154 THE EFFECT OF ZINC ON PORCINE IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT AFTER IN VITRO FERTILIZATION

2014 ◽  
Vol 26 (1) ◽  
pp. 191
Author(s):  
Y. Jeon ◽  
J. D. Yoon ◽  
L. Cai ◽  
S. U. Hwang ◽  
E. Kim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Inductively Coupled Plasma ◽  
In Vitro Maturation ◽  
Formation Rate ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Significant Difference

Zinc (Zn) is one of the abundant transition metals in biology and is an essential component of most cells. However, there are few reports about the effect of Zn in porcine oocytes. The objective was to investigate the effects of supplementary Zn during in vitro maturation (IVM) of porcine oocytes. We investigated nuclear maturation, intracellular glutathione (GSH) levels, reactive oxygen species (ROS) levels, and subsequent embryonic development after IVF. Before the experiment, Zn concentrations in IVM medium and body fluids were measured using inductively coupled plasma spectrophotometer (sensitivity: 1 μM) and treatment concentrations were determined. Zinc concentration was 12.6 μM in porcine plasma and 12.9 μM in porcine follicular fluid. We confirmed that Zn was not detected in IVM medium. A total of 541 cumulus–oocyte complexes (COC) were used for the evaluation of nuclear maturation. The COC were matured in TCM-199 medium supplemented with various concentrations of Zn (0, 6, 12, 18, and 24 μM). After 44 h of IVM, no significant difference was observed in all groups (metaphase II rate: 85.7, 88.7, 90.4, 90.3, and 87.2%, respectively). A total of 100 matured oocytes were examined for the effects of different Zn concentrations (0, 6, 12, 18, and 24 μM) on porcine oocyte intracellular GSH and ROS levels, which were measured through fluorescent staining and image analysis program. The groups of 12, 18, and 24 μM showed a significant (P < 0.05) increase in intracellular GSH levels (1.45, 1.67, and 1.78, respectively) compared with the control and 6 μM group (1.00 and 1.08, respectively). The intracellular ROS level of oocytes matured with 12, 18, and 24 μM (0.82, 0.68, and 0.55) were significantly (P < 0.05) decreased compared with the control and 6 μM groups (1.00 and 1.03, respectively). Finally, the developmental competence of oocytes matured with different concentrations of Zn (0, 6, 12, 18, and 24 μM) was evaluated after IVF. There were no significantly different in cleavage rates. However, cleavage patterns and blastocyst (BL) formation were different. Fragmented embryo ratio of the 12 μM group (14.9%) was significantly lower than that of the other groups (control, 6, 18, and 24 μM: 26.4, 17.8, 18.4, and 18.0%, respectively). Oocytes treated with 12 μM Zn during IVM had a significantly higher BL formation rate (28.2%) after IVF compared with the control (19.8%). In conclusion, these results indicate that Zn treatment as body fluid concentration during IVM improved the developmental potential of IVF in porcine embryos by increasing the intracellular GSH concentration and decreasing the ROS level. This work was supported, in part, by a grant from the Next-Generation Bio Green 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

189 THE EFFECTS OF RESVERATROL ON PORCINE OOCYTES IN VITRO MATURATION AND SUBSEQUENT DEVELOPMENT AFTER PARTHENOGENETIC ACTIVATION AND IN VITRO FERTILIZATION

2012 ◽  
Vol 24 (1) ◽  
pp. 207 ◽  
Author(s):  
S. S. Kwak ◽  
S. A. Jeong ◽  
Y. B. Jeon ◽  
S. H. Hyun
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
In Vitro Maturation ◽  
Formation Rate ◽  
Control Group ◽  
Developmental Potential ◽  
Parthenogenetic Activation ◽  
Nuclear Maturation ◽  
Significant Difference

