28 Short equilibration improves vitrification of invitro-produced bovine embryos using VitTrans device

2020 ◽  
Vol 32 (2) ◽  
pp. 140
Author(s):  
I. Martínez-Rodero ◽  
T. García-Martínez ◽  
M. López-Béjar ◽  
T. Mogas
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Field Conditions ◽  
Bovine Embryos ◽  
Fluid Medium ◽  
Apoptosis Index ◽  
Vitrification Solution

For the successful application of vitrification technology to field conditions, the procedures for the warming and transfer of the cryopreserved bovine embryos should be as simple as possible. The device VitTrans, designed by our group, enables warming/dilution of embryos and their transfer directly to recipient females in field conditions (Morato and Mogas 2014 Cryobiology 68, 288). VitTrans vitrification protocol consists of an incubation in equilibration solution during 12min followed by an exposure of 40s to vitrification solution. However, there are other reports using similar vitrification devices where equilibration length is shorter than ours. This study aimed to improve VitTrans methodology by comparing two vitrification protocols: short equilibration (SE) and long equilibration (LE). A total of 63 invitro-produced Day 7 blastocysts (IETS stage code 7) were randomly placed in an equilibration solution with 7.5% ethylene glycol + 7.5% dimethyl sulfoxide in holding medium (tissue culture medium-199 HEPES + 20% fetal calf serum) for either 3min (SE) or 12min (LE). Then, blastocysts were transferred to vitrification solution (15% ethylene glycol + 15% dimethyl sulfoxide + 0.5M sucrose in holding medium) for 40s, loaded onto the VitTrans device, plunged into liquid nitrogen, and covered with a 0.5mL straw. Fresh nonvitrified blastocysts (n=30) were set as control. For warming, the VitTrans was quickly submerged into a water bath at 45°C, while a syringe containing 0.3mL of diluting solution (0.5M sucrose in holding medium) at 45°C was injected through the hollow of the device. Blastocysts were then transferred to synthetic oviductal fluid medium and cultured for 24h at 38.5% in a 5% CO2 and 5% O2 environment in a humidified atmosphere. Re-expansion rates were recorded 3 and 24h after warming. Blastocysts were fixed and stained with SOX2 (Invitrogen) for inner cell mass (ICM) count, TUNEL (Roche) for apoptosis index assessment, and DAPI (Vector Laboratories) for total cell count (TCC). Images were captured using a Leica TCS SP5 confocal microscope (Leica Microsystems) and examined with Imaris 9.2 software (Oxford Instruments). Blastocyst survival rates were compared between groups using chi-squared test. Blastocyst TCC, ICM count, and apoptosis indices were analysed using analysis of variance. Significance was set at P ≤ 0.05. No differences were observed in re-expansion rate at 3h postwarming (61.3 and 59.4% for SE and LE, respectively). However, significantly higher re-expansion rates were found after 24h of culture for the blastocysts of the SE group (74.2%) when compared with the blastocysts of the LE group (65.7%). Blastocysts vitrified using the LE protocol produced the lowest TCC (115±5.9; P ≤ 0.05), whereas TCC of the SE (152±9.7) and fresh control (138±8.6) treatments were similar. No differences were found in ICM count among groups. Nevertheless, apoptosis index was higher (P ≤ 0.05) in both vitrification groups when compared with fresh control. These results indicate that short equilibration vitrification not only improves VitTrans outcomes but adds efficiency by taking less time to perform. Supported by MCIU, Spain (Project AGL2016-79802-P and Grant BES-2017-081962).

44 THE EFFECT OF SUCROSE CONCENTRATION FOR SINGLE-STEP DILUTION ON THE VIABILITY OF CRYOTOP-VITRIFIED IN VITRO-PRODUCED BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 115
Author(s):  
S. Kondo ◽  
K. Imai ◽  
O. Dochi
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Liquid Nitrogen ◽  
Sucrose Concentration ◽  
Calf Serum ◽  
Single Step ◽  
Bovine Embryos ◽  
Vitrification Solution ◽  
Phosphate Buffered Saline

