Oocyte activation procedures and influence of serum on porcine oocyte maturation and subsequent parthenogenetic and nuclear transfer embryo development

Zygote ◽  
2008 ◽  
Vol 16 (4) ◽  
pp. 279-284 ◽  
Author(s):  
E. García-Mengual ◽  
J. Alfonso ◽  
I. Salvador ◽  
C-C. Duque ◽  
M-A. Silvestre
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Oocyte Activation ◽  
Efficient Procedure ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Porcine Oocyte ◽  
Parthenogenetic Embryo ◽  
Nuclear Transfer Embryo ◽  
Parthenogenetic Embryos

SummaryThe viability of SCNT embryos is poor, with an extremely low cloned piglet production rate. In the present work, we studied the effect of three activation protocols based on ionomycin treatment (5 μM ionomycin for 5 min and incubated in 2 mM 6-DMAP for 3.5 h) or electric stimuli (two square wave electrical DC pulses of 1.2 kV/cm for 30 μs) combined or not with 6-DMAP on parthenogenetic embryo development. Oocytes activated by ionomycin plus 6-DMAP showed lower cleavage (47.2 vs. 78.5–81.5; p < 0.05) and blastocyst rates (11.3 vs. 29.2–32.1; p < 0.05) than those activated by electrical and electrical plus 6-DMAP treatments. Also, we studied the effect of addition of serum to maturation medium (0% vs. 10%) on nuclear maturation and further parthenogenetic and SCNT embryo development. We observed in the parthenogenetic embryos that cleavage rates in the serum-free group were significantly higher than in the serum-supplemented group (81.8 vs. 69.6% respectively; p < 0.05), although these differences were not detected in blastocyst rates or blastocyst nuclei numbers. Regarding SCNT embryos, no significant differences were observed in cleavage or blastocyst rates between different experimental groups of SCNT embryos. In conclusion, electrical pulse followed or not by 6-DMAP was found to be an efficient procedure to artificially activate MII porcine oocytes. Moreover, the addition of serum to oocyte maturation media did not seem to improve parthenogenetic or SCNT porcine embryo development.

Effects of Fructose in a Chemically Defined Maturation Medium on Oocyte Maturation and Parthenogenetic Embryo Development in Pigs

2017 ◽  
Vol 32 (3) ◽  
pp. 139-146
Author(s):  
Hyeji Shin ◽  
Minji Kim ◽  
Joohyeong Lee ◽  
Seung Tae Lee ◽  
Choon-Keun Park ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Maturation Medium ◽  
Parthenogenetic Embryo

Pioglitazone improves porcine oocyte maturation and subsequent parthenogenetic embryo development in vitro by increasing lipid metabolism

2019 ◽  
Vol 86 (9) ◽  
pp. 1245-1254 ◽  
Author(s):  
Sang‐Gi Jeong ◽  
Seung‐Eun Lee ◽  
Won‐Jae Kim ◽  
Yun‐Gwi Park ◽  
Jae‐Wook Yoon ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Porcine Oocyte ◽  
Parthenogenetic Embryo ◽  
Development In Vitro

scholarly journals Effect of adding growth factors during in vitro maturation on the developmental potentials of ewe oocytes selected by brilliant cresyl blue staining

2021 ◽  
Vol 14 (2) ◽  
pp. 452-456
Author(s):  
Mohamed Fathi ◽  
Amr F. Elkarmoty
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Blue Staining ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
Nuclear Maturation ◽  
Maturation Medium

Aim: Several factors had been concerned with the developmental competence of the sheep oocyte. This study aims to investigate the effect of adding growth factors (insulin-like growth factor 1 [IGF-1] and epidermal growth factor [EGF]) in the maturation medium of ewe oocytes selected based on brilliant cresyl blue (BCB) screening on in vitro maturation (IVM), fertilization, and pre-implantation embryo development. Materials and Methods: Cumulus-oocyte complexes (COCs) were obtained from the ovaries of slaughtered ewes by either aspiration or slicing techniques. COCs were in vitro matured in a medium containing IGF-1 and EGF (control group). For BCB screening, oocytes were stained and divided into BCB+ oocytes that matured in the same maturation conditions without adding growth factors (Group 2) or in the presence of growth factors (Group 3), and BCB– oocytes that matured in medium without growth factors (Group 4) or with growth factors (Group 5). Results: The supplementation of the maturation medium with growth factors during IVM of (BCB+) oocytes resulted in a significant increase in nuclear maturation rate (90.9%), fertilization rate (75.6%), and embryo developmental rates (60.0%, 46.7%, and 33.3% for cleavage, morula, and blastocyst, respectively). Conclusion: Culturing BCB+ oocytes in a maturation medium containing both EGF and IGF-1 showed a significant improvement in nuclear maturation, fertilization, and pre-implantation embryo development in vitro.

