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1.
The aim of this study was to investigate the effects of different vitrification solutions [EFS30 or EFS40 contains 30% (v/v) ethylene glycol (EG), 40% (v/v) EG; EDFS30 or EDFS40 contains 15% (v/v) EG and 15% (v/v) dimethyl sulfoxide (DMSO), 20% (v/v) EG and 20% (v/v) DMSO], equilibrium time during vitrification (0.5-2.5 min) and vitrification protocols [one-step straw, two-step straw and open-pulled straw (OPS)] on in vivo development of vitrified Boer goat morulae and blastocysts after embryo transfer. In the one-step straw method, the lambing rates of vitrified embryos in EFS30 (37.5%), EFS40 (40.5%) or EDFS30 (38.2%) group were similar to that of fresh embryos (57.5%) and conventional freezing method (46.7%) when the equilibrium time was 2 min. In the two-step straw method, the highest lambing rate was obtained when embryos were pretreated with 10% EG for 5 min and then exposed to EFS40 for 2 min (51.4%), showing similar lambing rates compared with fresh embryos (56.1%) or the embryos cryopreserved by conventional freezing method (45.2%). In the OPS method, the lambing rate in EFS40, EDFS30 or EDFS40 groups were similar to that (57.1%) of fresh embryos, or to that (46.0%) of embryos cryopreserved by conventional freezing method. The highest lambing rate (51.4%) of the group of OPS was obtained when the embryos were vitrified with EDFS30. In conclusion, either the two-step straw method in which embryos were pretreated in 10% EG for 5 min and then exposed to EFS40 for 2 min, or the OPS method in which embryos were pretreated in 10% EG + 10% DMSO for 30 s and then exposed to EDFS30 for 25 s was a simple and efficient method for the vitrification of Boer goat morulae and blastocysts.  相似文献   

2.
An efficient cryopreservation protocol for porcine morulae was investigated with three types of vitrification having different cooling rates (Exp. 1). Survival of embryos vitrified after removal of cytoplasmic lipid droplets was also examined by means of the minimum volume cooling (MVC) method (Exp. 2). In Exp. 1, the morula stage embryos were vitrified with a 0.25 ml plastic straw (ST-method), gel loading tip (GLT-method) and the MVC-method, respectively, and stored in liquid nitrogen after which they were warmed in sucrose solutions with cryoprotectants being subsequently removed in a stepwise manner. In Exp. 2, morulae were centrifuged with 7.5 microg/ml cytocharasin B at 12000 x g for 20 min to polarize the cytoplasmic lipid droplets that were then removed from the embryos by micromanipulation (delipation). Both those delipated at the morula stage and the intact embryos at the morula to blastocyst stages were vitrified by the MVC-method. In vitro survival of the vitrified embryos was assessed in both experiments by culturing in NCSU-23 + 10% FCS for 48 h. In vitro developments of vitrified embryos after warming to blastocysts were 20% (6/30) for the ST-method, 39% (18/46) for the GLT-method, and 60% (26/43) for the MVC-method. Embryo survival was further improved by vitrification after delipation (95%, 35/37) compared to intact vitrified morulae (24/42, 57%, P<0.001) and blastocysts (23/31, 74%, P<0.05). Moreover, the number of cells in blastocysts (92 +/- 25) derived from the delipated-vitrified morulae was comparable to those derived from intact control non-vitrified embryos (103 +/- 31). Our results demonstrate that vitrified porcine morulae have the highest survival when using the MVC-method in conjunction with delipation.  相似文献   

3.
用不同冷冻载体(玻璃管、塑料管和0.25 mL细管)及不同冷冻方法(程序化冷冻和玻璃化冷冻)对小鼠3.5 d~4 d桑椹胚和囊胚进行冷冻保存,并与不做任何冷冻保存处理直接培养进行对比。结果表明,使用玻璃管、塑料管和0.25 mL细管作为胚胎的承载材料进行玻璃化冷冻,效果差异不显著;采用程序化冷冻与OPS玻璃化冷冻法,对小鼠胚胎进行冷冻保存可以取得较好的结果。从而得出,用不同材质的冷冻载体进行玻璃化冷冻,可以获得与程序化冷冻相同的良好效果。  相似文献   