The present study investigated the effects of resveratrol (a phytoalexin with various pharmacological activities) during in vitro maturation (IVM) of porcine oocytes on nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, gene expression in matured oocytes and subsequent embryonic development after parthenogenetic activation (PA) and IVF. Data were analyzed with SPSS 17.0 using Duncan's multiple range test. In experiment 1, a total of 1146 cumulus–oocyte complexes (COC) were divided into 5 groups (0, 0.1, 0.5, 2.0 and 10.0 μM resveratrol). In the nuclear maturation after 44-h IVM, the groups of 0.1, 0.5 and 2.0 μM (83.0, 84.1 and 88.3%, respectively) had no significant difference compared to the control group (84.1%). The group of 10.0 μM decreased the nuclear maturation (75.0%) significantly (P < 0.05). In experiment 2, a total of 300 matured oocytes were examined for the effects of different resveratrol concentrations (0, 0.5, 2.0 and 10.0 μM) on porcine oocyte intracellular GSH and ROS levels. The groups of 0.5 and 2.0 μM showed a significant (P < 0.05) increase in intracellular GSH levels (1.3 and 1.3, respectively) compared with the control and 10.0 μM groups (1.0 and 1.0, respectively). The intracellular ROS level of oocytes matured with 2.0 μM resveratrol (0.4) was significantly (P < 0.05) decreased compared to other groups (control: 1.0; 0.5 μM: 0.6; and 10.0 μM: 0.7). In experiment 3, lower expression of apoptosis-related genes (Bax, Caspase-3 and Bak) was observed in matured oocytes treated with 2.0 μM resveratrol when compared with that of the control (P < 0.05). In experiment 4, a total of 728 oocytes were divided into 4 groups (control, 0.5, 2.0 and 10.0 μM) and examined subsequent to embryonic development after PA. Oocytes treated with 2.0 μM resveratrol during IVM had a significantly higher cleavage (CL) rate, blastocyst (BL) formation rate and total cell numbers (TCN) after PA compared with those of the control (2.0 μM: 96.6%, 62.1% and 49.1 vs control: 88.3%, 48.8% and 41.4, respectively) and the 10.0 μM groups (87.3%, 41.4% and 40.9, respectively). Oocytes treated with 0.5 μM resveratrol (87.2%, 50.5% and 48.6, respectively) during IVM had significantly higher TCN, but there were no differences in CL and BL formation rates. In experiment 5, a total of 935 oocytes in 3 groups (control, 2.0 and 10.0 μM resveratrol) were conducted in IVF. The BL formation rate and TCN were significantly higher in the group of 2.0 μM resveratrol (20.5% and 54.0, respectively) than the control (11.0% and 43.4, respectively) and 10.0 μM group (11.7% and 45.0, respectively), but there was no significant difference in CL rate. In conclusion, 2.0 μM resveratrol supplementation during IVM improved the developmental potential of PA and IVF in porcine embryos by increasing the intracellular GSH concentration, decreasing the ROS level and decreasing apoptosis-related gene expression during oocyte maturation. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ008121), Rural Development Administration, Republic of Korea.

302 EFFECT OF GROWTH DIFFERENTIATION FACTOR 8 ON PORCINE OOCYTE IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT AFTER PARTHENOGENETIC ACTIVATION AND IN VITRO FERTILIZATION

2015 ◽  
Vol 27 (1) ◽  
pp. 240
Author(s):  
J. D. Yoon ◽  
E. Lee ◽  
S.-H. Hyun
Keyword(s):  
Embryonic Development ◽  
In Vitro Maturation ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Vitro Fertilization ◽  
Treatment Groups ◽  
Nuclear Maturation ◽  
Intracellular Ros ◽  
Sperm Penetration