Supplementation with estradiol-17β improves porcine oocyte maturation and subsequent embryo development

2011 ◽  
Vol 95 (8) ◽  
pp. 2582-2584 ◽  
Author(s):  
Ji-Su Kim ◽  
Bong-Seok Song ◽  
Sang-Rae Lee ◽  
Seung-Bin Yoon ◽  
Jae-Won Huh ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Porcine Oocyte ◽  
Estradiol 17Β

150 DEVELOPMENT OF ZONA-FREE AND WITH ZONA PARTHENOGENETIC GOAT EMBRYOS IN DIFFERENT MEDIA

2010 ◽  
Vol 22 (1) ◽  
pp. 233
Author(s):  
M. K. Jena ◽  
D. Malakar ◽  
A. K. De ◽  
S. Garg ◽  
Y. S. Akshey
Keyword(s):  
Embryo Development ◽  
Chemical Activation ◽  
Formation Rate ◽  
Electrical Activation ◽  
Cleavage Rate ◽  
Maturation Medium ◽  
Parthenogenetic Embryo ◽  
Oviductal Fluid ◽  
Phosphate Buffered Saline

The present study was carried out to see the developmental efficiency of zona-free and with zona parthenogenetic goat embryos cultured in Research Vitro Cleave from Cook Australia (RVCL), Embryo Development Media (EDM), modified synthetic oviductal fluid (mSOF), and modified Charles Rosenkrans media (mCR2a). Zona-free embryos were cultured in 4 media, whereas with zona embryos were cultured in 3 media except mCR2a. Ovaries were collected from slaughterhouse and oocytes were isolated by puncturing the follicles in medium containing Dulbecco’s phosphate-buffered saline, 3% BSA, and 50 μg mL-1 gentamicin. Oocytes were matured in maturation medium containing TCM-199 (HEPES modified), 0.05 mg mL-1 Na pyruvate, 0.003 mg mL-1 L-glutamine, 5.5 mg mL-1 glucose, 3 mg mL-1 BSA, 5 μg mL-1 FSH, 10 μg mL-1 LH, 1 μg mL-1 estradiol-17β, 50 μg mL-1 gentamicin, and 10% FBS in 5% CO2 in air at 38.5°C. The COC (15 to 20 oocytes) were placed in 100-μL droplets of maturation medium and incubated in a CO2 incubator (5% CO2 in air) with maximum humidity at 38.5°C for 27 h. Matured oocytes were made cumulus free by treatment with hyaluronidase (0.5 mg mL-1) and zona-free by pronase (2 mg mL-1) in zona-free parthenogenesis. Then the oocytes were activated by 5 μM Ca ionophore for 5 min in a CO2 incubator and then treated with 2 mM 6-DMAP for 4 h. Activation was also done by electrical activation with DC 1.78 kV cm-1, 20 μs, and 2 pulses. Then the zona-free oocytes were kept for in vitro culture in 4 types of media such as RVCL, EDM, mSOF, andm CR2a for 7 days in 5% CO2 in air at 38.5°C. The cleavage rate andmorulae formation were observed in RVCL 40.95%, 13.95%, in EDM 46.92%, 14.75%, in mCR2a 56.66%, 5.88%, and in mSOF 48.23%, 14.63%, respectively. The cleavage rate and morulae formation were also found 55.9%, 14.63% during chemical activation and 32%, 12.5% in electrical activation. Hence, better result was found in chemical activation than electrical activation. For with zona parthenogenesis, the matured oocytes were chemically activated by 5 μM Ca ionophore for 5 min and 2 mM 6-DMAP for 4 h. Then the oocytes were cultured in RVCL, EDM, and mSOF in 100-μL micro-drops media for 7 days. The cleavage, morulae, and early blastocyst production rate were as follows: cleavage rate 75.68%, 72.03%, and 57.11%; morulae 44.61%, 30.29%, and 40.22%; and early blastocyst 17.49%, 11.88%, and 25.01% in RVCL, EDM, and mSOF, respectively. Hatched blastocyst formation rate was 6.75%, 5.48%, and 1.15% in RVCL, EDM, and mSOF, respectively. It could be concluded that zona-free parthenogenetic embryos were produced better in EDM medium and with chemical activation. With zona parthenogenetic embryo development was significantly (P < 0.05) higher in RVCL and EDM media.

