scholarly journals 113COMPARISON OF TWO ETHYLENE GLYCOL EQUILIBRATION TREATMENTS FOR THE QUICK FREEZING OF IN VITRO-PRODUCED BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 178
Author(s):  
A.C. Nicácio ◽  
R. Simões ◽  
C. Yamada ◽  
H.V.A. Caetano ◽  
M.R.B. Mello ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Liquid Nitrogen ◽  
Granulosa Cell ◽  
Cell Monolayer ◽  
Slow Freezing ◽  
Bovine Embryos ◽  
Treatment Groups ◽  
Quick Freezing ◽  
Hatching Rates

The aim of this study was to compare two ethylene glycol (EG) equilibration procedures for the quick freezing of in vitro-produced bovine embryos. Cumulus-oocyte complexes (COCs) were collected from slaughterhouse ovaries. COCs were matured in TCM199 containing 10% of bovine fetal serum, LH, FSH and E2, and fertilized. Presumptive zygotes were co-cultured in TCM199 with a granulosa cell monolayer, at 39°C in humidified atmosphere of 5% CO2 in air. Grade 1, expanded blastocysts (n=761) were selected 7 and 9 days after insemination and randomly distributed to one of eight treatment groups. In Equilibration Procedure 1, embryos were exposed to 10% EG for 5 min, and then to 17%, 22% or 28% EG for 60s (respectively referred to as EG 17, EG 22 and EG 28). In Equilibration Procedure 2, embryos were exposed to the same EG solutions as in Equilibration Procedure 1, but the period of exposure was 10min to 10% EG and 30 s to EG 17, EG 22 and EG 28. In Equilibration Procedure 3 (slow-freezing controls), embryos were exposed to 10% EG for either 5 or 10min and then cryopreserved by slow-freezing method at 1.2°C/min. In all treatment groups, EG solutions were prepared in PBS+0.2% BSA, and embryos were exposed to EG solutions at 22°C. Embryos were loaded into 0.25mL straws and heat-sealed. Straws were cooled in liquid nitrogen vapor for 2min, and then plunged into and stored in liquid nitrogen. Straws were thawed in room temperature air for 10s, and then in 25°C water for 20s. Thawed embryos were diluted by transferring them into 0.5ml of PBS+0.2% BSA+0.3M sucrose for 3min, and then 0.5mL of PBS+0.2% BSA for 3min. Embryos were co-cultured on granulosa cell monolayer in TCM199 and evaluated after 24h for blastocyst re-expansion (EXP), and again at 48, 72 and 96h for hatching (HAT). A total of 724 in vitro-produced bovine blastocysts were used as controls to determine hatching rates. The results are presented in the table. Embryos exposed to 10% EG for 10min (Equilibration Procedure 1) yielded significantly higher rates of blastocyst re-expansion and hatching when compared to embryos exposed for 5min (Equilibration Procedure 2, P<0.05). These results suggest that quick freezing of in vitro-derived bovine embryos may be an alternative to vitrification; however, additional studies are needed to optimize cryopreservation protocols and increase post-thaw survival. This project was supported by FAPESP (01/11266-4) Table 1 Effect of equilibration procedure on in vitro re-expansion and hatching rates of embryos cryopreserved by slow and quick freezing methods

scholarly journals 122COMPARISON OF TWO CRYOPROTECTANT DILUTION TREATMENTS FOR QUICK FROZEN IN VITRO-PRODUCED BOVINE EMBRYOS

2004 ◽  
Vol 16 (2) ◽  
pp. 183
Author(s):  
J.A. Visintin ◽  
A.C. Nicácio ◽  
C. Yamada ◽  
H.V.C. Amaral ◽  
R. Simões ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Granulosa Cell ◽  
Sucrose Solution ◽  
Cell Monolayer ◽  
Bovine Embryos ◽  
Treatment Groups ◽  
Nitrogen Vapor ◽  
Quick Freezing ◽  
Hatching Rates

