149 IN VITRO BOVINE EMBRYOS FROZEN BY DIRECT TRANSFER METHOD WITH ETHYLENE GLYCOL

2015 ◽  
Vol 27 (1) ◽  
pp. 166
Author(s):  
S. H. Kizil ◽  
M. Satilmis ◽  
N. Akyol ◽  
T. Karasahin
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
Hatching Rate ◽  
Direct Transfer ◽  
Embryo Freezing ◽  
Frozen Embryos ◽  
Transfer Method ◽  
Phosphate Buffered Saline ◽  
In Vitro Survival

The objective of this study was to search for capability of freezing by ethylene glycol direct transfer method of in vitro-produced cattle embryos. Fifty-six in vitro-produced good-quality cow embryos were frozen by direct transfer method with ethylene glycol in this study. Cattle ovaries were collected from a slaughterhouse and oocytes were aspirated from follicles with 2 to 8 mm diameters. Then oocytes were let for maturation of 20 to 22 h in 100-μL microdroplets of TCM-199 with 0.1 mM β-mercaptoethanol and 20% FCS. After 5 to 6 h of fertilization in Bracket Oliphant (BO), they were cultured for 7 days in 100 µL of CR1aa medium with 5% FCS under 5% CO2, 98% relative humidity, and 38.5°C in a CO2 incubator. Embryos were equilibrated for 15 min in room temperature in 1.8 M ethylene glycol + 0.1 M sucrose in Dulbecco's phosphate buffered saline (D-PBS) supplemented with 20% FCS. Embryos were then loaded individually into a 0.25-mL straw and placed directly into a cooling chamber of a programmable freezer with methyl alcohol precooled to –7°C. After 2 min, the straw was seeded and maintained at –7°C for 8 min more. Then it was cooled to –30°C at 0.3°C min–1 before plunging into liquid nitrogen. The frozen embryos were thawed by allowing the straw to stand in air for 5 to 6 s and then immersing them in a 30°C water bath for 10 s. After thawing, embryos were transferred into TCM-199 + 0.1 mM β-mercaptoethanol + 20% FCS medium to check in vitro survival rates at 48 h post-thawing. The re-expansion and hatching rate of blastocysts was 64.28% (36 blastocysts). This result indicated that ethylene glycol can be used effectively for cow embryo freezing as a suitable cryoprotectant for direct transfer method.

83 IMPROVED SURVIVAL TO ONE-STEP REHYDRATION OF VITRIFIED - WARMED VERSUS FROZEN - THAWED IN VITRO-PRODUCED BOVINE BLASTOCYSTS

2013 ◽  
Vol 25 (1) ◽  
pp. 189
Author(s):  
B. Trigal ◽  
E. Gómez ◽  
J. N. Caamaño ◽  
M. Muñoz ◽  
E. Correia ◽  
...  
Keyword(s):  
Survival Rates ◽  
Slow Freezing ◽  
Direct Transfer ◽  
Frozen Embryos ◽  
Intracellular Ice Formation ◽  
Intracellular Ice ◽  
One Step ◽  
Phosphate Buffered Saline

