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.

Effects of amino acids on development in vitro of cleavage-stage bovine embryos into blastocysts

1996 ◽  
Vol 8 (5) ◽  
pp. 835 ◽  
Author(s):  
T Pinyopummintr ◽  
BD Bavister
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Embryo Development ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Development In Vitro ◽  
Defined Culture

Effects of amino acids on early bovine embryo development in vitro were examined using a chemically-defined, protein-free culture medium. Bovine embryos produced in vitro were cultured from 18 h to 72 h post insemination in a simple medium containing lactate as the only energy source except for the amino acid treatments. Subsequently, embryos were transferred to TCM-199 supplemented with serum for blastocyst development to substantiate their developmental competence. Treatments were: (1) non-essential amino acids from TCM-199 (NEA); (2) essential amino acids from TCM-199 (EA); (3) NEA+EA; (4) Eagle's minimum essential medium amino acids (MEM AA); (5) 11 amino acids present in HECM-6 (11 AA); and (6) 0.2 mM glutamine (GLN). A higher proportion of embryos (percentage of inseminated ova) cleaved to the > or = 8-cell stage by 72 h post insemination in NEA (56.7%), EA (41.2%), 11 AA (40.3%) and GLN (51.1%) than in either NEA+EA (30.0%) or MEM AA (33.1%). However, after transfer to complex medium, embryos that had developed in EA, as well as those in MEM AA or NEA+EA, produced significantly fewer blastocysts (37.1%, 34.4% and 25.6% respectively) than those in NEA (56.7%), GLN (48.9%) or 11 AA (37.7%). The ability of blastocysts to hatch from their zonae pellucidae was also affected by amino acid treatment during cleavage stages. The present study indicated that the addition of NEA or GLN or 11 AA to a chemically-defined culture medium during the cleavage phase of bovine embryo development increases their subsequent ability to reach the blastocyst stage. These data have implications for understanding the nutritional needs of bovine embryos produced in vitro and for optimizing the composition of culture media to support their development.

58 STAGE–SPECIFIC EFFECTS OF THE PROGESTERONE RECEPTOR ANTAGONIST, RU486, ON EARLY BOVINE EMBRYO DEVELOPMENT

2012 ◽  
Vol 24 (1) ◽  
pp. 141
Author(s):  
C. M. O'Meara ◽  
T. Fair ◽  
P. Lonergan
Keyword(s):  
Progesterone Receptor ◽  
Receptor Antagonist ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Significant Decline ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Antagonist Ru486