The aim of this study was to compare the viability of in vitro-produced bovine embryos following quick freezing in ethylene glycol (EG) and subsequent dilution of EG by either a two- or a three-step procedure. Cumulus-oocyte complexes (COCs) were collected from slaughterhouse ovaries. COCs were matured in TCM199 containing 10% bovine fetal serum, LH, FSH and E2, and fertilized. Presumptive zygotes were co-cultured in TCM199 with a granulosa cell monolayer, at 39°C in humidified atmosphere of 5% CO2 in air. Grade 1, expanded blastocysts (n=544) were selected 7 and 9 days after insemination and randomly distributed to one of three EG equilibration treatment groups. Embryos were exposed to 10% EG for 10min, and then to 17%, 22% or 28% EG for 30s (respectively referred to as EG 17, EG 22 and EG 28). In all treatment groups, EG solutions were prepared in PBS+0.2% BSA, and embryos were exposed to EG solutions at 22°C. Embryos were loaded into 0.25-mL straws which were then heat-sealed. Straws were cooled in liquid nitrogen vapor for 2min, and then plunged and stored in liquid nitrogen. Straws were thawed in room temperature air for 10s, and then in 25°C water for 20s. The thawed embryos of the EG 17, EG 22 and EG 28 groups were randomly assigned to one of two EG dilution procedures. Two-step dilution consisted of transfer of embryos into PBS+0.2% BSA+0.3M sucrose solution for 3min, and then PBS+0.2% BSA for 3min. Three-step dilution consisted of transfer of embryos into PBS+10% EG+0.2% BSA+0.3M sucrose for 3min, PBS+0.2% BSA+0.3M sucrose for 3min, and then PBS+0.2% BSA for 3min. Embryos were co-cultured on a granulosa cell monolayer in TCM199 and evaluated after 24h for blastocyst re-expansion (EXP), and again at 48, 72 and 96h for hatching (HAT). A total of 724 in vitro-produced bovine blastocysts were used as controls to determine hatching rates. The results are presented in the Table. No significant differences were found between the two- and three-step dilution procedures (P>0.05) for in vitro-produced bovine embryos cryopreserved by quick freezing. This project was supported by FAPESP (01/11266-4). Table 1 In vitro re-expansion and hatching rates (%) of rapidly frozen embryos after two- or three-step dilution

127 IN VITRO DEVELOPMENT OF IVF CRYOPRESERVED BOVINE EMBRYOS SUPPORTED BY TCM-199 OR SOFaa CULTURE MEDIUM

2007 ◽  
Vol 19 (1) ◽  
pp. 181
Author(s):  
A. C. Nicacio ◽  
W. B. Feitosa ◽  
M. Rovegno ◽  
R. Simões ◽  
J. S. de A. Gonçalves ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Embryo Development ◽  
Culture Medium ◽  
Cell Monolayer ◽  
Slow Freezing ◽  
Bovine Embryo ◽  
Quick Freezing ◽  
Slow Group ◽  
Hatching Rates

