40 IN VITRO SURVIVAL RATES OF IN VIVO- AND IN VITRO-PRODUCED BOVINE EMBRYOS CRYOPRESERVED BY SLOW CONTROLLED FREEZING OR VITRIFICATION

2012 ◽  
Vol 24 (1) ◽  
pp. 132 ◽  
Author(s):  
P. Rodriguez Villamil ◽  
D. Lozano ◽  
G. A. Bó
Keyword(s):  
Solid Surface ◽  
Survival Rates ◽  
Slow Freezing ◽  
Embryo Cryopreservation ◽  
Bovine Embryos ◽  
Controlled Freezing ◽  
In Vitro Survival ◽  
Hatching Rates

Although slow programmable freezing is currently the standard method for bovine embryo cryopreservation, vitrification has become an alternative for in vitro-produced embryos. A study was designed to compare the in vitro survival rates of in vivo- and in vitro-produced bovine embryos with 1 of 2 commercially available methods of cryopreservation: slow freezing and solid surface vitrification. In vivo-produced Grade 1 blastocysts (n = 210) collected from superovulated donor cows 7 days post-insemination and in vitro-produced Grade 1 blastocysts (n = 122) from slaughterhouse oocytes, produced with the procedure described by Chaubal et al. (2007 Theriogenology 67, 719–728) were randomly allocated in 2 groups. Group 1 (slow freezing) embryos were exposed to 1.5 M ethylene glycol (ViGro EG; Bioniche Animal Health USA Inc., Pullman, WA, USA) for 5 min and loaded in 0.25-mL plastic straws. The straws were placed in a Freeze Control CL 5500 freezer (CryoLogic, Victoria, Australia) at –6.5°C, seeded and after 10 min of equilibration, cooled at –0.6°C min–1 until –CE°C, before plunging into liquid nitrogen. Group B (vitrification) embryos were exposed to a AE% EG+0.BEM trehalose solution for A min and then into C0% EG+AM trehalose solution for C0 sec at room temperature to be vitrified using the CVM system (CryoLogic). The CVM used a cryohook and the solutions with the embryos are exposed to a metal solid surface cooled at –AIF°C. The vitrification solution was chosen after a toxicity test in which several EG and trehalose combinations were tested (Rodriguez Villamil et al. Ith IRAC Symposium, Argentina B0AA). After at least 1 wk of storage, embryos in the slow freezing groups were thawed in water bath at C0°C for AB s, placed in holding medium for E min and then cultured in SOF. Vitrified embryos were placed directly in a 0.BE M sucrose solution for E min (at CG°C) and then cultured in SOF medium. Re-expansion and hatching rates were evaluated at BD and GB h, respectively. Data was analyzed by nonparametric tests with type of embryo and cryopreservation procedure as main effects, using the software Infostat (UNC, Argentina, B0A0). In vivo-produced embryos had higher (P < 0.0A) re-expansion (AGI/BB0, HA% vs FI/ABB, EF%) and hatching rates (AEI/BB0, GB% vs EC/ABB, DC%) than in vitro-produced embryos, regardless of cryopreservation method. However, re-expansion (DE/FC, GA%) and hatching (CI/FC, FB%) rates were higher (P < 0.0A) with in vitro-produced vitrified embryos than in vitro-produced embryos in the slow freezing group (re-expansion: BD/EI, D0% and hatching: AD/EI, BD%). Although similar re-expansion rates (IC/AA0, HE% vs HF/A00, HF%) were obtained with in vivo- produced embryos cryopreserved by the 2 systems, hatching rates tended to be lower (P = 0.0I) with in vivo-produced embryos that were vitrified compared with slow freezing (GH/AA0, GA% vs HA/AA0, HA%). In conclusion, solid surface vitrification improved the cryosurvival rates of in vitro-produced embryos compared with the conventional slow, controlled freezing procedure.

scholarly journals Cryopreservation of in vitro-produced bovine embryos by vitrification: In pursuit of a simplified, standardized procedure that improves pregnancy rates to promote cattle industry use

