In vitro comparisons of two cryopreservation techniques for equine embryos: slow-cooling and open pulled straw (OPS) vitrification.

Vitrification using open pulled straw (OPS) has provided encouraging results with embryos from other species. The aim of this study was to compare the survival of 6.5- and 6.75-day-old equine embryos after OPS vitrification and slow-cooling. Eighteen embryos were frozen using a slow-cooling method. Embryos were placed in modified PBS with increasing glycerol concentration (2.5%, 5%, 7.5% and 10% (v/v) 5 min each). Embryos were loaded into 0.25 ml straws then placed in a programmable freezer and subsequently plunged into liquid nitrogen. After thawing, cryoprotectant was removed by five steps with decreasing glycerol and sucrose concentrations. Twenty embryos were vitrified using the OPS method. Embryos were exposed to 7.5% dimethyl-sulfoxide (DMSO)+7.5% ethylene glycol (EG) for 3 min and in 18% DMSO+18% EG+0.4M sucrose for 1 min, loaded in OPS and plunged into liquid nitrogen. After warming, embryos were placed in decreasing sucrose concentrations. All embryos were cultured in synthetic oviduct fluid (SOF) medium for 3h and evaluated using 4',6-diamidino-2-phenylindole (DAPI) staining. The percentage of cells entering in S-phase (%SC) was evaluated by incorporation of BrdU. No significant differences were observed for mean diameter, morphological grade and percentage of degenerate embryos after 3h of culture for slow-cooling and OPS methods. The percentage of dead cells per embryo was similar for the two procedures (42+/-6 versus 46+/-9). The percentage of cells entering in S-phase did not differ significantly between the two procedures (27+/-5 versus 26+/-6). OPS vitrification may be as efficient as slow-cooling for the cryopreservation of equine embryos. However, these results should be confirmed by the transfer of OPS vitrified embryos to recipient mares.

Moussa M ，Bersinger I ，Doligez P ，Guignot F ，Duchamp G ，Vidament M ，Mermillod P ，Bruyas JF ... -  《THERIOGENOLOGY》

One-step dilution of open-pulled-straw (OPS)-vitrified mouse blastocysts in sucrose-free medium.

Embryos vitrified by the open-pulled-straw (OPS) method are only briefly exposed to cryoprotectants and not fully equilibrated with the cryoprotectant. That being the case, conceivably the post-thawing de- and rehydration processes may be omitted. This would render thawing and dilution in a single step and direct transfer to recipients possible without the need for a microscope and other laboratory equipment. Morphologically intact mouse blastocysts from superovulated 5- to 8-week-old virgin female NMRI mice were vitrified according to a protocol [6] slightly modified from the classical OPS-procedure of Vajta et al. [29] consisting of exposure to 10% dimethyl-sulfoxide (Me(2)SO)+10% ethylene glycol (EG) for 1 min, followed by 20% Me(2)SO+20% EG for 20s before loading into straws that are plunged into liquid nitrogen. In Group 1, 75 blastocysts were exposed to the standard thawing and dilution regimen involving exposure to three solutions of decreasing sucrose content (Control). In Groups 2, 3 and 4, 75 blastocysts each were transferred, in a single step, to medium at 37 degrees C containing 0.66, 0.33 or 0M sucrose, respectively. After 48 h of in vitro culture the proportion of hatched blastocysts was determined. In Group 1, this proportion amounted to 82.7%, in Groups 2, 3 and 4 to 76.0%, 73.3% and 78.7%, respectively (P>0.05). To examine their potential to continue development in vivo, OPS-vitrified blastocysts thawed according to the regimens of Groups 1 and 4 were transferred to recipients (10 embryos/recipient). In Group 1, 9/10 recipients got pregnant with 4.7+/-0.6 (mean+/-SEM) fetuses, in Group 4, 8/10 recipients with 5.0+/-0.5 fetuses. The overall embryo survival rate per group was 42% for Group 1 and 40% for Group 4. All fetuses were normally developed and viable and there were no significant differences between groups (P>0.05). It may be concluded that warming and transfer of OPS-vitrified mouse embryos in a single step in medium devoid of sucrose is feasible, which is tantamount to a substantial simplification of embryo transfer operations.

El-Gayar M ，Gauly M ，Holtz W 《-》

Viability of OPS vitrified sheep embryos after direct transfer.

The aim of this study was to evaluate the viability in the effect of open pulled straw (OPS) vitrification procedure of sheep embryos after direct transference. Embryos were produced in vivo and cryopreserved in slow freezing or OPS vitrification. The survival rates of cryopreserved embryos were compared to non-frozen standard pattern. In a first set of experiments, embryos at morula and blastocyst stages were dived in ethylene glycol (1.5 M) and frozen in an automatic freezer. After being thawed, they were directly or indirectly transferred to ewes recipient. A second group of embryos were drawn into OPS and plunged into liquid nitrogen after being exposed at room temperature for 1 min and 45 s in 10% EG plus 10% dimethyl sulphoxide (DMSO), then again for 30 s in 20% EG + 20% DMSO + 0.5 M sucrose. After being warmed, embryos were also directly transferred using a French mini straw as the catheter for the transplantation process or after in vitro dilution of cryoprotectants (two-step-process). No significant difference was observed among fresh, frozen or vitrified embryos on pregnancy rate (50.0%, 38.6% and 55.8%). However, when we evaluated only the direct transference, the pregnancy rate of OPS vitrified embryos was higher than that of frozen embryos (57.1% vs 34.8%) (p = 0.07). In addition, vitrified morulae had a higher pregnancy rate than the one with frozen embryos (64.0% vs 38.9%) (p = 0.07). Finally, our results indicate that OPS vitrification technique in association with direct transference improves the viability of sheep embryos with potential applications to field conditions.

