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<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 Temporal sensitivity of bovine embryos to culture environment after fertilization and the implications for blastocyst quality

Reproduction ◽  
2003 ◽  
pp. 337-346 ◽  
Author(s):  
P Lonergan ◽  
D Rizos ◽  
J Kanka ◽  
L Nemcova ◽  
AM Mbaye ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Blastocyst Quality ◽  
Gene Transcripts ◽  
Culture Environment ◽  
Bax Gene

The aim of this study was to examine the temporal sensitivity of bovine embryos to culture environment after fertilization to determine which period, if any, is most critical in determining blastocyst quality. Bovine zygotes produced in vitro were divided into six groups and cultured either in vitro (in synthetic oviductal fluid, SOF), in vivo (in the ewe oviduct) or in a combination of both systems. Development to the blastocyst stage, the ability of the blastocysts to withstand cryopreservation and the relative abundance of several gene transcripts were examined. Culture in SOF for either 2 or 4 days, followed by subsequent culture in the ewe oviduct, resulted in a significantly lower yield of blastocysts than did all other methods, the effect being most marked in embryos that were cultured in SOF for 4 days. In contrast, culture in vivo for the first 2 or 4 days after fertilization followed by culture in vitro did not have such a marked effect on blastocyst development. Blastocysts produced after culture in the oviduct for 6 days had the highest rates of survival over 72 h after warming (100% survival at 24 h; >95% survival at 72 h). The embryos that spent the last 4 days of culture in vivo also had relatively high rates of survival (100% at 24 h, 73.7% at 72 h). Blastocysts produced entirely in SOF had very low rates of survival after vitrification, with <40% viable at 24 h and <20% survival at 72 h. Blastocysts derived from embryos that spent the first 2 days in vivo and the last 4 days in vitro had the lowest rates of survival (6.7%), whereas those that spent the last 2 days only in SOF had intermediate rates of survival (40.6%). These differences were reflected in the relative abundance of transcripts for the Bax gene.

123 OPTIMIZATION OF CULTURE CONDITIONS FOR IN-VITRO-PRODUCED BOVINE EMBRYOS TO ENHANCE BLASTOCYST YIELD AND SURVIVAL FOLLOWING VITRIFICATION

2008 ◽  
Vol 20 (1) ◽  
pp. 142
Author(s):  
J. Block ◽  
L. Bonilla ◽  
P. J. Hansen
Keyword(s):  
Bovine Serum ◽  
Culture System ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Expansion Rate ◽  
Hatching Rate ◽  
Static System ◽  
Cleavage Rate ◽  
Static Culture