In vitro bovine embryo production is commercially applied around the world. However, the cryopreservation of these embryos is not yet possible, which raises difficulties for the expansion of this biotechnology. The aim of this work was to evaluate the influence of the culture media on embryo development after cryopreservation. Cumulus–oocyte complexes obtained from slaughterhouse bovine ovaries were in vitro-matured, fertilized, and cultured. A total of 600 expanded blastocysts (between 7 and 9 days of culture) were cryopreserved by slow freezing, quick freezing, or vitrification methods. For slow freezing (slow group), the embryos were exposed to 10% ethylene glycol (EG) for 10 min and cryopreserved at 1.2�C per minute. For quick freezing (quick group), the embryos were exposed to 10% EG for 10 min and to 20% EG + 20% glycerol (Gly) for 30 s. For vitrification (vitrification group), the embryos were exposed to 10% EG for 10 min and to 25% EG + 25% Gly for 30 s. The straws (quick and vitrification groups) were placed in nitrogen vapor (0.8 cm over the liquid nitrogen) for 2 min and then immersed in liquid nitrogen. The embryos were thawed in air for 10 s and in a water bath at 25�C for 20 s. For warming, embryos were washed in PBS + 0.2% BSA + 0.3 M sucrose for 3 min and in PBS + 0.2% BSA for 3 min. To evaluate development after thawing, the embryos were cultured on a granulosa cell monolayer with TCM-199 or SOFaa for 4 days. The embryos of the slow group showed re-expansion rates of 55.55% (55/99) and 29.00% (29/100), respectively, for TCM-199 and SOFaa. The quick group showed re-expansion rates of 4.85% (5/103) and 7.22% (7/97), and the vitrification group 10.89% (11/101) and 14.00% (14/100), respectively, for TCM-199 and SOFaa. The slow group showed hatching rates of 47.47% (47/99) and 11.00% (11/100), respectively; the quick group did not show hatching rates in either medium. The vitrification group showed hatching rates of 7.92% (8/101) and 6.00% (6/100), respectively, for TCM-199 and SOFaa. The results were analyzed by chi-square test, and all values were significant at P < 0.05. The slow group showed difference in re-expansion and hatching rates when the different media were compared. The quick and vitrification groups did not show differences in re-expansion and hatching rates when the different media were compared. The slow group showed higher re-expansion and hatching rates than the quick and vitrification groups. In conclusion, the culture medium influences embryo development after slow freezing, and the TCM-199 is more appropriate than SOFaa. This work was supported by FAPESP 04/05335-1

106 EFFECT OF GLYCEROL AND ETHYLENE GLYCOL ON THE DEVELOPMENT OF IN VITRO BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 161
Author(s):  
A. C. Nicacio ◽  
R. Simões ◽  
M. A. Peres ◽  
J. S. A. Gonçalves ◽  
M. E. O. D'Ávila Assumpção ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Cell Monolayer ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Chi Square ◽  
Chi Square Test ◽  
Hatching Rates

The aim of this study was to evaluate the viability of in vitro-produced bovine embryos after exposure to different cryoprotectant solutions and cryopreservation. Bovine ovaries were collected at slaughterhouse and oocytes were matured, fertilized, and cultured in vitro. The embryos were co-cultured on a granulosa cell monolayer in SOF + 5% FCS and nonessential amino acids. In Experiment 1, expanded blastocysts were exposed to 10% ethylene glycol (EG) solution for 10 min (Group EG) or to 10% EG solution for 10 min and to 20% EG + 20% glycerol (Gly) solution for 30 s (Group EG/Gly). Cryoprotectants were diluted with PBS + 0.2% BSA + 0.3 M sucrose and PBS + 0.2% BSA solutions, both for 3 min, and the hatching rate was evaluated after culture. In Experiment 2, after exposure, EG Group was cryopreserved by slow freezing procedure (1.2�C/min) and EG/Gly Group was vitrified on nitrogen vapor for 2 min. After thawing, cryoprotectants were diluted using PBS + 0.2% BSA + 0.3 M sucrose and PBS + 0.2% BSA solutions, both for 3 min; hatching rate was evaluated after culture. As a control group for both experiments, non exposed embryos were cultured and evaluated for hatching rate. In Experiment 1, the hatching rates were 59.72% (43/72) for control, 62.38% (63/101) for EG, and 69.00% (69/100) for EG/Gly groups. In Experiment 2, hatching rates were 59.72% (43/72) for control, 15.22% (7/46) for EG, and 0.00% (0/46) for EG/Gly groups. Results were analyzed by chi-square test. In Experiment 1, no differences were observed among groups (P > 0.05) and in Experiment 2, differences were observed among control, EG, and EG/Gly groups (P < 0.05). In conclusion, the cryoprotectants were not deleterious to the development of in vitro bovine embryos until hatching, but the cryopreservation procedures decreased embryo viability. This work was supported by FAPESP 04/05335-1.