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. The purpose of this study was to investigate the effects of GDF8 on in vitro porcine oocytes maturation and subsequent embryonic development after pathenogenetic activation (PA) and in vitro fertilization (IVF). We investigated nuclear maturation, intracellular glutathione (GSH), reactive oxygen species (ROS) levels, sperm penetration (SP) analysis, and subsequent embryonic development after PA and IVF. Each concentration (0, 1, 10, and 100 ng mL–1) of GDF8 was added in maturation medium during process of in vitro maturation. Data were analysed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science) mean ± s.e.m. After 44 h of IVM, no significant difference was observed on nuclear maturation from the different concentration (0, 1, 10, and 100 ng mL–1) of GDF8 treatment groups (85.5, 85.9, 89.4, and 87.6%, respectively) compared with the control (P > 0.05). The 10- and 100-ng mL–1 GDF8-treated groups showed a significant (P < 0.05) decrease in intracellular ROS levels compared with other groups. The embryonic developmental competence after PA was affected with GDF8 treatment during IVM. The 10- and 100-ng mL–1 treatment groups showed significantly (P < 0.05) higher cleavage rates (67.5 and 69.1%, respectively) compared with control group (53.7%). The 10- and 100-ng mL–1 treatment groups also showed significantly (P < 0.05) higher blastocyst formation rates (50.5 and 52.7%, respectively) compared with other groups (34.5 and 35.8%). The IVF embryonic developmental competence also was affected with GDF8 treatment during IVM. The 10-ng mL–1 treatment group showed a significantly (P < 0.05) higher blastocyst formation rates and total cell number compared with control (21.5 and 131.3 v. 15.0 and 92.6%, respectively). Also, in the sperm penetration assessment, the 10- and 100-ng mL–1 treatment groups showed higher mono spermy ratio and fertilization efficiency (32.7 and 27.1, 32.0 and 26.5 v. 22.6 and 19.7%, respectively) than control, which was significant (P < 0.05). In conclusion, the treatment with 10 ng mL–1 of GDF8 during IVM improved the PA and IVF porcine embryo developmental competence by decreasing the intracellular ROS levels.This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2013R1A2A2A04008751), Republic of Korea.

scholarly journals Development to Blastocyst Stage of Pig Oocytes Matured, Fertilized and Electroactivated In Vitro

2002 ◽  
Vol 45 (6) ◽  
pp. 547-556
Author(s):  
N. R. Mtango ◽  
M. D. Varisanga ◽  
D. Y. Juan ◽  
P. Wongrisekeao ◽  
T. Suzuki
Keyword(s):  
In Vitro Maturation ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Activation Rates ◽  
Oviduct Fluid

Abstract. This study was designed 1) to determine the effectiveness of two in vitro maturation (IVM) media (tissue culture medium [TCM] and modified synthetic oviduct fluid supplemented with amino acids [mSOFaa]), 2) to compare the effects of two in vitro fertilization (IVF) media (modified Tris-buffered medium [mTBM] and mSOFaa) on the developmental competence of pig oocytes, and 3) to test the activation ability of IVM pig oocytes matured in TCM or mSOFaa, electroactivated and cultured in mSOFaa. The nuclear maturation rates were similar between IVM media (91.0 % vs. 89.0 %). A similar result was obtained when the activation rates were 54.2 % in TCM and 56.0 % in mSOFaa, and the blastocyst rates were 7.9 % and 6.1 %, respectively. There was no significant difference between mSOFaa and mTBM in the percentage of embryos with two pronuclei 33.2 % vs. 13.8 % or polypronuclei 5.3 % vs. 13.4 %. The cleavage rate was the same in both media. The medium mSOFaa gave a significantly higher (P< 0.05) blastocyst rate than mTBM (12.7 % vs. 3.9 %). We concluded that mSOFaa can enhance in vitro maturation, fertilization and culture of pig oocytes.