2020 ◽  
Vol 36 (3) ◽  
pp. 251-270
Author(s):  
Van Do ◽  
Andrew Taylor-Robinson
Keyword(s):  
Slow Rate ◽  
Survival Rates ◽  
Slow Freezing ◽  
Embryo Cryopreservation ◽  
Human Embryos ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cattle Industry

The goal of cryopreservation is to retain the original stage of gametes and embryos after they have endured cooling and warming. Slow freezing is a standard method for in vivo-derived bovine embryo cryopreservation, threefifths of such embryos being frozen by this method globally. However, it is evident that slow freezing is not efficient for cryopreserving in vitro-produced bovine embryos. Hence, only one-third of in vitro-produced bovine embryos are cryopreserved. Vitrification is a preferred method for storage of human embryos; consequently, it has been explored as a novel means to store in vitro-produced bovine embryos, for which it shows considerable promise as an alternative to slow freezing. This is due to several reasons: vitrification is often less time-consuming than slow freezing; it does not need expensive slow rate freezing machines; and it has been proven to have comparatively higher survival rates. Yet, in the cattle industry vitrification continues to present shortcomings, such as possible toxicity of vitrification solutions and failure to standardize methods, which pose a challenge for its application to in vitro-produced bovine embryos. Therefore, determining the most suitable procedure is crucial to make vitrification more practical in commercial settings.

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 123VITRIFICATION OF IN VITRO-PRODUCED BOVINE BLASTOCYSTS BY ADDITION OF CRYOPROTECTANT IN ONE STEP

2004 ◽  
Vol 16 (2) ◽  
pp. 184
Author(s):  
D.J. Walker ◽  
L.F. Campos-Chillon ◽  
G.E. Seidel
Keyword(s):  
Ethylene Glycol ◽  
Embryo Quality ◽  
Survival Rates ◽  
Defined Medium ◽  
Embryo Cryopreservation ◽  
Equilibration Time ◽  
One Step ◽  
Hatching Rates

Vitrification combined with in-straw dilution may replace conventional cryopreservation of bovine embryos, but this requires further study for practicality. Our objectives were to compare three ethylene glycol concentrations (6, 7, and 8M) and two equilibration times (2.5 and 3.5min) for one-step addition of cryoprotectant. In vitro-matured oocytes from slaughterhouse ovaries, fertilized using sperm of 3 bulls, were cultured in chemically defined medium (CDM-1/CDM-2) plus FAF-BSA to produce 420 blastocysts. Day 7.5 embryos were placed into HCDM-2 (HEPES-buffered medium) and then transferred to a 6μL drop of vitrification solution (V) (6, 7, or 8M ethylene glycol, 0.5M galactose, and 18% w/v Ficoll 70 in HCDM-2). Immediately thereafter, 1cm column of DHCDM (0.5M galactose in HCDM-2) was drawn into a 0.25mL straw, followed by a 0.5cm column of air and another 7cm of DHCDM. Another 0.5cm column of air was aspirated before the 6μL of V (0.5cm) containing the embryos were aspirated; then 0.5cm of air followed. Finally, DHCDM was drawn until the first column came into contact with the cotton plug. Straws were then heat-sealed and plunged into liquid nitrogen slightly above the embryos after 2.5 or 3.5min equilibration. The rest of the straw was then submerged slowly. Straws were thawed in air for 10s and then in 37°C water for 20s. Straws were held at room temperature (24°C) for 4min before being expelled into HCDM-2. They were then placed into CDM-2+5% FCS for culture. Quality score (1=excellent, 2=fair, 3=poor), survival (S) as determined by expansion of blastocysts, and hatching (H) were assessed at 24 and 48h post-thaw. Data from 6 replicates (2/bull) were analyzed by ANOVA after arc sin transformation of percentage data. S and H responses were calculated as a percentage of non-frozen controls in the same replicate. Control survival and hatching rates were: 24S: 90%, 24H: 50%, 48S: 90%, 48H: 72%. Quality scores at both 24 and 48h were higher (P&lt;0.05) for 8M than 6M ethylene glycol (2.68 and 3.24 for 24h; 2.55 and 3.17 for 48h); values for 7M ethylene glycol were intermediate. Equilibration time had no effect on embryo quality (P&gt;0.1). Neither ethylene glycol concentration nor exposure time affected survival or hatching at 24 or 48h (P&gt;0.1). Survival rates (as a % of control) at 48h were: 8M: 57%, 7M: 55%, 6M: 36% and hatching: 8M: 39%, 7M: 30%, and 6M: 21%; 2.5min tended to be better than 3.5min for survival at 24h, hatching at 24h, survival at 48h, but not hatching at 48h (56% and 43%, 30% and 26%, 55% and 44%, 28% and 32% respectively). Higher concentrations of ethylene glycol proved beneficial in terms of embryo quality, with the same trend for survival and hatching rates. One-step addition of cryoprotectant for vitrification shows potential for simplifying embryo cryopreservation. However, further research is needed to produce more acceptable survival rates and to study vitrification of in vivo-produced embryos.

