133 CRISPR/Cas9 gene editing of invivo-fertilized bovine embryos via endoscopic oviductal flushing and electroporation of zygotes

2020 ◽  
Vol 32 (2) ◽  
pp. 193
Author(s):  
D. Miskel ◽  
L. Beunink ◽  
M. Poirier ◽  
V. Havlicek ◽  
F. Rings ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Gene Editing ◽  
Statistical Significance ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Bovine Embryos ◽  
Embryo Survival ◽  
Guide Rna ◽  
Endoscopic Method ◽  
Developmental Rates

In recent years, CRISPR/Cas9 has been used to efficiently edit the genomes of embryos in many animal models. Due to smaller anatomy, lower costs, and multiple ovulations, it is relatively simple to derive large numbers of invivo fertilized zygotes for gene editing experiments in small mammal models. In cattle, however, harvesting invivo fertilized zygotes generally requires a highly invasive surgical procedure. Here, we use the combination of a minimally invasive endoscopic method for harvesting invivo fertilized zygotes by oviductal flushing of superovulated heifers and the subsequent electroporation of zygotes with CRISPR/Cas9 ribonucleoproteins (RNP). After superstimulation of 21 heifers, on average 12 zygotes were flushed per animal with fetal bovine serum, then stored in synthetic oviductal fluid (SOFaa) before electroporation. Targeting exon 1 of the tyrosinase (Tyr) gene, zygotes were electroporated in 1-mm gap cuvettes (Biorad) in groups of ~20 in 20μL of OptiMEM media containing 3μM Cas9 RNP (IDT Cas9 protein pre-incubated with anti-Tyr guide RNA). Electroporation was performed in 3 replicates of 3 electrical potentials, namely 20, 25, and 30V using a Biojet CF 50. The other electroporation parameters were fixed at 5 repetitions of 2-ms square wave pulses at 100-ms intervals. The zygotes were than cultured under standard embryo culture conditions (SOFaa + 0.3% bovine serum albumin, 5% CO2, 5% O2, 39°C, humidified air). Embryo survival, cleavage, and developmental rates to the blastocyst stage were tracked. Statistical significance between groups was determined by pairwise one-way ANOVA using Sidak correction for multiple comparisons. Electroporation of invivo-derived zygotes using 20V yielded significantly higher survival (83.6% vs. 42.8% vs. 20.7% for 20, 25, and 30V, respectively), cleavage (65.6% vs. 37.9% vs. 40.0%), and developmental rates (47.5% vs. 21.4% vs. 16.5%) than 25 or 30V. There was no statistical difference between 25 and 30V. Subsequently, editing rates were determined using the T7 mismatch assay and verified with Sanger sequencing followed by sequence alignment and analysis using Tracking of Indels by Decomposition (TIDE) software (https://tide.nki.nl/). Although there was high variance between electroporation groups, blastocyst editing rates of up to 80.0% were achieved using 30V. To our knowledge, these are the first confirmed gene-edited bovine embryos produced from invivo fertilized zygotes. This method offers the ability to utilise the embryos of high-value cows or cows with known genotypes for genetic engineering experiments. In addition, given that electroporated bovine zygotes can be transferred back to the oviduct endoscopically, our future attempts will focus on genome editing in bovine embryos developed nearly completely within the physiological invivo environment.

58 EFFICIENCY OF OVUM PICKUP AND EMBRYO PRODUCTION IN VITRO IN CLONED CATTLE

2006 ◽  
Vol 18 (2) ◽  
pp. 137
Author(s):  
A. Lucas-Hahn ◽  
E. Lemme ◽  
K.-G. Hadeler ◽  
H.-G. Sander ◽  
H. Niemann
Keyword(s):  
Nuclear Transfer ◽  
Transvaginal Ultrasound ◽  
Statistical Significance ◽  
Blastocyst Stage ◽  
Ultrasound Guided ◽  
Embryo Production ◽  
Fetal Fibroblasts ◽  
Developmental Rates ◽  
In Vitro Embryo Production

