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).

Embryo development and sex ratio of in vitro-produced porcine embryos are affected by the energy substrate and hyaluronic acid added to the culture medium

2014 ◽  
Vol 26 (4) ◽  
pp. 570 ◽  
Author(s):  
Eva Torner ◽  
Eva Bussalleu ◽  
M. Dolors Briz ◽  
Marc Yeste ◽  
Sergi Bonet
Keyword(s):  
Hyaluronic Acid ◽  
Sex Ratio ◽  
Embryo Development ◽  
Culture Medium ◽  
Cell Number ◽  
Total Cell ◽  
Total Cell Number ◽  
Porcine Embryo ◽  
Preferential Loss

In the present study, the effects of replacing glucose with pyruvate–lactate and supplementing these in vitro culture (IVC) media with hyaluronic acid (HA) on porcine embryo development and sex ratio were examined. The in vitro-produced (IVP) porcine embryos were cultured in NCSU-23 medium with 0.0, 0.5 or 1.0 mg mL–1 HA, and with either 5.55 mM glucose (IVC-Glu) or pyruvate (0.17 mM)–lactate (2.73 mM) from 0 to 48 h post insemination (h.p.i.) and then with glucose from 48 to 168 h.p.i. (IVC-PL). Those embryos cultured with IVC-PL had significantly higher blastocyst rates (23.7 ± 1.5%) than those cultured with IVC-Glu (14.27 ± 2.75%). At 1.0 mg mL–1, HA tended to skew the sex ratio of blastocysts towards males in those embryos cultured in IVC-PL, and led to a significant decrease in the blastocyst rate compared with embryos cultured in the presence of 0.5 and 0.0 mg mL–1 HA and IVC-Glu (4.28 ± 0.28% vs 11.01 ± 1.42% and 10.14 ± 2.77%, respectively) and IVC-PL (14.37 ± 1.35% vs 20.96 ± 2.85% and 22.99 ± 1.39%, respectively). In contrast, there were no significant differences in the total cell number per blastocyst or in apoptosis rates. In conclusion, pyruvate and lactate were the preferred energy substrates in the early stages of IVP porcine embryos. Moreover, 1.0 mg mL–1 HA significantly decreased the percentage of blastocyst rates in both the IVC-Glu and IVC-PL groups, but only by a preferential loss of female embryos for those cultured in IVC-PL.

213 MATURATION OF OOCYTES WITH FOLLICULAR FLUID FROM GILTS CONSUMING HIGH FAT AND FRUCTOSE AFFECTS SUBSEQUENT EMBRYO DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 237
Author(s):  
R. Poole ◽  
V. McCracken ◽  
M. Rhoads ◽  
K. Lee
Keyword(s):  
Embryo Development ◽  
Follicular Fluid ◽  
Cell Number ◽  
Total Cell ◽  
Blastocyst Formation ◽  
High Fat ◽  
Total Cell Number ◽  
Treatment Groups ◽  
High Fructose

Infertility among women has become a growing issue in the world requiring a significant number to seek treatment by means of assisted reproductive technologies. One suggested reason for the fertility issue, which is known to specifically affect oocyte quality, is the modern diet. Previously, we have demonstrated that feeding a high-fructose diet to gilts led to poor reproductive tract characteristics and infertility. In this study, pre-pubescent gilts were fed either a high-fructose; high-fat diet (HFHF), with 15% beef tallow and 35% fructose; or an industry control diet (IND). Porcine follicular fluid (pFF) collected from these gilts was introduced into in vitro maturation systems to determine whether characteristics of the follicular fluid affect oocyte competence and embryo development. Follicles from ovaries, collected at a local abattoir, were aspirated by an 18 G needle attached to a 10-mL sterile syringe. Then selected cumulus‐oocyte complexes were maturated in vitro in a TCM-199 maturation media with cysteine, glucose, sodium pyruvate, epidermal growth factor (EGF), FSH, LH, and 20% pFF from treatment groups. Additionally, another group of oocytes, labelled follicle fluid free (FFF), were maturated in TCM-199 media without pFF. Three replicate experiments were conducted using a total of 365 oocytes, 124 FFF, 121 IND, and 120 HFHF. Oocytes were denuded by exposure to 0.1% hyaluronidase and oocytes that reached metaphase II (MII) were selected for in vitro fertilisation. After 5 h of co-incubation in modified Tween medium B with milk powder (mTBM)-based IVF media, presumable zygotes were transferred to porcine zygote medium-3 (PZM-3). Blastocyst frequency was recorded on Days 5 and 6. Day 6 blastocysts were stained with Hoechst for total cell number evaluation. The frequencies of blastocyst formation among the treatment groups were compared by a chi-squared test, and total cell numbers were compared by Student's t-test. Statistical significance was defined by P < 0.05. The frequency of oocytes reaching metaphase II (MII) were observed as 77.4% FFF, 72.7% IND, and 71.7% HFHF (P > 0.05), indicating the supplementation of pFF did not affect maturation. Day 5 blastocysts were observed at frequencies of 8.3% FFF, 6.8% IND, and 4.7% HFHF and did not differ. However, frequency of Day 6 blastocysts from HFHF group was tended to be lower compared with that of other groups; 12.5% FFF, 11.4% IND, and 4.7% HFHF (P = 0.06 and P = 0.1). Average total cell number of Day 6 blastocysts observed were 41.0 ± 9.1 FFF, 36.0 ± 8.9 IND, and 48.3 ± 10.6 HFHF. The total cell number from HFHF group tended to be higher than only that of IND group (P = 0.07). Based on these results, we concluded that the follicular fluid of females consuming HFHF diets did not have impact on nuclear maturation of oocytes but might affect oocyte competency, thus resulting in detrimental effects on subsequent development of embryos, especially blastocyst formation. Further studies will help us identify more specific effects of nutrition on oogenesis and subsequent embryo development.