Objectives were to identify modifications in culture conditions that improve blastocyst yield and cryosurvival. The objective of Experiment 1 was to determine effects of sequential culture and fructose on blastocyst yield. Embryos were cultured in modified SOF with 4 mg mL–1 bovine serum albumin (BSA) and 1.0 mm alanyl-glutamine in 5% (v/v) oxygen with or without 0.5 mm fructose in either a static or sequential culture system. For the sequential system, embryos >4 cells were selected and placed in fresh drops of medium at day 3 after insemination. Culture system and fructose did not affect cleavage rate or the proportion of embryos >4 cells on day 3. The proportion of >4 cell embryos that developed to the blastocyst stage was higher (P < 0.04) for static culture than for sequential culture (41.6 � 1.2 v. 30.6 � 1.2%) and there was a trend (P = 0.1) for the proportion of oocytes that developed to blastocyst at day 7 to be greater for static culture (26.8 � 1.2 v. 20.9 � 1.2%). In both culture systems, fructose increased (P < 0.03) blastocyst yield from embryos >4 cells (32.5 � 1.2 v. 39.7 � 1.2%) and tended (P < 0.06) to improve blastoocyst yield from oocytes (21.8 � 1.1 v. 25.3 � 1.1%). The objective of Exp. 2 was to evaluate whether blastocyst yield and survival after cryopreservation would be enhanced by BSA and hyaluronan. Embryos produced in vitro were cultured in 5% oxygen using a static system of modified SOF with or without 4 mg mL–1 BSA and with 0, 0.1, 0.5, or 1 mg mL–1 hyaluronan. Blastocyst and expanded blastocyst stage embryos on day 7 were vitrified (Campos-Chillon LF et al. 2006 Theriogenology 65, 1200–1214). Vitrified embryos were thawed and then cultured for 72 h in modified SOF containing 10% (v/v) fetal bovine serum and 50 µm dithiothreitol. Re-expansion rate was recorded at 24 and 48 h, and the proportion of embryos that hatched by 72 h of culture was recorded. There was no effect of BSA or hyaluronan on cleavage rate. Blastocyst yield from oocytes was increased (P < 0.0005) by BSA (15.3 � 1.1 v. 20.9 � 1.1%). Addition of hyaluronan at 1 mg mL–1 improved (P < 0.04) blastocyst yield (16.2 � 1.7 v. 21.2 � 1.7%), but there was no effect at lower concentrations. There were no interactions between BSA and hyaluronan. Re-expansion rate at 24 and 48 h after thawing was reduced (P < 0.007) by BSA (24 h: 39.1 � 3.6 v. 17.0 � 3.6%; 48 h: 45.6 � 3.8 v. 18.7 � 3.7%), and BSA tended (P < 0.06) to reduce hatching rate at 72 h (22.3 � 3.0 v. 9.8 � 3.0%). Treatment of embryos with hyaluronan did not affect re-expansion rate at 24 h but tended (P < 0.08) to increase re-expansion at 48 h. Moreover, hyaluronan increased (P < 0.05) hatching rate at 72 h after thawing (0 mg mL–1 – 9.8 � 4.2; 0.1 mg mL–1 – 16.9 � 4.5; 0.5 mg mL–1 – 23.4 � 4.1; 1.0 mg mL–1 – 14.2 � 4.1%). In conclusion, blastocyst yield was improved by addition of fructose, BSA, and hyaluronan to culture medium and by use of a static culture system. Hyaluronan also enhanced cryosurvival, but BSA was detrimental to blastocyst survival after vitrification. Support: USDA NRI 2006-55203-17390, BARD US-3551-04.

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.

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 229 EFFECT OF CULTURE CONDITIONS ON AQUAPORIN mRNA ABUNDANCE IN MOUSE BLASTOCYSTS

2005 ◽  
Vol 17 (2) ◽  
pp. 265
Author(s):  
H. Offenberg ◽  
P.D. Thomsen
Keyword(s):  
Blastocyst Stage ◽  
Culture Conditions ◽  
Mrna Abundance ◽  
Control Group ◽  
Mrna Levels ◽  
Blastocyst Formation ◽  
Gapdh Mrna ◽  
The Media