Progesterone plays a key role in the reproductive events associated with pregnancy establishment and maintenance. High concentrations of circulating progesterone in the immediate post-conception period are associated with an advancement of conceptus elongation, an associated increase in interferon-tau production and higher pregnancy rates in cattle. Progesterone-induced changes in the uterine environment are thought to be responsible for the reported advancement in conceptus elongation; however, the function of the progesterone receptor in embryos is not known. Therefore, the aim of the current study was to examine the effect of adding a progesterone receptor antagonist (mifepristone, RU486) at various stages of early embryonic development and at varying concentrations to examine the effects on subsequent embryo development in vitro. Bovine zygotes (n = 2902), 2-cell (n = 1991) and 8-cell (n = 1244) embryos, derived by in vitro maturation and fertilization, were cultured in synthetic oviduct fluid medium in the absence or presence of RU486 at concentrations ranging from 0.0004 to 20 μg mL–1. Cleavage rate (of zygotes), 8-cell development rate (of 2-cell embryos) and development to the blastocyst stage (for all cell stages) were recorded at Day 2, 3 and 8 post-insemination (day of IVF = Day 0), respectively. Cultures of zygotes in the presence of RU486 at concentrations of 0.004 and 0.04 μg mL–1 resulted in a decline in cleavage rate (62.5 ± 2.55% and 48.8 ± 5.07% for respective treatments vs controls without RU486 81.9 ± 5.97%; P ≤ 0.05). These same concentrations resulted in a significant decline in blastocyst development on Day 8 (18.8 ± 1.82% and 17.4 ± 4.85% for respective treatments compared to controls 35.1 ± 4.89%; P ≤ 0.05). Cultures at concentrations of 0.4 μg mL–1 resulted in a 10-fold decrease in blastocyst development (3.3 ± 1.3%; P ≤ 0.05) and concentrations in excess of 10 μg mL–1 completely ablated blastocyst development (P ≤ 0.05). Cultures of 2-cell embryos with RU486 at concentrations below 8 μg mL–1 had no effect on 8-cell rate or blastocyst development. However, cultures with RU486 at 10 μg mL–1 resulted in a significant decline in the proportion reaching the 8-cell stage (59.1 ± 4.59% vs 38.1 ± 2.13% for control and treated, respectively) and developing to the blastocyst stage (32.8 ± 4.68% vs 17.8 ± 3.77% for control and treated, respectively; P ≤ 0.05). Cultures with RU486 at a concentration of 20 μg mL–1 resulted in a dramatic effect in 8-cell rate (16.3 ± 2.55%; P ≤ 0.05) and prevented blastocyst development. Similarly, cultures of 8-cell embryos with RU486 at concentrations at or below 10 μg mL–1 had no effect on blastocyst development. However, cultures at concentrations of 15 or 20 μg mL–1 resulted in no blastocyst development. In conclusion, addition of the progesterone and glucocorticoid receptor antagonist RU486 to culture media has a clear stage-specific and concentration-dependent effect on bovine embryo development, which is more pronounced at earlier developmental stages. Supported by Science Foundation Ireland (07/SRC/B1156).

48 EFFECTS OF LIPID METABOLIC REGULATORS DURING BOVINE EMBRYO CULTURE ON BLASTOCYST DEVELOPMENT AND CRYOSURVIVAL

2014 ◽  
Vol 26 (1) ◽  
pp. 138 ◽  
Author(s):  
A. Ruiz ◽  
P. J. Hansen ◽  
J. Block
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Blastocyst Stage ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Phenazine Ethosulfate ◽  
Metabolic Regulators ◽  
Hatching Rates

The overall objective was to determine the effects of addition of lipid metabolic regulators during embryo culture on blastocyst development and survival following cryopreservation. For Experiment 1, embryos produced in vitro were cultured in 5% (vol/vol) oxygen in SOF-bovine embryo 1 (SOF-BE1) medium supplemented with or without 100 μM trans-10,cis-12 conjugated linoleic acid (CLA) and 0.3 μM phenazine ethosulfate (PES). Treatment with CLA began at the initiation of culture, whereas treatment with PES began at Day 3 after insemination. At Day 7 after insemination, the proportion of oocytes that developed to the blastocyst and advanced blastocyst (expanded, hatching, or hatched) stages was recorded. Blastocysts and expanded blastocyst-stage embryos were harvested and slow frozen in 1.5 M ethylene glycol. Embryos were thawed and then cultured for 72 h in SOF-BE1 medium containing 10% (vol/vol) fetal bovine serum and 50 μM dithiothreitol. Re-expansion and hatching rates were recorded at 24, 48, and 72 h. Addition of CLA had no effect on embryo development, whereas PES reduced (P < 0.01) development to the blastocyst (26.0 ± 0.8 v. 22.1 ± 0.8%) and advanced blastocyst (19.2 ± 0.9 v. 14.4 ± 0.9%) stages. Blastocysts cultured in the presence of CLA had higher (P < 0.05) re-expansion rates at 24, 48, and 72 h (50.8 ± 3.7 v. 65.7 ± 3.7%, 57.2 ± 4.0 v. 72.0 ± 4.05%, and 57.2 ± 4.0 v. 72.0 ± 4.0%, respectively). Addition of CLA tended (P < 0.07) to increase the hatching rate at 24 h and did increase (P < 0.05) the hatching rate at 48 h (12.4 ± 1.3 v. 16.2 ± 1.3% and 39.0 ± 3.2 v. 50.0 ± 3.2%, respectively). Treatment with PES had no effect on re-expansion rates but reduced (P < 0.05) hatching rates at 24 and 48 h (18.2 ± 1.3 v. 10.3 ± 1.3 and 50.2 ± 3.2 v. 38.8 ± 3.2%, respectively). There was no interaction between CLA and PES affecting embryo development or cryosurvival. For Experiment 2, embryos were produced in vitro as in Experiment 1 and cultured in SOF-BE1 medium with or without 3.03 mM L-carnitine (LC) and 10 μM forskolin (FK). Treatment with LC began at the initiation of culture and treatment with FK began at Day 6. All other methods were as described for Experiment 1. Addition of LC did not affect development to the blastocyst stage but reduced (P < 0.05) development to the advanced blastocyst stage (21.0 ± 1.2 v. 17.1 ± 1.2%). Treatment with FK had no effect on embryo development to the blastocyst or advanced blastocyst stages. Blastocysts cultured in the presence of LC had increased (P < 0.05) re-expansion rates at 24, 48, and 72 h (60.2 ± 2.0 v. 78.0 ± 2.0%, 62.9 ± 1.2 v. 83.3 ± 1.2%, and 63.0 ± 2.4 v. 82.8 ± 2.4%, respectively) and hatching rates at 48 and 72 h (48.6 ± 4.3 v. 64.1 ± 4.3% and 59.6 ± 3.0 v. 78.5 ± 3.0%, respectively). There was no effect of FK on cryosurvival and no interaction between LC and FK affecting embryo development or cryosurvival. In conclusion, blastocyst yield was not improved by any of the lipid metabolic regulators tested. Cryosurvival was enhanced by addition of CLA and LC but FK reduced survival following freezing. There were no additive effects of either CLA and PES or LC and FK for blastocyst yield or cryosurvival.Support was provided by USDA AFRI Grant 2010-85122-20623.