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.

Effects of changing culture medium on preimplantation embryo development in rabbit

Zygote ◽  
2021 ◽  
pp. 1-6
Author(s):  
Haixia Wang ◽  
Wenbin Cao ◽  
Huizhong Hu ◽  
Chenglong Zhou ◽  
Ziyi Wang ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Culture Medium ◽  
Cell Number ◽  
Apoptotic Index ◽  
Total Cell ◽  
Toxic Substances ◽  
Total Cell Number ◽  
Preimplantation Embryo Development

Summary Many studies have focused on the optimization of the composition of embryo culture medium; however, there are few studies involving the effect of a culture medium changing procedure on the preimplantation development of embryos. In this study, three groups were designed: a non-renewal group, a renewal group and a half-renewal group. The levels of reactive oxygen species (ROS), apoptotic index, blastocyst ratio and blastocyst total cell number were analyzed in each group. The results showed that the ROS level and the apoptotic index of blastocyst in the non-renewal group were significantly higher than in the renewal group and the half-renewal group (P < 0.05). The blastocyst ratio and blastocyst total cell number were significantly higher in the half-renewal group than that in non-renewal group and the renewal group (P < 0.05). These results demonstrated that the procedure of changing the culture medium influenced ROS level, apoptotic index, blastocyst ratio and total cell number of blastocysts. In addition, the result suggested that changing the culture medium may lead to a loss of important regulatory factors for embryos, while not changing the culture medium may lead to the accumulation of toxic substances. Half-renewal can alleviate the defects of both no renewal and renewal, and benefit embryo development. This study will be of high value as a reference for the optimization of embryo culture in vitro, and is very significant for assisted reproduction.

82 EARLY PORCINE EMBRYO ENERGY PREFERENCE AND SUBSEQUENT DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 170
Author(s):  
L. D. Spate ◽  
B. K. Redel ◽  
R. S. Prather
Keyword(s):  
Energy Source ◽  
Subsequent Development ◽  
Cell Number ◽  
Total Cell ◽  
Control Medium ◽  
Blastocyst Development ◽  
Total Cell Number ◽  
Cell Stage ◽  
Porcine Embryo

Early porcine embryo metabolism in vitro is not completely understood. It has been suggested that before embryo genome activation (4-cell stage), the preferred energy source of the embryo is pyruvate. In our porcine zygote culture medium (MU1), the energy sources are 0.2 mM pyruvate and 2.0 mM calcium lactate. Three experiments were performed with in vitro-matured and IVF embryos to examine the effect on blastocyst development after withholding pyruvate and/or lactate during the first 48 h of culture. In Experiment 1, embryos were cultured without lactate for 48 and then cultured to Day 6 in control medium containing lactate. Control embryos were cultured in medium with lactate starting after fertilization to Day 6. All data were analysed by using SAS 9.3 with a GENMOD procedure used for the blastocyst data and a GLM procedure used for the cell number data. On Day 6, the percentage of embryos that formed blastocysts was 30.2% for control and 26.5% for embryos cultured for 48 h without lactate (n = 490, 4 replications). The difference was not significant P > 0.05. In Experiment 2, embryos were cultured without pyruvate for 48 and then cultured to Day 6 in control medium containing pyruvate. Control embryos were cultured in medium with pyruvate starting after fertilization to Day 6. On Day 6, the percentage of embryos that formed blastocysts was 31.1% for control and 30.5% for embryos cultured for 48 h without pyruvate (n = 385, 3 replications). In Experiment 3, embryos were cultured in control medium for the first 48 h and then cultured to Day 6 in medium without pyruvate, thus forcing the embryos to use lactate instead of pyruvate. On Day 6, the percentage of embryos that formed blastocysts in the pyruvate free medium increased from 28.6%a ± 1.0 to 33.9%b ± 1.0; P ≤ 0.05 (n = 490, 4 replications) compared with the control and total cell number increased from 30.7a ± 1.5 to 41.3b ± 1.8 cells, respectively; P ≤ 0.05 (n = 65, 4 replications). The results from Experiments 2 and 3 were unanticipated as it was believed that the embryo would be more dependent on pyruvate for energy up to the blastocyst stage. We believed in Experiment 2 that from zygote to 4 cells the embryos were not as capable of using lactate and that removing the pyruvate would hinder further development. In Experiment 3, forcing the embryo to use lactate from Day 2 to Day 6 significantly improved blastocyst development and total cell number, suggesting that the embryo is not dependent on a specific energy source or that there are adequate pyruvate stores in the oocyte to 4-cell stage, to promote development to blastocyst. Funding was provided by Food for the 21st Century, the University of Missouri, and the National Institutes of Health (OD011140).