The aim of this study was to test sucrose concentrations for single-step dilution on the viability of vitrified in vitro-produced bovine embryos. Blastocysts (n = 173, 7 to 8 days after fertilization) were vitrified using the Cryotop (Kitazato, Tokyo, Japan) method placement by incubating the blastocysts in Dulbecco's phosphate buffered saline supplemented with 20% calf serum, 7.5% ethylene glycol, and 7.5% dimethyl sulfoxide for 3 min and then transferring into vitrification solution (Dulbecco's phosphate buffered saline supplemented with 20% calf serum, 16.5% ethylene glycol, 16.5% dimethyl sulfoxide, and 0.5 M sucrose). Each embryo was placed on a Cryotop with minimum volume of vitrification solution, and then the Cryotop was plunged into liquid nitrogen. Total time from placement in vitrification solution to plunging into liquid nitrogen was 1 min. The blastocysts were warmed by incubation in the single-step dilution medium for 5 min [0 M sucrose (n = 42), 0.25 M sucrose (n = 44), 0.5 M sucrose (n = 43), and 1.0 M sucrose (n = 44)] at 38.0°C. After dilution, the embryos were washed in TCM-199 supplemented with 20% calf serum and 0.1 mM β-mercaptoethanol and were cultured for 72 h in the same medium at 38.5°C in an atmosphere of 5% CO2. The rates of re-expanded blastocysts and hatched blastocysts were determined at 24 and 72 h after warming, respectively. Data were analysed using the chi-squared test. The percent of re-expanded blastocysts at 24 h after warming in dilution medium supplemented with any level of sucrose was significantly higher (P < 0.05) than in blastocysts warmed without sucrose (Table 1). The hatched blastocyst rate of embryos at 72 h after warming in dilution medium with 0.5 M sucrose was significant higher than that with no sucrose. There were no differences in hatched blastocyst rates between the sucrose concentrations supplemented to the dilution medium. These results suggest that embryos vitrified by the Cryotop method can be diluted in single-step dilution using 0.25, 0.5, or 1.0 M sucrose supplemented to the medium. Table 1.The effect of sucrose concentration for single-step dilution on the viability of Cryotop vitrified in vitro-produced bovine embryos

54 Effect of Different Cryopreservation Protocols for Sheep Embryonic Stem Cells

2018 ◽  
Vol 30 (1) ◽  
pp. 166
Author(s):  
N. Ibraimova ◽  
A. Seisenbayeva ◽  
Y. Toishibekov
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Fetal Calf Serum ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Embryonic Stem ◽  
Slow Freezing

Particular attention is required to improve cryopreservation of embryonic stem cells (ESC) and study their characteristics. Stem cells were obtained from the inner cell mass of Day 5-6 blastocysts. The ESC were then cultured on mTeSR™1 medium (Stemcell Technologies, Cambridge, MA, USA). We studied the survival of ESC after slow freezing and vitrification. Slow freezing was carried out using a Planer Kryo 360-3.3 freezer (Planer plc, Sunbury-on-Thames, United Kingdom), using various cryoprotectants: 1.5 M dimethyl sulfoxide (Me2SO), 1.5 M ethylene glycol (EG), or 1.5 M propylene glycol (PG). Six vitrification solutions (VS) were used to vitrify ESC: VS1 = 20% Me2SO + 20% EG + 0.5 M sucrose; VS2 = 20% Me2SO + 20% PROH + 0.5 M sucrose; VS3 = 20% EG + 20% PG + 0.5 M sucrose; VS4 = 20% Me2SO + 20% EG + 0.5 M sucrose + 10% FCS; VS5 = 20% Me2SO + 20% PROH + 0.5 M sucrose + 10% FCS; and VS6 = 20% EG + 20% PG + 0.5 M sucrose + 10% FCS. For the dehydration of cells and the addition of vitrification solutions, a 3-step equilibration was used. The proliferative properties of the cells were determined using an Apel PD-303S spectrophotometer (Apel Co. Ltd., Kawaguchi, Japan), using an MTT test (staining with methylthiazolyl-diphenyl tetrazolium). After slow freezing, the highest percentage of frozen–thawed cells proliferating was observed when using 1.5 M EG (P > 0.05). At the same time, the highest cell doubling after thawing was observed when using 1.5 M EG, and 1.5 M Me2SO. After vitrification, the highest percentage of proliferation was observed in the VS2 and VS4 groups (49.7 ± 3.2% and 53.2 ± 3.8%, respectively). It should be noted that the addition of fetal calf serum to the vitrification solution also increased the proliferation of ESC after vitrification and thawing.