The reproductive performance of cloned cattle was investigated by assessing the efficiency of transvaginal ultrasound-guided ovum pickup (OPU) and embryo production in vitro. Fetal fibroblasts from the endangered species, German Blackpied Cattle, had been used for nuclear transfer to produce three live cloned offspring (Lucas-Hahn et al. 2002 Theriogenology 57, 433). In the three cloned animals at 12–20 months of age, OPU was performed once per week and the total number of collected oocytes was recorded. In the case of Blondie, the procedure was terminated due to too small ovaries associated with insufficient function. Oocytes suitable for IVF were matured in vitro for 24 h and fertilized in vitro with the semen of a fertile bull. Oocytes derived from abbatoir ovaries were processed in parallel as controls. Embryos were in vitro-cultured in SOFaaBSA medium. Cleavage and developmental rates up to the morula/blastocyst stage were recorded in all groups. Statistical significance was tested using ANOVA and the Student-Newman-Keuls test. The results are presented in Table 1. Embryos from clones had lower cleavage and blastocyst rates compared to those derived from abattoir oocytes. However, results may have been confounded by potential OPU effects. Some of the blastocysts produced from Blacky (n = 5) and Paula (n = 2) were transferred to recipients. Two pregnancies resulted from the Paula transfers. The two male calves were delivered normally. After the completion of this experiment, all three cloned animals were artificially inseminated, became pregnant, delivered healthy calves, and are pregnant again at present. Further studies are needed to explore the fertility of cattle derived from somatic cloning. Table 1. OPU and in vitro embryo production in cloned cattle

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.

80 Biallelic CRISPR-Cas9 editing of gene associated with coat colour in microinjected bovine zygotes reaching the blastocyst stage

2019 ◽  
Vol 31 (1) ◽  
pp. 165
Author(s):  
M. Poirier ◽  
D. Miskel ◽  
F. Rings ◽  
K. Schellander ◽  
M. Hoelker
Keyword(s):  
Gene Editing ◽  
Fluorescent Protein ◽  
Survival Rates ◽  
Coat Colour ◽  
Blastocyst Stage ◽  
Transcription Activator ◽  
Guide Rna ◽  
Blastocyst Rate ◽  
Copa Gene

Successful genome editing of blastocysts using zygote microinjection with transcription activator-like effector nucleases has already been accomplished in cattle as well as a limited number of CRISPR-Cas9 microinjections of zygotes, mostly using RNA. Recent editing of the Pou5f1 gene in bovine blastocysts using CRISPR-Cas9, clarifying its role in embryo development, supports the viability of this technology to produce genome edited cattle founders. To further this aim, we hypothesise that editing of the coatomer subunit α (COPA) gene, a protein carrier associated with the dominant red coat colour phenotype in Holstein cattle, is feasible through zygote microinjection. Here, we report successful gene editing of COPA in cattle zygotes reaching the blastocyst stage, a necessary step in creating genome edited founder animals. A single guide RNA was designed to target the sixth exon of COPA. Presumptive zygotes derived from slaughterhouse oocytes by in vitro maturation and fertilization were microinjected either with the PX458 plasmid (Addgene #48138; n=585, 25ng µL−1) or with a ribonucleoprotein effector complex (n=705, 20, 50, 100, and 200ng µL−1) targeting the sixth exon of COPA. Plasmid injected zygotes were selected for green fluorescent protein (GFP) fluorescence at Day 8, whereas protein injected zygotes were selected within 24h post-injection based on ATTO-550 fluorescence. To assess gene editing rates, single Day 8 blastocysts were PCR amplified and screened using the T7 endonuclease assay. Positive structures were Sanger sequenced using bacterial cloning. For plasmid injected groups, the Day 8 blastocyst rate averaged 30.3% (control 18.1%). The fluorescence rate at Day 8 was 6.3%, with a GFP positive blastocyst rate of 1.6%, totaling 7 blastocysts. The T7 assay revealed editing in GFP negative blastocysts and morulae as well, indicating that GFP is not a precise selection tool for successful editing. In protein injection groups, the highest concentration yielded the lowest survival rates (200ng µL−1, 50.0%, n=126), whereas the lowest concentration had the highest survival rate (20ng µL−1, 79.5%, n=314). The Day 8 blastocyst rate reached an average of 25% across groups. However, no edited blastocysts were observed in the higher concentration groups (100,200ng µL−1). The highest number of edited embryos was found in the lowest concentration injected (20ng µL−1, 4/56). Edited embryos showed multiple editing events neighbouring the guide RNA target site ranging from a 12-bp insertion to a 9-bp deletion, as well as unedited sequences. Additionally, one embryo showed a biallelic 15-bp deletion of COPA (10 clones). One possible reason for the presence of only mosaic editing and this in-frame deletion could be that a working copy of COPA is needed for proper blastocyst formation and that a knockout could be lethal. Additional validation and optimization is needed to elucidate the functional role of COPA during early development and its modulation when creating founder animals.