Green RE ，Santos BF ，Sicherle CC ，Landim-Alvarenga FC ，Bicudo SD ... -  《-》

Vitrification of early-stage bovine and equine embryos.

The objectives of this study were to: (1) determine an optimal method and stage of development for vitrification of bovine zygotes or early embryos; and (2) use the optimal procedure for bovine embryos to establish equine pregnancies after vitrification and warming of early embryos. Initially, bovine embryos produced by in-vitro fertilization (IVF) were frozen and vitrified in 0.25mL straws with minimal success. A subsequent experiment was done using two vitrification methods and super open pulled straws (OPS) with 1- or 8-cell bovine embryos. In Method 1 (EG-O), embryos were exposed to 1.5M ethylene glycol (EG) for 5min, 7M ethylene glycol and 0.6M galactose for 30s, loaded in an OPS, and plunged into liquid nitrogen. In Method 2 (EG-DMSO), embryos were exposed to 1.1M ethylene glycol and 1.1M dimethyl sulfoxide (DMSO) for 3min, 2.5M ethylene glycol, 2.5M DMSO and 0.5M galactose for 30s, and loaded and plunged as for EG-O. Cryoprotectants were removed after warming in three steps. One- and eight-cell bovine embryos were cultured for 7 and 4.5 d, respectively, after warming, and control embryos were cultured without vitrification. Cleavage rates of 1-cell embryos were similar (P>0.05) for vitrified and control embryos, although the blastocyst rates for EG-O and control embryos were similar and higher (P<0.05) than for EG-DMSO. The blastocyst rate of 8-cell embryos was higher (P<0.05) for EG-O than EG-DMSO. Therefore, EG-O was used to cryopreserve equine embryos. Equine oocytes were obtained from preovulatory follicles. After ICSI, injected oocytes were cultured for 1-3 d. Two- to eight-cell embryos were vitrified, warmed and transferred into recipient's oviducts. The pregnancy rate on Day 20 was 62% (5/8) for equine embryos after vitrification and warming. In summary, a successful method was established for vitrification of early-stage bovine embryos, and this method was used to establish equine pregnancies after vitrification and warming of 2- to 8-cell embryos produced by ICSI.

Campos-Chillòn LF ，Suh TK ，Barcelo-Fimbres M ，Seidel GE Jr ，Carnevale EM ... -  《THERIOGENOLOGY》

In vitro assessment of a direct transfer vitrification procedure for bovine embryos.

We developed a simple vitrification technique for bovine embryos that could permit direct transfer. Embryos were produced in-vitro by standard procedures. The base medium for cryopreservation was a chemically defined medium similar to SOF + 25 mM Hepes and 0.25% fatty acid free bovine serum albumin (FAF-BSA) (HCDM2). In experiment 1, embryos were first exposed to 3.5M ethylene glycol (V1) for 1, 2 or 3 min at room temperature (20-24 degrees C), and then moved to 7 M ethylene glycol (V2) at 4 or 20-24 degrees C and loaded in 0.25-mL straws. After 45 s in 7 M ethylene glycol, straws were placed in liquid nitrogen. Embryos that were loaded at 20-24 degrees C had higher survival rates than those loaded at 4 degrees C (P<0.05). Exposure for 1 min was best for morulae, while 3 min was best for blastocysts. In experiment 2, blastocysts were handled at 24 degrees C and exposed to two concentrations of ethylene glycol in V1 (3.5 or 5 M) followed by V2 as in experiment 1, two warming temperatures (20 or 37 degrees C) and two post-warming holding times until culture (5 or 15 min). Exposure to 5 M ethylene glycol and warming at 37 degrees C was the optimal combination of procedures, and embryos survived well after 15 min in straws if warmed at 37 degrees C. In experiment 3, ethylene glycol concentration (3, 4 or 5 M) and exposure time (0.5 or 1 min) during two-step addition of cryoprotectant were studied for bovine morulae. In experiment 4, morulae were exposed to V2 for 30 or 45 s in HCDM2 or Vigro holding medium and then held in 22-24 degrees C air or 37 degrees C water post-warming. Experiment 5 was like experiment 4 except blastocysts were used. Overall survival rates of blastocysts in experiment 5 averaged 80% of non-vitrified controls after 48 h culture. The survival rates with in vitro-produced morulae in experiments 1, 3 and 4 were unacceptable. Vitrification solutions based on Vigro tended to result in higher survival than HCDM2 for blastocysts, but not morulae. In experiment 6, the survival rate in vitro of in vivo-produced morulae and blastocysts after two-step vitrification was nearly 100%. Our vitrification technique was very effective for in vitro produced blastocysts, but not for in vitro-produced morulae.

Campos-Chillòn LF ，Walker DJ ，de la Torre-Sanchez JF ，Seidel GE Jr ... -  《THERIOGENOLOGY》