scholarly journals Phospholipid composition and resistance to vitrification of in vivo blastocyst of a Brazilian naturalized porcine breed

2019 ◽  
Vol 71 (3) ◽  
pp. 837-847
Author(s):  
J.F.W. Sprícigo ◽  
L.O. Leme ◽  
A.L. Guimarães ◽  
J.C. Oliveira Neto ◽  
P.C.P. Silva ◽  
...  
Keyword(s):  
Phospholipid Composition ◽  
Embryo Cryopreservation ◽  
Blastocyst Development ◽  
Apoptotic Pathway ◽  
Maldi Tof ◽  
Porcine Embryo ◽  
Low Efficiency ◽  
Hatching Rates

ABSTRACT Piau porcine blastocysts were submitted to MALDI-TOF to identify the main phospholipids (PL). After that, in vivo blastocysts (D6) were vitrified (n=52), non-vitrified were used as control (n=42). After warming, blastocysts were in vitro cultured to assess re-expansion and hatching at 24 and 48 hours. Finally, at 48 hours, hatched blastocysts were submitted to RT-qPCR searching for BCL2A1, BAK, BAX and CASP3 genes. For MALDI-TOF, the ion intensity was expressed in arbitrary units. Blastocyst development was compared by Qui-square (P< 0.05). Among the most representative PL was the phosphatidylcholine [PC (32:0) + H]+; [PC (34:1) + H]+ and [PC (36:4) + H]+. Beyond the PL, MALDI revealed some triglycerides (TG), including PPL (50:2) + Na+, PPO (50:1) + Na+, PLO (52:3) + Na+ and POO (52:2) + Na. Re-expansion did not differ (P> 0.05) between fresh or vitrified blastocysts at 24 (33.3%; 32.7%) or 48 hours (2.4%; 13.5%). Hatching rates were higher (P< 0.05) for fresh compared to vitrified at 24 (66.7%; 15.4%) and 48 hours (97.6%; 36.0%). BAX was overexpressed (P< 0.05) after vitrification. In conclusion, Piau blastocysts can be cryopreserved by Cryotop. This study also demonstrated that the apoptotic pathway may be responsible for the low efficiency of porcine embryo cryopreservation.

44 IN VITRO AND IN VIVO SURVIVABILITY ON VITRIFIED EMBRYOS OBTAINED BY BOVINE SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 131
Author(s):  
K. Kaneyama ◽  
S. Kobayashi ◽  
S. Matoba ◽  
Y. Hashiyada ◽  
K. Imai ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Survival Rates ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Nuclear Transplantation ◽  
Embryo Cryopreservation