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.

109 INTRAFOLLICULAR GLUCOSE CONCENTRATION HAS AN INFLUENCE ON THE SEX OF BOVINE BLASTOCYSTS PRODUCED IN VITRO

2011 ◽  
Vol 23 (1) ◽  
pp. 160
Author(s):  
E. Abele ◽  
H. Stinshoff ◽  
A. Hanstedt ◽  
S. Wilkening ◽  
S. Meinecke-Tillmann ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Follicular Fluid ◽  
Glucose Concentration ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Bovine Embryos ◽  
Developmental Rates ◽  
Group 2 ◽  
Group 1

Several factors have been shown to alter the sex ratio of bovine embryos generated in vitro, i.e. the maturity of the oocyte at the time of insemination, the duration of sperm-oocyte co-incubation and the culture conditions after in vitro fertilization. It has been shown that the presence of glucose during in vitro culture reduced the development of female embryos to the blastocyst stage compared with controls cultured in the absence of glucose. The sex ratio of bovine embryos has also been linked with changes in the composition of the follicular fluid in which the oocyte undergoes growth and maturation, i.e. the intrafollicular testosterone concentration. However, no information is available regarding the effect of intrafollicular glucose concentration on the sex ratio of embryos after in vitro production (IVP). The purpose of this study was to determine whether different glucose concentrations in the follicular fluid at the time of cumulus–oocyte complex (COC) collection have an effect on the sex ratio of the resulting blastocysts after IVP. Ovaries from a local abattoir were transported to the laboratory within 2 h of slaughter. Follicles (3–8 mm) were individually dissected and the glucose concentration of each follicle was measured using a blood glucose monitoring system (Freestyle Freedom Lite, Abbott, Germany). Based on a glucose concentration, COC [low glucose: <1.1 mM (group 1) and high glucose: >1.1 mM (group 2)] were pooled in groups and used for blastocyst production employing standard protocols for IVP. Developmental rates were recorded at Day 3 (cleavage) and Day 7/8 (blastocyst stage). Total cell number of blastocysts was determined after Hoechst staining. Sex of the embryos was analysed via PCR using bovine X- and Y-chromosome specific primers. Developmental rates for COC stemming from follicles with different glucose concentrations did not show significant differences (P > 0.05) compared to each other [Cleavage rate: group 1: 81.8 ± 4.7% (93/117); group 2: 79.3 ± 4.9% (94/123); blastocyst rate: group 1: 35.6 ± 5.2% (38/117); group 2: 31.6 ± 5.2% (38/123)]. Total cell numbers were similar in embryos of both groups [Group 1: 117.7 ± 8.1 (n = 18); group 2: 117.2 ± 6.4 (n = 18)]. The overall sex ratio significantly differed (P < 0.05) from 1:1 in favour of females in both groups [Group 1: 85 v. 15% (n = 20); group 2: 63.6 v. 36.4% (n = 22)]. No significant difference (P > 0.05) in the overall sex ratio was detected in blastocysts produced under standard IVP conditions employed in the laboratory [without measurement of follicular glucose concentration, 55.0 v. 45.0%, (n = 20)]. In conclusion, under the conditions used in the present study, the intrafollicular glucose concentration from which the immature COC was collected affects the sex of the resulting embryo after IVP, favouring females. Further studies are needed to confirm these findings in living cows using the ovum pickup technique.

