Applying metabolomic analyses to the practice of embryology: physiology, development and assisted reproductive technology

2015 ◽  
Vol 27 (4) ◽  
pp. 602 ◽  
Author(s):  
Rebecca L. Krisher ◽  
Adam L. Heuberger ◽  
Melissa Paczkowski ◽  
John Stevens ◽  
Courtney Pospisil ◽  
...  
Keyword(s):  
Amino Acids ◽  
Culture Medium ◽  
Blastocyst Stage ◽  
Small Samples ◽  
Human Embryos ◽  
Blastocyst Development ◽  
Oct4 Expression ◽  
Complex Culture

The advent of metabolomics technology and its application to small samples has allowed us to non-invasively monitor the metabolic activity of embryos in a complex culture environment. The aim of this study was to apply metabolomics technology to the analysis of individual embryos from several species during in vitro development to gain an insight into the metabolomics pathways used by embryos and their relationship with embryo quality. Alanine is produced by both in vivo- and in vitro-derived human, murine, bovine and porcine embryos. Glutamine is also produced by the embryos of these four species, but only those produced in vitro. Across species, blastocysts significantly consumed amino acids from the culture medium, whereas glucose was not significantly taken up. There are significant differences in the metabolic profile of in vivo- compared with in vitro-produced embryos at the blastocyst stage. For example, in vitro-produced murine embryos consume arginine, asparagine, glutamate and proline, whereas in vivo-produced embryos do not. Human embryos produce more alanine, glutamate and glutamine, and consume less pyruvate, at the blastocyst compared with cleavage stages. Glucose was consumed by human blastocysts, but not at a high enough level to reach significance. Consumption of tyrosine by cleavage stage human embryos is indicative of blastocyst development, although tyrosine consumption is not predictive of blastocyst quality. Similarly, although in vivo-produced murine blastocysts consumed less aspartate, lactate, taurine and tyrosine than those produced in vitro, consumption of these four amino acids by in vitro-derived embryos with high octamer-binding transcription factor 4 (Oct4) expression, indicative of high quality, did not differ from those with low Oct4 expression. Further application of metabolomic technologies to studies of the consumption and/or production of metabolites from individual embryos in a complete culture medium could transform our understanding of embryo physiology and improve our ability to produce developmentally competent embryos in vitro.

144 USE OF A NOVEL BOVINE EMBRYO CULTURE MEDIUM TO IMPROVE BLASTOCYST DEVELOPMENT AND SURVIVAL FOLLOWING VITRIFICATION

2010 ◽  
Vol 22 (1) ◽  
pp. 231
Author(s):  
J. Block ◽  
L. Bonilla ◽  
P. J. Hansen
Keyword(s):  
Amino Acids ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Fresh Embryos ◽  
Hatching Rates

The objective of the present study was to determine whether culture of bovine embryos in a proprietary serum-free culture medium, Block-Bonilla-Hansen-7 (BBH-7), could improve development to the blastocyst stage and enhance survival following vitrification. For Exp. 1, embryos were produced in vitro and cultured in BBH-7 or modified synthetic oviductal fluid (mSOF; as in zygote 10:341 except with 10 μL mL-1 of nonessential amino acids, 20 μL mL-1 of essential amino acids, and 1 mg mL-1 of polyvinyl alcohol instead of albumin) in 5% (v/v) oxygen. Grade 1 expanded blastocysts were harvested at Day 7 post-insemination and vitrified using the open-pulled straw method (Vagta et al. 1998 Mol. Reprod. Dev. 51, 53-58). Vitrified embryos were thawed and cultured in vitro in either mSOF or BBH-7 supplemented with 10% fetal bovine serum and 50 μM dithiolthreitol. Re-expansion and hatching rates were recorded at 24, 48, and 72 h post-thaw. There was no effect of culture medium on cleavage rate. The proportion of oocytes that developed to the blastocyst and advanced blastocyst stages (expanded, hatching, and hatched) at Day 7 was higher (P < 0.001) for embryos cultured in BBH-7 than for embryos cultured in mSOF (41.9 ± 2.0 v. 14.7 ± 2.0% and 31.1 ± 1.3 v. 6.4 ± 1.3%, respectively). There was no effect of culture medium on re-expansion rates at 24, 48, and 72 h post-thaw or on hatching rates at 48 or 72 h. However, the proportion of embryos that were hatching or had hatched by 24 h post-thaw was higher (P < 0.001) for BBH-7 than for mSOF (59.0 ± 0.5 v. 26.7 ± 0.5%). For Exp. 2, late lactation and/or repeat breeder, lactating Holstein cows were synchronized for timed embryo transfer using the OvSynch-56 protocol. Embryos were produced in vitro and cultured in BBH-7 in 5% (v/v) oxygen. Vitrified embryos were produced as for Exp. 1. Fresh embryos were grade 1 expanded blastocysts harvested at Day 7 after insemination. A single embryo was transferred at Day 7 after putative ovulation to all cows with a corpus luteum confirmed by ultrasonography. Pregnancy was diagnosed at Day 28-30 of gestation by ultrasonography. There was no difference in the proportion of recipients that became pregnant after receiving either a fresh (7/18 = 39%) or vitrified (10/27 = 37%) embryo cultured in BBH-7. The results of the present study indicate that BBH-7 can be used to increase the proportion of oocytes that develop to the blastocyst stage. Moreover, the results demonstrate that vitrified embryos produced after culture in BBH-7 can achieve pregnancy rates similar to those obtained using fresh embryos. Support: USDA 2006-55203-17390 and Southeast Milk Checkoff Program.

