235 A COMPARISON OF PARTHENOGENETIC DEVELOPMENT OF PORCINE OOCYTES SYNCHRONISED DURING MATURATION BY CYCLOHEXIMIDE OR cAMP

2009 ◽  
Vol 21 (1) ◽  
pp. 215
Author(s):  
W. C. Chen ◽  
J. Zhu ◽  
P. Fisher ◽  
D. Amarnath ◽  
K. H. S. Campbell
Keyword(s):  
Time Window ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Chi Square ◽  
Parthenogenetic Development ◽  
High Level

In vitro maturation of porcine oocytes is characterized by a high level of asynchrony between oocytes. Previous studies reported that cycloheximide (CHX) and 3′, 5′-cyclic AMP (cAMP) synchronize porcine oocytes and improve development to blastocyst stage following IVF or have been used for somatic cell nuclear transfer (SCNT) (Ye et al. 2005 Biol. Reprod. 72(2), 399–406; Betthauser et al. 2000 Nat. Biotechnol. 18(10), 1055–1059). We previously reported that cAMP was more effective than CHX in synchronizing porcine oocyte maturation, producing MII oocytes in a shorter time window and providing a more homogenous population for future SCNT studies (Chen et al. 2008 SRF conference, 2008 abst, p34). Here we compared parthenogenetic development of porcine oocytes synchronized by these two treatments. Selected cumulus–oocyte complexes (COC) obtained from slaughtered gilts were randomly divided into three groups and cultured at 39°C, 5% CO2 in air in modified NCSU-23 medium (with 1 μm glutathione, 1 mm cysteine, 5 mg L–1 insulin, 10 ng mL–1 epidermal growth factor, 10% (v/v) porcine follicular fluid, 1% essential and 0.5% nonessential amino acids) ± hormones (10 IU mL–1 PMSG and 10 IU mL–1 hCG): (1) with hormones for the first 22 h and then without hormones until 44 h; (2) with hormones and 5 μg mL–1 CHX for 12 h, and then with hormones but no CHX until 44 h; (3) with hormones and 1 mm cAMP for 22 h, and then without hormones and cAMP until 44 h. Parthenogenetic development of cycloheximide and cAMP treated oocytes was compared by cleavage rate at 48 h postactivation (hpa) and blastocyst formation at 168 hpa. No significant differences were observed in the frequency of cleavage (96.7 ± 2.1% v. 81.4 ± 11.6% v. 84.5 ± 5.7%), development to blastocyst (28.3 ± 11.4% v. 27.1 ± 5.7% v. 32.8 ± 5.3%) between control, CHX or cAMP treated oocytes, respectively (chi-square test, P > 0.05). However, total cell number was significantly higher in the CHX group than cAMP group (42.7 ± 4.1 v. 31.8 ± 2.0, respectively; t-test, P < 0.05). The results demonstrate that synchronization of porcine oocytes by treatment with CHX or cAMP does not affect subsequent parthenogenetic development if judged by the blastocyst formation, although the meaning of the difference of total cell numbers between CHX and cAMP treatments is still unclear.

scholarly journals 141 BENEFICIAL EFFECT OF ETHYLENEDIAMINETETRAACETIC ACID COMBINED WITH HEMOGLOBIN ON PRE-IMPLANTATION DEVELOPMENT OF PORCINE IN VITRO PRODUCED EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 221
Author(s):  
J.H. Kim ◽  
G.S. Lee ◽  
H.S. Kim ◽  
S.H. Lee ◽  
D.H. Nam ◽  
...  
Keyword(s):  
Beneficial Effect ◽  
Embryo Development ◽  
Ethylenediaminetetraacetic Acid ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Porcine Embryo