153 ZINC INSUFFICIENCY DURING PORCINE OOCYTES IN VITRO MATURATION CAUSED MEIOTIC BLOCK AND DEVELOPMENTAL FAILURE

2014 ◽  
Vol 26 (1) ◽  
pp. 190
Author(s):  
E. Kim ◽  
Y. Jeon ◽  
J. D. Yoon ◽  
L. Cai ◽  
S. U. Hwang ◽  
...  
Keyword(s):  
Embryonic Development ◽  
In Vitro Maturation ◽  
Formation Rate ◽  
Treated Group ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Total Cell Number ◽  
Nuclear Maturation

The objective was to investigate the effects of zinc (Zn) insufficiency during in vitro maturation (IVM) of porcine oocytes. Zinc insufficiency was induced by treatment of Zn chelator, N,N,N′,N′-tetrakis-(2-pyridylmethyl)-ethylendiamine (TPEN). In experiment 1, we investigated the effect of duration of Zn insufficiency in IVM on oocytes maturation and subsequent embryonic development after parthenogenetic activation (PA). First, 10 μM TPEN was added to the IVM medium for 0, 7, 15, or 22 h. After TPEN treatment, 10 μM Zn were supplemented on IVM medium except in the 0 h group. Reductions in the nuclear maturation rates were dependent on TPEN duration. The 0-h-treated oocytes showed 83.9 ± 3.9% metaphase II (MII) rate; the 7-h-treated oocytes had significantly lower MII rate (44.8 ± 3.0%) than 0-h-treated oocytes. The majority of 15- and 22-h-treated oocytes were arrested at metaphase I (MI rate: 98.0 ± 1.0 and 97.2 ± 1.7%, MII rate: 0 and 0%, respectively). Embryonic developmental competence was similar to maturation results. Reduction in cleavage and blastocyst (BL) rates were also dependent on duration of TPEN treatment (cleavage rate: 65.3 ± 1.4, 42.6 ± 4.8, 2.6 ± 0.1, and 3.0 ± 1.6%; BL formation rate: 29.3 ± 2.8, 9.2 ± 1.5, 0, and 0% for 0, 7, 15, and 22 h). Total cell number of BL was also significantly different. Total cell number of BL in the 0-h-treated group (51.4 ± 4.5) was significantly higher than that in the 7-h-treated group (23.2 ± 1.6). In experiment 2, to confirm that the Zn insufficiency caused oocyte immaturities and loss of developmental competence in TPEN-treated oocytes, we investigated nuclear maturation and subsequent embryonic development following 3 groups: (1) non treatment (control); (2) 10 μM TPEN treatment during 22 h of IVM; (3) 10 μM TPEN + 10 μM Zn treatment during 22 h of IVM. Only TPEN-treated oocytes and TPEN+Zn-treated oocytes showed contrasting results. Oocyte maturation rates and subsequent embryonic development competence in TPEN with Zn-treated oocytes were similar to control (MII rate: 93.0 ± 1.2 and 92.7 ± 1.8%, BL formation rate: 42.0 ± 6.7 and 40.0 ± 7.5% for TPEN+Zn-treated oocytes and control). These results were significantly different compared with only TPEN-treated oocytes’ results (MII rate: 0.61 ± 0.61%, BL formation rate: 0%). In conclusion, Zn is an essential element for successful oocyte maturation and embryo development in porcine. Zinc insufficiency caused meiotic block and had lasting effects on early embryo development. This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

129 Embryo Production from Fully Grown and Growing Stage Oocytes in Japanese Black Calves

2018 ◽  
Vol 30 (1) ◽  
pp. 204
Author(s):  
S. Matoba ◽  
K. Takeda ◽  
Y. Ohkubo ◽  
M. Hirako ◽  
Y. Hirao
Keyword(s):  
Genetic Resources ◽  
Formation Rate ◽  
Developmental Competence ◽  
Breeding Value ◽  
Cox1 Gene ◽  
Embryo Production ◽  
Vitro Fertilization ◽  
Copy Numbers ◽  
Significant Difference

