Temporal expression of transcripts related to embryo quality in bovine embryos cultured from the two-cell to blastocyst stage in vitro or in vivo

2007 ◽  
Vol 74 (8) ◽  
pp. 972-977 ◽  
Author(s):  
Deirdre Corcoran ◽  
Dimitrios Rizos ◽  
Trudee Fair ◽  
Alex C.O. Evans ◽  
Patrick Lonergan
Keyword(s):  
Embryo Quality ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Temporal Expression

145 CHANGES IN OXYGEN COMSUMPTION RATES OF EMBRYOS IN KOREAN CATTLE

2010 ◽  
Vol 22 (1) ◽  
pp. 231
Author(s):  
C. Y. Choe ◽  
S. R. Cho ◽  
J. K. Son ◽  
S. H. Choi ◽  
C. Y. Cho ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Embryo Quality ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Significant Difference ◽  
Morula Stage

Oxygen consumption has been regarded as a useful indicator for assessment of mammalian embryo quality. This study was carried out to identify whether oxygen consumption rates measured in bovine embryos using SECM can be used as a standard criteria to evaluate bovine embryo quality. Oxygen consumption of bovine embryos at various developmental stages was measured and analyzed using SECM and ANOVA analysis, respectively. We found that the oxygen consumption significantly increased in blastocyst-stage embryos compared to other stage embryos (from 2-cell stage to morula stage), indicating that oxygen consumption reflects the cell number (5.2-7.6 × 1014 mol-1 s-1 v. 1.2-2.4 × 1014 mol-1 s-1, P < 0.05). There was no significant difference between 2-cell-stage embryos and 8-cell-stage embryos. In the morula-stage embryos, the oxygen consumption of in vivo derived embryos was significantly higher than that of in vitro produced embryos (4.0 × 1014 mol-1 s-1 v. 2.4 × 1014 mol-1 s-1, P < 0.05). However, there was no significant difference in consumption of oxygen by in vivo and in vitro derived bovine blastocyst-stage embryos (P > 0.05). Good-quality embryos with grade 1 or 2 showed significantly higher oxygen consumption than grade 3 or 4 embryos. These results showed that SECM could measure oxygen consumption in bovine embryos and the oxygen consumption could reflect embryonic development stage and embryo quality.

132 DIFFERENCES IN RESPIRATION RATES BETWEEN IN VIVO- AND IN VITRO-PRODUCED BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 174
Author(s):  
A. S. Lopes ◽  
S. E. Madsen ◽  
N. B. Ramsing ◽  
L. H. Larsen ◽  
T. Greve ◽  
...  
Keyword(s):  
Metabolic Stress ◽  
Average Diameter ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Outer Diameter ◽  
Bovine Embryos ◽  
Physiological Level ◽  
Respiration Rates