Developing a porcine embryo culture system is important for increasing the rates of implantation and pregnancy of somatic cell nuclear transfer (SCNT) embryos. Ethylenediaminetetraacetic acid (EDTA) was shown to inhibit glycolytic activity of cleavage stage embryos, thereby preventing the premature stimulation of glycolysis and enhancing development. However, EDTA should not be used for later-stage embryos as the inhibition of glycolysis reduces energy production at the blastocyst stage and significantly inhibits inner cell mass development. On the other hand, addition of a nitric oxide (NO) scavenger, hemoglobin (Hb), to the culture medium is known to promote embryo development to the blastocyst stage. This study was conducted to evaluate the beneficial effect of EDTA combined with Hb on pre-implantation development of porcine embryos in vitro. Porcine embryos produced by in vitro maturation and fertilization were cultured for 6 days in North Carolina State University (NCSU)-23 medium supplemented with EDTA or/and Hb. All data were subjected to one-way ANOVA and protected least significant difference (LSD) test using the general linear models (GLM) procedure of the statistical analysis system (SAS Institute, Inc., Cary, NC, USA) program to determine differences among experimental groups. Statistical significance was determined when the P value was less than 0.05. In Exp. 1, culturing porcine zygotes with 100 mM EDTA (n = 537) significantly increased cleavage rates (85.3%) at 48 h post-insemination compared to supplementing with 0, 1, or 10 mM EDTA (78.9, 79.7, or 78.2%, respectively). However, EDTA at these concentrations did not promote blastocyst formation compared to the control. In addition, no difference was observed in total cell numbers in blastocysts among the experimental groups (41.8, 42.6, 45.8, 44.5, respectively). In Exp. 2, in vitro-fertilized oocytes were cultured with 0, 1, or 10 mg/mL Hb. Culturing with Hb did not promote porcine embryo development, but significantly increased the total cell number of blastocysts obtained from 1 mg/mL Hb supplementation (n = 566) compared to that of the control (56.8 vs. 41.6). In Exp. 3, culturing embryos (n = 548) with 100 mM EDTA + 1 mg/mL Hb significantly improved rates of cleavage (84.0% vs. 75.2%) and blastocyst formation (19.2% vs. 12.7%), and the total number of cells in blastocysts compared to those of the control (58.4 vs. 42.3). In conclusion, our results demonstrated that EDTA or Hb have different roles in supporting in vitro pre-implantation development of porcine embryos; EDTA mainly stimulated early cleavage up to the 2- to 4-cell stage, and Hb promoted the total cell number of blastocysts. However, combined supplementation with these two chemicals improved cleavage, blastocyst formation, and total cell number in blastocysts. This study was supported by a grant from Korea Ministry of Science and Technology (Biodiscovery).

PSX-B-11 Comparing effects of FGF2 and IGF1 on the development competence of parthenogenetic activated bovine oocytes maturing in vitro

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 327-328
Author(s):  
Galina Singina
Keyword(s):  
Growth Factor ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Control Group ◽  
Total Cell Number ◽  
Developmental Potential ◽  
Maturation Medium ◽  
Bovine Oocytes

Abstract The oocyte quality acquired during in vitro maturation (IVM) are the main limitative factors affecting the embryo production. The aim of the present research was to study effects of fibroblast growth factor 2 (FGF2) and insulin-like growth factor 1 (IGF1) during IVM of bovine oocytes on their developmental potential after parthenogenetic activation. Bovine cumulus-oocyte complexes (COC; n = 1176) were cultured for 22h in either standard maturation medium (TCM-199 supplemented with 10% fetal calf serum (FCS), 0.2 mM sodium pyruvate, 10 μg/ml FSH and 10 μg/ml LH; Control) or maturation medium supplemented with different concentrations (5–160 ng/ml) of FGF2 and IGF1. After IVM, matured oocytes activated by sequential treatment with ionomycin followed by DMAP and cyclohexamide and then cultured up to the blastocyst stage. The obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using DAPI and TUNEL staining. The data from 4 replicates (77–91 oocytes per treatment) were analyzed by ANOVA. Cleavage rates of activated oocytes did not differ between groups and ranged from 63.7 to 68.1%. The addition of 10, 20 and 40 ng/ml of FGF2 to the IVM medium led to an increase in the yield of blastocysts [from 19.6±1.8% (Control) to 35.2±3.4, 29.8±1.9 and 31.1±2.1%, respectively (P&lt;0.05)] and in the total cell number in embryos that developed to the blastocyst stage (P&lt;0.05). Meanwhile, the blastocyst yield and the total cell number in blastocysts in the IGF1-treated groups were similar to that in the control group. No effects of both growth factors on the proportion of apoptotic nuclei in blastocysts (5.3–7.1%) were observed. Thus, FGF2 (but not IGF1) are able to maintain competence for parthenogenetic development of bovine COC during their maturation invitro. Supported by RFBR (18-29-07089) and the Ministry of Science and Higher Education of Russia.