Before fattening of Japanese Black female calves, the ovaries are sometimes removed and discarded. Production of embryos from the oocytes residing in such ovaries is beneficial for the rescue of genetic resources. The aim of this study was to establish an embryo production system using oocytes collected from the ovaries of calves just before fattening and to investigate the correlation between the developmental competence of oocytes and the onset of puberty. Ovaries were collected from Japanese Black calves (9.5 ± 0.1 months old, n = 30, 3 replicates) in a fattening farm and separated according to the presence or absence of corpus luteum as the indicator of puberty (CL+ and CL– groups, respectively). Immature fully grown oocytes (IM oocytes), ~120 μm in diameter, were aspirated from follicles of 2 to 6 mm in diameter (CL+; n = 132, CL–; n = 41) and cultured for 22 to 23 h for maturation (IVM). After in vitro fertilization (IVF) for 6 h (designated Day 0), the oocytes were cultured for 9 days (in vitro culture, IVC) (Matoba et al. 2014 J. Dairy Sci. 97, 743-753). Growing oocytes, ~100 μm in diameter, were also collected by dissecting the follicles smaller than 1 mm in diameter. The growing oocytes were cultured for 14 days on membrane inserts for in vitro growth (IVG) (Hirao et al. 2013 Biol. Reprod. 89, 1-11). Then, IVG oocytes (CL+; n = 29, CL–; n = 32) were subjected to IVM, IVF, and IVC. Presumptive zygotes were cultured individually in microwells in culture dishes. Mitochondrial DNA (mtDNA) copy numbers (COX1 gene) of oocytes were examined (Takeda et al. 2010 Mitochondrion 10, 137-142). A comparison was made between the oocytes derived from calf ovaries and those of oocytes collected from cow ovaries by transvaginal ovum pick-up or by aspiration of the ovaries obtained at a local slaughterhouse. In IM oocytes, the rate of embryos developed to the blastocyst stage on Day 7 to 9 was higher in the CL+ group than in the CL– group (38.9 ± 2.6 v. 10.0 ± 7.1%, respectively; P < 0.05, t-test). However, IVG oocytes were compared, there was no significant difference in the blastocyst formation rate between the CL+ or CL– groups (34.7 ± 5.2 v. 19.8 ± 10.1%, respectively). The mtDNA copy numbers of matured oocytes were similar between IM and IVG oocytes irrespective of the maturity of the donor animals. In conclusion, we demonstrated the possibility of embryo production by IVM/IVF/IVC using fully grown and growing oocytes that are present in the ovaries of calves before fattening. Puberty positively affected the developmental competence of IM oocytes but the effect was not significant in IVG oocytes. Utilisation of both fully grown oocytes and growing oocytes may double the chance of rescuing genetic resources of high-breeding-value calves. This study was partly supported by grants from the Ito Foundation. We thank staff at Mie-Katoubokujou for allowing access to calves’ ovaries.

208 EFFECT OF GDF8 AND SB-431542 ON PORCINE OOCYTE DURING IN VITRO MATURATION AND SUBSEQUENT EMBRYONIC DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 235
Author(s):  
J. D. Yoon ◽  
E. Lee ◽  
S.-H. Hyun
Keyword(s):  
Treatment Group ◽  
Embryonic Development ◽  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Transforming Growth Factor ◽  
Formation Rate ◽  
Transforming Growth Factor Β ◽  
Nuclear Maturation ◽  
Porcine Oocyte

