scholarly journals 154 EFFECTS OF BLASTOMERE BIOPSY AND OXYGEN CONCENTRATION DURING CULTURE ON THE DEVELOPMENT AND INTERFERON-TAU SECRETION OF BOVINE EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 227
Author(s):  
K.M. Johnson ◽  
X. Alvarez ◽  
H.M. Kubisch
Keyword(s):  
Genetic Diagnosis ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Optimal Time ◽  
Human Embryos ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Interferon Tau ◽  
And Control

Pre-implantation genetic diagnosis has become a powerful tool in human in vitro fertilization. Yet, for ethical reasons, controlled experiments assessing the effects of embryo biopsies on human embryos are difficult to perform. Therefore, a series of experiments was performed to examine the effects of blastomere biopsies on subsequent development of IVF-derived bovine embryos cultured under either atmospheric or low (5%) oxygen. Embryos were generated by IVF of in vitro-matured oocytes and cultured in CR1aa medium containing 5% bovine fetal serum. The first experiment was designed to assess the optimal time for blastomere removal. One blastomere was removed from each of a large number of embryos at either 48 or 72 h after IVF. Biopsy at 48 h resulted in 17.2% (10/58) of embryos proceeding to the blastocyst stage, which was significantly lower than when biopsies were performed at 72 h (37.5% (33/88), P < 0.05). In the second experiment, embryos were cultured under either atmospheric or 5% O2 following blastomere removal. Biopsies at 72 h had no effect on rate of blastocyst formation with 38.5% (110/286) of controls and 33.7% (60/178) of biopsied embryos proceeding to the blastocyst stage. However, culture under 5% O2 significantly increased the number of blastocysts from 29.9% (69/231) to 43.3% (101/233). This effect was significant in both biopsied and control embryos. In contrast, staining of blastocysts for cell numbers did not reveal any effects of biopsies or culture conditions. In the final experiment, biopsies were again performed at 72 h and embryos were cultured as previously. Blastocysts were collected and cultured individually for 48 h in 50-μL-medium droplets in their respective O2 concentrations after which time the medium was assayed for concentration of interferon-tau (IFN-τ). Previous work has shown that IFN-τ secretion can be affected by genetic and environmental factors. Reduced O2 concentration again significantly increased blastocyst formation from 24.9 to 36.9% (49/197 vs. 75/203, P < 0.05), while blastomere removal had no effect on development. IFN-τ secretion did not differ between biopsied and control blastocysts, although culture under atmospheric O2 resulted in significantly increased IFN-τ concentration in medium droplets (12,690.1 ± 2715.6 vs. 4726.8 ± 2488.5 pM, P < 0.05). This work was supported by a grant (HMK) from the National Institutes of Health (HD 36421).

scholarly journals 136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
Author(s):  
C.E. Ferguson ◽  
T.R. Davidson ◽  
M.R.B. Mello ◽  
A.S. Lima ◽  
D.J. Kesler ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Direct Role ◽  
Treatment Groups ◽  
And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

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 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.

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.

237 THE EFFECTS OF BULLS AND X-SORTING OF SPERM ON THE ACCURACY OF NONINVASIVE CRITERIA TO PREDICT BLASTOCYST FORMATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 208
Author(s):  
S. Matoba ◽  
T. Somfai ◽  
T. Nagai ◽  
M. Geshi
Keyword(s):  
Formation Rate ◽  
Calf Serum ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Blastocyst Development ◽  
Chi Square