335 EFFECT OF VITAMIN E ON THE DEVELOPMENT OF BOVINE OOCYTES AFTER CHEMICAL ACTIVATION

2007 ◽  
Vol 19 (1) ◽  
pp. 283
Author(s):  
J. I. Park ◽  
Y. Jang ◽  
E. S. Lee
Keyword(s):  
Vitamin E ◽  
Apoptotic Cell ◽  
Chemical Activation ◽  
Calf Serum ◽  
Cell Number ◽  
Total Cell ◽  
Chi Square ◽  
Cell Numbers ◽  
Humidified Air

Oxidative stress is known to induce apoptotic cell death by reactive oxygen species (ROS) generated from in vitro culture systems. This study was conducted to evaluate the effect of Vitamin E (VitE), as antioxidant, on development of bovine embryos activated in vitro. Bovine ovaries were collected from slaughtered cows at a local abattoir. Oocytes were aspirated from follicles 3-8 mm in diameter and transferred to maturation medium: tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal calf serum, 100 mg/mL-1 l-cysteine, 20 mg/mL-1 sodium pyruvate, gonadotropins (250 IU each of eCG and hCG/mL), 10 mg/mL-1 epidermal growth factor, and 100 �M VitE. Oocytes were cultured at 38.9�C in 5% CO2 in humidified air. After 22 hours of culture, oocytes with polar bodies were selected and subjected to activation treatments. Oocytes were exposed to calcium ionomycin (5 �M for 5 min), followed by incubation with 6-DMAP (2 mM) for 3.5 hours in medium supplemented with or without VitE (100 �M). After activation, oocytes were cultured in mSOF medium containing 0.8% BSA at 38.9�C in 5% CO2, 5% O2 in humidified air for 7–8 days. Cell numbers were counted by the number of nuclei of blastocysts stained with Hoechst 33342, and apoptosis was detected by TUNEL assay using a MK500 kit (Takara Bio, Inc., Otsu, Shiga, Japan). Total cell and apoptotic cell number were determined under a fluorescence microscope. Data were analyzed using Student&apos;s t-test and chi-square test. The cleavage and blastocyst rates were significantly higher (P &lt; 0.05) after activation with VitE (78.1&percnt; and 16.3&percnt;, n &equals; 80) than without VitE (66.7&percnt; and 11.0&percnt;, n &equals; 60). Total cell numbers were also significantly higher (P &lt; 0.05) in blastocysts after activation with VitE (143.0 &plusmn; 34.02, n &equals; 21) than in those without VitE (127.63 &plusmn; 40.25, n &equals; 20). However, the percentage of TUNEL-positive (apoptotic) cells was similar between blastocysts activated with VitE (5.38 &plusmn; 2.22) and those without VitE (6.76 &plusmn; 1.98). The results of the present study demonstrate that vitamin E added to activation medium promoted further development of activated embryos, although its role in the alleviation of apoptosis remains unclear.