4.
This study was conducted to examine the utility of vitrification for bovine embryos with low‐quality grade, and simple cryoprotectants dilution method for practitioners. In Experiment 1, survival of frozen embryos was compared with that of vitrified embryos using minimum volume cooling (MVC). Then, vitrified embryos were used to confirm the optimum sucrose concentration in Experiment 2. The survival rates of embryos that had been vitrified following diluted cryoprotectants with the one‐step in‐straw method were compared with those of fresh control embryos in Experiment 3. Frozen‐thawed or vitrified‐warmed blastocysts were cultured with TCM‐199 supplemented with 100 μmol/L beta‐mercaptoethanol +5% fetal bovine serum at 38.5°C in an atmosphere of 5% CO2 in air, their survival after 24 hr were compared. The development to term of fair quality in vivo embryos after vitrification was examined in Experiment 4. Results show that survival rates of frozen‐thawed embryos were lower (p < .05) than that of vitrified‐warmed ones. When vitrified embryos were warmed in 0.3 mol/L sucrose in straws, their survival rate was 100%. The total cell numbers of vitrified‐warmed embryos were comparable to those of fresh control embryos. The six calves from 13 vitrified embryos were delivered in Experiment 4. These results indicate that MVC vitrification following one‐step cryoprotectants dilution is utilized to preserve low‐quality bovine embryos.  相似文献   

5.
Solid surface vitrification (SSV) was compared with in-straw vitrification for cryopreservation of biopsied mouse embryos. Eight-cell stage embryos were zona drilled and one blastomere was removed. Developed morulae or blastocysts were vitrified in microdrop (35% EG + 5% PVP + 0.4 M trehalose) or in straw (7.0 M EG + 0.5 M sucrose). Following recovery, embryos were cultivated in vitro or transferred into recipients. Cryopreservation had an effect not only on the survival of biopsied embryos but also on their subsequent development in vitro. Cryosurvival of biopsied morulae vitrified in straw was significantly inferior to SSV. The post-warm development of biopsied and non-biopsied morulae was delayed on Day 3.5 and 4.5 in both vitrification groups. A delay in development was observed on Day 5.5 among vitrified non-biopsied blastocysts. The percentage of pups born from biopsied morulae or blastocysts following cryopreservation did not differ from that of the control. No significant differences could be detected between methods within and between embryonic stages in terms of birth rate. The birth rate of biopsied embryos vitrified in straw was significantly lower compared to the non-biopsied embryos. The novel cryopreservation protocol of SSV proved to be effective for cryopreservation of morula- and blastocyst-stage biopsied embryos.  相似文献   

6.
Mammalian embryos are most commonly cryopreserved in liquid nitrogen; however, liquid nitrogen is not available in special environments, such as the International Space Station (ISS), and vitrified embryos must be stored at −80°C. Recently, the high osmolarity vitrification (HOV) method was developed to cryopreserve mouse 2-cell stage embryos at −80°C; however, the appropriate embryo is currently unknown. In this study, we compared the vitrification resistance of in vivo-derived, in vitro fertilization (IVF)-derived, and intracytoplasmic sperm injection (ICSI)-derived mouse 2-cell embryos against cryopreservation at −80°C. The ICSI embryos had lower survival rates after warming and significantly lower developmental rates than the in vivo and IVF embryos. Further, IVF embryos had a lower survival rate after warming, but a similar rate to the in vivo embryos to full-term development. This result was confirmed by simultaneous vitrification of in vivo and IVF embryos in the same cryotube using identifiable green fluorescent protein-expressing embryos. We also evaluated the collection timing of the in vivo embryos from the oviduct and found that late 2-cell embryos had higher survival and developmental rates to full-term than early 2-cell embryos. Some early 2-cell embryos remained in the S-phase, whereas most late 2-cell embryos were in the G2-phase, which may have affected the tolerance to embryo vitrification. In conclusion, when embryos must be cryopreserved under restricted conditions, such as the ISS, in vivo fertilized embryos collected at the late 2-cell stage without long culture should be employed.  相似文献   

