• Journal of Embryo Transfer
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• v.32 no.3
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• pp.111-122
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• 2017

The objective of this experiment was to explore the effects of Roscovitine (Rosco) prior to in vitro maturation (IVM) of immature pig oocyte. Brilliant cresyl blue test has been used to select the good quality of oocyte. Specifically, the effects of Rosco exposure on nuclear and cytoplasmic maturation, diameter, intracellular glutathione (GSH) and reactive oxygen species (ROS), and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), and gene expression levels in SCNT embryos have been measured. Cumulus oocyte complexes (COCs) have been exposed in $75{\mu}M$ of Rosco for 22 and 44 h. The COCs that were matured in the IVM for 44 h without Rosco used as control group. Diameter of matured porcine oocytes 44 h culture with Rosco was significantly lower than 22 h culture with Rosco and control groups. GSH was higher in control group than 22 h and 44 h with Rosco but reduction of ROS in 22 h than 44 h with Rosco. In PA, exposure with Rosco 44 h oocytes group has been significantly lower than 22 h and control group in rates of maturation, cleavage and blastocyst formation. Similarly, in SCNT embryos rates of maturation, cleavage and formation of blastocyst have been also significantly lower in 44 h Rosco treated group than other two groups. SCNT embryos treated with Rosco 22 h showed greater expression levels of POU5F1, DPPA2 and NDP52Il mRNA compared with other two groups. Our results demonstrate that Rosco treatment with 22 h prior to IVM improves the development competence of porcine oocyte.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.139-146
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• 2017

The objective of this study was to determine the effect of fructose that was supplemented to a chemically defined in vitro maturation (IVM) medium on oocyte maturation and embryonic development after parthenogenesis in pigs. The base medium for in vitro maturation (IVM) was porcine zygote medium (PZM) that was supplemented with 0.05% (w/v) polyvinyl alcohol (PVA) or 10% (v/v) porcine follicular fluid (pFF). In the first experiment, when immature pig oocytes were matured in a chemically defined medium that was supplemented with 5.5 mM glucose or with 1.5, 3.0 and 5.5 mM fructose, 3.0 mM fructose resulted in a higher nuclear maturation (91.5%) than 1.5 and 5.5 mM fructose (81.9 and 81.9%, respectively) but showed a similar result with 5.5 mM glucose (94.2%). However, there was no significant differences among groups in the embryo cleavage (89.4-92.4%), blastocyst formation (37.5-41.1%), and mean cell number of blastocyst (30.8-34.2 cells). Fructose at the concentration of 3.0 mM (1.08 pixels/oocyte) resulted in a higher intra-oocyte glutathione (GSH) content than 1.5 and 5.5 mM fructose (1.00 and 0.87 pixels/oocytes, respectively) while the cumulus cell expansion was not influenced. In the second experiment, effect of individual and combined supplementation of a chemically defined maturation medium with 5.5 mM glucose or 3.0 mM fructose was examined. No significant effect was found in the nuclear maturation (86.3-92.6%). Embryo cleavage was significantly increased by the combined supplementation with glucose and fructose (95.2%) compared to that with 3.0 mM fructose only (85.7%) while blastocyst formation (37.3-42.8%) and embryonic cell number (33.3-34.1 cells) were not altered. Effect of supplementation of pFF-containing medium with glucose and fructose + glucose was examined in the third experiment. No significant effect by the supplementation with glucose and fructose or glucose alone was observed in the nuclear maturation of oocytes (90.7-94.1%) and blastocyst formation (51.0-56.5%). Our results demonstrate that 3.0 mM fructose was comparable to 5.5 mM glucose in supporting in vitro oocyte maturation and embryonic development after parthenogenesis and could be used as an alternative energy source to glucose for in vitro maturation of pig oocytes.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.11
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• pp.1565-1572
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• 2015

Porcine embryonic stem cells (pESCs) have become an advantageous experimental tool for developing therapeutic applications and producing transgenic animals. However, despite numerous reports of putative pESC lines, deriving validated pESC lines from embryos produced in vitro remains difficult. Here, we report that embryo aggregation was useful for deriving pESCs from in vitro-produced embryos. Blastocysts derived from embryo aggregation formed a larger number of colonies and maintained cell culture stability. Our derived cell lines demonstrated expression of pluripotent markers (alkaline phosphatase, Oct4, Sox2, and Nanog), an ability to form embryoid bodies, and the capacity to differentiate into the three germ layers. A cytogenetic analysis of these cells revealed that all lines derived from aggregated blastocysts had normal female and male karyotypes. These results demonstrate that embryo aggregation could be a useful technique to improve the efficiency of deriving ESCs from in vitro-fertilized pig embryos, studying early development, and deriving pluripotent ESCs in vitro in other mammals.