Development of bovine embryos cultured in CR1aa and IVD101 media using different oxygen tensions and culture systems

2010 ◽  
Vol 58 (4) ◽  
pp. 465-474 ◽  
Author(s):  
Tamás Somfai ◽  
Yasushi Inaba ◽  
Yoshio Aikawa ◽  
Masaki Ohtake ◽  
Shuji Kobayashi ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Culture Media ◽  
Cell Mass ◽  
Calf Serum ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Cell Numbers ◽  
Culture Systems ◽  
Significant Difference

The aim of the present study was to optimise the culture conditions for the in vitro production of bovine embryos. The development of in vitro fertilised bovine oocytes in CR1aa supplemented with 5% calf serum and IVD101 culture media were compared using traditional microdrops and Well of the Well (WOW) culture systems either under 5% or 20% oxygen tension. After 7 days of culture, a significantly higher blastocyst formation rate was obtained for embryos cultured in CR1aa medium compared to those cultured in IVD101, irrespective of O 2 tensions and culture systems. The blastocyst formation in IVD101 was suppressed under 20% O 2 compared to 5% O 2 . Despite their similar total cell numbers, higher rates of inner cell mass (ICM) cells were observed in blastocysts developed in IVD101 medium than in those developed in CR1aa, irrespective of O 2 tensions. There was no significant difference in blastocyst formation, total, ICM and trophectoderm (TE) cell numbers between embryos obtained by microdrop and WOW culture systems irrespective of the culture media and O 2 tensions used. In conclusion, CR1aa resulted in higher blastocyst formation rates irrespective of O 2 tension, whereas IVD101 supported blastocyst formation only under low O 2 levels but enhanced the proliferation of ICM cells.

scholarly journals Stereomicroscopic and histological examination of bovine embryos following extended in vitro culture

2005 ◽  
Vol 17 (8) ◽  
pp. 799 ◽  
Author(s):  
Natalie I. Alexopoulos ◽  
Gábor Vajta ◽  
Poul Maddox-Hyttel ◽  
Andrew J. French ◽  
Alan O. Trounson
Keyword(s):  
In Vitro Culture ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Calf Serum ◽  
Collagen Gel ◽  
Embryonic Cells ◽  
Bovine Embryos ◽  
Term Survival

Attempts to support survival of mammalian embryos after hatching have met with limited success, although some mouse studies have reported growth at the post-implantation stage. The aim of the present research was to establish and characterise an in vitro culture system that could support extended growth and differentiation of bovine embryos. Abattoir-derived oocytes were matured and fertilised in vitro. Presumptive zygotes were cultured in modified synthetic oviduct fluid (SOFaaci) medium supplemented with 5% cow serum (CS). On Day 9, single hatched blastocysts (n = 160) were randomly allocated to SOFaaci supplemented with either 5% bovine serum albumin, 5% CS, 5% fetal calf serum (FCS) or SOF only and cultured on a collagen gel substrate for up to 45 days. Embryos were evaluated at various time-points until complete disaggregation or the total disappearance of embryonic cells. Blastocyst viability post hatching was severely compromised in protein-free SOFaaci medium. Addition of FCS generated increased embryonic growth for the longest time period (Day 45) when compared to the other groups. Long-term survival of embryonic cells was observed stereomicroscopically by the proliferation and development of three-dimensional tubular structures to 85% confluence in culture. Haematoxylin and eosin staining of morphological structures obtained from all treatment groups revealed embryos displaying trophoblast, inner cell mass and hypoblast development to varying degrees. Regardless of treatment, extended in vitro culture did not result in development comparable with that described for in vivo embryos. In the present work, however, there was evidence of extended culture of bovine embryos beyond that achieved previously. However, further research is required to identify the exact requirements for extended in vitro culture for bovine embryos.