7.
绵羊玻璃化冷冻胚胎直接移植试验研究   总被引：1，自引：0，他引：1  
应用EFS40玻璃化液对6.5～7日龄的绵羊胚胎进行玻璃化冷冻及解冻后直接移植试验.结果:桑椹胚、囊胚冷冻解冻后移植的妊娠率分别为37.50%(3/8)和54.55%(6/11),胚胎存活率分别为33.33%(3/9)和50.00%(6/12),差异均不显著(P＞0.05);胚胎解冻后用0.5 mol/L蔗糖脱防冻剂与直接用胚胎存放液脱除防冻剂的妊娠率分别为44.44%(4/9)和50.00%(5/10),胚胎存活率分别为40.00%(4/10)、45.45%(5/11)差异不显著(P＞0.05);10枚解冻后的胚胎细管内脱防冻剂后,直接装管移植给8只受体,妊娠率为50.00%(4/8),胚胎成活率为40.00%(4/10),与同期常规冷冻解冻组相比无显著差异(P＞0.05).  相似文献   

8.
The present study was conducted to examine post-thaw in vitro developmental competence of buffalo embryos cryopreserved by cytoskeletal stabilization and vitrification. In vitro produced embryos were incubated with a medium containing cytochalasin-b (cyto-b) in a CO2 incubator for 40 min for microfilament stabilization and were cryopreserved by a two-step vitrification method at 24℃ in the presence of cyto-b. Initially, the embryos were exposed to 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in a base medium for 4 min. After the initial exposure, the embryos were transferred to a 7 µl drop of 25% EG and 25% DMSO in base medium and 0.3 M sucrose for 45 sec. After warming, the embryos were cultured in vitro for 72 h. The post-thaw in vitro developmental competence of the cyto-b-treated embryos did not differ significantly from those vitrified without cyto-b treatment. The hatching rates of morulae vitrified without cyto-b treatment was significantly lower than the non-vitrified control. However, the hatching rate of cyto-b-treated vitrified morulae did not differ significantly from the non-vitrified control. This study demonstrates that freezing of buffalo embryos by cytoskeletal stabilization and vitrification is a reliable method for long-term preservation.  相似文献   

9.
The capacity of different vitrification media and methods was tested onto in vivo and in vitro produced bovine morula/blastocysts and their ultrastructure and survival studied post-thawing. Two vitrification solutions were finally selected, named 40 ES (40% ethylene glycol in PBS containing 0.5 M sucrose) and 35 EFS (composed of 35% (v/v) ethylene glycol in PBS containing 0.5 M/l sucrose and 30% (w/v) Ficoll 70). The straws were either precooled or not precooled in nitrogen vapour, plunged and stored in LN2 for 10–25 days, and then thawed in a 20° C waterbath. The content of the straws was rediluted in 1M sucrose solution in PBS and later cocultured with BOEC for 48 h. The overall survival rates for in vitro and in vivo embryos were 36% (12 of 33) and 20% (3 of 15) after 24 h and 21% (7 of 33) and 33% (5 of 15 ) after 48 h. The survival rates for precooled embryos were significantly higher than for not precooled (48% vs 13% after 24 h and 44% vs 4% after 48 h) when tested across vitrification media. The in vitro-produced embryos presented an ultrastructure similar to the pre-freeze state, irrespective of the vitrification media used. The in vivo developed embryos showed a rather modified post-thaw ultrastructure, with clear signs of osmotic changes at both the trophoblastic and embryonic cells. The results indicated that in vitro and in vivo developed bovine embryos can survive vitrification using ethylene glycol as a cryoprotectant.  相似文献   