It is known that culture conditions can alter gene expression of the pre-implantation embryo. We have previously shown that aquaporins (AQPs) are expressed in the mouse embryo and that they are involved in the passage of water across the trophoblast cells during blastocyst formation. This study was conducted to investigate whether AQP mRNA abundance is altered by culturing embryos in vitro compared to in vivo developed embryos. Furthermore we wanted to investigate if AQP mRNA abundance was influenced by the osmolality of the media. It is possible to compare the effect of hyperosmolality that the embryo may be able to compensate for by adding glycerol which can cross some AQPs, compared to the addition of sucrose which can not cross the membranes. Mouse embryos were obtained by superovulating B6D2F1 mice followed by culture of the flushed presumptive zygotes in KSOM to the blastocyst stage (in vitro) or by flushing blastocysts from the uterus (in vivo). For the study of the influence of osmolality on AQP mRNA abundance, zygotes were flushed and cultured to the compacted 8-cell stage and then placed in media of increasing osmolality, using either glycerol or sucrose. The osmolalities of the media were 243 (control), 300, 350, and 400 mOsm. Embryos were cultured to the blastocyst stage and frozen in liquid nitrogen. Embryonic RNA was extracted using a Dynabeads mRNA Capture kit (Dynal, Oslo, Norway). Real time PCR was performed on embryonic cDNA on a Lightcycler (Roche Diagnostics, 2650 Hvidovre, Denmark) using aquaporin-specific primers and primers for β-actin and GAPDH. The results of the quantitative RT-PCR analysis showed that in vitro-cultured embryos had a lower mRNA abundance for AQP 8, 9, and 11 compared to the in vivo-developed embryos but that the AQP 3 mRNA abundance was unaltered. Analysis of the housekeeping genes showed that GAPDH mRNA levels were unchanged in vitro, whereas β-actin was up-regulated in vitro. The osmotically challenged embryos showed the following blastocyst rates compared to the controls: glycerol 300: 100%; glycerol 350: 100%; glycerol 400: 100%; sucrose 300: 100%; sucrose 350: 78%; and sucrose 400: 0%. Thus, glycerol up to 400 mOsm had no effect on blastocyst rates, whereas addition of sucrose reduced blastocyst formation, with a total inhibition at 400 mOsm. Analysis of the mRNA abundance showed a reduction of AQP 8 in the glycerol solutions. The level was reduced to 30% of the control group at 300 mOsm, to 27% at 350 mOsm and to 8% at 400 mOsm. There was no corresponding reduction of AQP 8 mRNA abundance in sucrose solutions. Further, AQP 3, 7, 9, and 11 mRNA levels as well as β-actin and GAPDH mRNA levels were unaltered in the osmotically challenged embryos. In conclusion, this study shows that embryonic culture affects the abundance of several AQPs and that compensation of a glycerol-induced osmotical challenge induces down-regulation of AQP 8 expression. Embryos tolerate high glycerol concentrations better than high sucrose concentrations but the possible role of AQP 8 in this process is unclear at present.

Growth and viability of human blastocysts in vitro

2000 ◽  
Vol 8 (3) ◽  
pp. 241-287 ◽  
Author(s):  
GM Jones
Keyword(s):  
Blastocyst Stage ◽  
Culture Conditions ◽  
Cleavage Stage ◽  
Embryo Viability ◽  
Early Cleavage ◽  
Uterine Endometrium ◽  
Stage Of Development ◽  
Uterine Lavage

The transfer of a blastocyst established the first human clinical pregnancy following in vitro fertilization (IVF). Nine years later Cohen et al. reported pregnancies resulting from the transfer of cryopreserved human blastocysts. However, it was another six years before the first report of births resulting from the transfer of human blastocysts produced in vitro appeared in the medical literature. In the intervening period clinics have opted to transfer embryos at the early cleavage stage to the uterus, despite the fact that in vivo the embryo does not enter the uterus until two to three days later at the morula to blastocyst stage of development. The viability and potential for implantation of blastocysts is high, as indicated by the finding that more than 60% of in-vivo-derived blastocysts, recovered by uterine lavage following artificial insemination of fertile donors, implant and develop into viable fetuses when transferred to recipients. This is in stark contrast to the 10–20% of in-vitro-produced embryos transferred at the early cleavage stage of development that result in a live-birth. This reduction in viability following transfer of in-vitro-derived early cleavage stage embryos may have several possible explanations: (1) a failure of implantation due to poor synchronization between the embryo and the uterine endometrium; (2) a hostile environment in the uterus for early cleavage stage embryos; (3) sub-optimal in vitro culture conditions which result in a reduction in embryo viability; (4) the assumption that all oocytes retrieved in an IVF cycle have an equal ability to develop into viable embryos; and (5) the failure to identify the most viable embryo in a cohort. Certainly, improving culture conditions and laboratory techniques for developing high quality blastocysts routinely in vitro will not only address many of the above questions but will also improve the quality and viability of earlier stages of embryo development.