171 Effect of progesterone and prolactin on the developmental competence of bovine oocytes during the terminal phase of in vitro maturation

2019 ◽  
Vol 31 (1) ◽  
pp. 210
Author(s):  
G. Singina ◽  
E. Shedova ◽  
T. Taradajnic ◽  
V. Konnova ◽  
E. Tsyndrina
Keyword(s):  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Terminal Phase ◽  
Cleavage Rate ◽  
Total Cell Number ◽  
One Step ◽  
Group 2 ◽  
The One

To date, considerable progress has been achieved in in vitro production (IVP) technologies in cattle; however, developmental potentials of oocytes matured in vitro remain low compared with in vivo-matured oocytes. Thus, a better understanding of different aspects of oocyte maturation may allow us to increase the embryo development rate. Our study was aimed to assess the effects of progesterone (P4) and prolactin (PRL) on the bovine oocyte developmental competence. Bovine cumulus-enclosed oocytes (CEO) were matured using either one-step or two-step maturation conditions. For the one-step protocol, CEO were cultured for 24h in TCM-199 supplemented with 10% fetal calf serum (FCS), 10μg mL−1 porcine FSH, and 10μg mL−1 ovine LH (standard medium). For the two-step procedure, CEO were first cultured for 16h in the standard medium (n=1263) and then transferred to 1 of 3 experimental media and cultured for additional 8h in either absence or presence of either P4 (50 ng mL−1) or bovine PRL (50ng mL−1). The 3 media tested in the two-step maturation were (1) TCM-199 containing 10% FCS (group 1), (2) TCM-199 containing 3mg mL−1 BSA (group 2), or (3) Fert-TALP medium supplemented with 6mg mL−1 BSA (group 3). Fert-TALP was selected because it can potentially be used throughout maturation and fertilization. Following in vitro maturation, all oocytes underwent an IVF/in vitro culture procedure as described previously (Singina et al. 2014 Reprod. Fertil. Devel. 26, 154). The embryo development was evaluated at Days 2 and 7 for cleavage and blastocyst rates. In addition, obtained blastocysts were fixed, and the total cell number and the level of apoptosis were determined using 4′,6-diamidino-2-phenylindole (DAPI) and TUNEL staining. The data from 4 to 5 replicates (113-159 oocytes per treatment) were analysed by ANOVA. For oocytes matured for 24h in the one-step culture, the cleavage rate, blastocyst rate, total cell number, and apoptotic nuclei per blastocyst were 66.1±1.1, 23.7±2.0, 71.4±9.1, and 4.8±1.2%, respectively. For the two-step culture, the cleavage rate did not differ from that of the one-step culture system, ranging from 64.8 to 76.5%. Also, no effects of the two-step systems were observed on total cell number (63.0-78.8) or the proportion of apoptotic nuclei (3.3-5.3%) at the blastocyst stage. The culture of CEO in group 1 (without the supplements) had a reduced blastocyst rate (17.4±0.4%; P&lt;0.05) compared with the standard one-step maturation group, and the addition of P4 (but not PRL) improved the blastocyst yield (P&lt;0.05). Furthermore, when P4 (but not PRL) was added to group 2 and group 3 media, blastocyst rates increased significantly (32.9±3.1 and 32.8±2.7%, respectively) compared with those of the one-step group (P&lt;0.05), but did not differ from those of untreated groups 2 and 3 (26.2±2.7 and 30.0±3.0%, respectively). Our data indicate that P4 supplementation during the terminal phase of two-step IVM can enhance the developmental competence of bovine oocytes and that the nature of this effect depends on the composition of IVM medium, whereas no effect of PRL supplementation was observed. The study was supported by RFBR (No. 17-29-08035).

Activation of in vivo- and in vitro-derived porcine oocytes by using multiple electrical pulses

1999 ◽  
Vol 11 (8) ◽  
pp. 457 ◽  
Author(s):  
Christopher G. Grupen ◽  
Paul J. Verma ◽  
Zhong Tao Du ◽  
Stephen M. McIlfatrick ◽  
Rodney J. Ashman ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Single Treatment ◽  
Blastocyst Formation ◽  
Parthenogenetic Development ◽  
Electrical Pulses ◽  
Number Of Cells