In vitro development of equine nuclear transfer embryos: effects of oocyte maturation media and amino acid composition during embryo culture

Zygote ◽  
2003 ◽  
Vol 11 (1) ◽  
pp. 77-86 ◽  
Author(s):  
Y.H. Choi ◽  
Y.G. Chung ◽  
S.C. Walker ◽  
Westhusin M.E. ◽  
K. Hinrichs
Keyword(s):  
Amino Acids ◽  
Embryo Culture ◽  
Nuclear Transfer ◽  
Culture Medium ◽  
Essential Amino Acids ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Embryo Culture Medium ◽  
Igf I

This study was conducted to evaluate the effects of insulin-like growth factor I (IGF-I) and other media factors during oocyte maturation, and the presence of different compositions of amino acids in embryo culture medium, on the development of equine embryos. Oocytes recovered from slaughterhouse-derived ovaries were matured in vitro for 24 h and those with a polar body were subjected to intracytoplasmic sperm injection (ICSI) or nuclear transfer with adult fibroblasts (NT). For ICSI embryos, there were no significant differences in rates of morphological cleavage, cleavage with normal nuclei or average nucleus number at 96 h post-ICSI between the absence and presence of IGF-I in maturation medium, or between embryos cultured in G1.2 or a modified CZB medium (CZB-C). Embryos produced by interspecies NT (equine donor cells into bovine cytoplasts) also showed no difference in cleavage rate or average nucleus number whether cultured in G1.2 or in CZB-C. The rates of cleavage, cleavage with normal nuclei and average nucleus number of equine NT embryos were not significantly different among oocytes matured in M199 with FSH in the presence or absence of IGF-I, or in EMMI medium, which contains IGF-I, epidermal growth factor, steroid hormones, FSH and LH. There were no differences in development of equine NT embryos cultured in any of three amino acid treatments (with or without non-essential amino acids, or containing taurine, hypotaurine and cysteine only). The cleavage rate and average nucleus number of parthenogenetically activated oocytes (treated similarly to NT oocytes but not enucleated or subjected to donor cell injection) were significantly (p < 0.05) higher than those for NT embryos. These results indicate that the presence of IGF-I or of EMMI medium during in vitro maturation of equine oocytes does not have a beneficial effect on their developmental competence as assessed at 96 h. Presence or absence of non-essential amino acids in embryo culture medium does not affect development of NT embryos within the first 96 h of culture. Factors associated with enucleation or nuclear transfer decrease the developmental competence of equine NT embryos. CZB-C medium may be used for culture of equine embryos with results similar to those obtained with G1.2 medium, thus providing a base medium that may be modified for further study of culture requirements of equine embryos.