Erratum to: 264 EXOGENOUS LINOLENIC ACID IN OOCYTE MATURATION MEDIA PROMOTES NUCLEAR MATURATION AND PARTHENOGENETIC PREIMPLANTATION EMBRYONIC DEVELOPMENT IN THE GOAT

2013 ◽  
Vol 25 (1) ◽  
pp. 280
Author(s):  
A. Veshkini ◽  
A.-A. Khadem ◽  
M. Soleimani ◽  
R. Jahanbin ◽  
M. Salehi ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Linolenic Acid ◽  
Oocyte Maturation ◽  
Mass Spectrometry Analysis ◽  
Control Group ◽  
Parthenogenetic Activation ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Wide Range ◽  
And Control

Dietary intakes of polyunsaturated fatty acids are thought to mediate a wide range of actions in reproductive tissues. This includes the effects on ovarian follicle and corpus luteum functions via improved energy efficiency as well as providing precursors for the synthesis of signalling molecules such as steroids and prostaglandins. An appropriate level of α-linolenic acid (ALA) in the oocyte maturation medium has been shown to induce molecular changes associated with oocyte maturation and embryo developmental competence. In that light, we hypothesised that supplementation of exogenous ALA to maturation media could enhance nuclear maturation and embryonic development in the goat. A preliminary experiment was executed to measure the level of ALA in antral follicles by gas chromatography/mass spectrometry analysis. Our results revealed that the concentration of ALA in follicular fluids ranged from 0.006 to 0.02 mg mL–1 (21.5 to 71.8 µM, with a mean of ~50 µM). To test the effect of ALA on the competence of goat oocytes to complete meiotic maturation to metaphase II and sustain embryonic development, ovaries were obtained from a local abattoir. Cumulus–oocyte complexes were recovered by the slicing method followed by selection of oocytes with a homogenous cytoplasm and at least three layers of compact cumulus cells. The cumulus–oocyte complexes were placed in maturation media supplemented with 50 µM ALA. Oocytes in the control group were incubated in the same maturation medium without ALA. In vitro maturation (IVM) was performed in a humidified atmosphere containing 5% CO2, 5% O2, and 90% N2 at 38.5°C for 24 h. After IVM, several oocytes from the treatment (n = 170) and control (n = 166) groups were stained with Hoechst and were evaluated in relation to their metaphase-II rate. Other groups of oocytes from both the treatment (n = 70) and control (n = 61) groups were subjected to parthenogenetic activation by applying 1 min of exposure to 2.5 µM ionomycin followed by 2 mM 6-DMAP treatment for 3 h. After activation, oocytes were cultured in CR1aa medium for 7 days under the conditions stated above. Four replications were performed. Differences in developmental rates were analysed for significance by one-way ANOVA using SAS version 8.0 (SAS Institute Inc., Cary, NC, USA), considering P < 0.05 to be significant. As a result, supplementation of the maturation media with ALA did not appear to affect cumulus expansion. In contrast, IVM of goat oocytes in the presence of ALA resulted in a significantly higher maturation rate compared with maturation without ALA supplementation (66.4% v. 57.9%). Likewise, addition of ALA to the IVM medium significantly increased the rate of cleavage (60.1% v. 52.4%) and blastocyst formation (22.6% v. 14.9%), calculated from the activated oocytes. Collectively, the results of our study show that supplementation of IVM media with 50 µM ALA promotes nuclear maturation, increases cleavage rate, and results in higher blastocyst rate in goat oocytes after parthenogenetic activation. Thus, providing appropriate levels of ALA in maturation media could have beneficial effects on embryo development and reproductive efficiency in the goat.