140 PRODUCTION OF RECONSTRUCTED IN VITRO PRODUCED BOVINE EMBRYOS BY INNER CELL MASS AND TROPHECTODERM AGGREGATION IN VITRO

2011 ◽  
Vol 23 (1) ◽  
pp. 174
Author(s):  
I. P. Emanuelli ◽  
E. Razza ◽  
C. M. Barros ◽  
M. F. G. Nogueira
Keyword(s):  
In Vitro Culture ◽  
Fetal Calf Serum ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Bos Indicus ◽  
Calf Serum ◽  
Cell Aggregation ◽  
Bovine Embryos ◽  
Advanced Stages

The efficiency of embryonic chimerism tends to decrease when embryos in advanced stages of development, such as morulae and blastocysts, are used. To perform the inner cell mass (ICM) transfer to a trophectoderm (TE) receptor, it is essential to use embryos at an advanced stage and blastocoel presence. This method of embryo reconstruction has been performed only by the micromanipulator microinjection method (Zheng et al. 2005 Zygote 1, 73–71; Loi et al. 2007 Trends Biotechnol. 25, 195–200; Roth et al. 1989 Biol. Reprod. 41, 675–682; and Murakami et al. 2006 Cloning Stem Cells 8, 51–60). This study aimed to validate a manual procedure to reconstruct embryos using the method of ICM and TE approximation in the presence of phytohemagglutinin. Bos indicus ovaries from the abattoir were used to obtain 230 cumulus–oocyte complexes (COC; quality I and II). The COC were matured in 90-μL drops of TCM-199 bicarbonate supplemented with 10% fetal calf serum (FCS) and incubated in vitro for 22 to 24 h. Fertilization occurred in TALP-IVF medium, and the COC were incubated for 18 h. Presumptive zygotes were transferred to SOF medium to in vitro culture. Incubation conditions were 38.5°C and 5% CO2 in air. In vitro produced (IVP) embryos 8.5 days after fertilization were used for the experiment. Ninety-three hatching or hatched blastocysts were put into 3-μL microdrops of protein-free HEPES-buffered SOF (HSOF) medium to hold the embryos on the dish bottom and to allow handmade sections of ICM and TE. The section was performed with a microblade (Ultra-Sharp Splitting Blade, Bioniche, Bogart, GA, USA) under a stereomicroscope (35× magnification). Seventy half-structures from 35 different blastocysts were obtained to form pairs (ICM+TE). Each pair was transferred to drops with 500 μg mL–1 of phytohemagglutinin-L (3 min) before the approximated pairs were transferred to SOF medium in cell aggregation well-of-the-well (WOW) micro-wells (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264) to in vitro culture (38.5°C; 5% O2 and 5% CO2). The aggregation rate was 25.7% (9/35) and all the reconstructed blastocysts by aggregation (24 h) re-expanded after 48 h of culture. The technique of handmade ICM and TE section and posterior aggregation in the presence of an agglutinating agent was feasible for the structural and functional reconstruction and re-expansion of the blastocyst produced. Financial support: FAPESP, Brazil (06/06491-2, 07/07705-9, 09/10679-5, and 09/04888-0).

96 EFFICIENT PRODUCTION OF MONOZYGOTIC TWIN BOVINE EMBRYOS USING BLASTOMERE SEPARATION TECHNIQUE WITH COMMERCIAL WELL OF THE WELL CULTURE DISH

2016 ◽  
Vol 28 (2) ◽  
pp. 178
Author(s):  
Y. Hashiyada ◽  
Y. Aikawa ◽  
S. Sugimura ◽  
H. Matsuda ◽  
M. Ohtake ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Statistical Significance ◽  
Cell Mass ◽  
Calf Serum ◽  
Monozygotic Twin ◽  
Efficient Production ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Culture Dish ◽  
Cell Stage