The current protocols used to activate pig nuclear transfer embryos are less efficient than those used for other species. To address this problem, the effect of multiple sets of electrical pulses on the parthenogenetic development of in vivo- and in vitro-derived porcine oocytes was examined. Each set of pulses consisted of two 1.5 kV cm–1 DC pulses of 60 s duration each, administered 1 s apart. For in vivo-derived oocytes, application of a second set of pulses 30 min after the first set increased the proportion of oocytes that developed to the blastocyst stage compared with a single treatment (51 v. 34%). Application of a third set of pulses 30 min after the second set reduced the rate of blastocyst formation compared with two sets of pulses. In contrast, the rate of blastocyst formation was greater with one set of pulses compared with two sets for in vitro matured oocytes (31 v. 16%). Additional sets of electrical pulses did not affect the number of cells in blastocysts obtained from either group of oocytes compared with a single treatment. In summary, the study demonstrates that the application of a second set of activating pulses 30 min after the first set is beneficial to in vivo-derived oocytes, but detrimental to in vitro matured oocytes, in terms of their ability to develop parthenogenetically to the blastocyst stage.

scholarly journals 152EFFECT OF REPLACEMENT OF PYRUVATE/LACTATE IN CULTURE MEDIUM WITH GLUCOSE ON PREIMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 198
Author(s):  
N.W.K. Karja ◽  
S. Medvedev ◽  
D. Fuchimoto ◽  
A. Onishi ◽  
M. Iwamoto ◽  
...  
Keyword(s):  
Energy Source ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Culture Period ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Energy Substrates ◽  
Anova Test

Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) reported that replacement of pyruvate and lactate with glucose, as energy substrates, at 48h of culture in IVC medium enhanced the quality of IVP porcine blastocysts. However, the exact time during early cleavage stages when the utilization of glucose as an energy source is optimal has not yet been determined. The purpose of this study was to examine the effects of glucose supplementation at different times of culture on the developmental competence of IVP porcine embryos. Porcine cumulus-oocytes complexes were matured in modified NCSU-37 solution and fertilized in vitro according to Kikuchi et al. All cultures were performed at 38.5°C, 5% O2, 5% CO2, and 90% N2. In experiment 1, after being fertilized (Day 0), putative zygotes (1158 in 6 trials) were cultured in NCSU-37 supplemented with 0.4% BSA, 0.17mM sodium pyruvate, and 2.73mM sodium lactate (IVC-pyr/lac). Embryos (30–50 in each group) were then transferred into NCSU-37 supplemented with 0.4% BSA and 5.55mM D-glucose (IVC-glu) at 24, 48, 72, 96, or 118h of culture. As control groups, putative zygotes (391) were cultured in IVC-pyr/lac or IVC-glu for the whole culture period. In experiment 2, after being fertilized, putative zygotes (543 in 4 trials, 30–50 in each group) were cultured in IVC-pyr/lac, and then were transferred into IVC-glu at 48h, 53h, 58h, or 63h of culture, because glycolytic activity of in vitro-derived porcine embryos was reported to increase around the 8-cell stage, and some embryos develop to that stage before 72h of culture in experiment 1. All embryos were cultured for 6 days, and then development to the blastocyst stage and number of cells per blastocyst were assessed. When IVF embryos were cultured in IVC pyr/lac for 24h or 48h and subsequently in IVC-glu until day 6 in experiment 1, the rates of blastocyst formation were significantly higher (P&lt;0.05, ANOVA test) than those of embryos cultured in IVC-pyr/lac for the whole culture period (24.4% and 23.0% v. 14.5%, respectively). However, when IVC pyr/lac was replaced with IVC-glu, there were no significant differences between the energy source replacement groups and the glucose-only group in terms of the proportions of cleavage, development to the blastocyst stage and mean cell number per blastocyst (P&gt;0.05, ANOVA test) (15.2%–24.4%, and 16.8%, respectively). Replacement of pyruvate and lactate with glucose at 58h of culture in experiment 2 significantly enhanced the rate of blastocyst formation (P&lt;0.05, ANOVA test) but not the mean cell number compared with zygotes in which the replacement was done at 48, 53, and 63h of culture (31.3% v. 20.6%, 20.8%, and 21.1%, respectively) (P&lt;0.05, ANOVA test). In conclusion, replacement of pyruvate and lactate with glucose as energy substrates was optimal at 58h of culture for the in vitro development of pig embryos to the blastocyst stage.