In vitro and in vivo culture effects on mRNA expression of genes involved in metabolism and apoptosis in bovine embryos

2005 ◽  
Vol 17 (8) ◽  
pp. 775 ◽  
Author(s):  
Hiemke M. Knijn ◽  
Christine Wrenzycki ◽  
Peter J. M. Hendriksen ◽  
Peter L. A. M. Vos ◽  
Elly C. Zeinstra ◽  
...  
Keyword(s):  
Glucose Metabolism ◽  
Mrna Expression ◽  
Critical Period ◽  
Glucose Transporter ◽  
Expression Patterns ◽  
Culture Conditions ◽  
Glut 4 ◽  
Gene Transcripts

Bovine blastocysts produced in vitro differ substantially from their in vivo-derived counterparts with regard to glucose metabolism, level of apoptosis and mRNA expression patterns. Maternal embryonic genomic transition is a critical period in which these changes could be induced. The goals of the present study were twofold: (1) to identify the critical period of culture during which the differences in expression of gene transcripts involved in glucose metabolism are induced; and (2) to identify gene transcripts involved in apoptosis that are differentially expressed in in vitro- and in vivo-produced blastocysts. Relative abundances of transcripts for the glucose transporters Glut-1, Glut-3, Glut-4 and Glut-8, and transcripts involved in the apoptotic cascade, including BAX, BCL-XL, XIAP and HSP 70.1, were analysed by a semiquantitative reverse transcription–polymerase chain reaction assay in single blastocysts produced in vitro or in vivo for specific time intervals, that is, before or after maternal embryonic transition. Whether the culture environment was in vitro or in vivo affected the expression of glucose transporter transcripts Glut-3, Glut-4 and Glut-8. However, the critical period during culture responsible for these changes, before or after maternal embryonic transition, could not be determined. With the exception of XIAP, no effects of culture system on the mRNA expression patterns of BAX, BCL-XL and HSP 70.1 could be observed. These data show that expression of XIAP transcripts in expanded blastocysts is affected by in vitro culture. These findings add to the list of bovine genes aberrantly expressed in culture conditions, but do not support the hypothesis that maternal embryonic transition is critical in inducing the aberrations in gene expression patterns studied here.

175 BOVINE EMBRYO DEVELOPMENT AND MRNA EXPRESSION IN BLASTOCYSTS CULTURED IN ISOLATED MOUSE OVIDUCTS MAINTAINED IN SOF OR KSOM

2006 ◽  
Vol 18 (2) ◽  
pp. 195
Author(s):  
D. Rizos ◽  
B. Pintado ◽  
J. de la Fuente ◽  
P. Lonergan ◽  
A. Gutierrez-Adan
Keyword(s):  
Embryo Development ◽  
Relative Abundance ◽  
Culture Medium ◽  
Embryo Quality ◽  
Blastocyst Stage ◽  
Glut 1 ◽  
Gene Transcripts ◽  
Culture Environment ◽  
Chi Square Analysis

It is well known that modification of the post-fertilization culture environment of mammalian pre-attachment embryos can affect blastocyst quality, manifested in terms of morphology, cryotolerance, and relative abundance of certain gene transcripts. Culture of in vitro-produced bovine zygotes in the ewe oviduct leads to the development of blastocysts of a quality similar to those derived totally in vitro (Rizos et al. 2002 Biol. Reprod. 66, 589-595). However, such a system has disadvantages from a practical and animal welfare point of view. The isolated mouse oviduct (IMO) culture system is a potential alternative and has been successfully used in the in vitro culture of mouse, rat, hamster, and pig embryos from the one-cell stage to the morula/blastocyst stage. The aim of this study was to examine (1) the development of bovine zygotes in the IMO maintained in two different media (SOF and KSOM) in organ culture, and (2) the quality of the resultant blastocysts assessed in terms of the relative abundance of transcripts for several genes that have been previously implicated in embryo quality. Mouse oviducts were isolated from adult Swiss females (CD1, Harlan) the day after mating with an intact male. Approximately 10-15 presumptive bovine zygotes, produced by in vitro oocyte maturation and fertilization, were transferred to the ampullae of the isolated oviducts and were cultured in Transwell plates (Costar, Corning, NY, USA) over 1.1 mL of culture medium (SOF, n = 241 or KSOM, n = 320) at 39�C in an atmosphere of 5% CO2 in air at maximum humidity. A control group of embryos was cultured in droplets (25 �L) of the same culture medium and conditions in parallel (SOF, n = 278, KSOM, n = 225). Five replicates (=days of bovine ovary collection) were carried out. Following 6 days of culture, embryos were recovered from the oviducts/culture drops and blastocysts were snap-frozen in liquid nitrogen. Quantification of all gene transcripts was carried out by real time quantitative RT-PCR. Data on embryo development were analyzed by chi-square analysis and differences in transcript abundance by ANOVA. Culture in the IMO did not affect the proportion of zygotes developing to the blastocyst stage compared to the respective control droplets (SOF: 21.0 vs. 21.9%; KSOM: 22.0 vs. 22.2%). Culture in the IMO in SOF resulted in an increase (P d 0.05) in the abundance of transcripts for Oct-4 and SOX and reduced abundance of Glut-1, Na/K transporter, Cx43, and survivin, compared to control embryos. In contrast, culture in the IMO in KSOM resulted in increased abundance of transcripts for Glut-1, Cx43, Oct-4, and survivin and a reduced expression of Na/K transporter and SOX. Transcripts for G6PDH, IFN, and E-Cad were unaffected by culture environment. In conclusion, culture in the IMO leads to alterations in the relative abundance of transcripts that have been previously associated with embryo quality following culture in the ewe oviduct. However, the effect is dependent on the basal medium used.