Vitrification allows cryopreservation of embryos while avoiding the detrimental effects derived from the intracellular ice formation during slow freezing. However, while slow freezing allows direct transfer of embryos, vitrification usually requires 1 or 2 rehydration steps after warming. The aim of this work was to analyze survival rates and quality of vitrified or slow frozen in vitro-produced (IVP) embryos, after warming/thawing by one-step procedures. Bovine blastocysts were produced in vitro, and on Day 7 and 8 excellent- and good-quality expanded blastocysts were selected for slow freezing (n = 175) or vitrification (n = 176) in 4 replicates. Slow freezing was performed in phosphate buffered saline containing ethylene glycol (1.5 M) and sucrose (0.1 M). Embryos were placed in a Biocool chamber (Biocool II, FTS® Systems Inc.) at –7°C for 5 min and seeded. After 5 min, embryos were cooled at –0.3°C min–1 until –32°C and plunged in LN2. Embryos were thawed in a water bath at 37°C for 30 s. Vitrification was performed in fibreplugs as previously described (Trigal et al. 2012 Theriogenology 10.1016/j.theriogenology.2012.06.018). For warming, embryos were incubated for 5 min in TCM199–HEPES, 20% FCS, and 0.25 M sucrose. Thawed and warmed embryos were washed and subsequently cultured in mSOFaaci + 6 g L–1 BSA + 10% FCS for 48 h. Re-expansion (RE) (at 2, 24, and 48 h in culture) and hatching rates (HR; at 24 and 48 h in culture) were recorded. Total cells were counted in blastocysts that hatched at 24 and 48 h after fixation and bisbenzimide staining. Data were analyzed by ANOVA and are presented as least squares means ± standard error. No differences were found within RE at 2 and 24 h, and HR at 24 h (2-h RE: 94.1 ± 4.9 v. 95.4 ± 4.9; 24-h RE: 92.6 ± 4.9 v. 94.5 ± 4.9; HR: 21.0 ± 7.0 v. 20.6 ± 7.0, for slow frozen and vitrified embryos, respectively; P > 0.05). However, at 48 h, vitrified embryos hatched at higher rates than did slow-frozen embryos (53.6 ± 10.2 v. 32.5 ± 10.2; P < 0.05). Vitrified embryos (143.5 ± 12.7) had higher (P < 0.05) cell numbers than did slow-frozen embryos (106.1 ± 9.6) after hatching. Our results show that vitrification of IVP embryos in fibreplugs followed by a one-step warming is a promising candidate procedure to replace slow freezing for direct transfer on field. These results must be completed with embryo transfers to analyze pregnancy rates. RTA2011-00090 (FEDER-INIA) is acknowledged. Muñoz, Trigal, and Correia are sponsored by RYC08-03454, Cajastur, and FPU2009-5265, respectively.

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).

scholarly journals High in vitro survival rate of sheep in vitro produced blastocysts vitrified with a new method and device

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Sergio Ledda ◽  
Jen M. Kelly ◽  
Stefano Nieddu ◽  
Daniela Bebbere ◽  
Federica Ariu ◽  
...  
Keyword(s):  
Survival Rate ◽  
Survival Rates ◽  
Blastocyst Stage ◽  
Hatching Rate ◽  
High Survival ◽  
Apoptotic Cells ◽  
Embryo Survival ◽  
New Device ◽  
In Vitro Survival

Abstract Background To advance the use of embryo vitrification in veterinary practice, we developed a system in which embryo vitrification, warming and dilution can be performed within a straw. Ovine in vitro produced embryos (IVEP) were vitrified at either early (EBs: n = 74) or fully expanded blastocyst stage (FEBs: n = 195), using a new device named “E.Vit”, composed by a 0.25-mL straw with a 50-μm pore polycarbonate grid at one end. Embryos at each stage (EBs and FEBs) were vitrified by either Two-step (TS) or Multi-step (MS; 6 different concentrations of vitrification solutions) protocol. Non-vitrified embryos (n = 102) were maintained in in vitro culture as a control. Warming consisted of placing the straws directly into 1.5 mL tubes containing a TCM-199 solution with three decreasing concentrations of sucrose. Blastocyst re-expansion, embryo survival and hatching rate were evaluated at 2, 24 and 48 h post warming. The number of apoptotic cells was determined by TUNEL assay. Results Blastocyst re-expansion (2 h) after warming was higher (P < 0.05) in FEBs group, vitrified with the MS and TS methods (77.90% and 71.25%, respectively) compared with the EBs group (MS: 59.38% and TS: 48.50%, respectively). Survival rates of vitrified FEBs after 24 h IVC were higher (P < 0.001) in both methods (MS and TS) than vitrified EBs (MS: 56.25%; TS: 42.42%) and was higher (P < 0.05) in the MS method (94.19%) compared with those in TS (83.75%). After 48 h of culture the hatching rate for FEBs vitrified in MS system (91.86%) was similar to control (91.89%), but higher than FEB TS (77.5%) and EBs vitrified in MS (37.5%) and TS (33.33%). Number of apoptotic cells were higher in EBs, irrespective of the system used, compared to FEBs. The number of apoptotic cells in FEBs vitrified with MS was comparable to the control. Conclusions A high survival rate of IVP embryos can be achieved by the new “E.Vit” device with hatching rates in vitro comparable with control fresh embryos. This method has the potential for use in direct embryo transfer in field conditions.