Growth differentiation factor-8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. SB-431542 (SB) is a specific inhibitor of transforming growth factor-β superfamily type I activin receptor-like kinase (ALK) receptors such as ALK4, ALK5, and ALK7. The purpose of this study is investigation of the effects of GDF8 and SB on porcine oocytes during in vitro maturation and subsequent embryonic development. We first performed ELISA to detect GDF8 concentrations in follicular fluid for each size of follicle; sizes were as follows: small (<3 mm), medium (>3 mm and <6 mm), and large (>6 mm) follicle. After detection of the GDF8 concentration in follicular fluid, we investigated the effect of GDF8 and SB treatment during in vitro maturation (IVM) on nuclear maturation, intracellular glutathione (GSH), and reactive oxygen species (ROS) levels, and embryonic development after IVF and parthenogenetic activation (PA). Data were analysed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science, IBM, New York, NY, USA) mean ± SEM. The ELISA result showed different concentrations of GDF8 for each grade of follicular fluid: small, 0.479 ng mL–1; medium, 0.668 ng mL–1; and large, 1.318 ng mL–1. During the IVM process, 1.318 ng mL–1 of GDF8 and 5 ng mL–1 of SB were added to the maturation medium as control, SB, SB+GDF8, and GDF8 treatment groups. After 44 h of IVM, GDF8 group (90.4%) showed a significantly higher nuclear maturation rate than control and SB+GDF8 groups (85.4 and 81.7%). The SB group (78.9%) showed significantly reduced nuclear maturation rate compared with control (P < 0.05). The GDF8 treatment group showed a significant decreased intracellular ROS and increased GSH levels compared with other groups (P < 0.05). The SB+GBF8 treatment group showed a significantly better cytoplasmic maturation than the SB treatment group. In the PA embryonic development analysis, the GDF8 treatment group showed a significantly higher blastocyst formation rate compared with other groups (47.9, 37.2, 46.4, and 58.7% respectively; P < 0.05). In the IVF embryonic development analysis, the GDF8 treatment groups showed significantly higher blastocyst formation rate compared with the SB group (28.2 and 42.2%, respectively; P < 0.05). In conclusion, treatment with GDF8 during porcine oocyte IVM improved the embryonic developmental competence via increased cytoplasmic maturation and led to better oocyte maturation from the ALK receptor inhibition by SB.

scholarly journals Porcine follicular fluid derived from > 8 mm sized follicles improves oocyte maturation and embryo development during in vitro maturation of pigs

Zygote ◽  
2020 ◽  
pp. 1-6
Author(s):  
Ji-Eun Park ◽  
Sang-Hee Lee ◽  
Yong Hwangbo ◽  
Choon-Keun Park
Keyword(s):  
Embryonic Development ◽  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Developmental Competence ◽  
Control Group ◽  
Cumulus Expansion ◽  
Treatment Groups ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

Summary The aim of the present study was to investigate the effects of porcine follicular fluid (pFF) from large-sized (LFF; >8 mm in diameter) and medium-sized (MFF; 3–6 mm in diameter) follicles on the maturation and developmental competence of porcine oocytes. Cumulus–oocyte complexes (COCs) were collected from follicles 3–6 mm in diameter. The collected COCs were incubated for 22 h with LFF or MFF (in vitro maturation (IVM)-I stage) and were incubated subsequently for 22 h with LFF or MFF (IVM-II stage). Cumulus expansion was confirmed after the IVM-I stage and nuclear maturation was evaluated after the IVM-II stage. Intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured and embryonic development was evaluated. Relative cumulus expansion and GSH levels were higher in the LFF group compared with in the MFF group after the IVM-I stage (P < 0.05). After the IVM-II stage, the numbers of oocytes in metaphase-II were increased in the LFF group and GSH content was higher in all of the LFF treatment groups compared with in the MFF treatment groups during both IVM stages (P < 0.05). ROS levels were reduced by LFF treatment regardless of IVM stage (P < 0.05). Blastocyst formation and the total numbers of cells in blastocysts were increased in all LFF treatment groups compared with the control group (P < 0.05). These results suggested that pFF from large follicles at the IVM stage could improve nucleic and cytoplasmic maturation status and further embryonic development through reducing ROS levels and enhancing responsiveness to gonadotropins.