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).

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.

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).

194 ANTIOXIDATIVE EFFECT OF BROWN ALGAE PHLOROTANNINS ON REACTIVE OXYGEN SPECIES GENERATION AND EMBRYO DEVELOPMENT OF PORCINE PARTHENOGENETIC EMBRYOS UNDER OXIDATIVE AND HEAT-STRESSED CONDITIONS

2007 ◽  
Vol 19 (1) ◽  
pp. 213
Author(s):  
M. Takahashi ◽  
K. Nagayama ◽  
M. Sakatani ◽  
S. Kobayashi ◽  
K. Morishita ◽  
...  
Keyword(s):  
Embryo Development ◽  
Brown Algae ◽  
Cell Number ◽  
Total Cell ◽  
Oxygen Species ◽  
Blastocyst Formation ◽  
Antioxidative Effect ◽  
Total Cell Number ◽  
Intracellular Ros ◽  
O2 Concentration

We investigated the antioxidative effect of brown algae phlorotannins on reactive oxygen species (ROS) generation and embryo development of parthenogenetically activated porcine embryos under oxidative and heat-stressed conditions. Cumulus–oocyte complexes (COCs) were aspirated from follicles on the surface of porcine ovaries collected from an abbattoir. COCs were matured in NCSU-23 containing 10% (v/v) porcine follicular fluid and hCG during the first 22 h, followed by an extra 22 h of culture in hormone-free NCSU-23. After 44 h of maturation, oocytes were denuded of cumulus cells and used for parthenogenetic activation. Oocytes were activated by single 100-�s pulse of 1.5 kV cm-1 DC in 1-mm electrodes. Activated oocytes were cultured for 5 h in NCSU-23 containing BSA, EGF, and 5 �g mL-1 cytochalasin B. Embryos were then cultured for 7 days in PZM-5 medium that was a slightly modified version of the PZM-4 medium reported by Yoshioka et al. (2002 Biol. Reprod. 60, 112–119). In Experiment 1, after parthenogenetic activation, embryos were cultured for 7 days at 38.5�C under 5% O2, 5% CO 2, and 90% N2 (defined as 5% O2) as a control. Embryos were also cultured under 5% CO2 in air (defined as 20% O2) with or without 100 ng mL-1 brown algae phlorotannins extracted from Ecklonia kurome. The number of embryos developed to the blastocyst stage was observed on Day 6. The total cell number of Day 7 blastocysts was counted by DAPI staining of nuclei. On Day 2, intracellular ROS levels of individual embryos were measured with fluorescent dyes (222,722-dichlorodihydrofluorescein diacetate). In Experiment 2, on Day 1 or 2, embryos cultured in 5% O2 concentration at 38.5�C were exposed to 41.5�C for 6 h with or without 100 ng mL-1 phlorotannins and cultured at 38.5�C until Day 7. After 6 h of heat-shock on Day 1 or Day 2, intracellular ROS levels were measured as described in Experiment 1. Statistical analysis was carried out by ANOVA. In Experiment 1, the rate of blastocyst formation and the total cell number were significantly decreased (P &lt; 0.05) when embryos were cultured under 20% O2 compared to 5% O2. In contrast, addition of phlorotannins significantly increased the rate of blastocyst formation under high O2 concentration. ROS levels were also significantly increased by higher O2 concentration. In contrast, addition of phlorotannins significantly reduced the ROS levels. In Experiment 2, heat-shock to embryos on Days 1 and 2 significantly (P &lt; 0.05) decreased the rate of blastocyst formation compared to the control. In contrast, addition of phlorotannins significantly (P &lt; 0.05) increased embryo development and decreased the intracellular ROS levels of heat-stressed embryos. These results indicate that oxidative and heat stress conditions decrease embryo development and increase the level of intracellular ROS. However, addition of phlorotannins promotes embryo development by decreasing the oxidative stress.

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