260 EVALUATION OF PORCINE IN VITRO PRODUCTION WITH THE ADDITION OF CHITOSAN TO CULTURE MEDIA

2015 ◽  
Vol 27 (1) ◽  
pp. 219 ◽  
Author(s):  
F. García ◽  
Y. Ducolomb ◽  
S. P. Miranda-Castro ◽  
J. F. De la Torre-Sánchez ◽  
S. Romo
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Culture Media ◽  
Biological Properties ◽  
In Vitro Production ◽  
Porcine Oocyte ◽  
Main Component ◽  
Vitro Production ◽  
Positive Effect

Chitosan is a partially deacetylated polymer obtained from the alkaline deacetylation of chitin, which is a glucose-based unbranched polysaccharide widely distributed in nature as the main component of exoskeletons of crustaceans and insects. Chitosan has a variety of physicochemical and biological properties resulting in numerous applications. In addition to its lack of toxicity and allergenicity, its biocompatibility, biodegradability, and bioactivity make it a very attractive substance for diverse applications as a biomaterial in pharmaceutical and medical fields. Chitosan stimulates cell growth and it has been used in fibroblast culture, increasing cell proliferation. For these reasons, it is important to evaluate if this polymer has a positive effect on embryo production. The aim of this study was to evaluate porcine oocyte maturation and embryo development, comparing the effect of supplementing different concentrations of chitosan to the maturation (MM) and development media (DM). Cumulus-oocyte complexes (COC) were aspirated from ovarian follicles of slaughtered sows. The COC were matured in supplemented TCM-199 (MM) and incubated for 44 h. All incubations were performed at 38.5°C, with 5% CO2 in air and humidity at saturation. After maturation IVF was performed, frozen-thawed semen from the same boar was used and gametes were co-incubated in MTBM for 7 h. Then, putative zygotes were cultured in NCSU-23 (DM) for 144 h. The following experiments were performed: 1) addition of 0 (control), 35, 50, 100, and 150 ppm chitosan to the MM (n = 1353), 2) addition of 0, 50, 100, and 150 ppm chitosan to the DM (n = 739), 3) addition of 0, 50, 100, and 150 ppm of chitosan to the MM first and then the same concentrations to the DM (n = 702). When chitosan was added to the MM, the highest percentage of matured oocytes (metaphase II) was obtained in the 50 ppm treatment (87%, P < 0.05) when compared with the control, 100, and 150 ppm groups (78, 78, and 82%, respectively). Regarding the percentage of blastocysts, there were no differences when comparing the treatment and the control groups (ranging from 12 to 13%). After addition of chitosan to the putative zygotes in the DM, the percentage of morulae in the 150 ppm treatment was significantly increased with regard to the other groups (54 v. 46%, respectively, P < 0.05). When adding chitosan to both MM and DM, there was no effect on embryo development. It is concluded that the addition of chitosan to the MM at a concentration of 50 ppm significantly improved oocyte maturation and a concentration of 150 ppm in the DM increased the percentage of morulae. Chitosan had a positive effect on oocyte maturation and embryo development. These results justify further investigations to find out if chitosan can be useful as a supplement for chemically defined media.

scholarly journals 311EFFECT OF OOCYTE MATURATION MEDIA ON THE SPEED OF MEIOTIC PROGRESSION AND BLASTOCYST DEVELOPMENT OF BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 275
Author(s):  
D. Fischer ◽  
J. Bordignon ◽  
C. Robert ◽  
D. Betts
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Immunofluorescence Microscopy ◽  
Chromosomal Abnormalities ◽  
Culture Media ◽  
Blastocyst Formation ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
Meiotic Progression