In vitro-produced (IVP) bovine embryos differ (e.g. morphology and physiology) from their in vivo counterparts. Oxygen consumption is an indicator of the overall metabolic activity of a single embryo. Therefore, the aim of this study was to determine and compare respiration rates of in vivo- and in vitro-produced bovine day 7 embryos. Diameters of these two embryo types were also compared. In vivo embryos (n = 28) were recovered from 8 superovulated Holstein Frisian cows on day 7 following AI, while IVP embryos (n = 160; Holm et al. 1999 Theriogenology 52, 683-700) were used on day 7 after fertilization. Embryos were measured (outer diameter) and morphologically evaluated (Quality 1 to 4, IETS Manual, 1998). Only transferable in vivo embryos were used (i.e. excluding Quality 4). Respiration rates were measured on each embryo by Nanorespirometer technology (Lopes et al. 2005 Reprod. Fertil. Develop. 17, 151). Data were analyzed using Proc Mixed, and values are presented as mean � SEM. Values with different superscripts differ significantly (P < 0.05). The average respiration rates were 0.82 � 0.06a nL/h for in vivo vs. 1.37 � 0.06b nL/h for IVP embryos. The average respiration rates for the different morphological qualities were as follows (nL/h, numbers in brackets): IVP: 2.1 � 0.08a (38), 1.37 � 0.07b (55), 1.08 � 0.07c (48) and 0.62 � 0.11d (19) for Quality 1, 2, 3, and 4, respectively. In vivo: 1.17 � 0.21b,c,e (6), 0.80 � 0.15c,d,e (12), and 0.64 � 0.16d,f (10) for Quality 1, 2, and 3, respectively. The average diameter (mm) of in vivo and IVP embryos was 0.157 � 0.002a and 0.176 � 0.002b, respectively. Respiration rates were directly related to embryo diameter; larger embryos were associated with higher respiration rates (y = 17.55 � 1.32 nL/h � mm, n = 188). Respiration rates of in vivo embryos were significantly lower than those of IVP embryos, regardless of quality. This difference could reflect an effect of the culture conditions on IVP embryos because media components affect embryo metabolism. Moreover, the different ages (day 7 for IVP vs. approximately Day 6.5 for in vivo embryos, because in vivo embryos are less than 7 days after fertilization at recovery) and stages (IVP: up to expanded blastocyst stage; in vivo: morula or early blastocyst stage) could have influenced the results and also partly explain the smaller diameter of the in vivo embryos. Finally, respiration rates decreased proportionately to the morphological quality within embryo type, indicating that morphological differences are reflected at the physiological level. In conclusion, this study further outlines metabolic differences between in vivo and IVP bovine embryos. Whether such differences are a manifestation of metabolic stress associated to the separation from the natural environment or reflect suboptimal culture conditions is yet to be determined. ASL is supported by FCT, Portugal.

159 USE OF PHYTOHEMAGGLUTININ-L, AS AN AGGLUTINATOR AGENT, TO PRODUCE CHIMERAS OF IVF BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 238
Author(s):  
I. P. Emanuelli ◽  
B. F. Agostinho ◽  
M. P. M. Mancini ◽  
C. M. Barros ◽  
M. F. G. Nogueira
Keyword(s):  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Exact Test ◽  
Fisher's Exact Test ◽  
Stage Of Development ◽  
Fisher’S Exact Test

Embryonic chimeras have been used as a tool to understand embryogenesis and organogenesis, as well as to prove, in vivo, the pluripotency of the embryonic stem cells. One of the techniques used to obtain embryonic chimeras is aggregation, which can be performed with intact or half-embryos and in different stages of the development, produced by in vivo or in vitro systems and in different wells. However, its efficiency tends to reduce when advanced stages, such as morulae and blastocysts, are used. The aim of this work was to evaluate the effect of the treatment with an agglutinating agent (phytohemagglutinin-L; PHA) in the percentage of chimeras produced with IVF bovine embryos. Bovine ovaries (from abattoir) were used to obtain 270 COC that were matured in drops (90 μL) of TCM-199 bicarbonate medium, supplemented with 10% of FCS, and incubated in vitro for 22 to 24 h. The fertilization occurred in TALP-IVF medium, and the COC were maintained in the incubator for 18 h. After fertilization, the presumptive zygotes were transferred to SOF culture medium to in vitro culture. In vitro maturation, fertilization, and culture were performed under 38.5°C, 5% CO2 in air and saturated humidity. The chimerism by aggregation was tested between 2 intact (zona-free) 8- to 16-cell stage embryos in the presence (G1, n = 16) or absence of PHA (G2, n = 14) and between one half-morula and one half-blastocyst with (G3, n = 15) or without PHA (G4, n = 12). The embryos in groups G1 and G3 were treated with PHA in a concentration of 500 μLg mL-1 for 3 min. After PHA treatment, the pairs of embryos were allocated in wells, under previously described culture conditions, until expanded blastocyst stage could be observed (Day 7 of culture). At 24 h of culture, embryonic aggregation pairs were first evaluated to detect only cohesive masses of cells. The results (chimerism rate) were 62.5%, 42.9%, 40.0%, and 25.0%, respectively, for groups G1, G2, G3, and G4. There were no significant differences neither among groups (chi-square, P = 0.252) nor between G1 and G2 (P = 0.464), G3, and G4 (P = 0.683; Fisher’s exact test). Main effects as use of PHA (G1 + G3 v. G2 + G4, P = 0.284) and stage of embryos (G1 + G2 v. G3 + G4, P = 0.183; Fisher’s exact test) were not statistically significant. However, when all groups were compared, the power of the performed test (0.354) was below the desired power of 0.800 (i.e. one must be cautious in over-interpreting the lack of difference among them). In the conditions of this study, it was concluded that the treatment with PHA did not increase the rate of aggregation in the embryonic chimera production, even for half-embryos in advanced stage of development (morulae and blastocysts). Granted by FAPESP, Brazil: 06/06491-2 and 07/07705-9 (MFGN) and 07/04291-9 (MPMM).