Although many studies have been conducted on somatic cell nuclear transfer, there are only a few reports on cryopreservation of reconstructed embryos after nuclear transplantation. The objective of this study was to examine in vitro or in vivo development of vitrified blastocysts obtained by nuclear transfer. Nuclear transfer was carried out according to the procedure of Goto et al. (1999 Anim. Sci. J. 70, 243–245), and conducted using abattoir-derived oocytes and cumulus cells derived by ovum pickup from Holstein and Japanese Black cows. Embryos were vitrified as described by Saito et al. (1998 Cryobiol. Cryotech. 43, 34–39). The vitrification solution (GESX solution) was based on Dulbecco's PBS containing 20% glycerol (GL), 20% ethylene glycol (EG), 0.3 M sucrose (Suc), 0.3 M xylose (Xyl), and 3% polyethylene glycol (PEG). The blastocysts were equilibrated in three steps, with 10% GL, 0.1 M Suc, 0.1 M Xyl, and 1% PEG for 5 min (1); with 10% GL, 10% EG, 0.2 M Suc, 0.2 M Xyl, and 2% PEG for 5 min (2) and GESX solution (3). After transfer to GESX, equilibrated embryos were loaded to 0.25-mL straws and plunged into liquid nitrogen for 1 min. The vitrified blastocysts were warmed in water (20°C) and diluted in 0.5 M and 0.25 M sucrose for 5 min each. Equilibration and dilution procedures were conducted at room temperature (25–26°C). After dilution, the vitrified blastocysts were cultured in TCM-199 supplemented with 20% fetal calf serum and 0.1 mM β-mercaptoethanol at 38.5°C under gas phase of 5% CO2 in air. In Experiment 1, survival rates after vitrification were compared between the nuclear transfer and the IVF blastocysts. Survival rates of vitrified nuclear transfer blastocysts (n = 60, Day 8) at 24 and 48 h were 70.0% and 56.7%, respectively, and those of vitrified IVF blastocysts (n = 41) were 82.9% and 82.9%, respectively. There were no significant differences in survival rates at 24 and 48 h between the two groups. In Experiment 2, one (VIT-single) or two (VIT-double) vitrified and one (nonVIT-single) or two (nonVIT-double) nonvitrified reconstructed blastocysts per animal were transferred into Holstein dry cows. The result of Experiment 2 is shown in Table 1. This experiment demonstrated that the vitrification method in this study can be used for cloned embryo cryopreservation but the production rate should be improved. Table 1. Comparison of survival rates of vitrified or nonvitrified cloned embryos after transfer

193 IMPROVED POST-THAW SURVIVAL OF BOVINE EMBRYOS PRODUCED IN SERUM-FREE IN VITRO PRODUCTION SYSTEM

2016 ◽  
Vol 28 (2) ◽  
pp. 227
Author(s):  
M. Nõmm ◽  
E. Mark ◽  
O. Sarv ◽  
S. Kõks ◽  
Ü. Jaakma
Keyword(s):  
Survival Rates ◽  
Slow Freezing ◽  
In Vitro Fertilisation ◽  
Ministry Of Education ◽  
In Vitro Production ◽  
Freezing And Thawing ◽  
Embryo Survival ◽  
Serum Free

Over a few decades the bovine in vitro embryo production (IVP) systems have been improving rapidly. Still, the goal to produce the same quality embryos in vitro as in vivo has not yet been reached. The FCS is usually added to media during IVP to provide growth factors and energy sources. Currently, serum-free culture systems are often preferred due to the lower risk of contamination and prevention of the development of large offspring syndrome. The aim of this study was to establish whether complete elimination of FCS from the bovine IVP system has an effect on blastocyst rates, embryo quality, and embryo survival rates after slow freezing. We replaced our conventional in vitro maturation (IVM) medium [tissue culture medium-199, 10% (v/v) FCS, 10 µg mL–1 epidermal growth factor (EGF), 1500 U mL–1 serum gonadotropin and chorionic gonadotropin (PG600), Na-pyruvate 0.5 mM, gentamycin sulfate 50 µg mL–1 and l-glutamine 1 mM] with SOF (SOFaaci) supplemented with 0.4% fatty acid-free BSA fraction V, 10 µg mL–1 EGF, and 1500 U mL–1 PG600. Matured cumulus-oocyte complexes (COC) from both experimental groups (total of 1145 from serum-free IVP and 687 from our conventional IVP system) were used for in vitro fertilisation and culture. Blastocyst rates were similar in the serum-free and our usual IVP protocol, 18 and 22%, respectively. Seventy-seven Grade 1 (according to IETS) Day 7 blastocysts from the serum-free IVP system and 80 Grade 1 Day 7 blastocysts from our conventional IVP system were frozen in 1.5 M ethylene glycol and 0.1 M sucrose containing cryopreservation medium. The post-thaw survival rates after 24 h of culture and evaluated as percentages of re-expanded embryos were 63.6% for the serum-free IVP and 46.3% for the conventional IVP system (P < 0.05, Z Test for 2 population proportions). These results indicate that it is possible to have a completely serum-free bovine IVP system and based on the slow freezing and thawing results the quality of serum-free IVP embryos might be better than of the embryos matured in our conventional maturation media. However, more experiments and increased sample sizes are needed to confirm the results. This study was supported by Project 3.2.0701.12–0036 of Archimedes Foundation, AP 2.4 of CCRMB, and institutional research funding (IUT 08–01) of the Estonian Ministry of Education and Research.