Different preferences of IVF and SCNT bovine embryos for culture media

Zygote ◽  
2012 ◽  
Vol 22 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Xian-rong Xiong ◽  
Li-jun Wang ◽  
Yong-sheng Wang ◽  
Song Hua ◽  
Xiang-dong Zi ◽  
...  
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Blastocyst Stage ◽  
Apoptotic Index ◽  
Expression Level ◽  
Rt Pcr ◽  
Treatment Combination ◽  
Blastocyst Development ◽  
Cleavage Rate

SummaryThe preference of fertilized (IVF) and somatic cell nuclear transfer (SCNT) presumptive zygotes for different media when cultured in vitro to the blastocyst stage was evaluated in this study. The experiment comprised two zygote production methods (IVF and SCNT) × two culture media (mSOF and G1.5/G2.5) factorial design in which culture droplets that contained approximate 30 presumptive zygotes formed the experimental plots for the assessment of cleavage and blastocyst development. There were 15 to 20 replicates (culture droplets) per treatment combination. Sub-samples 30 to 41 of the blastocysts produced were assessed for cell number and cell apoptosis. A further 10 blastocysts per treatment combination were used for quantitative real-time polymerase chain reaction (RT-PCR) to evaluate the relative abundance of Hsp70 and Bax mRNA. Presumptive zygotes produced by IVF were developmentally more competent than SCNT zygotes in terms of cleavage rate (66.9 vs. 57.0%; P < 0.05) and blastocyst development rates (blastocysts of presumptive zygotes 29.7 vs. 24.8%; blastocysts of cleaved zygotes 44.4 vs. 36.6%; P < 0.05). Over both zygote production systems, however, the results were similar whether culture was in mSOF or in G1.5/G2.5 media for cleavage rate (63.2 vs. 62.4%; P > 0.05) and blastocyst development rate (blastocysts of presumptive zygotes 26.4 vs. 25.7%; P > 0.05; blastocysts of cleaved zygotes 41.8 vs. 41.2%; P > 0.05). There was, however, a significant interaction between the method of zygote production and culture medium for the apoptotic index of blastocysts. The interaction was such that IVF-produced zygotes cultured in mSOF had a lower apoptotic index compared with those cultured in G1.5/G2.5 (4.7 ± 1.2% vs. 9.8 ± 0.9%; P < 0.05) whereas SCNT zygotes had a higher apoptotic index when cultured in mSOF compared with those cultured in G1.5/G2.5 (11.9 ± 1.5% vs. 4.5 ± 1.2%; P < 0.05). Moreover, RT-PCR analysis showed that embryos from IVF-produced zygotes cultured in mSOF had a lower expression level of stress-related and apoptosis genes (Hsp70 and Bax) than those cells cultured in G1.5/G2.5 medium, while SCNT-derived embryos cultured in mSOF had a higher expression level of these genes than those embryos cultured in G1.5/G2.5 medium. The results of this study show that bovine IVF- and SCNT-produced presumptive zygotes have different nutrient requirements for in vitro culture to the blastocyst stage of development. IVF-derived zygotes have a preference for mSOF as the culture medium whereas the G1.5/G2.5 medium is more suitable for the culture of bovine SCNT-derived zygotes.