EVALUATION OF DEVELOPMENT AFTER CRYOPRESERVATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 136
Author(s):  
A. C. Nicacio ◽  
R. Simões ◽  
M. A. Peres ◽  
J. S. A. Gonçalves ◽  
F. F. Paula-Lopes ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Granulosa Cells ◽  
Embryo Cryopreservation ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Quick Freezing ◽  
Group 10 ◽  
Hatching Rates

The inefficiency of embryo cryopreservation protocols limits the broad use of in vitro production (IVP) of bovine embryos. The aim of this work was to identify the damage caused by cryopreservation and embryo culture of IVP bovine embryos after thawing by in vitro development before and after cryopreservation. Cumulus–oocyte complexes were in vitro-matured, fertilized, and cocultured on granulosa cells in SOF with amino acids (SOFaa) supplemented with FCS. Expanded blastocysts (n = 600) harvested on Days 7 to 9 were submitted to controlled freezing [controlled group: 10% ethylene glycol (EG) for 10 min and 1.2°C min–1 cryopreservation], quick-freezing [quick group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s], or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. The embryos of the control group were not exposed to cryoprotectant or cryopreservation method, and the hatching rate was evaluated on Day 12 post-insemination. The straws (quick and vitrification groups) were first placed on nitrogen vapor (0.8 cm over the liquid nitrogen) for 2 min and then immersed in liquid nitrogen. Embryos were thawed in air for 10 s followed by a 25°C water bath for 20 s. Embryos were rehydrated in PBS + 0.2% BSA + 0.3 m sucrose and PBS + 0.2% BSA for 3 min each. To evaluate development of frozen–thawed embryos, they were cocultured on granulosa cells in TCM-199 or SOFaa both supplemented with FCS for 4 days. Hatching rate of the control group was 46.1%. Data were analyzed by PROC MIXED model of SAS System for Windows®. For TCM-199, the controlled group hatching rate was 44.65 ± 5.94%, quick group did not hatch, and vitrification group showed hatching rates of 9.4 ± 6.8%. For SOFaa, the controlled group hatching rate was 11.6 ± 3.4%, embryos submitted to the quick group did not hatch, and the vitrification group showed hatching rates of 8.7 ± 4.5%. Values were significant at P < 0.05. The controlled group showed a difference compared with the other groups of cryopreservation in both media (TCM-199 and SOFaa). However, TCM 199 showed high rates of re-expansion and hatching. In conclusion, the culture medium influences embryo development after cryopreservation, and TCM-199 is more appropriate than SOFaa. Financial support by FAPESP (04/05335-1).

49 SUCCESSFUL CRYOPRESERVATION USING LOW ETHYLENE GLYCOL CONCENTRATION FOR IN VITRO-PRODUCED BOVINE EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 132
Author(s):  
M. Takayama ◽  
S. Sato ◽  
Y. Nishimura ◽  
K. Imai ◽  
O. Dochi
Keyword(s):  
Ethylene Glycol ◽  
Calf Serum ◽  
Bovine Embryos ◽  
Embryo Survival ◽  
Lower Survival Rate ◽  
Treatment Groups ◽  
Chi Squared ◽  
Hatching Rates