141 BIOPSY TECHNIQUE IN BOVINE EMBRYOS PRODUCED IN VITRO AT EARLY STAGES OF DEVELOPMENT: EVALUATION OF QUALITY AND DEVELOPMENT CAPACITY

2008 ◽  
Vol 20 (1) ◽  
pp. 151
Author(s):  
J. Polisseni ◽  
M. O. Guerra ◽  
R. V. Serapião ◽  
M. M. Pereira ◽  
I. M. Folhadella ◽  
...  
Keyword(s):  
Preimplantation Genetic Diagnosis ◽  
Embryo Quality ◽  
Genetic Diagnosis ◽  
Blastocyst Stage ◽  
Chromosome Abnormalities ◽  
Control Group ◽  
Bovine Embryos ◽  
Blastocyst Rate ◽  
Number Of Cells

One of the causes of embryo mortality is chromosome abnormalities that occur during gametogenesis, fertilization, and embryo early development. Thus, a combination of morphological standards and techniques of molecular analyses could identify abnormal embryos. Preimplantation genetic diagnosis (PGD) is an emergent technology for use with farm animal embryos. With this procedure, blastomeres are removed by the biopsy of embryos at the 8- to 16-cell stage to provide cells for analyses of chromosome abnormalities prior to transfer. The aim of this study was to evaluate the effect of biopsy in bovine 8- to 16-cell embryos fertilized in vitro on embryo quality and subsequent development in vitro. A group of 706 oocytes were obtained from slaughterhouse ovaries, matured, and fertilized in vitro at 38.8�C with 95% humidified air and 5% CO2. The zygotes were semi-denuded and cultured in CR2aa medium under the same conditions as for in vitro fertilization. The rate of cleavage was 78.20%. Three days after fertilization, part of the 8- to 16-cell (298/706) embryos were distributed randomly across two groups: control (n = 103) and biopsy (n = 92) of blastomeres, and then returned to in vitro embryo culture to evaluate development until the blastocyst stage and the capacity to hatch. The amount of cells removed was one-fourth of the embryo. The blastocyst rate was evaluated on Day 8 after fertilization and the hatching rate on Day 10. Embryo morphology and quality were evaluated as previously described in the International Embryo Transfer Society manual (1998). To evaluate overall quality, embryos were stained on the 10th day of culture and the blastomeres were counted with the imaging software AxioVision 3.1 (Carl Zeiss, Feldbach, Switzerland). The blastocyst rate was analyzed by treatment groups with the chi-square test and the number of cells/embryo was analyzed by ANOVA with SAS (SAS Institute, Inc., Cary, NC, USA). The percentage of 8- to 16-cell embryos that developed to the blastocyst stage was similar (P > 0.05) between the control (66.0%, 68/103) and the biopsied (53.3%, 49/92) groups. Furthermore, no difference was noted in the hatching rates between the control group and the biopsied group (42.6%, 29/42 v. 44.9%, 22/49, respectively). Overall, no impact was detected on embryo quality from embryo biopsy with no difference in mean (�SE) blastocyst cell number between the control group (blastocysts: 67.1 � 3.1; expanded blastocysts: 100.7 � 6.9; hatched blastocysts: 189.9 � 16.1) and the biopsied group (blastocysts: 61.1 � 5.5; expanded blastocysts: 121.87 � 10.6; hatched blastocysts: 187.3 � 18.5). In conclusion, the biopsy used on 8- to 16-cell bovine IVF-derived bovine embryos does not affect the subsequent embryo development and number of cells/embryo or blastocyst, showing that it can be used to provide genetic material for preimplantation genetic diagnosis without affecting embryo quality. This work was supported financially by FAPEMIG.