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).

88 EFFECT OF VITRIFICATION PROCEDURE ON SURVIVAL RATE OF BOVINE EMBRYOS PRODUCED IN VITRO

2011 ◽  
Vol 23 (1) ◽  
pp. 149
Author(s):  
E. Y. Herrera ◽  
C. de Frutos ◽  
R. Laguna-Barraza ◽  
A. Gutierrez-Adan ◽  
D. Rizos
Keyword(s):  
Survival Rate ◽  
Survival Rates ◽  
Calf Serum ◽  
Basal Medium ◽  
Bovine Embryos ◽  
Oviduct Fluid ◽  
Bovine Oocytes ◽  
Cryopreservation Method ◽  
Hatching Rates

Vitrification as a cryopreservation method has many advantages compared with slow freezing. Many variables in the vitrification process exists that influence the survival rates of vitrified oocytes and embryos. These include the cryoprotectants (type, concentration, and duration of exposure), the temperature of the vitrification solution at exposure, the device used for vitrification, and the quality and developmental stage of embryos. It is worthwhile to mention that vitrification protocols successfully used in bovine oocytes and embryos have been used also with human oocytes and embryos. Vitrification is relatively simple, requires no freezing equipment, and relies on the placement of the embryos in a very small volume of vitrification medium that must be cooled at extreme rates not obtainable in regular enclosed straws. The aim of the present study was to evaluate the efficiency of 4 different vitrification protocols on the survival rate of in vitro produced (IVP) bovine embryos. Blastocysts were produced by a standard IVP procedure following in vitro maturation, fertilization, and culture in synthetic oviduct fluid supplemented with 5% fetal calf serum (FCS). On Day 7 (Day of IVF = Day 0), a total of 297 blastocysts were vitrified using (i) the open pulled straw (OPS) in 20% DMSO and 20% ethylene glycol (EG) in a basal medium of TCM-199 with HEPES supplemented with 20% FCS; (ii) the modified OPS, in 20% DMSO, 20% EG, and 0.5 M sucrose in a basal medium of phosphate buffer saline (PBS) supplemented with 20% FCS; (iii) the cryoloop, in 15% DMSO, 15% EG, 10 mg mL–1 Ficoll 70, and 0.65 M sucrose in a basal medium of PBS supplemented with 20% FCS; and (iv) in 0.25 straws in 20% glycerol, 20% EG, 0.3 M sucrose, 3% polyethylene glycol, and 0.3 M xylose in a basal medium of PBS. After warming, embryos were placed in culture for additional 24 h. Re-expansion and hatching rates were measured at 2 and 24 h after warming. Data were analysed by 1-way ANOVA. At 2 h post-warming, the re-expansion of blastocysts vitrified with cryoloop was significantly higher compared with OPS, modified OPS, and the 0.25 straw methods (54.08 ± 15.53 v. 10.40 ± 3.00, 22.67 ± 9.20, and 8.82 ± 2.15, respectively; P ≤ 0.028). At 24 h post-warming, only embryos from cryoloop and modified OPS were still alive with a survival rate of embryos vitrified with cryoloop significantly higher than that of those vitrified with modified OPS (48.45 ± 17.56 v. 3.75 ± 3.75, respectively; P ≤ 0.007). Hatching rates at 24 h post-warming were not different between cryoloop and modified OPS groups (5.63 ± 4.40 and 1.25 ± 1.25, respectively). These results clearly demonstrate that embryo cryotolerance is affected by the method used for cryopreservation. Moreover, cryoloop vitrification was found to be more effective than OPS and 0.25 straw methods for the cryopreservation of bovine embryos.