Differential effects of culture and nuclear transfer on relative transcript levels of genes with key roles during preimplantation

Zygote ◽  
2006 ◽  
Vol 14 (1) ◽  
pp. 81-87 ◽  
Author(s):  
P.N. Moreira ◽  
R. Fernández-Gonzalez ◽  
M.A. Ramirez ◽  
M. Pérez-Crespo ◽  
D. Rizos ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Culture Medium ◽  
Cumulus Cell ◽  
Blastocyst Stage ◽  
Mrna Levels ◽  
Differential Effects ◽  
Glut 1 ◽  
Mouse Somatic Cell

It is well known that the preimplantation culture environment to which embryos are exposed influences the expression of developmentally important genes. Recently, it has been reported that MEMα, a culture medium commonly used for somatic cells, allows high rates of preimplantation development and development to term of mouse somatic cell nuclear transfer (SCNT) embryos. The objective of this study was to compare the differential effects of this medium and of the nuclear transfer procedure on the relative mRNA abundance of several genes with key roles during preimplantation. The relative mRNA levels of nine genes (Glut 1, Glut 5, G6PDH, Bax, Survivin, Gpx 1, Oct4, mTert and IGF2bp1) were quantified at blastocyst stage on cumulus cell cloned embryos cultured in MEMα, as well as on in vivo cultured and MEMα cultured controls. Only three of the nine transcripts analysed (Glut 5, Gpx 1 and Igf2bp1) were significantly down-regulated at blastocyst stage in in vitro produced controls. However, most genes analysed in our MEMα cultured cloned embryos showed altered transcription levels. Interestingly, between cloned and in vitro produced controls only the transcription levels measured for Glut 1 were significantly different. This result suggests that Glut 1 may be a good marker for embryo quality after cumulus cell nuclear transfer.

92 Culture method for long-distance transport of bovine embryos derived from IVF before blastulation using microtubes

2019 ◽  
Vol 31 (1) ◽  
pp. 172
Author(s):  
T. Yamanouchi ◽  
H. Matsuda ◽  
K. Ogata ◽  
Y. Hashiyada
Keyword(s):  
Culture Medium ◽  
Liquid Paraffin ◽  
Calf Serum ◽  
Petri Dish ◽  
Culture Method ◽  
Optimal Number ◽  
Blastocyst Development ◽  
Long Distance