scholarly journals 245RELATIVE ABUNDANCE OF HSP 70 MRNA IN BOVINE EMBRYOS PRODUCED IN VITRO USING DIFFERENT EMBRYO/VOLUME RATIOS IN CULTURE

2004 ◽  
Vol 16 (2) ◽  
pp. 243
Author(s):  
A.T.D. Oliveira ◽  
C. Gebert ◽  
R.F.F. Lopes ◽  
H. Niemann ◽  
J.L. Rodrigues
Keyword(s):  
Relative Abundance ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Gene Transcripts

In spite of in vitro embryo production systems having been greatly improved over recent years, employing a variety of culture conditions (media, protein sources, gas atmosphere, etc.), we still do not know much about the real necessity of embryos to develop under the same conditions as occur in vivo. These differences between in vivo and in vitro culture at preimplantation embryonic stages can produce deviations in gene expression and in normal fetal development (large offspring syndrome). Heat shock proteins (Hsp) are engaged in cell response to regulatory signals or perturbations in the microenviroment and can be used as a sensitive indicator of stress caused by suboptimal culture conditions (Wrenzycki et al., 2001Hum. Reprod. 16, 893–901). Hsp act as chaperones in facilitating protein folding and assembly and stabilize damaged proteins to prevent aggregation of fragments, thereby allowing repair or degradation. The aim of the present study was to investigate the effects of different embryo/volume ratios on bovine embryo development and the relative abundance of Hsp 70.1 gene transcripts. In this experiment, oocytes were isolated from slaugterhouse ovaries and matured, fertilized and cultured in groups of 5, 10, 20 or 30 per each drop of 100μL. The oocytes were matured in TCM 199 supplemented with 0.4% BSA. After maturation, oocytes were fertilized in TALP medium, using frozen/thawed sperm, selected using a percoll density gradient. The zygotes were cultured to the morula or Day 7 blastocyst stage employing SOF supplemented with 0.4 % BSA. Developmental check points were cleavage rate (Day 3pi), blastocyst formation (Day 8pi) and hatching (Day 11pi). A semi-quantitative RT-PCR assay was used to determine the relative levels of gene transcripts in single embryos at morula (Day 6) and blastocyst (Day 7) stages (Wrenzycki et al., 2001 Biol. Reprod. 65, 309–317). Data of cleavage, blastocyst formation and hatching rates were analyzed using chi-square test. Relative abundance (RA) of Hsp 70.1mRNA were compared in tested groups using ANOVA followed a Tukey test. Differences at P&lt;0.05 were considered significant. Results show that no significative difference in hatching rate per blastocyst produced was detected among the four groups. Cleavage rate and blastocyst formation were significantly higher in groups with 5, 10 and 20 embryos compared with drops containing 30 embryos. Hsp transcripts were detected in morula and blastocyst stages in all groups. In morula stage, no differences were observed in the RA of Hsp 70.1mRNA among groups with 5, 10, 20 and 30 embryos cultured per drop. However, in blastocyst stage, the RA was significantly increased in the group with 20 embryos per drop as compared to the group with 5 embryos. The results show that different embryo/volume ratios in culture influence not only cleavage rate, blastocyst formation and hatching rate, but also expression of Hsp 70.1 gene. Further studies changing other culture conditions and using in vivo-derived bovine embryos will aid in elucidating which culture systems are ideal to produce bovine embryos in vitro. This research was supported by CAPES/DAAD program and CNPq.

scholarly journals 243TEMPORAL DIVERGENCE IN THE PATTERN OF MRNA EXPRESSION IN BOVINE EMBRYOS CULTURED FROM THE ZYGOTE TO BLASTOCYST STAGE IN VITRO OR IN VIVO