• Korean Journal of Animal Reproduction
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• v.19 no.1
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• pp.43-48
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• 1995

The purpose of this study was to examine the suvival rates of cryopreserved porcine embryos collected on Day 6 (Day 0=onset of estrus) with various cryprotectants. Eighty two embryos at different stages, ranged from expanded blastocyst to hatched blastocyst, were allocated to 6 experimental groups in different combinations of cryoprotectants glycerol, egg yolk and/or trehalose. Porcine embryos were cryopreserved using conventinal slow freezing precedures. The embryos were equilibrated with one of the freezing solutions, cooled from 25 to -7$^{\circ}C$ at 1$^{\circ}C$/min, seeded at -7$^{\circ}C$ frozen to -36$^{\circ}C$ at 0.5$^{\circ}C$/min, and then plunged into liquid nitrogen. The frozen embryos were thawed by immersion in 37$^{\circ}C$ water and the cryoprotectants were removed by dilution with 0.5M sucrose solution. Embryonic survival was estimated from the normal development of embryos for 12, 24 and 48hrs culture. Then the embryos were stained and their cell nuclei was counted. The survival rates of morphological embryos were significantly higher in group I (10% glycerol) or group IV (10% glycerol＋10% egg yolk＋0.5M trehalose) than those in other groups, although the nuclei number was quite higher in embryos treated with 10% glycerol and 0.25M trehalose at expended blastocyst. However, hatched blastocysts showed higher viability and nuclei number treated with either egg york or trehalose, but the survival rates after 48hrs of culture were quite low. These results indicate that egg yolk and trehalos as a supplement to freezing solutions can be useful to the cryopreservation ofn porcine embryos.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.2
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• pp.86-92
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• 2019

The aim of this study was to investigate effects of hyaluronidase during IVM on oocyte maturation, oxidative stress status, expression of cumulus expansion-related (PTX, pentraxin; GJA1, gap junction protein alpha 1; PTGS2, prostaglandin-endoperoxide synthase 2) and fatty acid metabolism-related (FADS1, delta-6 desaturase; FADS2, delta-5 desaturase; PPARα, peroxisome proliferator-activated receptor-alpha) mRNA, and embryonic development of porcine oocytes. The cumulus-oocyte complexes (COCs) were incubated with 0.1 mg/mL hyaluronidase for 44 h. Cumulus expansion was measured at 22 h after maturation. At 44 h after maturation, nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured. Gene expression in cumulus cells was analyzed using real time PCR. The cleavage rate and blastocyst formation were evaluated at Day 2 and 7 after insemination. In results, expansion of cumulus cells was suppressed by treatment of hyaluronidase at 22 h after maturation. Intracellular GSH level was reduced by hyaluronidase treatment (p < 0.05). On the other hand, hyaluronidase increased ROS levels in oocytes (p < 0.05). Only PTGS2 mRNA was enhanced in COCs by hyaluronidase (p < 0.05). Population of oocytes reached at metaphase II stage was higher in control group than hyaluronidase treated group (p < 0.05). Both of cleavage rate and blastocyst formation were higher in control group than hyaluronidase group (p < 0.05). Our present results showed that developmental competence of porcine oocytes could be reduce by hyaluronidase via inducing oxidative stress during maturation process and it might be associated with prostaglandin synthesis. Therefore, we suggest that suppression of cumulus expansion of COCs could induce oxidative stress and decrease nuclear maturation via reduction of GSH synthesis and it caused to decrease developmental competence of mammalian oocytes.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.7
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• pp.903-912
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• 2012

In Erhualian and Yorkshire reciprocal cross $F_1$ pig populations, we examined the mRNA expression characteristic of liver-derived IGF-1, IGF-1R, IGF-2, IGF-2R and IGFBP-3 during the embryonic and postnatal developmental periods (E50, E70, E90, D1, D20, D70, D120 and D180). Our results demonstrated that the IGF-system genes mRNA levels exhibited an ontogenetic expression pattern, which was potentially associated with the porcine embryonic development, postnatal growth, organogenesis and even the initiation and acceleration of puberty. The expression pattern of IGF-system genes showed variation in the reciprocal cross ($F_1$ YE and EY pigs). This study also involved the expression features of imprinted genes IGF-2 and IGF-2R. The parent-of-origin effect of imprinted genes was reflected by their differential expression between the reciprocal crosses populations. The correlation analysis also indicated that the regulatory network and mechanisms involved in the IGF system were a complex issue that needs to be more fully explored. A better understanding of IGF system components and their interactive mechanisms will enable researchers to gain insights not only into animal organogenesis but also into somatic growth development and even reproduction.