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.

scholarly journals 324SPHINGOSINE-1-PHOSPHATE PROTECTS CULTURED BOVINE OOCYTES FROM PHYSIOLOGICALLY RELEVANT THERMAL STRESS

2004 ◽  
Vol 16 (2) ◽  
pp. 282 ◽  
Author(s):  
Z. Roth ◽  
P.J. Hansen
Keyword(s):  
Thermal Stress ◽  
Heat Shock ◽  
Blastocyst Stage ◽  
Caspase Activity ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Developmental Potential ◽  
Oocyte Competence ◽  
Cell Death Pathway

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that can block the sphingomyelin cell-death pathway by suppressing ceramide-induced apoptosis. The present study was performed to test whether S1P protects oocytes from heat shock during in vitro maturation. Cumulus-oocyte complexes obtained by slicing follicles were placed in maturation medium with or without 50nM S1P and cultured at 38.5°C (CON) or 41°C (41C) for the first 12h of maturation. Incubation during the last 10h of maturation (22-h total maturation time), fertilization, and embryonic development were performed at 38.5°C and 5% (v/v) CO2. Blastocyst development was recorded at 8 days post-insemination (dpi) and activity of group II caspases in 8-day blastocysts was determined using a fluoroprobe, PhiPhiLux-G1D2 (OncoImmunin, Gaithersburg, MD, USA). Data were analysed by least-squares ANOVA with the GLM procedure of SAS. Percentage data were subjected to arcsin transformation before analysis. Exposure of oocytes to thermal stress during the first 12h of maturation reduced cleavage rate (P&lt;0.01) and the number of oocytes developing to the blastocyst stage (P&lt;0.04). There was a temperature x S1P interaction for cleavage rate (P&lt;0.03) because S1P blocked effects of thermal stress on cleavage rate. Without S1P, the percentage of oocytes that cleaved by 3 dpi were 83.6±2.7% and 65.8±2.7% for CON and 41C, respectively. In the presence of S1P, percent cleavage was 86.7±2.7% and 83.9±2.7% for CON and 41C, respectively. There was a trend (P=0.06) for a temperature x S1P interaction for percent oocytes developing to blastocyst stage because S1P blocked effects of heat shock on development. Without S1P, the percentages of oocytes that developed to the blastocyst stage were 28.7±3.0% and 15.2±3.0% for CON and 41C, respectively. In the presence of S1P, percent blastocysts were 24.3±3.4% and 23.9±3.0% for CON and 41C, respectively. When development was expressed as percentage of cleaved embryos, however, there were no effects of temperature, S1P, or temperature x S1P on percent development to the blastocyst stage. Blastocyst caspase activity was not affected by temperature or S1P. In summary, exposure to physiologically relevant thermal stress during the first 12h of maturation has a deleterious effect on oocyte competence and this effect can be reduced by S1P. The fact that heat shock reduced the percentage of oocytes but not the percentage of cleaved embryos that became blastocysts suggests that oocytes that survive effects of heat shock and cleave have normal potential to develop to the blastocyst stage. Moreover, since heat shock did not affect caspase activity, it is likely that blastocysts from heat-shocked oocytes have normal developmental potential, at least as determined by caspase activity. Support: BARD FI-330-2002 and USDA Grants 2002-35203-12664 and 2001-52101-11318.

Amino acid metabolism of bovine blastocysts derived from parthenogenetically activated or in vitro fertilized oocytes

1998 ◽  
Vol 10 (3) ◽  
pp. 279 ◽  
Author(s):  
Y. G. Jung ◽  
T. Sakata ◽  
E. S. Lee ◽  
Y. Fukui
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Metabolism ◽  
Culture Medium ◽  
Acid Metabolism ◽  
Essential Amino Acids ◽  
Fluid Medium ◽  
Vitro Fertilization ◽  
Oviduct Fluid