2004 ◽  
Vol 16 (2) ◽  
pp. 242
Author(s):  
P. Lonergan ◽  
D. Rizos ◽  
A. Gutierrez-Adan ◽  
P.M. Moreira ◽  
B. Pintado ◽  
...  
Keyword(s):  
Growth Factor ◽  
Relative Abundance ◽  
Transcript Abundance ◽  
Blastocyst Stage ◽  
Insulin Like Growth Factor ◽  
Bovine Embryos ◽  
Interferon Tau ◽  
Cell Stage

The objective of this study was to examine the time during the post-fertilization culture period that gene expression patterns of in vitro cultured bovine embryos diverge from those of their in vivo cultured counterparts. Presumptive bovine zygotes were produced by IVM/IVF of immature oocytes collected from the ovaries of slaughtered animals. At approximately 20h post-insemination (hpi), presumptive zygotes were randomly divided into two culture groups, either in vitro in synthetic oviduct fluid or in vivo, and transferred into the ewe oviduct. Embryos were recovered from both systems at approximately 30hpi (2-cell), two (4-cell), three (8-cell), four (16-cell), five (early morula), six (compact morula) or seven (blastocyst) days pi and snap-frozen for the analysis of transcript abundance using real-time PCR. The transcripts studied were interferon-tau, apoptosis regulator box-a (Bax), connexin 43, sarcosine oxidase, glucose transporter 5, mitochondrial Mn-superoxide dismutase, insulin-like growth factor II, and insulin-like growth factor-I receptor, most of which are known from our previous work to be differentially transcribed in blastocysts derived from culture in vitro or in vivo. Analysis was done on pools of 10 embryos. Data were analyzed using one-way repeated measures ANOVA. The relative abundance of the transcripts studied varied throughout the preimplantation period and was strongly influenced by the culture environment. For example, transcripts for interferon-tau were detected from the 8-cell stage onwards in in vitro-cultured embryos but not until the early morula stage in those cultured in vivo. Levels of this transcript increased significantly at the compact morula and blastocyst stages in both groups but were significantly higher (P&lt;0.05) in in vitro-cultured embryos at both stages. mRNA for Bax was not detected before the 8-cell stage in in vitro cultured embryos and not until the 16-cell stage in in vivo cultured embryos. The abundance of this transcript increased significantly thereafter up to the blastocyst stage in both groups. The level of expression was significantly higher (P&lt;0.05) at all stages of development in in vitro-cultured embryos than those cultured in vivo. The relative abundance of Cx43 transcripts decreased in both in vitro- and in vivo-cultured embryos at the 8- to 16-cell stage. Levels remained low thereafter in the in vitro-cultured embryos but significantly increased in those cultured in vivo. Transcript abundance was significantly higher in in vivo cultured embryos from Day 4 onwards with a ten-fold difference presence at the blastocyst stage. Differences also existed for the other transcripts studied. These data demonstrate that changes in transcript abundance in blastocyst stage embryos are in many cases a consequence of perturbed transcription earlier in development. Depending on the transcript, these differences may be evident in as short as 10h of culture.

Effect of Two-step Vitrification on Developmental Competence of in vitro and in vivo Produced Bovine Embryos

2010 ◽  
Vol 79 (9) ◽  
pp. S55-S61 ◽  
Author(s):  
Jaroslava Hlavicová ◽  
Miloslava Lopatářová ◽  
Svatopluk Čech
Keyword(s):  
Developmental Stages ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Holstein Friesian ◽  
Quality Grade ◽  
Friesian Cattle