• Journal of Embryo Transfer
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• v.24 no.1
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• pp.65-69
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• 2009

The aim of the present study was to investigate the cleavage pattern, its developmental ability and apoptosis of porcine embryo in vitro. Morphology data on a total of 919 embryos were analyzed retrospectively. Forty-eight hours after insemination, embryos were classified into five groups based on the cleavage state as follows; 1 cell, 2 cell, 4 cell, 5 to 8 cell and fragmentation. These groups were cultured another 120 hours and then evaluated for blastocyst formation. Blastocyst formation rates were significantly higher in 4 cell (42.5%) and 5 to 8 cell (48.6%) cleaving groups than in other groups (p<0.05). On the other hand, 2 cell and fragmentation groups produced 4.9% and 3,9% blastocysts, respectively. And we could verify that in the event of 2 cell block and fragmentation of embryo. To analyze the apoptotic frequency in preimplantation development of porcine IVF embryos, all cells of each blastocyst were performed by TUNEL assay. There were no significantly differences in the total cell numbers of embryos and apoptotic cell rate in blastocysts among the each classified groups. Data suggest that 4 cell and 5 to 8 cell cleaving embryos at 48 hour after insemination have high developmental competence, and may be an useful parameter to predict the development of preimplantation embryos and to study using preimplanation embryonic research.

• Journal of Animal Reproduction and Biotechnology
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• v.39 no.3
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• pp.169-178
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• 2024

Background: Typical difficulties encountered during in vitro fertilization (IVF) to produce embryos in pigs include poor pronucleus formation and poor-quality fertilized embryos because of high polysperm invasion. In this study, we evaluated the effects of supplementation with apple seed extract (ASE) and coculture systems on porcine in vitro-fertilized embryo culture. Methods: Slaughterhouse-derived ovaries were used to obtain cumulus-oocyte complexes (COCs). COCs were conventionally used to perform IVF. We examined the differences in apoptosis and metabolism during development following addition of ASE to normal culture and coculture systems. Matrix metalloproteinases (MMPs), cell development-related factors, and apoptotic proteins were compared in porcine embryos produced under different conditions. Results: The expression of genes related to insulin-like growth factor (IGF) signaling was increased in the coculture system. In the ASE group, early apoptosis and necrosis were reduced in fertilized embryos and the late survival rate increased. Supplementation of the coculture system with ASE led to increased expression of BCL-2 and decreased expression of Casp-3 in the cytoplasm, thereby lowering the apoptosis rate and inducing MMP expression. In addition, compared with the extract-supplemented group in normal culture, the activity of MMP-2 decreased in the coculture system supplemented with ASE, activity of MMP-9 increased, and the expression of dynactin p62 and BrdU in the cytoplasm was higher than that in the other groups. Conclusions: The coculture system increased the activity of the embryonic cytoplasm compared with the non-coculture system. Supplementation with ASE may induce cell activity and inhibit the expression of apoptotic factors.

• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.167-172
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• 2012

The key regulators of apoptosis are the interacting protein of the Bcl-2 family. Bcl-2, an important member of this family, blocks cytochrome C release by sequestering pro-apoptotic BH3-only proteins such as Bid, Bad, Bax and Bim. The pro-survival family members (Bcl-2, Bcl-XL, Bcl-W) are critical for cell survival, since loss of any of them causes cell death in certain cell type. However, its role during early porcine embryonic development is not sufficient. In this study, we traced the effects of Bcl-2 inhibitor, ABT-737, on early porcine embryonic development. We also investigated several indicators of developmental potential, including gene expression (apoptosis-related genes) and apoptosis, which are affected by ABT-737. Porcine embryos were cultured in the PZM-3 medium with or without ABT-737 for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without ABT-737 ($14.7{\pm}3.0$ vs $30.3{\pm}4.8%$, p<0.05). TUNEL assay showed that the number of containing fragmented DNA at the blastocyst stage increased in the ABT-737 treated group compared with control (4.7 vs 3.7, p<0.05). The mRNA expression of the pro-apoptotic gene Bax increased in ABT-737 treated group (p<0.05), whereas expressions of the anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-XL, Bcl-W) decreased (p<0.05). Also, expressions of the ER stress indicator genes (GRP78, XBP-1 and sXBP-1) increased in ABT-737 treated group (p<0.05). In conclusion, Bcl-2 is closely associated with of apoptosis- and ER stress-related genes expressions and developmental potential in pig embryos.

• Proceedings of the KSAR Conference
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• 2001.10a
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• pp.55-55
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• 2001

Monosaccaride hexose, may have different role in embryo development of different species. Glucose, fructose and galactose are glycolysible substrates but their effect on embryo development is not identical. Glucose has negative effect to early embryonic stage in several species whereas it is inevitable after compaction. For fructose, it can support blastocyst formation in hamster, mouse and bovine embryo. Effect of galactose is known as detrimental even at a low concentration while glucose has adverse effect only at high concentration in hamster. (omitted)