In vitro-produced (IVP) bovine embryos tend to have a lower survival rate after cryopreservation than in vivo embryos do. Therefore, the freezing medium (FM) and concentration of cryoprotectant are very important factors. This study was to investigate the effect of 1.2 M ethylene glycol (EG) with 0.1 M sucrose (SUC) on survival of IVP embryos after freezing. The COC were matured in 25 mM HEPES-buffered TCM199 (TCM199) supplemented with 5% calf serum (CS) and 0.02 AU mL−1 FSH. Oocytes (20 to 25) were cultured in 100-μL droplets of maturation medium for 20 h. After 6 h of gamete co-culture (5 × 106 sperm/mL), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% CS for 9 days (fertilization = Day 0). Only the expanded blastocysts from Days 7 to 9 were used in this experiment and separated into 3 treatment groups. The first and second groups were frozen in Dulbecco’s phosphate-buffered saline (D-PBS) supplemented with 20% CS, 0.1 M SUC, and 1.2 or 1.5 M EG (groups 1.2 or 1.5 M EG), respectively. The third group was D-PBS supplemented with 20% fetal calf serum (FCS), 0.25 M SUC, and 1.4 M glycerol (group GLY). In each group, embryos were equilibrated with their FM for 10 min and loaded into 0.25-mL straws individually. These straws were placed into the cooling chamber of a programmable freezer precooled to −7°C. After 2 min, the straws were seeded and then held for a further 13 min at −7°C. Then, the straws were cooled to −30°C at −0.3°C/min before being plunged into liquid nitrogen. The cryopreserved embryos were thawed by allowing the straws to stand in air for 7 s and then warming them in a 30°C water bath for 20 s. The thawed embryos were washed twice using 38°C D-PBS supplemented with 20% FCS. Subsequently, they were immersed in the same medium, held at 38°C for 10 min, and then each embryo was cultured in 20-μL droplets of TCM199 supplemented with 20% FCS and 0.1 mM β-mercaptoethanol for 72 h. The rates of embryos developing to the re-expanded and hatching blastocyst stages were determined 72 h after thawing. All data were analysed by the chi-squared test with Yates’ correction. The re-expanded and hatching rates of frozen-thawed embryos after 72 h in culture were not significantly different between 1.2 M EG (n = 39: 71.8% and 69.2%), 1.5 M EG (n = 38: 76.3% and 63.2%), and 1.4 M GLY (n = 37: 75.7% and 64.9%) groups (P > 0.05). Survival and hatching rates according to embryo quality were also not significantly different between 1.2 M EG (good n = 18: 88.9% and 88.9%; fair n = 21: 57.1% and 52.4%), 1.5 M EG (good n = 19: 89.5% and 84.2%; fair n = 19: 63.2% and 42.1%), and 1.4 M GLY (good n = 18: 77.8% and 66.7%; fair n = 19: 73.7% and 63.2%) (P > 0.05). In conclusion, cryoprotectant type and concentration did not affect embryo survival or development after cryopreservation in this study. Therefore, the ethylene glycol concentration used for the cryopreservation of IVP embryos can be reduced.

Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos

Zygote ◽  
2011 ◽  
Vol 20 (2) ◽  
pp. 117-122 ◽  
Author(s):  
Alessandra Corallo Nicacio ◽  
Renata Simões ◽  
Fabiola Freitas de Paula-Lopes ◽  
Flavia Regina Oliveira de Barros ◽  
Maria Angelica Peres ◽  
...  
Keyword(s):  
Culture Conditions ◽  
Slow Freezing ◽  
Control Group ◽  
Hatching Rate ◽  
Rapid Freezing ◽  
Bovine Embryos ◽  
Group 10 ◽  
Controlled Freezing ◽  
Hatching Rates

SummaryThe aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7–9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen–thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows®. Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.

scholarly journals 97 ASSESSMENT OF VIABILITY OF IN VITRO PRODUCED BOVINE EMBRYOS FOLLOWING VITRIFICATION BY CVM OR SLOW FREEZING WITH ETHYLENE GLYCOL AND TRIPLE TRANSFER

2005 ◽  
Vol 17 (2) ◽  
pp. 199 ◽  
Author(s):  
B. Peachey ◽  
K. Hartwich ◽  
K. Cockrem ◽  
A. Marsh ◽  
A. Pugh ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
Slow Freezing ◽  
High Survival ◽  
Bovine Embryos ◽  
Embryo Survival ◽  
Frozen Embryos ◽  
Morula Stage ◽  
Fresh Embryos