Monozygotic twin embryos can be produced using the technique of blastomere separation and well of the well (WOW) dish having handmade micro-wells by the needle depression (Tagawa et al. 2008). We have recently reported that developed commercial WOW dish enhances embryo competence in individual culture (Sugimura et al. 2010). The present study was conducted to evaluate the availability of commercial WOW dish for production of monozygotic twin embryos in bovine. Embryos were produced using oocytes from ovaries collected at an abattoir by IVM, IVF, and IVC. For each culture, TCM-199 supplemented with 5% calf serum (CS), Brackett-Oliphant solution supplemented with 10 mg mL–1 BSA, and CR1aa containing 5% CS were used. To evaluate the adaptability of dishes on culture of isolated blastomeres from different cell stage, 2- (n = 63), 4- (n = 94), 8- (n = 137), and 10- to 14- (n = 116) cell stages were obtained on 24–27 h, 30–36 h, 48–54 h, and 48–54 h from the beginning of fertilization, respectively. The zona pellucida was removed by exposure of 0.25% pronase, followed by gentle pipetting by inspiration and expiration in the IVC medium. Then, two halves separated from the original number of blastomeres were randomly allocated to the conical micro-wells of commercial dish (Dai Nippon Printing, Tokyo, Japan) or created micro-wells by pressing the bottom of the dish with an eyeleteer (control). The approximate diameter and depth of each 25 wells in a commercial dish was 287 and 168 μm, and each 20 wells in the control were 800 and 600 μm. The blastomeres were cultured in wells covered with a droplet of 2.5 μL well–1 IVC medium until Day 8 (IVF = Day 0). Expanded blastocysts (n = 28) derived from tetra-blastomeres of 8-cell stage were stained to determine the number of the inner cell mass (ICM) and trophectoderm (TE) in each group. Statistical significance of the blastocyst formation rates and the number of cells were analysed by the chi-square test and the Student’s t-test, respectively. In the 2-cell stage, blastocyst formation rate in commercial dish tended to be higher than that in the control (60.0% v. 46.1%). The rate of monozygotic blastocyst pairs in commercial dish was higher compared with the control (48.0% v. 26.3%, P < 0.05). In the 4-cell stage, rates of blastocyst formation (50.0% v. 33.0%, P < 0.05) and the pairs (39.5% v. 12.5%, P < 0.01) in the commercial dish, both were higher compared with the control. In the 8-cell stage, there were no differences between two groups in rates of blastocyst formation (53.1% v. 59.0%) and the pairs (41.8% v. 48.7%), similarly in the 10- to 14-cell stage (47.9% v. 56.8% and 36.2% v. 40.9%, respectively). The ICM, TE, and total cell numbers were not different between the commercial and the control dish (28.0 ± 3.2 v. 26.0 ± 3.8, 64.6 ± 4.3 v. 76.0 ± 7.9, and 92.6 ± 6.2 v. 102.0 ± 11.0, respectively). These results indicate that separated blastomeres could be developed to blastocysts efficiently and stably regardless of embryo cell stage with a commercial WOW culture dish.

scholarly journals Base editing in bovine embryos reveals a species-specific role of SOX2 in regulation of pluripotency

2021 ◽  
Author(s):  
Lei Luo ◽  
Yan Shi ◽  
Huanan Wang ◽  
Zizengchen Wang ◽  
Yanna Dang ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Rna Seq ◽  
Bovine Embryos ◽  
Base Editing ◽  
Sox2 Expression ◽  
Nanog Expression ◽  
Species Specific ◽  
Lineage Specific Genes

The emergence of the first three lineages during development are orchestrated by a network of transcription factors, which are best characterized in mice. However, the role and regulation of these factors are not completely conserved in other mammals, including human and cattle. Here, we establish a gene inactivation system by introducing premature codon with cytosine base editor in bovine embryos with a robust efficiency. Of interest, SOX2 is universally localized in early blastocysts but gradually restricted into the inner cell mass in cattle. SOX2 knockout results in a failure of the establishment of pluripotency. Indeed, OCT4 level is significantly reduced and NANOG was barely detectable. Furthermore, the formation of primitive endoderm is compromised with few SOX17 positive cells. Single embryo RNA-seq reveals a dysregulation of 2074 genes, among which 90% are up-regulated in SOX2-null blastocysts. Intriguingly, more than a dozen lineage-specific genes, including OCT4 and NANOG, are down-regulated. Moreover, SOX2 expression is sustained in the trophectoderm in absence of CDX2 in bovine late blastocysts. Overall, we propose that SOX2 is dispensable for OCT4 and NANOG expression and disappearance of SOX2 in the trophectoderm depends on CDX2 in cattle, which are all in sharp contrast with results in mice.