282 GENE EXPRESSION PATTERNS AND QUALITY OF BOVINE EMBRYOS PRODUCED UNDER DIFFERENT OXYGEN CONCENTRATIONS

2007 ◽  
Vol 19 (1) ◽  
pp. 256
Author(s):  
W. J. Son ◽  
M. K. B. ◽  
Y. J. Jeong ◽  
S. Balasubramanian ◽  
S. Y. Choe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Expression Patterns ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Blastocyst Development ◽  
Glut 1

Various factors are known to influence the survival and development of in vitro-produced embryos, including co-culture with somatic cells, antioxidants, and O2 tension. Studies in several species report that embryo development and quality were enhanced at low O2 concentrations. This study compared the effects of 2 O2 concentrations on IVP embryo development, embryo quality, and gene expression to those of in vivo counterparts. Cumulus–oocyte complexes were matured in vitro in TCM-199 with hormones and 10% FCS, and inseminated in TALP medium. Presumptive zygotes were cultured in SOF medium under either 5% or 20% O2 in air. In triplicate, sets of 5 embryos at the 2-cell, 4-cell, 8-cell, 16-cell, morula, and Day 7 blastocyst stages were used for analyzing the expression patterns of apoptotic (Bax and Bcl2), metabolism (Glut-1 and Glut-5), stress (Sox, Hsp70, and G6PDH), compaction (Cx43), oxidation (PRDX5, NADH, and MnSOD), and implantation (VEGF and IFN-tau) genes using real-time quantitative PCR. The expression of each gene was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Statistical analysis was performed with Bonferroni and Duncan tests by ANOVA (P &lt; 0.05). Cleavage rates did not differ among groups. Blastocyst and hatched blastocyst development in 5% O2 was significantly (P &lt; 0.05) higher than in 20% O2. Total cell number of in vivo blastocysts was significantly (P &lt; 0.05) higher than that of IVP blastocysts. ICM ratio and apoptosis of in vivo blastocysts were significantly (P &lt; 0.05) lower than for IVP blastocysts. The relative abundances (RAs) of Glut-1, Glut-5, MnSOD, NADH, PRDX5, Cx43, Bcl2, and IFN-τ were significantly (P &lt; 0.05) higher in in vivo embryos, whereas the RAs of Sox, G6PDH, Hsp70, Bax, and VEGF were significantly (P &lt; 0.05) lower than for IVP counterparts. In conclusion, culture at 5% O2 concentration resulted in higher rates of development to the blastocyst stage, higher total cell numbers, and decreased apoptosis. Furthermore, differences in expression of genes including Glut-1, Glut-5, Sox, G6PDH, Hsp70, Bax, Bcl2, Cx43, PRDX5, NADH, MnSOD, VEGF, and IFN-τ may prove useful in determining optimal culture conditions. This work was supported by ARPC (204119-03-SB010), Republic of Korea.

250 HUMAN EXHALED AIR CAN EFFECTIVELY SUPPORT IN VITRO MATURATION OF PORCINE OOCYTES AND SUBSEQUENT IN VITRO DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 223
Author(s):  
Z. B. Cao ◽  
L. C. Sui ◽  
S. F. Ji ◽  
J. W. Chen ◽  
T. Gui ◽  
...  
Keyword(s):  
Formation Rate ◽  
Cell Number ◽  
Total Cell ◽  
High Oxygen ◽  
Blastocyst Formation ◽  
Total Cell Number ◽  
Nuclear Maturation ◽  
Air Atmosphere ◽  
Significant Difference