The uptake and synthesis of 19 amino acids by fresh or frozen–thawed bovine blastocysts produced by parthenogenesis (PT) or in vitro fertilization (IVF) were compared in the present study. Fresh blastocysts, 180 h after IVF or PT activation, and frozen–thawed blastocysts, 168 h old and cultured for 12 h post-thawing, were cultured in synthetic oviduct fluid medium (SOFM) containing polyvinyl alcohol (PVA) with both essential and non-essential amino acids (EAA and NEAA, respectively) (Medium 1: M1) or SOFM containing PVA with only EAA (Medium 2: M2). In Experiment 1, when fresh or frozen–thawed PT blastocysts were cultured in M1, the uptake of glutamate (in fresh only), aspartate and arginine, and the synthesis of glutamine and alanine were significantly enhanced. In the culture with M2, serine, asparagine, glutamate, glutamine, glycine, arginine and alanine were significantly taken up. It was found that the glutamine concentrations was significantly higher (P < 0.001) in the culture medium drops containing embryos than in the drops without embryos. In Experiment 2, when PT blastocysts were cultured in M1, the uptake of aspartate and synthesis of alanine were greater (P < 0.01) than those by IVF blastocysts. When M2 was used, a significant (P < 0.01) production of serine, asparagine, glutamate, glutamine and alanine, and the uptake of arginine by PT blastocysts were observed. In Experiment 3, when IVF blastocysts were cultured in M1, fresh blastocysts depleted more aspartate and glutamate, and produced more glutamine and alanine than frozen–thawed blastocysts. When cultured in M2, frozen–thawed blastocysts depleted more threonine (P < 0.01) than fresh blastocysts. These results indicate that the uptake and synthesis of amino acids were different in fresh or frozen–thawed bovine blastocysts derived from PT or IVF. These differences in amino acid metabolism may be related to the viability of the blastocysts.

125 EXAMINING CRITERIA FOR EXTENDING BOVINE BLASTOCYST SURVIVAL IN VITRO

2008 ◽  
Vol 20 (1) ◽  
pp. 143
Author(s):  
F. N. T. Cooke ◽  
T. M. Rodina ◽  
P. J. Hansen ◽  
A. D. Ealy
Keyword(s):  
Conditioned Medium ◽  
Culture System ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Blastocyst Development ◽  
Interferon Tau ◽  
Cell Numbers ◽  
Bovine Blastocyst

Most of the current culture procedures used for bovine in vitro embryo production terminates at the blastocyst stage. Developing procedures for extending embryo lifespan beyond this phase will provide a valuable tool for understanding events that occur during the second week of pregnancy in cattle. The overall objective of the present studies was to identify culture conditions required to support bovine blastocyst development beyond its initial formation. In the first study, individual day 8 blastocysts (day 0 = day of IVF) were cultured until day 11 in 30 µL microdrops of Potassium Simplex Optimized Medium-Bovine Embryo 2 containing 0.1 mm non-essential amino acids or Tissue Culture Medium 199 (M199). Both media were supplemented with 5% [v/v] fetal bovine serum (FBS) and incubations were in an atmosphere of either 5 or 21% (v/v) oxygen. A medium by oxygen interaction (P = 0.007) occurred when assessing cell number on day 11. Blastocysts cultured in M199 and in a 5% O2 environment had greater (P < 0.002) cell numbers (536 � 49) than blastocysts incubated in other conditions (339 � 28). Conditioned medium from blastocysts incubated in 21% O2 contained greater (P < 0.05) concentrations of bioactive interferon-tau (IFNT) than blastocysts incubated in 5% O2 regardless of medium type (70.5 � 28 v. 17.2 � 2.6 ng mL–1). In a follow-up study, blastocysts could remain morphologically viable through day 11 in M199 containing at least 2.5% FBS. To examine whether oxidative stress was responsible for the increase in IFNT production under 21% O2, blastocysts were incubated under a 5% O2 atmosphere in M199 containing 2.5% FBS and increasing concentrations of tert-butylhydroperoxide (tBH), a membrane-permeable oxidative agent. Addition of e3 nm tBH decreased cell numbers but did not increase IFNT concentrations in conditioned medium. To examine whether blastocysts could survive beyond day 11 in culture, day 11 blastocysts were transferred to 400 �L of M199 with 20% FBS under 5% oxygen and cultured from day 11 to 20–21 post-IVF. Half of the medium was replaced every 2–3 days. On day 13–14, 16.6 � 6.1% of blastocysts showed initial signs of degeneration. A portion of blastocysts (32.9 � 9.6%) began attaching to plates on days 13–15 and produced outgrowths that appeared viable on days 20–21. All of the non-attached blastocysts degenerated by day 17–18. No blastocyst elongation was detected. In conclusion, a culture system was developed that sustains blastocyst viability and IFNT production in vitro to day 11. Although this culture system allowed blastocyst survival until day 14, normal conceptus development (i.e. elongation/filamentation) was not achieved. Nonetheless, the culture system provides a useful tool for examining the initial stages of blastocyst development and IFNT production from individual bovine embryos.