The aim of this study was to establish the effect of two-step vitrification on survival rate of bovine embryos produced in vitro (method A) and in vivo (method B) from Holstein-Friesian cattle. The embryos suitable for vitrification were frozen by a two-step technique, using increasing concentrations of dimethyl sulphoxide (DMSO) and ethylene glycol (EG). After thawing, the quality grade and developmental stage of embryos was assessed. In vitro developmental competence of embryos of different quality grade obtained by method B (n = 82) was significantly higher (p < 0.001) compared to method A (n = 98). The best results were detected when we vitrified the embryos of the grade 1 quality; namely, the hatched blastocyst stage was reached by 6.9% (2/29) of embryos retrieved by method A and by 36.7% (11/30) of embryos retrieved by method B (p < 0.01). In the case of developmental competence of embryos at different developmental stages we reached significantly better results (p < 0.001) when we vitrified the embryos produced by method B (n = 84) in comparison with method A (n = 67). We noted a higher hatching rate at the stage of expanded blastocyst; namely, the hatched blastocyst stage was reached by 7.4% (2/27) of embryos produced by method A and by 30.8% (8/26) of embryos produced by method B (p < 0.05). In general, the hatched blastocyst stage was reached by 15.1% (50/331) of all thawed embryos retrieved by method A and B. In conclusion, when we applied two-step vitrification on the grade 1 quality embryos at the stage of expanded blastocyst produced in vitro or at the stage of morula produced in vivo we achieved the highest hatching rates.

scholarly journals Effects of supplementation with heat shock cognate 17 KDA protein (HSC70) on in vitro bovine embryo viability

2021 ◽  
Author(s):  
Aimé Jazmín Garza Arredondo ◽  
Diana Elisa Zamora Ávila ◽  
Uziel Castillo Velásquez ◽  
Gustavo Moreno Degollado ◽  
José Fernando De La Torre Sánchez ◽  
...  
Keyword(s):  
Heat Shock ◽  
Blastocyst Stage ◽  
Vital Role ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Stage Of Development

Abstract Endogenous heat shock cognate 71 kDa protein (HSC70) has a vital role in early embryonic development. This study assessed the effects of exogenous HSC70 on bovine embryo development and expression of genes associated with apoptosis. Expression analyses of HSPA1A, HSPA8, Bcl-2, and Bax genes were performed in bovine embryos in vivo on day 7 of development. Subsequently, expression of HSPA1A and HSPA8 were associated with apoptotic genes (Bcl-2 and Bax) in cultured bovine embryos in vitro that were supplemented with various concentrations (0 or control group, 50, and 100 ng) of HSC70. The results indicated that the control group (0 ng) in vitro embryos had higher expression of HSPA8, Bax, and Bcl-2 genes, compared with the vivo embryos (P < 0.01). In vitro-produced embryos supplemented with 50 ng or 100 ng HSC70 had higher expression of HSPA1A, HSC70, Bcl-2, and Bax genes, compared with the control group (P < 0.01). Embryos supplemented with 100 ng had greater expression of the HSPA8 gene compared with the control group and the group supplemented with 50 ng. However, embryos supplemented with 50 ng had better characteristics (i.e., stage of development and quality) than the control and 100-ng groups. In conclusion, supplementation of in vitro culture medium with HSC70 promoted development to the blastocyst stage and improved blastocyst quality.

Comparative glucose and fructose incorporation and conversion by in vitro Produced bovine embryos

Zygote ◽  
1996 ◽  
Vol 4 (2) ◽  
pp. 85-91 ◽  
Author(s):  
Catherine Guyader-Joly ◽  
Chaqué Khatchadourian ◽  
Yves Ménézo
Keyword(s):  
Free Amino ◽  
Vero Cells ◽  
Carbon Dioxide Production ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Cell Numbers ◽  
By Products

SummaryWe have investigated the quality of bovine IVM/IVF embryos co-cultured on Vero cells. Blastocyst cell numbers are very similar to those obtained in vivo, and higher than those obtained by co-culture with oviduct cells. The metabolism and conversion of fructose and glucose are not equivalent even though carbon dioxide production is similar and increasing from morula to blastocyst. Formation of free amino acids and incorporation into proteins are higher and faster for glucose than for fructose, but this conversion is rather stable with embryonic growth. Moreover, the by-products formed are not the same. Glucose at physiological concentrations (i.e.2 mM)seems to be a more appropriate fuel for the burst of embryonic development at the blastocyst stage in preparation for hatching.

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