The objective of the present study was to examine the feasibility of culturing porcine oocytes and embryos in vitro using the human exhaled lung air atmosphere. In Experiment 1, the effects of lung air atmosphere on nuclear maturation of prepubertal gilt oocytes and subsequent development in vitro of parthenogenetic-activated and somatic-cell-cloned embryos were explored. Abattoir-derived prepubertal gilt cumulus–oocyte complexes (COC) were matured in TCM-199 supplemented with 10 IU mL–1 of eCG, 10 IU mL–1 of hCG, 10 ng mL–1 of epidermal growth factor, and 10% porcine follicular fluid (pFF) for 40 to 44 h at 38.5°C, 100% humidity, and 5% CO2+20% O2 (high oxygen tension) or human exhaled air encapsulated in plastic, airtight bags (lung air) or 5% CO2+7% O2 (low oxygen tension) in the incubator. Nuclear maturation was evaluated by the presence of the 1st polar body. For parthenogenetic activation, denuded oocytes with the 1st polar body were selected and stimulated with a single 1.6-kV/cm, 100-μs direct current pulse followed by culture in porcine zygote medium-3. For NT, denuded metaphase II oocytes were enucleated, and then the donor cell was directly injected into the perivitelline space. After NT, reconstructed couplets were fused and activated electrically followed by treatment in 7.5 μg mL–1 of cytochalasin B and 10 μg mL–1 of cycloheximide for 4 to 6 h before culture in porcine zygote medium-3. We found no significant difference among groups in terms of nuclear maturation rate (66.5% v. 60.2%, 63.2%), cleavage rate (94.8% v. 94.2%, 85.2%), blastocyst formation rate (39.5% v. 40.3%, 32.5%), and total cell number (37 v. 38, 32). Moreover, as for porcine cloned embryo, no significant difference between the lung-air and high-oxygen (20% O2) groups was observed in the cleavage rate (88.3% v. 80.3%), blastocyst formation rate (7.3% v. 10.7%), and total cell number (34 v. 36). The above results indicated that porcine oocytes can be matured in vitro safely and efficiently using the human exhaled lung air atmosphere. In Experiment 2, in vitro developmental competence of porcine zona-free parthenogenetically activated embryos cultured in a lung air, low oxygen (5% O2), or high oxygen (20% O2) tension gas environment was studied. We found no obvious difference among the 3 groups regarding the rates of cleavage (83.0%, 83.6%, 82.8%), but blastocyst formation rate (26.8% v. 48.6%, 48.2%) and total cell number (23 v. 34, 29) in lung air were lower than those in the rest of the groups (P < 0.05). The results show that lung air could be an alternative for preparing a gas environment for in vitro culture of porcine zona-free parthenotes, although not an ideal alternative. Taken together, porcine oocytes and embryos can be cultured in vitro safely and efficiently using the human exhaled lung air atmosphere. Z. B. Cao and L. C. Sui contributed equally to this work. X. R. Zhang and Y. H. Zhang are the corresponding authors. This work was supported by NSFC (30700574), 863 (2008AA101003).

83 IN LOW OXYGEN CULTURE, IS HYPOTAURINE NECESSARY FOR IN VITRO DEVELOPMENT OF PORCINE EMBRYOS?

2016 ◽  
Vol 28 (2) ◽  
pp. 171
Author(s):  
J. A. Benne ◽  
L. D. Spate ◽  
B. M. Elliott ◽  
R. S. Prather
Keyword(s):  
Embryo Culture ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Free Radical Scavengers ◽  
Total Cell Number ◽  
In Vitro Development ◽  
Significant Difference ◽  
Vitro Development