Previously, an early first cleavage and a second cleavage after IVF with a normal cleavage pattern defined by even blastomeres without fragments or protrusions was found to be a potent marker for the selection of embryos with high developmental competence (Sugimura et al. 2012 PLoS ONE 7, e36627). The aim of this study was to investigate the effects of bulls and X-sorting of sperm on the ability of these simple noninvasive markers to predict the potency of bovine IVF embryos to develop to the blastocyst stage in vitro. Immature oocytes were matured in TCM199 supplemented with 0.02 armour unit mL–1 FSH and 5% calf serum at 38.5°C in 5% CO2 and 95% air for 22 to 23 h. After maturation, oocytes were inseminated with either of non-sorted frozen-thawed sperm from 3 bulls (A–C) or X-sorted sperm of bull A. Putative zygotes were cultured (IVC) in CR1aa medium supplemented with 5% calf serum and 0.25 mg mL–1 linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2 for 216 h. Embryo kinetics were observed individually by time-lapse cinematography (CCM-1.3Z; Astec, Fukuoka, Japan; Sugimura et al. 2010 Biol. Reprod. 83, 970–978). First and second cleavage kinetics and pattern were categorized according to Sugimura et al. (2012). For each bull, blastocyst development from embryos possessing the following 3 selection markers was compared: (marker 1) the first cleavage within 28 h after IVF, (marker 2) marker 1 combined with 2 even blastomeres without fragments or protrusions, and (marker 3) marker 2 combined with the second cleavage within 50 h after IVF with ≥6 even blastomeres without fragments or protrusions, respectively. Data were analysed by the Yates' corrected chi-square test. A total of 823 oocytes were used in at least 3 replications. When non-sorted sperm was used for IVF, there was not difference (P > 0.05) in total blastocyst formation rates on Day 8 (Day 0 = IVF) among bulls (ranging between 49.5 and 60.8%); however, blastocyst formation rate of embryos generated from X-sorted sperm of bull A (39.5%) was lower (P < 0.05) compared with other groups despite of similar cleavage rates. Embryos having marker 3 criteria developed to the blastocysts stage at significantly higher rates than those having marker 1 criteria in case of non-sorted sperm of bulls A, B, C, and X-sorted sperm of bull A (75.9, 87.0, 90.0, and 75.0% v. 59.5, 62.2, 63.6, and 46.3%, respectively). In groups produced from non-sorted sperm of bulls A, B, C, and X-sorted sperm of bull A, blastocyst development rates of embryos with marker 2 criteria (73.7, 75.0, 90.0, and 65.8%, respectively) were higher (P < 0.05) than those of embryos having marker 1 criteria but did not differ significantly from those with marker 3 criteria. Our results reveal that a first cleavage within 28 h after IVF to 2 even blastomeres without fragments or protrusions are potent predictive markers of the developmental competence of bovine embryos to the blastocyst stage regardless of bulls and sperm sorting.Research was partly supported by JSPS KAKENHI (26450388).

54 ESTABLISHMENT OF PREGNANCY BY OVINE NUCLEAR TRANSFER EMBRYOS RECONSTRUCTED USING CAFFEINE-TREATED IN VITRO-MATURED OOCYTES AS CYTOPLAST RECIPIENTS

2006 ◽  
Vol 18 (2) ◽  
pp. 135 ◽  
Author(s):  
J.-H. Lee ◽  
M. Marfil ◽  
M. Panarace ◽  
M. Medina ◽  
K. H. S. Campbell
Keyword(s):  
Nuclear Transfer ◽  
Animal Health ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Interferon Tau ◽  
Chi Square ◽  
Developmental Potential ◽  
Donor Nucleus ◽  
Chi Square Test