10.
The aim of this study was to determine the optimum conditions for vitrifying in vitro produced day 7 porcine embryos using different vitrification devices and blastocoele collapse methods. Firstly embryos were collapsed by micro-pipetting, needle puncture and sucrose with and without conducting vitrification. In the next experiment, non-collapsed embryos were vitrified in an open device using either superfine open-pulled straws (SOPS) or the CryoLoopTM system, or vitrified in a closed device using either the CryoTipTM or Cryo BioTM’s high security vitrification system (HSV). The post-thaw survival of embryos vitrified in the open devices did not differ significantly (SOPS: 37.3%; CryoLoopTM: 37.3%) nor did the post-thaw survival of embryos vitrified in the closed devices (CryoTip™: 38.5%; HSV: 42.5%). The re-expansion rate of embryos that were collapsed via micro-pipetting (76.0%) did not differ from those that were punctured (75.0%) or collapsed via sucrose (79.6%) when vitrification was not performed. However, embryos collapsed via sucrose solutions (24.5%) and needle puncture (16.0%) prior to vitrification were significantly less likely to survive vitrification than the control (non-collapsed) embryos (53.6%, P < 0.05). The findings show that both open and closed vitrification devices were equally effective for the vitrification of porcine blastocysts. Collapsing blastocysts prior to vitrification did not improve survival, which is inconsistent with the findings of studies in other species. This may be due to the extremely sensitive nature of porcine embryos, and/or the invasiveness of the collapsing procedures.  相似文献   

11.
Generating techniques to enhance the success of blastomere separation is important for bovine economy, because it increases the number of transferable embryos. This study aimed to identify the optimum cryoprotectants for the vitrification of bovine embryos and the separation of blastomeres at different stages. In experiment 1, expanded blastocysts were vitrified in two different vitrification solutions, either (1) ethylene glycol (EG) + propylene glycol (PG) or (2) EG. The survival rate of blastocysts in the EG + PG was higher than that of the EG. In experiment 2, intact two‐cell and eight‐cell stage embryos were vitrified in the same solutions used in experiment 1. The EG + PG produced more dead embryos than the EG (P < 0.05). In the EG, the rate of blastocyst formation was similar for the vitrified two‐ and eight‐cell embryos and the non‐vitrified ywo‐cell embryos. In experiment 3, separated blastomeres of two‐ and eight‐cell embryos were vitrified in EG. There was no difference in the rate of blastocyst formation and total number of cells between the two vitrified groups. In summary, at the blastocyst stage, EG + PG was superior, based on both survival rates and cell numbers; however, at the 2–8 cell stage, the use of EG alone was better than the other groups.  相似文献   

12.
转基因兔胚胎玻璃化冷冻保存的研究   总被引：4，自引：0，他引：4  
在２５℃条件下,将兔体外受精精子载体转基因兔桑椹胚置于含有４０％乙二醇、１８％Ｆｉｃｏｌ、０．３ｍｏｌ蔗糖的ｍＰＢＳ溶液（ＥＦＳ４０）中平衡２分钟,然后直接投入液氮,成功地进行了玻璃化冷冻保存。解冻后桑椹胚发育至囊胚和孵化囊胚的比例分别为６５．８１％和３９．２４％,与未经冷冻的鲜胚发育比例（７１．０５％和４３．４２％）相比,没有明显的差异。７８枚经玻璃化冷冻和解冻的桑椹胚移植给５只受体,其中２只妊娠,共产下８只活仔兔。  相似文献   

13.
In this study the efficacy of the combination of glycerol (GLY) and ethylene glycol (EG) as cryoprotectants in a vitrification method developed for direct embryo transfer was evaluated by in vitro development of in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos after vitrification. The IVF and SCNT blastocysts were vitrified in either 40% GLY, 30% GLY + 10% EG, or 20% GLY + 20% EG using French straws. After warming, the straws were held vertically for 1 min without shaking and were then placed horizontally for 5 min to dilute the cryoprotectants. After washing, the vitrified-warmed embryos were cultured in vitro for 72 h. There were no differences among the vitrification solutions with respect to the rates of vitrified-warmed IVF and SCNT embryos surviving and developing to the hatched blastocyst stage. However, the rates of development to the hatched blastocyst stage of the SCNT embryos vitrified with 40% GLY tended to be higher than those vitrified with 30% GLY + 10% EG or 20% GLY + 20% EG (26% vs. 7-8%, respectively). The development rates to the hatched blastocyst stage of the IVF and SCNT embryos vitrified with solution containing EG were significantly lower (P<0.05) than those of non-vitrified embryos. These results suggest that use of the combination of GLY and EG as cryoprotectants had no beneficial effect on the viability of embryos after in-straw dilution. However, this method is so simple that it can be used for practical direct transfer of vitrified embryos in the field.  相似文献   