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 119CRYOPRESERVATION OF BIOPSIED BOVINE EMBRYOS PRODUCED IN VITRO USING ARABINOGALACTAN AND 1.5M ETHYLENE GLYCOL

2004 ◽  
Vol 16 (2) ◽  
pp. 182
Author(s):  
B. Shangguan ◽  
N. Yang ◽  
R. Vanderwal ◽  
M.D. Darrow
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
Field Trials ◽  
Bovine Embryos ◽  
Embryo Survival ◽  
Ag Addition ◽  
Freezing Medium ◽  
Frozen Embryos

Arabinogalactan (AG) in combination with 1.5M ethylene glycol (EG) has been used successfully in cryopreserving biopsied in vivo bovine embryos (Darrow, 2002 Theriogenology 57(1), 531). This study was undertaken to investigate the efficiency of AG addition in a freezing medium (FM) to cryopreserve biopsied bovine embryos produced in vitro (IVP). Blastocysts of grade 1 were collected at Days 7 and 8 post-insemination. After biopsy with a small blade, embryos were transferred to CR1aa medium and cultured for 2 hours (h) before being frozen. In experiment 1, a group of unbiopsied embryos were handled in a manner similar to that used for the biopsied embryos. Embryos were frozen using either 1.5M EG+0.1M sucrose (EG+) (AB Technology, Pullman, WA, USA) or a FM containing 1.5M EG and different concentrations of AG (AG1, 2 and 3, courtesy of AB Technology). Embryos remained in FM for 10 (exp.1), 5 (exp.2), 5 and 10 (exp.3) or 5, 10, and 20 (exp.4) minutes before being loaded into a freezer and cooled down to −35°C at 0.3°C/min. Frozen embryos were thawed (35°C, 20 seconds) and cultured in CR1aa at 38.5°C for 3 days. Embryo survival rates (S%) were recorded at 24, 48 and 72h post-thawing. Data were compared with t-test or ANOVA procedures using SigmaStat 3.0. Results from exp.1 (Table) indicate that biopsied and unbiopsied embryos survived well in EG+ or AG2. While the biopsy procedure did not affect the post-thaw S% of embryos in either FM, no significant differences were observed between embryos frozen with EG+ and AG2 (P=0.055). Reducing or increasing AG concentration in FM by 2-fold (AG1 and 3, respectively) did not significantly affect the post-thaw S% at 24h (EG+, 80.0%, n=133; AG1, 83.3%, n=135; AG2, 71.4%, n=137 and AG3, 75.0%, n=135; P=0.217, exp.2). However, shortened exposure from 10 to 5 minutes to AG2 resulted in an improvement in S% at 24h, from 35.7% (n=80) to 61.4% (n=82, P&lt;0.05; exp.3). When AG1 (=0.5×AG2) was used in the FM the S% at 24h after different exposure times was not significant (5 minutes, 77.8%, n=179; 10 and 20 minutes, 66.7%, n=179 and 183; P=0.472, exp.4). This study demonstrates that addition of AG to the FM effectively sustains the viability of biopsied IVP embryos during freezing and any potential harmful impact of AG on embryo survival can be minimized by reducing AG concentration or the time of embryo exposure to AG prior to freezing. Further studies are needed to determine optimal AG concentration. Currently, field trials are underway to evaluate the ability of AG medium to promote pregnancies from frozen, biopsied IVP embryos. Table 1 Post-thaw survival rates of biopsied IVP embryos frozen in ethylene glycol with sucrose (EG+) and a FM containing arabinogalactan (AG2). Data are means±SEM

134 CONCEPTION RATES OF IN VITRO-PRODUCED BOVINE EMBRYOS CRYOPRESERVED IN 6% GLYCEROL AND TRANSFERRED BY THE DIRECT METHOD

2007 ◽  
Vol 19 (1) ◽  
pp. 184
Author(s):  
N. Takada ◽  
S. Hayasaka ◽  
K. Chiba
Keyword(s):  
Ethylene Glycol ◽  
Conception Rate ◽  
Hatching Rate ◽  
Glycerol Solution ◽  
Direct Transfer ◽  
Bovine Embryos ◽  
Conception Rates ◽  
Chi Square Analysis