In embryos reconstructed by somatic cell nuclear transfer (SCNT), components of the oocyte cytoplasm are capable of reprogramming the somatic genome to control subsequent development. Although the mechanisms that control nuclear reprogramming are unknown, we have previously hypothesized that the occurrence of nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC) in the donor nucleus are beneficial. In previous studies we have demonstrated that treatment of ovine oocytes with caffeine (10 mM), a protein phosphatase inhibitor, increased the activities of both MPF and MAPK in enucleated oocytes (Lee and Campbell 2004 Reprod. Fertil. Dev. 16, 125) and additionally resulted in a significant increase in the occurrence of NEBD and PCC in donor nuclei. Furthermore, SCNT embryos reconstructed following caffeine treatment had significantly increased cell numbers at the blastocyst stage. More recently we have demonstrated that the use of caffeine treated ovine oocytes as cytoplast recipients can regulate the expression of several developmentally important genes in SCNT embryos, including Oct-4 and interferon-tau (Choi et al. 2006 Reprod. Fertil. Dev. 18, in press). This study was designed to establish the developmental potential of NT embryos reconstructed using caffeine treated oocytes as cytoplast recipients. Ear skin fibroblast cells established from a Merino ram were quiesced in DMEM containing 0.1% fetal bovine serum (FBS) for 3 days. Oocyte maturation and embryo reconstruction and culture were performed as previously described (Lee and Campbell 2004 Reprod. Fertil. Dev. 16, 125) with the exception that ovaries from Merino � Romney Marsh cross ewes were stimulated with FSH sponge (Folltropin�-V; Bioniche Animal Health, Beltsville, Ontario, Canada) and were collected at slaughter on Day 13 following sponging. Blastocyst stage embryos were surgically transferred to the uterine horn of synchronized Merino � Romney Marsh cross recipients (three blastocysts per recipient). Recipient ewes were scanned by ultrasonography at Days 30, 60, and 90 following embryo transfer. All data were analyzed by chi-square test. There were no differences in fusion (145/167; 86.8% vs. 174/205; 84.9%), cleavage (123/145; 84.8% vs. 135/174; 77.6%), or the development to blastocyst (33/145; 22.8% vs. 34/174; 19.5%) between control SCNT embryos and caffeine treated SCNT embryos. However, although the frequency of pregnancy between control and caffeine-treated NT groups (5/15; 33.3% vs. 7/14; 50.0%) at 30 days was not significantly different, control SCNT embryos showed significantly lower pregnancies (1/15; 6.7%) than caffeine treated SCNT embryos (4/14; 28.6%) at both 60 and 90 days. In conclusion, embryos reconstructed using caffeine-treated cytoplasts can induce pregnancy at the same frequency as untreated controls; furthermore, the results suggest that SCNT embryos produced in this way are more able to maintain pregnancy.

232 EFFECT OF MEIOTIC ARREST USING BUTYROLACTONE I AND ROSCOVITINE ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 264
Author(s):  
R. R. D. Maziero ◽  
M. D. Guastali ◽  
M. J. Sudano ◽  
D. M. Paschoal ◽  
P. N. Guasti ◽  
...  
Keyword(s):  
Linear Models ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Negative Effect ◽  
Vitro Production ◽  
Co2 Atmosphere

Butyrolactone I (BL-I) and roscovitine (ROS) are selective inhibitors of the cyclin-dependent kinases, and both have been shown to reversibly inhibit meiotic resumption in cattle oocytes without having a negative effect on subsequent development to the blastocyst stage. The aim of this study was to evaluate the in vitro production of bovine embryos in the presence of BL-I (Enzo Life Sciences International Inc., Plymouth Meeting, PA, USA) and ROS (Sigma-Aldrich, St. Louis, MO, USA). For that, Nellore oocytes were matured in TCM-199 with Earle’s salts + 10% FCS, FSH, and LH, in a 5% CO2 atmosphere. To delay meiosis, the oocytes were maintained for 6 h in medium in the presence of 25 µM BL-I + 6.25 µM ROS. The oocytes were then cultured for 18 h in agent-free medium to resume meiosis, completing 24 h of maturation. After 24 h of maturation (Day 0), oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand), in a 5% CO2 atmosphere. Semen was selected through Percoll gradient and the concentration was adjusted to 2 × 106 sperm mL–1. The presumed zygotes were cultured inside a bag in 90-µL droplets of SOFaa + 0.6% BSA + 2.5% FCS in a 5% CO2, 5% O2, and 90% N2 atmosphere until Day 7, when blastocyst rate was evaluated. Five replicates were done. Data were analysed with ANOVA, followed by Tukey’s test using the general linear models procedure (PROC GLM) of SAS, version 9.2 (SAS Institute Inc., Cary, NC, USA). The level of significance adopted was 5%. No statistical differences were observed in blastocyst production rate: control, 33.3 ± 3.74%; BL-I + ROS, 38.0 ± 4.5%. Thus, the presence of BL-I + ROS in the prematuration medium of bovine oocytes did not compromise their subsequent developmental competence. The next steps of the present study are evaluating the kinetics of blastocyst formation and detecting apoptotic cells in situ by TUNEL, which will show that these substances do not compromise embryonic quality.