Vitrification has become the method of choice for the preservation of in vitro derived embryos of a number of species, and several methods of vitrification have been developed. One such method, the cryoLogic vitrification method (CVM) yields high survival rates of warmed embryos (Lindemans W et al. 2004 Reprod. Fertil. Dev. 16, 174 abst). In this study, the post-warm viability of bovine IVP embryos following either vitrification using CVM or slow freezing using ethylene glycol (EG) was compared. In addition, the survival of embryos following triple transfer to synchronized recipients was measured and the embryo (“e”) and recipient (“r”) contributions to embryo survival was determined using the “er” model for embryo survival (McMillan WH et al. 1998 Theriogenology 50, 1053–1070). Bovine IVP methods were those of van Wagtendonk et al. 2004 Reprod. Fertil. Dev. 16, 214 (abst). On day 7 of culture (Day 0 = IVF), Grade 1 and 2 embryos that had reached at least the late morula stage were selected for vitrification (20% DMSO, 20% ethylene glycol) or freezing in 1.5 M ethylene glycol + 0.1 M sucrose (0.5°C/min to −35°C). Following storage in LN2 for at least 24 h the embryos were thawed, the cryoprotectant removed, and the embryos cultured for 72 h in mSOF medium under 5% CO2, 7% O2, 88% N2. The number of hatching embryos was recorded at 24-h intervals. In addition, blastocyst and expanded blastocyst embryos were thawed and immediately transferred nonsurgically to recipients (three embryos of the same grade to each recipient) on Day 7 of a synchronized cycle (Day 0 = heat). The recipients were ultrasound-scanned for the presence of, and number of, fetuses on Days 35 and 62, respectively. The invitro assessment of 148 CVM and 230 EG frozen embryos indicated that more vitrified than EG embryos hatched by 72 h (73% vs. 62%; CVM vs. EG, χ2 = 4.5, P < 0.05). Overall, more Grade 1 embryos hatched than Grade 2 (74% vs. 60%, χ2 = 7.2, P < 0.01). CVM embryos (105) were triple-transferred to 35 recipients, and EG embryos (30) were triple-transferred to 10 recipients. Recipient pregnancy rates at Day 62 were 80% and 70%, respectively. Overall embryo survival was 38.5% (41% for CVM and 30% for EG). The overall calculated “e” and “r” values were 0.39 and 1.0 (“e”: 0.42 and 1.0, and “r”: 0.31 and 1.0, respectively, CVM and EG groups). Survival rates of CVM embryos to Day 62 (41%) were slightly lower than that previously obtained for fresh embryos produced using an identical IVP procedure (44% – van Wagtendonk AM 2004).

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

50 SURVIVAL OF SEXED IVF-DERIVED BOVINE EMBRYOS FROZEN AT DIFFERENT PREIMPLANTATION STAGES OF DEVELOPMENT

2017 ◽  
Vol 29 (1) ◽  
pp. 132
Author(s):  
L. Ferré ◽  
C. Fresno ◽  
M. Kjelland ◽  
P. Ross
Keyword(s):  
Cytochalasin B ◽  
Cell Monolayer ◽  
Cumulus Cell ◽  
Slow Freezing ◽  
Treatment Level ◽  
Bovine Embryos ◽  
Low Oxygen ◽  
Stages Of Development ◽  
Cell Layers