In vitro-produced (IVP) embryos are more easily damaged by cryopreservation than in vivo-derived embryos. Therefore, transportation of fresh IVP embryos in a manner that can maintain viability is necessary. This study was conducted to determine the preferable culture conditions for transport of embryos at 5 days post-insemination (dpi) in 1.5-mL microtubes. Cumulus-oocyte complexes derived from an abattoir were matured and then inseminated with frozen-thawed semen. Presumptive zygotes were cultured in mCR1aa (CR1)+5% calf serum (CS) until use. In Exp. 1, embryos with 5 blastomeres at 5 dpi were randomly assigned to 1 of 3 groups: 25mM Hepes-CR1aa (H-CR1)+5% CS or 25mM Hepes-M199 (H-M199)+5% CS in air, or CR1 in 5% CO2. Embryos were cultured in microdrops overlaid with liquid paraffin in a petri dish for 48h at 38.5°C. In Exp. 2, the optimal number of embryos to culture per microtube was assessed. Presumptive zygotes were cultured in groups of 20, 40, or 80 in 1mL of CR1 covered with liquid paraffin in microtubes in an incubator at 38.5°C in 5% CO2 until 7 dpi. For Exp. 3, culture of embryos in microtubes in a portable incubator was tested. At 5 dpi, 5-cell embryos (n=17 to 36 per microtube) were statically cultured in 1mL of CR1 or H-CR1 in microtubes in a portable incubator set at 38.5°C for 48h. The CR1 was pre-equilibrated in an incubator in 5% CO2 for 24h before use. Embryos were harvested from microtubes after 48h and were then cultured in microdrops of CR1 overlaid with liquid paraffin in a petri dish in an incubator at 38.5°C in 5% CO2 until 8 dpi. In Exp. 4, embryos (n=29 to 39 five-cell embryos per microtube) were transported in a portable incubator by land for 1000km over a period of 44h using the same conditions as in Exp. 3. Control embryos were statically cultured in microdrops of CR1 in an incubator in 5% CO2. Statistical analyses were carried out by ANOVA (Exp. 1 and 2), t-test (Exp. 3), or Fisher’s exact test (Exp. 4). In Exp. 1, there was no effect (P&gt;0.05) of culture medium on blastocyst development at 7 dpi (27.6±2.3, 25.7±7.2, and 17.3±2.9% for CR1, H-CR1, and H-M199, respectively). In Exp. 2, blastocyst development at 7 dpi was not affected (P&gt;0.05) by the number of presumptive zygotes cultured per microtube (43.6±8.3, 42.4±4.0, and 39.9±2.9% for 20, 40, and 80 presumptive zygotes, respectively). In Exp. 3, blastocyst development at 8 dpi was not affected (P&gt;0.05) by culture medium (60.7±7.4 and 53.1±4.4% for CR1 and H-CR1, respectively); however, the pH of CR1 changed from 7.5 to 8.1 at 48h after culture. In Exp. 4, blastocyst development at 8 dpi was not affected (P&gt;0.05) by transport (57.1, 64.4, and 75.5% for CR1, H-CR1, and control, respectively). These results indicate that IVP embryos harvested at 5 dpi can be transported by portable incubator with no effect on embryo development to the blastocyst stage. This work was supported by grants from the Project of the Bio-oriented Technology Research Advancement Institution, NARO (the special scheme project on advanced research and the development for next-generation technology).

Some effects of genotype and composition of the culture medium on the development of mouse zygotes in vitro

1993 ◽  
Vol 5 (4) ◽  
pp. 405 ◽  
Author(s):  
ZF Du ◽  
RG Wales
Keyword(s):  
Culture Medium ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Reciprocal Crosses ◽  
Maternal Genome ◽  
Zygote Development ◽  
Mouse Zygotes ◽  
Better Than

The effects of EDTA and the presence of glucose and glutamine in CZB medium on the development of mouse zygotes of different genotype were investigated. Although 30-80% of zygotes (depending on the cross) passed the 2-cell stage in EDTA-free medium, the addition of a low concentration of EDTA was necessary in these experiments to obtain blastocysts in culture. In reciprocal crosses between outbred (Qs), inbred (DBA/2) and hybrid (B10D2F1) stock, there was evidence of a strong influence of the maternal genome on zygote development, with those from B10D2F1 females performing best irrespective of sire. A paternal influence on development was also evident but the most successful sire varied with the genotype of female used and reciprocal crosses differed greatly in the ability of the resultant zygote to develop in culture. For zygotes recovered from Qs females, CZB medium containing glucose and glutamine supported development to the blastocyst stage better than did medium devoid of these substrates. Tests with embryos from B10D2F1 females indicated that the presence of glucose for the whole or for part of the incubation period stimulated blastocyst development. However, the addition of glutamine to the medium in these tests had no significant effect on the development of blastocysts.