scholarly journals 72EFFECTS OF GENE EXPRESSION IN BOVINE EMBRYOS RECONSTRUCTED WITH FIBROBLASTS TRANSFECTED WITH LUCIFERASE GENE ON THE SUBSEQUENT DEVELOPMENT

2004 ◽  
Vol 16 (2) ◽  
pp. 157
Author(s):  
K. Saeki ◽  
T. Tamari ◽  
A. Kasamatsu ◽  
K. Shirouzu ◽  
S. Taniguchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fusion Gene ◽  
Calcium Ionophore ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Luciferase Gene ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Transfected Cells

During embryo development, embryonic gene activation (EGA) is one of the first critical events. Inappropriate EGA results in failure of further development. We have reported that gene expression in bovine embryos reconstructed with fibroblasts begins at 48 hours postfusion (hpf) and reaches a maximum level at 60hpf as detected by their bioluminescence following injection of chicken β-actin/firefly luciferase fusion gene (β-act/luc+) into their nuclei (Saeki et al., 2001 Theriogenology 55, 289). In the present study, effects of gene expression in embryos reconstructed with bovine fibroblasts transfected with luciferase gene on their subsequent development to the blastocyst stage were examined. Cultured bovine fibroblasts taken from an ear of a female calf were transfected with plasmid containing β-act/luc+/IRES/EGFP and neor using GeneJammer (StrataGene, La Jolla, CA, USA). Neomycin-resistant cells were selected by culturing with G418. Then, EGFP-positive colonies were further selected under fluorescence microscopy to obtain stably transfected cells. The transfected cells were cultured for several passages. Growing (50 to 60% confluence, GCs) and serum-starved cells (SCs) were used as donor cells. In vitro-matured bovine oocytes derived from slaughterhouse ovaries were enucleated at 20h post maturation. Enucleated oocytes were electrofused with the cells, and activated with calcium ionophore and cycloheximide. Luminescence in the embryos was detected with an imaging photon counter at 0 and 60hpf. Luminescence-positive (P) and -negative (N) embryos were cultured separately at each detection time. Embryos were cultured until 168hpf, and examined for cleavage and blastocyst development. Experiments were repeated 3 times, and totals of 91 and 123 embryos were reconstructed with GCs and SCs, respectively. Data were analyzed with Fisher’s PLSD test following ANOVA by Stat View software (Ver. 5.0). At 0hpf, luminescence was detected in 55 and 4% of embryos reconstructed with GCs and SCs, respectively. At 60hpf, luminescence was detected in 47 and 28% of P and N embryos with GCs, and 17 and 40% of P and N embryos with SCs at 0hpf, respectively. Cleavage rates were not different among groups (P&gt;0.05). Blastocysts were obtained only from the groups of embryos that were N at 0hpf and P at 60hpf (8% with GCs and 17% with SCs). No embryos in the other groups developed to the blastocyst stage. These results suggest that appropriate gene expression in embryos reconstructed with somatic cells is important for their subsequent development and that detecting the reporter gene expression can be used for selection of viable cloned embryos.