88 SURVIVAL AFTER FREEZING OF IN VITRO AND IN VIVO BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 203
Author(s):  
S. R. Cho ◽  
S. H. Choi ◽  
C. Y. Choe ◽  
J. J. Son ◽  
H. J. Kim ◽  
...  
Keyword(s):  
Cell Number ◽  
Water Bath ◽  
Bovine Embryos ◽  
Estrous Cycles ◽  
Embryo Freezing ◽  
Frozen Embryos ◽  
In Vitro Survival ◽  
Medium Culture

The present study was conducted to investigate the survivability of post-thawed bovine embryos for direct transfer. Bovine ovaries were collected at a local slaughterhouse. The cumulus-oocyte complexes (COC) were aspirated from 2 to 8 mm antral follicles using a syringe with an 18-gauge needle. Selected COC were washed in HEPES-buffered tissue culture medium (TCM-199) supplemented with 5% FBS. Sets of 15 COC were matured for 22 h in 50-μL droplets of TCM-199 supplemented with 5% FBS, 10 μg mL-1 of LH, 10 μg mL-1 of FSH, that had been previously covered with mineral oil and equilibrated in an atmosphere of 5% CO2, 5% O2, and 90% N2 at 39°C. Mature COC were fertilized with frozen-thawed semen treated with BO medium (Brackett and Oliphants Biol. Reprod. 12, 260-274). All oocytes and embryos were placed in CR1aa medium culture system for in vivo embryo production. The Korean native cows that were between days 8 and 12 of their estrous cycles were superovulated with 28 mg of porcine follicle stimulating hormone (FSH, Antorine-R10; Kawasaki Mitaka Pharmaceutical, Tokyo, Japan) in twice daily i.m. injections, with a gradual decrease over 4 days. For embryo freezing, Day 7 and 8 blastocysts were equilibrated for 15 min in 1.5 M, and 1.8 M ethylene glycol(EG) was used as a cryoprotectant. Embryo was loaded into 0.25 mL straw and directly into a cooling chamber (CL-863, USA) and kept at -7°C for 10 min, including time for seeding, and further cooled to -35°C at -0.3°C. After 2 min at this temperature, they were plunged into liquid nitrogen. Thawing was performed by keeping straws at room temperature for 10 s, followed by immersion in water bath at 35°C and 37°C. Embryos were evaluated at 24, 48, and 72 h post thawing. Embryos that survived were recorded as either blastocysts that had expanded or hatched at 24 h or had hatched at 72 h. All of the results were analyzed by ANOVA using the STATVIEW program. After frozen the blastocysts cultured without serum, better survivability for frozen embryos was seen in the 1.8 M EG with 0.5% BSA (bovine serum albumin) group than 1.5 M EG with 0.5% BSA (75.7 v. 72.7). The survivability of frozen-thawed embryos was significantly higher in the 37°C water bath than 35°C (85.7% v. 70.8%). However, there was no difference in the total cell number of thawed embryos (142 ± 13 v. 137 ± 12), and chromosome abnormality higher than in vivo frozen-thawed embryos. In conclusion, the results suggest that the thawing temperature at 37°C may be optimal for better in vitro survival of frozen-thawed embryos produced in vitro and in vivo. Furthermore, the data suggest that embryo freezing system may provide reasonable conditions for embryo transfer.

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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