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.

scholarly journals Lysophosphatidic Acid Signaling in Late Cleavage and Blastocyst Stage Bovine Embryos

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Ana Catarina Torres ◽  
Dorota Boruszewska ◽  
Mariana Batista ◽  
Ilona Kowalczyk-Zieba ◽  
Patricia Diniz ◽  
...  
Keyword(s):  
Lysophosphatidic Acid ◽  
Culture Medium ◽  
Marker Gene ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Human Embryos ◽  
Lipid Mediator ◽  
Bovine Embryos ◽  
Pluripotency Marker

Lysophosphatidic acid (LPA) is a known cell signaling lipid mediator in reproductive tissues. In the cow, LPA is involved in luteal and early pregnancy maintenance. Here, we evaluated the presence and role of LPA in bovine early embryonic development. In relevant aspects, bovine embryos reflect more closely the scenario occurring in human embryos than the mouse model. Transcription of mRNA and protein expression of enzymes involved in LPA synthesis (ATX andcPLA2) and of LPA receptors (LPAR1–4) were detected in Days 5 and 8in vitroproduced embryos. Embryonic LPA production into culture medium was also detected at both stages of development. Supplementation of culture medium with LPA (10−5 M) between Days 2 and 8 had no effect on embryo yield and quality and on blastocyst relative mRNA abundance of genes involved in prostaglandin synthesis (PTGS2,PGES, andPGFS) and steroidogenesis (3βHSD). However, LPA treatment affected transcription levels of embryo quality markers, decreasingBAX(apoptotic) and increasingBCL2(antiapoptotic) andIGF2R(growth marker) gene transcription levels. Blastocyst transcription ofOCT4(pluripotency marker) was not affected by LPA stimulation. In conclusion, LPA is an early bovine embryonic autocrine/paracrine signaling mediator, and LPA action may be relevant in early embryo-maternal interactions leading to embryonic survival.

Glucose and glycine synergistically enhance the in vitro development of porcine blastocysts in a chemically defined medium

2012 ◽  
Vol 24 (3) ◽  
pp. 443 ◽  
Author(s):  
Tomomi Mito ◽  
Koji Yoshioka ◽  
Shoko Yamashita ◽  
Chie Suzuki ◽  
Michiko Noguchi ◽  
...  
Keyword(s):  
Culture Medium ◽  
Survival Rates ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Atp Content ◽  
Blastocyst Development ◽  
In Vitro Development ◽  
Defined Culture ◽  
Vitro Development

In the present study, the effects of glucose and/or glycine on the in vitro development of Day 5 (Day 0 = IVF) porcine blastocysts were determined. The addition of 2.5–10 mM glucose to the chemically defined culture medium porcine zygote medium (PZM)-5 significantly increased blastocyst survival rates compared with those of blastocysts cultured in the absence of glucose. The addition of 5 and 10 mM glycine to PZM-5 containing 5 mM glucose significantly enhanced the development to hatching and the number of hatched blastocysts compared with no addition of glycine. However, the addition of glycine to PZM-5 with no glucose did not improve blastocyst development. The ATP content of Day 6 blastocysts cultured with glucose was significantly higher than that of blastocysts cultured in the absence of glucose, regardless of glycine supplementation. The diameter and total cell numbers were significantly greater, and the apoptotic index was significantly lower, in Day 6 blastocysts cultured with both glucose and glycine. These results indicate that glucose is an important energy source for the porcine blastocyst and that glucose and glycine act synergistically to enhance development to the hatching and hatched blastocyst stage in vitro.

86 IN VITRO-MATURED GILT OOCYTES CAN HAVE EQUAL OR BETTER DEVELOPMENTAL COMPETENCE THAN SOW OOCYTES WITH NEW MATURATION MEDIA

2017 ◽  
Vol 29 (1) ◽  
pp. 150 ◽  
Author(s):  
L. D. Spate ◽  
S. L. Murphy ◽  
J. A. Benne ◽  
A. Giraldo ◽  
D. Hylan ◽  
...  
Keyword(s):  
Growth Factor ◽  
Somatic Cell ◽  
Culture Media ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Development In Vitro ◽  
Humidified Air