scholarly journals 100EFFECTS OF USING MICRO-PIPETTE TIPS OF DIFFERENT DIAMETERS AND VOLUME OF VITRIFICATION SOLUTION ON THE VIABILITY OF IVM BOVINE OOCYTES AFTER VITRIFICATION

2004 ◽  
Vol 16 (2) ◽  
pp. 171
Author(s):  
Y. Inaba ◽  
O. Dochi ◽  
H. Koyama
Keyword(s):  
Ethylene Glycol ◽  
Calf Serum ◽  
Cleavage Rate ◽  
Chi Square ◽  
Pipette Tip ◽  
Blastocyst Rate ◽  
Vitrification Solution ◽  
Different Diameters ◽  
And Control ◽  
Bovine Oocytes

The objective of this study was to investigate the effects of the diameters of micro-pipette tips and the volume of vitrification solution (VS) on viability of IVM bovine oocytes after vitrification. COCs were aspirated from 2–5mm follicles of ovaries obtained at a local abattoir. COCs were matured for 19h in TCM-199 supplemented with 5% calf serum (CS) and 0.02mgmL−1 FSH at 38.5°C in an atmosphere of 5% CO2 in air. The matured oocytes were then vitrified on the basis of Kuwayama and Kato (2000 J. Assist. Reprod. Genet. 17, 477 abst). Matured oocytes were first exposed to 7.5% ethylene glycol (EG) and 7.5% DMSO in holding medium (HM; Dulbecco’s PBS supplemented with 20% CS) for 3min, and then equilibrated for 1min in 15% EG, 15% DMSO, and 0.5M sucrose in HM. Ten oocytes were loaded into each micro-pipette tip (MidAtlantic Diagnostics, Inc., Marlton, NJ, USA), and directly plunged into liquid nitrogen. Warming was performed by placing the narrow end of the micro-pipette tips directly into HM containing 0.5M sucrose; the tips maintained in this medium for 5min. After washing in HM, oocytes underwent an additional 3h of maturation. They were then subjected to IVF (Day 0). After IVF, morphologically intact oocytes were cultured. Oocytes matured for 20–21h were used as a control. The cleavage rate at Day 3 and blastocyst rate at Day 7 to 9 were based on the number of cultured oocytes, and analyzed using the chi-square method. In experiment 1, the oocytes were vitrified with 0.5μL of VS in micro-pipette tips with 150-, 200-, or 275-μm inner diameters (ID) (100 eggs per tip size). The number of morphologically intact oocytes was 64 (150μm), 62 (200μm), and 54 (275μm). The cleavage rates of morphologically intact oocytes at Day 3 of 150μm (45.3%) and 200-μm tips (45.2%) were significantly lower than that of 275-μm tips (53.7%) and the control (63.6%) (P&lt;0.05). The blastocyst rate of morphologically intact oocytes at Day 7 to 9 of 150-μm (9.4%) and 275-μm tips (14.8%) were significantly lower than that of the control (33.0%) (P&lt;0.05), and that of 200-μm tips (19.4%) also showed a tendency of being lower than that of the control (P&lt;0.1). In experiment 2, the oocytes were vitrified with 0.3 (70 eggs), 0.5 (60 eggs), or 1μL (60 eggs) of VS in micro-pipette tips with 200-μm ID. The number of morphologically intact oocytes was 40 (0.3μL), 32 (0.5μL), and 28 (1μL). The cleavage rates of morphologically intact oocytes at Day 3 of the 0.3μL (45.0%), 0.5μL (37.5%), and 1μL solutions (35.7%) were significantly lower than that of the control (67.6%) (P&lt;0.05). However, there were no differences in the blastocyst rate of morphologically intact oocytes at Day 7 to 9 among 0.3μL (15.6%), 0.5μL (28.1%), and 1μL solutions (17.9%), and control (23.9%). These results suggest that the viability of IVM bovine oocytes after vitrification may be improved by using micro-pipette tips with 200-μm ID and containing 0.5μL of VS.

scholarly journals A shorter equilibration period in the VitTrans in-straw bovine embryo vitrification method improves post-warming outcomes