22 Vitrification of bovine embryo using antifreeze polyamino acid

2019 ◽  
Vol 31 (1) ◽  
pp. 137
Author(s):  
T. Fujikawa ◽  
Y. Gen ◽  
S.-H. Hyon ◽  
C. Kubota
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
Basal Medium ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Chi Square ◽  
Embryo Survival ◽  
Polyamino Acid

Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound and a polyamino acid with a known functional resemblance to antifreeze proteins. We previously reported that CPLL is an effective cryoprotectant for bovine cells, sperm, and slow-frozen embryos. In this study, we investigated CPLL as a cryoprotectant for vitrified bovine embryos. We developed bovine embryos in vitro and vitrified them at the blastocyst stage. Embryos were equilibrated (3min) and vitrified (1min). Vitrified embryos were cryopreserved in LN (Cryotop® device; Kitazato Corp., Tokyo, Japan) for at least 1 week, thawed with a 0.3M sucrose warming solution, and then cultured in a basal medium (Gibco® medium 199, Grand Island, NY, USA; supplemented with 100µM 2-mercaptoethanol, 10% fetal bovine serum, and antibiotics) at 38.5°C in a humidified atmosphere (5% CO2, 5% O2, 90% N2). We evaluated the embryos morphologically for survival and hatched rate at 0, 24, 48, and 72h post-thawing. In control, the equilibration solution (ES) consisted of 7.5% (vol/vol) dimethyl sulfoxide (DMSO) and 7.5% (vol/vol) ethylene glycol, and the vitrification solution (VS) consisted of 16.5% (vol/vol) DMSO and 16.5% (vol/vol) ethylene glycol and 0.5M sucrose. In this study, CPLL was added to ES and VS at various concentrations instead of DMSO. The CPLL was added at 16.5, 11.0, 5.5, and 2.2% (wt/vol) to VS; respectively, these solutions were named P16.5, P11.0, P5.5, and P2.2. The ES was used 45% CPLL of VS each. Embryos underwent the above procedure concurrently, with testing replicated at least 3 times. We evaluated 88, 34, 38, 44, and 28 embryos with each solution (control, P16.5, P11.0, P5.5, and P2.2, respectively). Results were analysed statistically with a chi-square test and residual analysis, regarding P&lt;0.05 as significant. Survival rates were significantly greater in P11.0 at 24h post-thawing (55.7% v. 89.5%; P&lt;0.05) and in P11.0 and P5.5 at 48h post-thawing (47.7% v. 78.9% and 47.7% v. 79.5%, respectively; P&lt;0.05) relative to controls but showed no significant differences at 0h post-thawing. Hatched rates were significantly greater in P11.0 and P5.5 through 72h post-thawing relative to controls (44.7% v. 22.7% and 52.3% v. 22.7%, respectively; P&lt;0.05). The CPLL improved post-thawing embryo survival and hatched rates when applied during vitrification, thus demonstrating cryoprotective effectiveness. We conclude that CPLL acts as a low-toxicity cryoprotectant for vitrified bovine embryos, and our results are consistent with previous reports of protective CPLL effects for cells and cell membranes.

98 EXPERIMENTAL TRANSFER OF BOVINE IVF-DERIVED 32-CELL STAGE EMBRYOS INTO THE UTERUS: ENVIRONMENTAL EFFECTS ON DEVELOPMENTAL CHARACTERISTICS

2016 ◽  
Vol 28 (2) ◽  
pp. 179
Author(s):  
M. Hoelker ◽  
D. Salilew-Wondim ◽  
F. Rings ◽  
D. Tesfaye ◽  
K. Schellander
Keyword(s):  
Culture Media ◽  
Uterine Cavity ◽  
Blastocyst Stage ◽  
Considerable Proportion ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Developmental Rates ◽  
Uterine Environment