Vitrification of in vitro matured oocytes of Mangalica and Large White pigs

2008 ◽  
Vol 56 (3) ◽  
pp. 399-410 ◽  
Author(s):  
Erika Varga ◽  
Erzsébet Gajdócsi ◽  
Brigitta Petz Makkosné ◽  
Ildikó Salamon ◽  
Ágnes Bali Papp
Keyword(s):  
Embryonic Development ◽  
Developmental Competence ◽  
Large White ◽  
Normal Morphology ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Maturation Rate ◽  
Morula Stage ◽  
Potential Tool

The breeding of Mangalica, a native pig breed in Hungary, had been started in 1833, but this pig breed almost became extinct in Hungary in the past decades. In 1991, the number of sows was only 200. Although in these days the existing Mangalica population consists of more than 6000 animals representing different colour variations, the preservation of this traditional pig breed is still very important. Vitrification is a potential tool for the preservation of gametes and embryos of these animals. The aim of this study was to investigate the effects of vitrification on the developmental competence of Mangalica (M) and Large White (LW) oocytes following fertilisation. The oocytes were vitrified by the Open Pulled Straw (OPS) method using different concentrations of ethylene glycol and dimethyl sulphoxide as cryoprotectants. After rehydration the oocytes underwent in vitro fertilisation; the resultant zygotes were then cultured in vitro for four days to assess embryonic development. In the first experiment, in vitro maturation of M and LW oocytes was compared. No significant difference was observed in the nuclear maturation rate of LW and M oocytes. In the second experiment, the sensitivity of oocytes to vitrification was examined by evaluating oocyte morphology after thawing. A higher percentage of LW oocytes showed normal morphology compared to M oocytes, indicating that Mangalica oocytes are more sensitive to cryoprotectants than Large White oocytes. After warming and in vitro fertilisation, more than 50% of the oocytes started embryonic development and by the end of the incubation period morula stage embryos had developed in both groups. The results show that the OPS vitrification technique is well suited to preserve Mangalica oocytes and from these oocytes morula embryos can be produced.

155 EFFECT OF CO-CULTURE WITH CUMULUS-DERIVED SOMATIC CELLS DURING IN VITRO MATURATION ON PORCINE CUMULUS–OOCYTE COMPLEXES AND SUBSEQUENT EMBRYONIC DEVELOPMENT AFTER IN VITRO FERTILIZATION

2014 ◽  
Vol 26 (1) ◽  
pp. 191 ◽  
Author(s):  
J. D. Yoon ◽  
L. Cai ◽  
S. U. Hwang ◽  
Y. Jeon ◽  
E. Kim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Mrna Expression ◽  
In Vitro Maturation ◽  
Somatic Cells ◽  
The Other ◽  
Control Group ◽  
Cortical Granules ◽  
Developmental Potential ◽  
Significant Difference