2020 ◽  
Author(s):  
Iris Martínez-Rodero ◽  
Tania García-Martínez ◽  
Erika Alina Ordóñez-León ◽  
Meritxell Vendrell-Flotats ◽  
Carlos Olegario-Hidalgo ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Survival Rates ◽  
Field Conditions ◽  
Bovine Embryo ◽  
Direct Transfer ◽  
Treatment Groups ◽  
Cell Counts ◽  
Hatching Rates

Abstract Background VitTrans is a device that enables the vitrification and warming/dilution of in vitro produced bovine embryos followed by their direct transfer to recipient females in field conditions. This study sought to improve the VitTrans method by comparing two equilibration times: short (SE: 3 min) and long (LE: 12 min). Outcome measures recorded in vitrified D7 and D8 expanded blastocysts were survival and hatching rates, differential cell counts, apoptosis rate and gene expression. Results While survival rates at 3 h and 24 h post-warming were reduced (P < 0.05) after vitrification, hatching rates of D7 embryos vitrified after SE were similar to those obtained in fresh non-vitrified blastocysts. Hatching rates of vitrified D8 blastocysts were lower (P < 0.05) than of fresh controls, regardless of treatment. Total cell counts, and inner cell mass and trophectoderm cell numbers were similar in hatched blastocysts derived from D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values, regardless of treatment. The rate of apoptotic cells was significantly higher in both treatment groups when compared to fresh controls, although apoptosis rates were lower using the SE than LE protocol. No differences emerged in expression of the genes BAX, AQP3, CX43 and IFNτ between blastocysts vitrified after SE or LE, whereas a significantly higher abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE compared to LE. Conclusions The VitTrans device combined with a shorter exposure to the equilibration medium improves vitrification/warming outcomes facilitating the direct transfer of vitrified embryos under field conditions.

scholarly journals Three-Dimensional Live Imaging of Bovine Preimplantation Embryos: A New Method for IVF Embryo Evaluation

2021 ◽  
Vol 8 ◽  
Author(s):  
Yasumitsu Masuda ◽  
Ryo Hasebe ◽  
Yasushi Kuromi ◽  
Masayoshi Kobayashi ◽  
Kanako Urataki ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Three Dimensional ◽  
Preimplantation Embryos ◽  
Infrared Light ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Developmental Potential

Conception rates for transferred bovine embryos are lower than those for artificial insemination. Embryo transfer (ET) is widely used in cattle but many of the transferred embryos fail to develop, thus, a more effective method for selecting bovine embryos suitable for ET is required. To evaluate the developmental potential of bovine preimplantation embryos (2-cell stage embryos and blastocysts), we have used the non-invasive method of optical coherence tomography (OCT) to obtain live images. The images were used to evaluate 22 parameters of blastocysts, such as the volume of the inner cell mass and the thicknesses of the trophectoderm (TE). Bovine embryos were obtained by in vitro fertilization (IVF) of the cumulus-oocyte complexes aspirated by ovum pick-up from Japanese Black cattle. The quality of the blastocysts was examined under an inverted microscope and all were confirmed to be Code1 according to the International Embryo Transfer Society standards for embryo evaluation. The OCT images of embryos were taken at the 2-cell and blastocyst stages prior to the transfer. In OCT, the embryos were irradiated with near-infrared light for a few minutes to capture three-dimensional images. Nuclei of the 2-cell stage embryos were clearly observed by OCT, and polynuclear cells at the 2-cell stage were also clearly found. With OCT, we were able to observe embryos at the blastocyst stage and evaluate their parameters. The conception rate following OCT (15/30; 50%) is typical for ETs and no newborn calves showed neonatal overgrowth or died, indicating that the OCT did not adversely affect the ET. A principal components analysis was unable to identify the parameters associated with successful pregnancy, while by using hierarchical clustering analysis, TE volume has been suggested to be one of the parameters for the evaluation of bovine embryo. The present results show that OCT imaging can be used to investigate time-dependent changes of IVF embryos. With further improvements, it should be useful for selecting high-quality embryos for transfer.

Sign in / Sign up

Export Citation Format

Share Document