The ability to freeze in vitro-produced bovine embryos with a high post-thaw viability is still problematic and hampers logistics of on-farm embryo transfer. The objectives of this experiment were to compare different stages of development, freezing methods, and addition of cytoskeletal stabilisers (cytochalasin-B) before freezing. Ovaries were collected from an abattoir and oocytes aspirated from 2- to 6-mm follicles. Cumulus-oocyte complexes containing compact and complete cumulus cell layers were selected and matured in groups of 50 in 400 µL of M199 medium supplemented with ALA-glutamine (0.1 mM), Na pyruvate (0.2 mM), gentamicin (5 µg mL−1), EGF (50 ng mL−1), ovine FSH (50 ng mL−1), bLH (3 µg mL−1), cysteamine (0.1 mM), and 10% fetal bovine serum (FBS) for 22 to 24 h. Fertilization (Day 0) was done using female sex-sorted semen selected with a discontinuous density gradient and diluted to a final concentration of 1 × 106 sperm/mL. Synthetic oviductal fluid (SOF)-FERT medium was supplemented with fructose (90 µg mL−1), penicillamine (3 µg mL−1), hypotaurine (11 µg mL−1), and heparin (20 µg mL−1). After 18 h, presumptive zygotes were denuded and cultured in groups of 15 to 20 in 50-µL drops of SOF-BSA for 7 days. On Day 3.5 post-fertilization, 3% FBS was added. Low oxygen tension (5% O2) was used for culture. Morulae were selected at Day 5.5–6, blastocysts at Day 6–6.5, and expanded blastocysts at Day 6.5–7. Embryo harvesting for each stage was performed from a dedicated drop/dish and discarded in order to avoid further embryo stage collections. Grade 1 morulae, blastocysts, and expanded blastocysts were selected for freezing and placed randomly into 2 groups: slow-freezing and vitrification. Before freezing, half of the embryos from each stage were exposed to cytochalasin-B for 45 min. The slow freezing protocol consisted of 1.5 M ethylene glycol (EG) + 20% FBS + 0.4% BSA, and the cooling rate was 0.5°C/min. Slow-frozen embryo thawing was performed by exposing the 0.25-mL straws to air (23°C) for 10 s and then underwater at 35°C for 1 min. The vitrification (Cryo-Top) medium was 15% (vol/vol) EG + propylene glycol. Vitrified embryos were thawed in a solution of H199 + 20% FBS and 0.25 M sucrose at 39°C. Thawed embryos from both groups were cultured in SOF-BSA + 10% FBS under cumulus/granulosa cell monolayer co-culture. Embryo assessment involved post-thaw survival (0 h), re-expansion, and hatching of the zona pellucida (72 h). Three replicates were performed for each treatment level. Fisher’s l.s.d. test with Bonferroni correction was used to determine treatment differences (P < 0.05). The post-thaw survival, re-expansion, and hatching results showed that either expanded blastocysts (84.7 ± 3.2%, 74.1 ± 3.9%, and 60.9 ± 4.4%) or blastocysts (81.7 ± 3.5%, 69.6 ± 4.2%, and 55 ± 4.6%) were preferred (P < 0.05) embryo stages for cryopreservation compared with morulae (67.6 ± 4.4%, 52.5 ± 4.6%, and 33.2 ± 4.3%). Vitrification and cytochalasin-B pre-freezing exposure (61.3 ± 3.6% and 56.6 ± 3.8%) provided better (P < 0.05) hatching results compared with slow-freezing and without cytochalasin-B (37.8 ± 3.6% and 42.5 ± 3.7%).

scholarly journals A Shorter Equilibration Period Improves Post-Warming Outcomes after Vitrification and in Straw Dilution of In Vitro-Produced Bovine Embryos

Biology ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 142
Author(s):  
Iris Martínez-Rodero ◽  
Tania García-Martínez ◽  
Erika Alina Ordóñez-León ◽  
Meritxell Vendrell-Flotats ◽  
Carlos Olegario Hidalgo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Survival Rates ◽  
Bovine Embryos ◽  
Treatment Groups ◽  
Apoptosis Rate ◽  
Cell Counts ◽  
Hatching Rates

This study was designed to the optimize vitrification and in-straw warming protocol of in vitro-produced bovine embryos by comparing two different equilibration periods, short equilibrium (SE: 3 min) and long equilibrium (LE: 12 min). Outcomes recorded in vitrified day seven (D7) and day eight (D8) expanded blastocysts were survival and hatching rates, cell counts, apoptosis rate, and gene expression. While survival rates at 3 and 24 h post-warming were reduced (p < 0.05) after vitrification, the hatching rates of D7 embryos vitrified after SE were similar to the rates recorded in fresh non-vitrified blastocysts. The hatching rates of vitrified D8 blastocysts were lower (p < 0.05) than of fresh controls regardless of treatment. Total cell count, and inner cell mass and trophectoderm cell counts were similar in hatched D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values regardless of treatment. The apoptosis rate was significantly higher in both treatment groups compared to fresh controls, although rates were lower for SE than LE. No differences emerged in BAX, AQP3, CX43, and IFNτ gene expression between the treatments, whereas a significantly greater abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE. A shorter equilibration vitrification protocol was found to improve post-warming outcomes and time efficiency after in-straw warming/dilution.

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