Usually, in vitro-produced bovine embryos are cultured in vitro in static culture systems for 7 to 9 days in media composed according the oviducal fluid although it is well accepted that around Day 4.5–5 the bovine embryo enters the uterine cavity, providing environmental conditions different from the oviduct. Therefore, one has to raise the question whether changing culture media properties after Day 5 of culture could have beneficial effects on early development of bovine embryos. To answer that question, we transferred bovine IVF derived 32-cell stage embryos into the uterine cavity of synchronized recipients. All embryos had been matured and fertilized under routine standard conditions and were cultured in synthetic oviducal fluid supplemented with essential and nonessential amino acids (SOFaa) supplemented with either 0.3% fatty acid free bovine serum albumin (BSAfaf/Uterus) or 10% serum (serum/uterus) at 38.5°C, 5% O2, and 5% CO2 in humidified air prior transfer into the uterine environment, allowing further development to the blastocyst stage within the physiological environment prior recollection at Day 7 by routine uterine flushing followed by comparison with statically in vitro-developed embryos cultured in media supplemented with serum (serum/serum group) or BSAfaf (BSAfaf/BSAfaf group). All in all, a total of 1031 in vitro-derived 32-cell stage embryos were transferred to 21 synchronized Simmental recipient heifers. Of these, a total of 680 embryos (66%) could be recollected at Day 7. Embryos of the serum/serum group reached a higher blastocyst rate compared with embryos of the BSAfaf/BSAfaf group (68% v. 41%; P < 0.05, ANOVA, Tukey test), whereas the developmental rate to the blastocyst stage did not differ after 9 days of in vitro culture, indicating higher developmental kinetics of bovine 32-cell stage embryos when culture media is supplemented with serum. Moreover, embryos of the serum/uterus group reached significantly lower developmental rates to the blastocyst stage until Day 7 compared with embryos of the serum/serum group (12.9% v. 68.4%). Likewise, embryos in the BSAfaf/uterus group reached significantly lower developmental rates to the blastocyst stage until Day 7 compared with embryos in the BSAfaf/BSAfaf group (16.0% v. 40.1%). When allowed to develop for additional 48h in vitro, developmental rates to the blastocyst stage at Day 9 were still higher in BSAfaf/BSAfaf treatment compared with the BSAfaf/uterus treatment (91.4% v. 74.4%) and the serum/serum treatment compared with the serum/uterus treatment (92.5% v. 56.0%). Taken together, the results of our study demonstrate that uterine transfer of bovine 32-cell stage embryos results in reduction of developmental kinetics as well as lower developmental rates compared with embryos statically cultured in vitro. That might indicate, that a considerable proportion of bovine 32-cell stage embryos might not be able to adapt to the uterine environment.

scholarly journals 118VITRIFICATION OF PRONUCLEAR-STAGE MOUSE EMBRYOS ON ALUMINUM FOIL FLOATING ON LIQUID NITROGEN

2004 ◽  
Vol 16 (2) ◽  
pp. 181 ◽  
Author(s):  
H. Sagirkaya ◽  
F. Ergin ◽  
H. Bagis ◽  
S. Arat
Keyword(s):  
Liquid Nitrogen ◽  
Culture Media ◽  
Aluminum Foil ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Mouse Embryos ◽  
Morphological Appearance ◽  
Development Rates ◽  
Developmental Rates ◽  
And Control

The cryopreservation of pronuclear-stage embryos has a special importance in transgenic technology, cloning, and human-assisted reproductive technology. The objective of this study was to investigate the efficiency of a vitrification method modified in our lab for pronuclear-stage embryos. In experiment I, groups of 10 pronuclear-stage mouse embryos were cultured in 20μL drops of three different culture media (G1.3/G2.3, CZB and M16) covered with mineral oil (Sigma M-8410, St. Louis, MO, USA). Twenty-four hours later, embryos cultured in G1.3 were transferred into G2.3 medium. In experiment II, 25–30 pronuclear-stage embryos were transferred into a 50-μL drop of equilibration medium containing 4% ethylene glycol (EG, Sigma E-9129) in TCM-199 (Sigma M-2520) supplemented with 10% FCS at 37°C for 12–15min; then they were rinsed three times in 30-μL drops of vitrification medium containing 35% EG, 5% polyvinylpyrrolidone (PVP, Sigma P-0930) and 0.4M trehalose (Sigma T-0167) in TCM-199 supplemented with 10% FCS at 37°C for 20–30s. Embryos rinsed in vitrification solution were aspirated into a micropipette as a 1–2-μL drop containing 25–30 embryos and dropped onto aluminum foil floating on liquid nitrogen (LN2). Vitrified droplets were stored in cryovials in LN2. Warming was performed by moving the vitrified droplets into 0.3M trehalose in TCM-199 supplemented with 10% FCS at 37°C. Embryos having normal morphological appearance under stereomicroscope examination were cultured in G1.3/G2.3 medium. Differences in the two experiments were analyzed by one-way ANOVA. In experiment I, development rates to the blastocyst stage were 26%, 10% and 4% for G1.3/G2.3, CZB and M16 media, respectively. The highest development rate in experiment I was obtained in G1.3/G2.3 culture media (P&lt;0.05). Therefore, G1.3/G2.3 media were used for culturing of vitrified-warmed and control embryos. In experiment II, the rate of embryos having normal morphology was 98.5%. There were no significant differences between the development rates of vitrified (13.1%) and control (18.7%) embryos to the blastocyst stage (P&gt;0.05). Although the vitrification method resulted in a high survival rate based on the morphological appearance, developmental rates of vitrified and control embryos were found to be lower than expected and reported previously by other researchers. We believe that the low developmental rates in this study were due to our culture conditions but not our vitrification method. Therefore, it could be concluded that this vitrification method is an efficient one for pronuclear-stage embryo cryopreservation and better development rates could be obtained by improving the culture conditions. This study was supported by a grant from TUBITAK, Turkey (VHAG-1908-102V048). F. Ergin is a young volunteer researcher.