99 In Vitro Development of Alpaca Embryos Obtained by Bisection

2018 ◽  
Vol 30 (1) ◽  
pp. 189
Author(s):  
L. Landeo ◽  
R. S. Molina ◽  
M. E. Zuñiga ◽  
T. R. Gastelu ◽  
C. Sotacuro ◽  
...  
Keyword(s):  
Amino Acids ◽  
Streptomyces Griseus ◽  
Petri Dish ◽  
Cumulus Cells ◽  
Essential Amino Acids ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Morula Stage

The objective of this study was to evaluate the in vitro developmental competence of alpaca embryos bisected at different embryonic stages. Gametes were obtained from ovaries and testes collected from a local abattoir. Cumulus-oocyte complexes (COC) were recovered (n = 120) by aspiration of ovarian follicles using a 5-mL syringe with an 18-gauge needle. Then, COC with at least 3 layers of cumulus cells and a homogeneous cytoplasm were matured in TCM-199 supplemented with 10% FCS, FSH (0.02 IU [JM1] [P2] [P3]), and 0.01 mg mL−1 oestradiol 17β [JM4] for 26 h at 38.5°C and 5% CO2 in air. After in vitro maturation, COC were placed in a 30-mL Petri dish containing FERT-TALP solution for 30 min. Then, epididymal alpaca spermatozoa (3 × 106 mL−1) were added to the dish and co-incubated with the COC for 20 h at 38.5°C and 5% CO2 in air. Motile epididymal sperm were selected by swim-up method centrifuged for 15 min at 350 × g in 2 mL of SPERM-TALP supplemented with 6 mg mL−1 of fatty-acid-free BSA. Sperm pellet was extended and culture in 5% CO2 in air at 38.5°C for 45 min. Thirty-three viable embryos at different stages [2-cells (n = 6), 8-cells (n = 15), and morulae (n = 12)] were bisected into approximately equal halves using a micro-surgical blade. The embryos were previously treated with 2 mg mL−1 of protease from Streptomyces griseus (P 8811, Sigma, St. Louis, MO, USA) for 2 min to remove the zona pellucida. After bisection, the demi-embryos were cultivated in in vitro culture (IVC) medium containing 0.036 mg mL−1 sodium pyruvate, 0.146 mg mL−1 l-glutamine, 1% essential amino acids, 0.5% nonessential amino acids, and supplemented with 10% FCS using the well-of-the-well system. The demi-embryos were incubated for 7 days (changing the media every 48 h) in 5% CO2 in air at 38.5°C. Additional embryos (n = 60) were obtained using the same conditions described above and used as a control group (unmanipulated). We obtained 66 demi-embryos [2-cells (n = 12), 8-cells (n = 30), and morulae (n = 24)] after bisection that were considered for IVC. From 12 demi-embryos bisected at 2-cell and 30 bisected at 8-cell stages, 3 (25%) and 30 (100%) reached the morula stage respectively. However, they did not develop any further. Interestingly, 18 demi-embryos bisected in morula reached the blastocyst stage (80%). For unmanipulated embryos, we obtained 42% (25/60), 35% (21/60), 32% (19/60), and 28% (17/60) of cleavage, morulae, and blastocyst and hatched blastocyst rates, respectively. In conclusion, alpaca embryos bisected at earlier stages (less than 8-cell) are not suitable to produce blastocysts. The earliest stage to produce blastocyst from bisected alpaca embryos is the morula stage.

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