86 BIRTH OF HEALTHY CALVES AFTER INTRAFOLLICULAR OOCYTE TRANSFER

2015 ◽  
Vol 27 (1) ◽  
pp. 136
Author(s):  
M. Hoelker ◽  
A. Kassens ◽  
E. Held ◽  
C. Wrenzycki ◽  
U. Besenfelder ◽  
...  
Keyword(s):  
Lipid Content ◽  
Ex Vivo ◽  
Survival Rates ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
In Vitro Production ◽  
Negative Effects ◽  
Uterine Flushing

The in vitro production (IVP) of bovine embryos is a well-established technique that has been available for nearly 20 years. However, there remain major differences between IVP-derived blastocysts and their in vivo-derived counterparts. Many studies have pointed out that most of these differences are due to the in vitro developmental environment. To circumvent these negative effects due to in vitro culture conditions, a new method – intrafollicular oocyte transfer (IFOT) – was established in the present study. Using modified ovum pick-up (OPU) equipment, in vitro-matured oocytes derived from slaughterhouse ovaries were injected into the dominant preovulatory follicle of synchronised heifers (follicular recipients) enabling subsequent ovulation, in vivo fertilization, and in vivo development. A total of 810 in vitro-matured oocytes were transferred into 14 heifers. Subsequently, 222 embryos (27.3%) were recovered after uterine flushing at Day 7. Based on the number of cleaved embryonic stages, 64.2% developed to the blastocyst stage, which did not differ from the IVP-derived embryos (58.2%). Interestingly, lipid content of IFOT-derived blastocysts did not differ from the fully in vivo-produced embryos, whereas IVP-derived blastocysts showed significantly higher lipid droplet accumulation compared with fully in vivo-derived and IFOT-derived blastocysts (P < 0.05). Accordingly, IFOT blastocysts showed significantly higher survival rates after cryopreservation than complete IVP-derived embryos (77% v. 10%), which might be attributed to a lower degree of lipid accumulation. In agreement, transfer of frozen-thawed IFOT blastocysts to synchronized recipients (uterine recipients) resulted in much higher pregnancy rates compared with transfer of IVP-derived blastocysts (42.1 v. 13.8%) but did not differ from frozen-thawed ex vivo blastocysts (52.4%). Of these presumed IFOT pregnancies, 7 went to term, and microsatellite analysis confirmed that 5 calves were indeed derived from IFOT, whereas 2 were caused by fertilization of the follicular recipient's own oocyte after AI. Taken together, IFOT-derived blastocysts closely resemble in vivo-derived blastocysts, confirming earlier suggestions that the ability to develop to the blastocyst stage is already determined in the matured oocyte, whereas the quality in terms of lipid content and survival rate after cryopreservation is affected by the environment thereafter. However, to the best of our knowledge, this is the first study reporting healthy calves after intrafollicular transfer of in vitro-matured oocytes.

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