The purpose of this study was to investigate the effects of co-culture with cumulus-derived somatic cells (CSC) during porcine in vitro maturation (IVM) and subsequent embryonic development after IVF. The CSC were cultured in Dulbecco's modified Eagle medium for 48 h with various numbers of cumulus-derived somatic cells (0.0, 2.5, 5.0, and 10.0 × 104), and then cultured in TCM-199 for 4 h before the oocytes were added. Cumulus-oocytes complexes from 3- to 6-mm follicles were matured in 500 μL of TCM-199, with eCG and hCG, for 22 h, and then cultured in M199 without hormones for 22 h. Each experiment consisted of at least 4 replicates. Statistical analyses were carried out using SPSS 17.0 software (SPSS Inc., Chicago, IL). Percentage data were compared by one-way ANOVA, followed by Duncan's multiple range test. Data were presented as means ± s.e.m. Differences were considered to be significant if the P-value was 0.05. After IVM, no significant difference (P < 0.05) was observed in nuclear maturation rate among the 0.0, 2.5, 5.0, and 10.0 × 104 groups (88.0 ± 2.37, 81.5 ± 2.17, 87.0 ± 1.98 and 86.0 ± 1.93%, respectively). The 2.5 × 104 group showed a significant (P < 0.05) increase in intracellular glutathione (GSH) levels compared with that of the other groups. Intracellular reactive oxygen species (ROS) levels of mature oocyte in all groups showed no significant differences. The developmental competence of matured oocytes in all groups was evaluated after IVF. The 2.5 and 5.0 × 104 groups showed significantly (P < 0.05) high cleavage rates (60.0 ± 4.7 and 64.52 ± 5.9%, respectively) compared with the 0 and 10.0 × 104 groups (43.15 ± 5.0 and 53.8 ± 5.0%, respectively). The 2.5 × 104 group showed a significantly (P < 0.05) higher BL formation rate (35.7 ± 2.9) than control group (21.0 ± 3.8%, respectively), and higher total cell number (127.25 ± 7.7) compared with the 0 and 10 × 104 groups (89.3 ± 4.0 and 92.6 ± 3.7, respectively). In the analysis of gene expression, IVF-BL derived from the 2.5 and 5.0 × 104 groups showed higher (P < 0.05) mRNA expression of PCNA, which is an essential component of the DNA replication and repair machinery and POU5F1 has been used to evaluate developmental potential in embryos. The 10.0 × 104 group showed higher (P < 0.05) mRNA expression of caspase-3 and Bak as known pro-apoptotic factors, compared with the control group IVF-BL. The results of cortical granules distribution which leads digesting sperm receptor proteins ZP2 and ZP3 to block polyspermy, showed that the 2.5 × 104 group was increased significantly (P < 0.05) compared with the other co-culture groups (13.7 ± 6.1, 29.2 ± 9.5, 18.3 ± 0.8 and 19.52 ± 5.3, respectively). In conclusion, co-culture with 2.5 × 104 cumulus-derived somatic cells during IVM improved the developmental potential of porcine IVF embryos by increasing the intracellular GSH level and distribution of cortical granules during oocyte maturation. This work was supported, in part, by a grant from the Next-Generation BioGreen 21 Program (No. PJ00956901), Rural Development Administration, and the National Research Foundation of Korea Grant funded by the Korean Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

scholarly journals Effect of Interleukin-7 on In Vitro Maturation of Porcine Cumulus-Oocyte Complexes and Subsequent Developmental Potential after Parthenogenetic Activation

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 741
Author(s):  
Dongjin Oh ◽  
Joohyeong Lee ◽  
Eunhye Kim ◽  
Seon-Ung Hwang ◽  
Junchul-David Yoon ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Developmental Competence ◽  
Control Group ◽  
Developmental Potential ◽  
Parthenogenetic Activation ◽  
Nuclear Maturation ◽  
Interleukin 7

Interleukin-7 (IL-7) is a cytokine essential for cell development, proliferation and survival. However, its role in oocyte maturation is largely unknown. To investigate the effects of IL-7 on the in vitro maturation (IVM) of porcine oocytes, we analyzed nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, and subsequent embryonic developmental competence after parthenogenetic activation (PA) under several concentrations of IL-7. After IVM, IL-7 treated groups showed significantly higher nuclear maturation and significantly decreased intracellular ROS levels compared with the control group. All IL-7 treatment groups exhibited significantly increased intracellular GSH levels compared with the control group. All oocytes matured with IL-7 treatment during IVM exhibited significantly higher cleavage and blastocyst formation rates after PA than the non-treatment group. Furthermore, significantly higher mRNA expression levels of developmental-related genes (PCNA, Filia, and NPM2) and antioxidant-related genes (GSR and PRDX1) were observed in the IL-7-supplemented oocytes than in the control group. IL-7-supplemented cumulus cells showed significantly higher mRNA expression of the anti-apoptotic gene BCL2L1 and mitochondria-related genes (TFAM and NOX4), and lower transcript levels of the apoptosis related-gene, Caspase3, than the control group. Collectively, the present study suggests that IL-7 supplementation during porcine IVM improves oocyte maturation and the developmental potential of porcine embryos after PA.

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