O-223 Fatty acid supplementation into warming solutions improve the developmental competence of mouse, bovine, and human oocytes and embryos after vitrification

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
K Ohata ◽  
K Ezoe ◽  
T Miki ◽  
S Kouraba ◽  
N Fujiwara ◽  
...  
Keyword(s):  
Lipid Content ◽  
Formation Rate ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Human Embryos ◽  
Control Group ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Intracellular Lipid ◽  
Mouse Oocytes

Abstract Study question Does fatty acid (FA) supplementation into vitrification and warming solutions influence the developmental competence of oocyte and embryo after vitrification and warming? Summary answer FA supplementation during the warming process improves the developmental competence of vitrified-warmed mouse oocytes and embryonic-morphologies after vitrification at the cleavage-stage in bovines and humans. What is known already Vitrified metaphase II stage oocytes exhibit a diminished ability to develop into blastocysts and live births. Previous studies have shown reduction in intracellular lipid content as one of the factors associated with reduced developmental competence of oocytes after vitrification as the intracellular lipid content of oocytes is affected by vitrification. FAs derived from break down of lipids are primarily transferred to the mitochondria, where it plays a crucial role in cellular metabolism. However, the effects of FA supplementation in warming solutions on the cytoplasmic lipid content and subsequent embryo development are unknown. Study design, size, duration A chemically defined FA mixture was added to the vitrification and/or warming solutions. Oocytes collected from C57BL6/N (n = 80) were randomly divided into three groups (fresh, n = 634; non-FA (control), n = 961; FA, n = 1,686), and were vitrified-warmed with/without FA. Lipid composition, developmental competence, and gene expression levels were compared among the groups. Bovine embryos (fresh, n = 420; control, n = 524; FA, n = 492) and discarded human day-2 embryos (control, n = 87; FA, n = 92) were used to examine the developmental competence of embryos. Participants/materials, setting, methods Lipids in the ooplasm were stained with Nile red and the fluorescence intensity was analysed. The developmental competence of mouse oocytes was examined by performing intracytoplasmic sperm injection. Expressions of FA metabolism-related genes were measured. The bovine embryos were vitrified at the four-cell stage and cultured to the blastocyst stage after warming. Cryopreserved discarded human embryos were warmed and cultured. The obtained blastocysts were then placed on fibronectin-coated dishes to examine the outgrowth formation. Main results and the role of chance Lipid content of mouse oocytes was significantly lower in the control group compared to that in the fresh group (P &lt; 0.05). On the contrary, lipid contents of FA and fresh groups were comparable (P = 0.24). Blastocyst formation rate was significantly higher in the FA group than that in the control group (55.7% and 44.8%, respectively; P &lt; 0.05). To examine the optimal timing for FA supplementation, FA was added to the vitrification solution (FAvit), warming solution (FAthaw), and/or both solutions (FAvit-thaw). Blastocyst formation rate was significantly higher in the FAthaw group than that in the control group (59.8% and 50.0%, respectively; P &lt; 0.05). The mRNA expressions of Acaa2 and Hadha in mouse embryos were significantly higher in the FAthaw group compared to that in the control group (P &lt; 0.05). Moreover, FA supplemented warming solutions significantly improved the blastocyst formation rate in bovines (control, 53.5%; FAthaw, 64.5%; P &lt; 0.05). Developmental rate to the expanded blastocyst stage was slightly improved in human embryos (control, 53.7%; FAthaw, 63%; P = 0.38) and the proportion of Grade A in inner cell mass and trophectoderm was significantly higher in the FAthaw group than that in the control group (P &lt; 0.05). There were no differences in the outgrowth abilities between the control and FAthaw groups. Limitations, reasons for caution Since the experiments of the current study on human embryos were performed in vitro using discarded embryos, in vivo developmental ability was not evaluated. Therefore, to validate the application of our findings in human assisted reproductive technologies, further clinical trials (ART) are warranted. Wider implications of the findings FA supplementation into the warming solutions improved the developmental competence of vitrified–warmed oocytes and cleaved embryos by activating the β-oxidation pathway. These results indicate that FA supplementation into warming solutions is a potential strategy to improve clinical outcomes in human ART. Trial registration number not applicable

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