embryos produced by NT associated with lower implantation rate, increase abortion rate and production of abnormal fetuses.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.1
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• pp.55-63
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• 2001

Objectives: This study was carried out to evaluate the effects of embryonic stage, cryoprotectant, and freezing-thawing method on the rates of survival and development of the cryopreserved mouse early embryo and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Materials and Methods: Two to eight cell embryos were obtained from oviducts of mated $F_1$ hybrid female mice superovulated by pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG). Two-step 1,2-propanediol (PROH), dimethylsulfoxide (DMSO) and 4-step PROH DMSO were used as cryoprotectant and dehydration and rehydration method of embryos, and slow-cooling or rapid-cooling method was used as frozen program. The survival rates of embryos were measured after thawing and rehydration, and the developmental rates of embryos were compared and observed during culturing embryos for 24, 48, 72, 96 hrs. Results: As for the survival and development rates of embryos according to embryonic stage, the survival rate of 2 cell stage in PROH and DMSO was significantly higher than 4-8 cell (64.5% versus 62.1 %,79.7% versus 73.2%) (p<0.01, p<0.01), but the development rates of 4-8 cell embryos in PROH and DMSO were significantly higher than 2 cell embryos for whole culture period (p<0.01) and the development rates of 4-8 cell embryos in PROH were significantly higher than 2 cell embryos in DMSO (p<0.01). As for the survival and development rates of embryos according to cryoprotectant, the survival rate of 2 cell embryo in DMSO was significantly higher than that in PROH (74.4% versus 64.5%) (p<0.01), whereas the development rate of embryos was not differ till 24 hrs. The developmen1 rate from morular to hatching blastocyst, however, was significantly higher in PROH than in DMSO during 48 hr (p<0.01). The survival rate of 4-8 cell embryo was 62.1% in PROH and 73.2% in DMSO. The development rates of embryo in PROH were significantly higher for whole culture periods (p<0.01, 0.05). In respect to the effect of freezing and thawing program on the survival and development rates of embryos, method of slow cooling and rapid thawing was more effective than that of rapid cooling and rapid thawing. Conclusions: The survival rate of embryo in 2 cell stage was higher than in 4-8 cell stage, and PROH appears more effective cryoprotectant than DMSO because PROH showed better development rates of embryos in 2 and 4-8 cell stage. Moreover, slow cooling and rapid thawing method was considered as the best cryopreservation program.

• Clinical and Experimental Reproductive Medicine
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• v.19 no.2
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• pp.125-131
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• 1992

The possible effect of human follicular fluid(hFF) on the growth and development of fertilized oocytes and embryos is important because the fallopian tubes are exposed to FF after follicular rupture and the processes of fertilization and embryo cleavage occur inside the fallopian tubes. Previously, it was suggested that human FF might adversely affect on the development of early mouse embryos. In order to investigate the effect of hFF on the development of embryos, early mouse embryos were cultured in media containing various protein sources as bovine serum albumin(BSA), fetal cord serum(FCS) and FF. And we evaluated the development of early mouse embryos in terms of the morphology, cleavage rate, and cell count of blastcysts. There were no significant differences in the morula and blstocyst formation rates of 2-cell mouse embryos cultured in the media containg three different protein sources and three different concentrations of FF. The blastocyst formation rate of 1-cell mouse embryo cultured in FF group was significantly higher than that cultured in BSA group(P<0.05). The morula and blastocyst formation rates of 2-cell mouse embryos of the group cultured in the media containing FF were comparable with those of other two groups, in addition, the cell count of blastocysts of FF group in the 2-cell embryo culture was higher than those of BSA group and HCS group(P<0.01), and this finding was also noted in 1-cell embryo culture. There was no difference in the morula and blastocyst formation rates of the 2-cell mouse embryos cultured in the media containing different concentrations of FF. These results suggest that mature human follicular fluid has no inhibitory activity on the development of early mouse embryos even in high concentration and may be a good protein source which is positively associated with the development of mouse embryos in vitro especially in 1 cell embryo culture.