For decades it has been known that reactive oxidative species (ROS) form during in vitro embryo culture. A buildup of ROS can be detrimental to individual cells in the embryo and lead to a decrease in development and quality. To overcome oxidative stress in culture systems, additives, such as taurine and/or hypotaurine, have been used. In the pig, taurine or hypotaurine addition is deemed necessary for normal in vitro development. Another commonly used technique to reduce ROS is to culture embryos in a lowered oxygen environment (e.g. 5%). Porcine zygote medium 3 (PZM3) base culture medium is used in the following experiments and contains 5 mM hypotaurine, which is one of the most costly additives in the medium. The objective of this experiment was to determine if hypotaurine is still necessary if the embryos were cultured in 5% O2 from the zygote to the Day 6 blastocyst stage. In Experiment 1, oocytes were matured for 44 h and fertilized in vitro. After fertilization, presumptive zygotes were then transferred to 500 µL of MU-1 medium (PZM3 with 1.69 mM arginine) that either contained or did not contain hypotaurine for overnight culture at 20% O2. On Day 1, the same embryo culture plates were moved to 5% O2, 5% CO2, and 90% N2 and cultured to Day 6. The percent blastocyst stage was determined, and total cell number was counted in 3 of the 5 replicates in order to give us an indication of the embryo quality. The percent blastocyst in the controls (+hypotaurine) was 34.4% ± 2.8 and not different from the no hypotaurine (32.9% ± 2.2; N = 830; 5 replications; P > 0.10). Furthermore, total cell number was not different between the two groups (30.8 ± 1.5 v. 33.6 ± 1.8, respectively, N = 146; 3 replications; P > 0.10). In Experiment 2, the same experiment was repeated in somatic cell nuclear transfer derived embryos, which may be more sensitive to ROS due to the micromanipulation procedure. Wild type fetal fibroblast cells were used as donor cells. There was no significant difference in development to the blastocyst stage due to the presence or absence of hypotaurine (17.7% ± 2.5 v. 11.8% ± 2.3, respectively; N = 454; 4 replications; P = 0.07). All blastocyst data were analysed using the GENMOD procedure in SAS 9.4 (SAS Institute Inc., Cary, NC, USA), and cell number data were analysed using the PROC GLM also with SAS 9.4. These data show that porcine embryos can be efficiently cultured to the blastocyst stage without adding any oxygen free radical scavengers to the media when culturing in reduced oxygen atmosphere. Further studies include evaluating term development via embryo transfers and measuring ROS production of these embryos. Funding was provided by Food for the 21st Century and the National Institutes of Health (U42 OD011140).

Tolerance of lamb and mouse oocytes to cryoprotectants during vitrification

Zygote ◽  
2018 ◽  
Vol 27 (1) ◽  
pp. 36-45
Author(s):  
Jaqueline Sudiman ◽  
Alice Lee ◽  
Kheng Ling Ong ◽  
Wu Zi Yuan ◽  
Sarah Jansen ◽  
...  
Keyword(s):  
Cell Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cortical Granule ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Parthenogenetic Activation ◽  
Mouse Oocytes ◽  
Different Response

SummaryMouse and lamb oocytes were vitrified with, or exposed to, different cryoprotectants and evaluated for their effects on their survival and developmental competence after in vitro fertilization (IVF) and activation treatments. Control oocytes remained untreated, whilst the remainder were exposed to three different combinations of vitrification solutions [dimethyl sulfoxide (DMSO) + ethylene glycol (EG), EG only, or propanediol (PROH) + EG] and either vitrified or left unfrozen (exposed groups). Oocytes in the control and vitrified groups underwent IVF and developmental competence was assessed to the blastocyst stage. In lambs, survival rate in vitrified oocytes was significantly lower than for oocytes in the exposed groups (P <0.05). Blastocyst development was low in vitrified oocytes compared with controls (<6% vs 38.9%, P <0.01). Parthenogenetic activation was more prevalent in vitrified lamb oocytes compared with controls (P <0.05). No evidence of zona pellucida hardening or cortical granule exocytosis could account for reduced fertilization rates in vitrified lamb oocytes. Mouse oocytes demonstrated a completely different response to lamb oocytes, with survival and parthenogenetic activation rates unaffected by the vitrification process. Treatment of mouse oocytes with DMSO + EG yielded significantly higher survival and cleavage rates than treatment with PROH + EG (87.8% and 51.7% vs 32.7% and 16.7% respectively, P <0.01), however cleavage rate for vitrified oocytes remained lower than for the controls (51.7% vs 91.7%, P <0.01) as did mean blastocyst cell number (33 ± 3.1 vs 42 ± 1.5, P <0.05). From this study, it is clear that lamb and mouse show different tolerances to cryoprotectants commonly used in vitrification procedures, and careful selection and testing of species-compatible cryoprotectants is required when vitrifying oocytes to optimize survival and embryo development.

Sign in / Sign up

Export Citation Format

Share Document