Ethylene glycol has been used as the standard cryoprotectant for direct transfer of bovine embryos due to its high permeability. But Merton et al. reported that cryoprotectivity of glycerol for bovine embryos was superior to that of ethylene glycol (2001 Theriogenology 55, 312 abst). We previously reported that nonsurgical transfer of in vivo-derived bovine embryos cryopreserved in a lower concentration (5%) of glycerol and thawed by stepwise method resulted in a 55.4% conception rate, whereas direct transfer without removal of cryoprotectant showed only a 45.1% conception rate (Takada et al. 2005 Jpn. J. Embryo Transfer 27, 59–64). In this experiment, survival and conception rates of in vitro-produced (IVP) bovine embryos cryopreserved in 6% glycerol solution (GLY) were compared to those of embryos cryopreserved in 10% ethylene glycol plus 0.1 M sucrose solution (EG). Cumulus–oocyte complexes were matured and fertilized according to Numabe et al. (2000 Theriogenology 54, 1409–1420). Presumed zygotes were cultured in mSOF supplemented with 5% calf serum (CS) and 0.25% linoleic acid albumin at 38.5�C under 5% CO2, 5% O2, 90% N2 for 7 days. At the expanded blastocyst stage, embryos were placed in GLY or EG in PBS supplemented with 20% CS for 15 min at room temperature and loaded into 0.25-mL straws. Straws were placed directly into an alcohol freezer. When the cryoprotectant was GLY, straws were seeded at -4.0�C, held for 10 min, cooled at 0.5�C min to -30.5�C, and then plunged into liquid nitrogen. When the cryoprotectant was EG, the seeding point was -7.5�C, and the plunging point was -34.0�C, but the rest of the protocol was the same as for GLY. In Exp. 1, thawing in both groups was done in a 30�C water bath, and the contents were directly rehydrated in PBS with 20% CS. Thawed embryos were cultured in mSOF with 5% CS for 24 h to assess embryo survival rate, based on the re-expansion of the blastcoele and on their hatching ability. In Exp. 2, embryos in both groups were thawed and transferred to synchronous recipients without removing the cryoprotectant. Data were analyzed using chi-square analysis. In Exp. 1, the developmental rates of post-thaw embryos were similar in GLY (46/52, 88.5%) and EG (45/52, 86.5%); however, the hatching rate was significantly higher (P &lt; 0.05) in embryos cryopreserved in EG (26/52, 50.0%) than in GLY (15/52, 28.8%). In Exp. 2, the conception rates of embryos were similar in both groups, GLY (7/15, 46.7%) and EG (6/15, 40.0%). In conclusion, after direct rehydration of embryos, the developmental ability of IVP bovine embryos cryopreserved in EG was superior to that of embryos cryopreserved in GLY in vitro. However, conception rates in vivo were similar in both groups. These results suggest that a lower concentration of glycerol might be still useful as a cryoprotectant for direct transfer of IVP bovine embryos.

scholarly journals 107TOXICITY OF ETHYLENE GLYCOL ON FROZEN AND THAWED IVP EMBRYOS IN DIRECT TRANSFER METHOD

2004 ◽  
Vol 16 (2) ◽  
pp. 175 ◽  
Author(s):  
S. Matoba ◽  
K. Imai ◽  
Y. Mimaki ◽  
M. Narita ◽  
M. Tagawa ◽  
...  
Keyword(s):  
Survival Rate ◽  
Ethylene Glycol ◽  
Calf Serum ◽  
Holding Time ◽  
Morphological Development ◽  
Direct Transfer ◽  
Holding Period ◽  
Development Data ◽  
Transfer Method