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.

120 EMBRYO SURVIVAL FOLLOWING LIPID-BASED TRANSFECTION OF 1-CELL STAGE BOVINE EMBRYOS WITH SMALL INTERFERING RNA (siRNA) FRAGMENTS AND/OR DNA

2006 ◽  
Vol 18 (2) ◽  
pp. 168 ◽  
Author(s):  
M. Bertolini ◽  
L. R. Bertolini ◽  
S. G. Petkov ◽  
K. R. Madden ◽  
J. D. Murray ◽  
...  
Keyword(s):  
Embryo Development ◽  
Small Interfering Rna ◽  
Blastocyst Stage ◽  
Qualitative Assessment ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Embryo Survival ◽  
Interfering Rna ◽  
Higher Doses

The RNA interference (RNAi) technology is a powerful tool for studies in functional genomics. The aim of this study was to evaluate the effects of a cationic lipid-based small interfering RNA (siRNA) and/or DNA delivery to 1-cell-stage bovine embryos on survival to the blastocyst stage. In vitro-produced (IVP) embryos were generated according to Bertolini et al. 2002 (Theriogenology 58, 973), and cloned embryos were produced by the handmade cloning technique (Vajta et al. 2003 Biol. Reprod. 68, 571) using green fluorescent protein (GFP)-expressing fibroblast cells as nuclear donors. Lipofections were performed on zona-free 1-cell-stage IVP embryos at 24–28 h post-fertilization by exposure to 1% (v/v) Lipofectamine 2000 (Invitrogen Co., CA, USA), 0.002% (w/v) GFP plasmid (pEFGP-N1, Clontech Laboratories, CA, USA) and/or various doses of siRNA GFP-specific siRNA oligonucleotide (Invitrogen) or DNA methyltransferase 1 (Dnmt1)-specific siRNA fragments for 60 min at 39°C, according to 5 treatment groups: (1) zona-intact IVP embryos (controls), (2) zona-free control embryos (controls for embryo development after zona removal), (3) embryos treated with GFP + GFP-siRNA at 0, 50, 100, 200, 400, or 800 nm, (4) embryos treated with Dnmt1-siRNA at 0, 50, 100, 250, or 500 nm, and (5) cloned embryos (positive controls for GFP expression). After treatment, embryos were in vitro-cultured in a WOW culture system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256) for 7 days. Cleavage and developmental rates to at least 8-cell and to blastocyst stages were assessed at 48, 96, and 168 h post-fertilization (hpf), respectively. Data were analyzed by the chi-square test. Cleavage rates in embryos treated with higher doses of siRNA were lower than in all other groups (Table 1). Embryo survival to at least 8-cell stage at 48 h, based on cleavage, was similar among all treatments (data not shown), but survival to blastocyst stage was affected by higher doses of GFP- or Dnmt1-siRNA (Table 1). After a qualitative assessment by fluorescence microscopy at 168 hpf, 40 to 63% of GFP-transfected blastocysts showed various levels of fluorescence, irrespective of the siRNA treatments. Fragments of siRNA are known to be short-lived in cultured cells, although we are still uncertain of their behavior and effects in early bovine embryos. We are currently analyzing the effectiveness of the siRNA transfection in the early IVP and clone embryo. In conclusion, liposome transfection of 1-cell-stage embryos did not affect survival and development to the blastocyst stage. However, survival followed an siRNA dose-response effect, with doses higher than 400 nm appearing to be detrimental to embryo development, with a developmental arrest at or close to the embryonic genome activation period. Table 1. Developmental rate of bovine embryos following lipid-based transfection at the 1-cell-stage

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