Environment is crucial for in vitro development of gametes and embryos. The recent progression of culture media towards defined conditions brought to surface the impact of different medium supplements on oocyte and embryo development. In this work we evaluate the effect of various oocyte culture media on bovine oocyte maturation and subsequent embryo development. Bovine cumulus-oocyte complexes were recovered from slaughterhouse ovaries and matured in vitro in either TCM-199 (Gibco) or SOF (Synthetic Oviduct Fluid) media supplemented with BSA (fatty acid-free) or serum (fetal bovine serum). Oocytes from each treatment group were denuded and fixed at 18, 20, 22, 24, 26 and 28h post-maturation (p.m.). Oocyte meiotic progression was monitored in each of the groups (n=28–40 oocytes/group) by immunofluorescence microscopy of chromatin. Oocytes matured in SOF showed a slower rate of meiotic progression when compared to the other groups, with the highest percentage of oocytes reaching the MII stage by 28h p.m. (60.71% SOF-BSA, 71.43% SOF-Serum). The fastest developmental rate was observed in oocytes matured in TCM-serum (77.15% at 24h p.m.) followed by oocytes matured in TCM-BSA (74.29% at 26h p.m.). In order to evaluate the effect of nuclear maturation on chromosome segregation, chromosomal organization of MII oocytes was evaluated by immunofluorescence microscopy within each media group (n=26–31 oocytes/group) at 18, 22 and 26h p.m.. No chromosomal abnormalities were found at 18h p.m.. Both media supplemented with BSA induced lower frequencies of chromosomal abnormalities (0 to 3.23%) and (3.57 to 7.69%) for SOF and TCM, respectively, when compared to their serum-supplemented counterparts (7.14 to 11.54%) and (10 to 10.71%) for SOF and TCM, respectively at 22 and 26h p.m.. Remarkably, the maturation medium and its supplements influenced the speed of blastocyst development. For this experiment, oocytes were matured in TCM-BSA, TCM-Serum, SOF-BSA or SOF-serum, fertilized in vitro in a TALP-base media supplemented with BSA and cultured in SOF-BSA. Blastocyst development was assessed at 7, 8 and 9 days of culture. Cleavage rates were similar between the groups (84–90%), whereas development rates to blastocyst stage varied among treatment groups. Maturation in SOF-BSA induced a delay in blastocyst formation that reached its highest percentage only on day 9 of culture (30.8%); moreover, blastocyst development was carried over until Day 12. When oocytes were matured in the presence of serum, the number of blastocysts did not increase after Day 8 of culture (26.6%, TCM-serum). These results provide evidence of a severe impact of oocyte culture media on the nuclear maturation of oocytes and their subsequent embryonic development after IVF. Moreover, the difference in the rate of oocyte maturation and blastocyst formation emphasizes the necessity for reviewing and adapting current protocols to new systems such as SOF-BSA. [Research funded by NSERC and OMAF of Canada.]

Glucose in a maturation medium with reduced NaCl improves oocyte maturation and embryonic development after somatic cell nuclear transfer and in vitro fertilization in pigs

Zygote ◽  
2021 ◽  
pp. 1-8
Author(s):  
Yongjin Lee ◽  
Hanna Lee ◽  
Joohyeong Lee ◽  
Seung Tae Lee ◽  
Geun-Shik Lee ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Somatic Cell ◽  
Oocyte Maturation ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Blastocyst Formation ◽  
Nuclear Maturation ◽  
Maturation Medium ◽  
Glucose Supplementation

Summary This study was conducted to examine whether glucose in maturation medium containing reduced NaCl could improve oocyte maturation and embryonic development in pigs. The base medium was bovine serum albumin-free porcine zygote medium (PZM)-3 containing 10% (v/v) pig follicular fluid (FPZM) or 0.1% (w/v) polyvinyl alcohol (PPZM). Using each medium, the effects of NaCl concentrations (108 and 61.6 mM) and 5.56 mM glucose supplementation (designated as PZM108N, PZM108G, PZM61N, and PZM61G, respectively) were examined using a 2 × 2 factorial arrangement. When oocytes were matured in FPZM, glucose supplementation improved nuclear maturation compared with no supplementation, regardless of the NaCl concentrations. FPZM61G showed a higher blastocyst formation compared with FPZM108N and FPZM108G after parthenogenesis (PA). Blastocyst formations of somatic cell nuclear transfer (SCNT) embryos derived from FPZM61N and FPZM61G were higher compared with those of oocytes from FPZM108N. When oocytes were matured in PPZM, glucose added to PPZM108 and PPZM61 increased nuclear maturation compared with no supplementation. However, glucose added to PPZM108 did not alter embryonic development after PA. Additionally, oocytes matured in PPZM61G showed a higher blastocyst formation compared with those from PPZM61N. In SCNT, blastocyst formation was not influenced by glucose supplementation of PPZM108, but was increased by maturation in glucose-supplemented PPZM61. In embryonic development of in vitro fertilization (IVF), oocytes matured in medium with reduced NaCl and glucose showed significantly higher blastocyst formation compared with those matured in PPZM108G. Our results demonstrated that glucose in maturation medium containing 61.6 mM NaCl increased oocyte maturation and embryonic development after PA, SCNT, and IVF.

Effect of Actin Polymerization Inhibitor During Oocyte Maturation on Parthenogenetic Embryo Development and Ploidy in Capra hircus

2013 ◽  
Vol 51 (11-12) ◽  
pp. 944-953 ◽  
Author(s):  
R. Ranjan ◽  
R. K. Singh ◽  
T. Yasotha ◽  
Manish Kumar ◽  
Gopal Puri ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Actin Polymerization ◽  
Capra Hircus ◽  
Polymerization Inhibitor ◽  
Parthenogenetic Embryo
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