It has long been thought that oocytes obtained from sows yielded a higher level of developmental competence compared with oocytes obtained from prepubertal gilts. Because gilt-derived oocytes are more readily available to our laboratory and they are less developmentally competent, we hypothesised that by making alterations to our maturation system we could improve the developmental competence of the gilt-derived oocytes to that of their sow-derived counterparts. We performed 2 experiments that evaluated the ability of each source of oocyte to develop to the blastocyst stage, using altered maturation media. The first experiment focused on the developmental ability of each source of oocytes, through IVF and culture. The second experiment again focused on the developmental competence of each oocyte source but through somatic cell NT. For both experiments, the sow-derived oocytes were obtained from Desoto Biosciences and the gilt ovaries were collected from Smithfield Inc. in Milan, Missouri. Both sets of oocytes were in vitro matured in M199 supplemented with 0.57 mM cysteine, 5 μg mL−1 LH and FSH, and 10 ng mL−1 epidermal growth factor; however, the gilt derived media was altered to contain 40 ng mL−1 fibroblast growth factor 2 and 20 ng mL−1 insulin-like growth factor and leukemia inhibitory factor. Additionally, the maturation media for the sow-derived oocytes contained the addition of 5 μg mL−1 insulin and 10% follicular fluid. In the first experiment we performed IVF on oocytes from the 2 sources as per our laboratory standard IVF procedure, co-incubating the oocytes with 0.25 × 106 porcine semen for 4 h, followed by washing and moving the oocytes to MU2 culture media at 38.50°C in 5% CO2, humidified air overnight. After overnight culture the presumptive zygotes were transferred to the same conditions with 5% CO2, 5% O2, and 90% N2. After an additional 5 days, blastocyst development was assessed. The gilt oocytes yielded 39.3a ± 7.2% blastocyst, and the sow oocytes had a blastocyst rate of 24.9b ± 6.9%, with an n of 389 and 313, respectfully. Statistical analysis was performed by using Genmod in SAS 9.4. In the second experiment, using standard laboratory protocol for somatic cell NT, we activated both sets of oocytes with 200 μM thimerosal for 10 min followed by 30-min incubation with 4 mM dithiothreitol. The embryos were co-incubated for 15 h with 500 nM Scriptaid in the MU2 culture media in 5% CO2, humidified air; then these embryos were also moved to 5% CO2, 5% O2, and 90% N2 and cultured to Day 6. The sow oocytes produced a blastocyst percentage of 38.6%, and the gilt oocyte group had a blastocyst percentage of 43.5%, with an n of 290 and 285, respectfully. There was no difference statistically between these treatments. Both gilt and the sow oocyte sources have yielded live piglets at this time. We concluded that the maturation system used for our gilt-derived oocytes resulted in equal or better development in vitro compared with the sow-derived oocytes. Follow-up experiments evaluating in vivo development are needed for a complete comparison. This work was funded by Food for the 21st Century University of MO, and the NIH U42OD011140.

Investigations of oocyte in vitro maturation within a mouse model

Zygote ◽  
2004 ◽  
Vol 12 (1) ◽  
pp. 1-18 ◽  
Author(s):  
Boon Chin Alexis Heng ◽  
Ng Soon Chye
Keyword(s):  
In Vitro Maturation ◽  
Culture Medium ◽  
Germinal Vesicle ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Standard Duration ◽  
Maturation Rate ◽  
Meiotic Competence

This study attempted to develop a ‘less meiotically competent’ murine model for oocyte in vitro maturation (IVM), which could more readily be extrapolated to human clinical assisted reproduction. Oocyte meiotic competence was drastically reduced upon shortening the standard duration of in vivo gonadotrophin stimulation from 48 h to 24 h, and by selecting only naked or partially naked germinal vesicle oocytes, instead of fully cumulus enclosed oocyte complexes. With such a less meiotically competent model, only porcine granulosa coculture significantly enhanced the oocyte maturation rate in vitro, whereas no significant enhancement was observed with macaque and murine granulosa coculture. Increased serum concentrations and the supplementation of gonadotrophins, follicular fluid and extracellular matrix gel within the culture medium did not enhance IVM under either cell-free or coculture conditions. Culture medium conditioned by porcine granulosa also enhanced the maturation rate, and this beneficial effect was not diminished upon freeze–thawing. Enhanced IVM in the presence of porcine granulosa coculture did not, however, translate into improved developmental competence, as assessed by in vitro fertilization and embryo culture to the blastocyst stage.

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