Ethylene glycol (EG) is a cryoprotectant which is highly permeable to mammalian embryos. But the toxicity of this cryoprotectant for embryos after thawing has not been investigated. The aim of this study was to determine the toxicity of EG to embryos frozen and thawed by a direct transfer method. In vitro-produced Day 7 blastocysts (n=529) of grade 1 quality were used in this study. Embryos were frozen in 1.5MEG in Dulbecco’s PBS (DPBS) supplemented with 0.1M sucrose, 4mgmL−1 BSA and 20% fetal calf serum (FCS). Embryos were transferred into freezing medium, loaded into 0.25-mL straws and kept for more than 15min for equilibration; then the straws were plunged into a −7°C methanol bath of a programmable freezer for 1min, seeded at −7°C, held at −7°C for 14min, cooled to −30°C at the rate of −0.3°Cmin−1 and then plunged into liquid nitrogen. The straws were thawed by holding in air for 6sec, and then placed in water at 30°C for 15s. After thawing, the straws were held for 0, 10, 20, 30 and 60min (holding time) at either 38.5 or 26.0°C. Ethylene glycol was removed from the embryos by placing them into DPBS supplemented with 20% CS at 38.5°C more than 20min. The embryos were cultured in TCM-199 supplemented with 20% FCS and 0.1mM β-mercaptoethanol under a gas phase of 5% CO2 in air at 38.5°C for 72h. Viability of embryos was evaluated at 0-, 24-, 48- and 72-h incubation by their morphological development. Data were analyzed by ANOVA. There was no significantly difference in the survival rate of thawed embryos held at 38.5°C or 26.0°C for the same holding periods. The survival rate of the thawed embryos held at 38.5°C decreased significantly when the holding period exceeded 30min compared with no holding period after 24- and 72-h culture (P&lt;0.05, respectively). On the other hand, the survival rate of the thawed embryos held at 26.0°C decreased significantly when the holding time was 60min compared with less than 20min of holding after 24-h culture, and less than 10min after 72-h culture (P&lt;0.05, respectively). Therefore, toxicity of EG was observed when thawed embryos were held for 30 and 60min at 38.5°C and 60min at 26.0°C. These results suggest the toxicity of EG in direct transfer methods can be avoided by transferring the embryos within 20min after thawing. Table 1

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.

83 VITRIFICATION OF IMMATURE AND IN VITRO-MATURED HORSE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 149 ◽  
Author(s):  
L. Bogliolo ◽  
F. Ariu ◽  
I. Rosati ◽  
M. T. Zedda ◽  
S. Pau ◽  
...  
Keyword(s):  
Survival Rate ◽  
Ethylene Glycol ◽  
Survival Rates ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Hoechst 33342 ◽  
Nuclear Maturation ◽  
And Control ◽  
Cryopreservation Protocol

Few attempts have been carried out to cryopreserve equine oocytes, and an effective cryopreservation protocol is not defined yet. Studies were conducted to compare the viability of immature and in vitro-matured horse oocytes vitrified by the minimal volume cooling (MVC) cryotop vitrification method (Kuwayama et al. 2005 Reprod. BioMed. Online 11, 300–308). Oocytes were recovered from slaughterhouse ovaries and divided, on the basis of the morphology of cumulus cells, into cumulus-expanded (CE) and cumulus-compacted (CC) oocytes. Groups of CC and CE oocytes were vitrified immediately after recovery [germinal vesicle (GV) stage] or matured in vitro (IVM) and cryopreserved at the MII stage as follows: oocytes were incubated 30 min in TCM-199 + 20% FCS + 10% ethylene glycol (EG) + 10% DMSO, followed by 20 min in TCM-199 + 20% FCS + 20% EG + 20% DMSO + 0.25 M sucrose, loaded in cryotops (2 µL), and plunged into liquid nitrogen. Warming was performed at 38.5°C by washing the oocytes in TCM-199 + 20% FCS with decreasing sucrose concentrations (1.25 M, 0.62 M, 0.31 M). After warming oocytes cryopreserved at the GV stage were matured in vitro for 24 h (CE) or 36 h (CC) in TCM-199 + 10% FCS + FSH, LH each at (0.1 UI/mL) + cysteamine, fixed, and stained with glycerol-Hoechst 33342 to assess nuclear maturation. Oocytes vitrified at the MII stage were in vitro cultured for 2 h to evaluate their morphological survival on the basis of the presence of an intact zona pellucida and membrane. Nonvitrified oocytes undergoing the same maturation protocol were used as controls. Results (Table 1) indicated that the survival rate of oocytes vitrified at the GV stage, after IVM, was similar between CE and CC oocytes (43.6% vs 42.6%). Significantly (P < 0.01) higher numbers of vitrified CE MII oocytes (52.9%) survived, compared to CC (34.8%), after 2-h culture. The percentages of viable MII oocytes from CE and CC GV vitrified oocytes were 43.6% and 40.9% respectively and were comparable to those from vitrified MII oocytes (CE, 52.9%; CC, 34.8%) and control oocytes (CE, 56.4%; CC, 53.3%). In conclusion, the results of this study showed that vitrification by the MCV Cryotop method of horse oocytes at either the GV or the MII stage allows a similar number of viable mature oocytes to be recovered. Table 1. Maturation and survival rates of immature and mature equine oocytes vitrified by the MCV Cryotop method

31 IN VITRO SURVIVAL AND PREGNANCY OUTCOME OF ZONA-FREE TRANSGENIC GOAT CLONED EMBRYOS AFTER OVIDUCTAL TRANSFER TO FEMALE RECIPIENTS ON DAY 1 OF DEVELOPMENT

2013 ◽  
Vol 25 (1) ◽  
pp. 163
Author(s):  
C. Feltrin ◽  
N. Mohamad-Fauzi ◽  
L. H. Aguiar ◽  
S. G. Neto ◽  
L. T. Martins ◽  
...  
Keyword(s):  
Pregnancy Outcome ◽  
Embryo Transfer ◽  
Cell Fusion ◽  
Cell Nucleus ◽  
Zona Pellucida ◽  
Cell Injection ◽  
Transfer Method ◽  
Nucleus Transfer ◽  
In Vitro Survival

Survival to term following embryo transfer is usually low after cloning by somatic cell nuclear transfer (SCNT). The aims of this study were to evaluate the effects of the zona pellucida (presence or absence) and the cell nucleus transfer method (cell fusion or cell injection) on the in vitro survival and pregnancy outcome of Day-1 goat cloned embryos transferred into the oviduct of recipient females. In vitro-matured goat oocytes from slaughterhouse ovaries were polar body selected, with a group of oocytes subjected to enzymatic zona pellucida removal. Zona-free (ZF) and zona-intact (ZI) oocytes were enucleated by micromanipulation procedures (Oback and Wells 2003 Cloning Stem Cells 5, 3–12; Keefer et al. 2000 Biol. Reprod. 66, 199–203). Somatic nucleus donor cells from 3 transgenic females for mammary gland expressing human lysozyme (Maga et al. 2003 Trans. Res. 12, 485–496) were either fused (CF) with enucleated ZI and ZF oocytes or injected (CI) into enucleated ZI oocytes, with the assessment of fusion or injection survival rates performed after 60 min. Two direct-current (DC) pulses were used to induce fusion in the ZI group (2 kV cm–1 each for 10 µs) and in the ZF group (1.0 kV cm–1 for 20 µs). Embryo reconstruction using ZI oocytes was done by micromanipulation. Zona-free (CF) or ZI (CF or CI) reconstructed cloned embryos were chemically activated in ionomycin/DMAP, followed by in vitro culture for 12 h prior to the surgical embryo transfer into the oviducts of synchronous female recipients (Day 1). Pregnancy diagnosis was carried out on Day 30 of gestation by ultrasonography. Survival after cell fusion, cell injection, and embryo transfer were compared by the χ2 test, for P < 0.05. After 16 replications, 1047 in vitro-matured oocytes obtained from 208 does were used for embryo reconstruction (Table 1). In vitro survival was higher in ZI oocytes that were injected with somatic cells than in ZI oocytes or ZF subjected to cell fusion. Pregnancy rates were similar between groups, irrespective of the cell nucleus transfer method or the presence or not of the zona pellucida, but the overall efficiency (fetal survival/recipients) was higher in the ZI-CF group. Currently, 2 ongoing pregnancies carrying 3 cloned concepti from ZI oocytes fused to somatic cells are in late gestation (>110 days). In conclusion, the cell injection method promoted higher survival and, consequently, better efficiency than cell fusion for the reconstruction of goat cloned embryos. However, the zona removal did not affect subsequent in vivo embryo development, as the transfer of zona-free embryos into the oviducts of synchronous recipients resulted in similar pregnancy rates than with zona-intact embryos. Table 1.In vitro survival and pregnancy outcome of goat cloned embryos after embryo reconstruction and transfer to female recipients on Day-1 of development

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