14.
小鼠桑椹胚简易玻璃化冷冻技术再探讨   总被引：12，自引：0，他引：12  
本试验继小鼠扩张囊胚玻璃化冷冻保存成功后，在室温（２５℃）下利用不同浓度的ＥＦＳ玻璃化溶液，对小鼠的桑椹胚简易玻璃化冷冻技术进行再探讨。结果是胚胎在１０％ＥＧ溶液中预先处理５分钟，再移入事先配置好含有ＥＦＳ３０的０．２５ｍｌ塑料细管中１分钟平衡后直接投入液氮中冷冻，解冻后获得的发育率最高（９４％）。冻胚移植后妊娠率和产仔率分别为５６％（９／１６）及４２％（４９／１１６）。与对照组相比差异不显著（Ｐ＞０．０５）  相似文献   

15.
In vitro development of embryos collected from the same gilts mated at first and third estrus was compared. Embryos from one to eight cells were collected from gilts 36 to 48 h after detection of estrus. Embryos were cultured for 8 d in Whitten's medium in a humidified atmosphere of 5% CO2 in air at 37 degrees C and were observed daily. No differences were detected among percentages of one- to eight-cell embryos developing into morulae from gilts in first or third estrus (P greater than .05). Similar percentages of one- to two-cell embryos from gilts mated at first and third estrus developed into blastocysts (45.8 and 55.2%, respectively), expanded blastocysts (10.4 and 24.1%, respectively) and hatching blastocysts (4.2 and 3.4%, respectively; P greater than .05). Fewer three- to eight-cell embryos from gilts in first estrus than from gilts in third estrus developed into blastocysts (63.4 and 91.1%) and expanded blastocysts (14.6 and 55.6%; P less than .01). Similar percentages of embryos with abnormal morphology were observed among morulae developing from one- to eight-cell embryos collected from gilts mated at first and third estrus (14.9 and 9.9%, respectively; P greater than .05). In contrast, more morphologically abnormal embryos were observed among blastocysts developing from gilts mated at first estrus than at third estrus (31.2% and 14.0%, respectively; P less than .05). The results suggest that the reduced in vitro development of embryos collected from gilts mated at first estrus may be due to an aberration in blastocoel formation and expansion.  相似文献   

16.
Equine embryos can remain viable for 12 to 24 hours when cooled and stored at 5°C.1 Cryopreservation of embryos would allow for long-term preservation of genetic material and more efficient management of embryo recipients. This study compared pregnancy rates after transfer of equine embryos vitrified within 1 hour of collection or cooled for 12 to 19 hours before vitrification. Mares (N = 40) were superovulated using equine follicle-stimulating hormone (eFSH). Embryos were recovered 6.5 days after ovulation or 8 days after human chorionic gonadotropin. Forty morulae or early blastocysts with a grade of 1 to 2 and <300 mm in diameter were randomly assigned to 1 of 2 treatments: Group 1 (n = 20), washed 4 times in a commercial holding medium and then vitrified; Group 2 (n = 20), washed 3 times and then stored in the same holding medium at 5°C to 8°C in a passive cooling device for 12 to 19 hours before being vitrified. To thaw, embryos were warmed by holding the straw in air at room temperature for 10 seconds and then submerged in a water bath (20°C to 22°C) for an additional 10 seconds. The contents of the straw were transferred directly into a recipient that had ovulated 4 to 6 days previously. There were no differences (P > .05) in embryo diameter, grade, or morphology score between treatment groups before vitrification. Pregnancy rates (day 16) were not different (P > .05) between embryos vitrified immediately after collection (15 of 20; 75%) and embryos cooled for 12 to 19 hours before vitrification (13 of 20; 65%). Based on these results, small equine embryos (<300 mm) can be stored at 5°C to 8°C for 12 to 19 hours before vitrification without a significant loss of viability.  相似文献   

17.
This study examined the effects of different vitrification medium compositions and exposure times (2, 4 and 6min) on the post-thaw development of buffalo embryos produced in vitro (IVP). The compositions were (1) 40% ethylene glycol (EG); (2) 25% glycerol (G)+25% EG, and (3) 25% EG+25% dimethylsulfoxide (DMSO). The base medium was 25mM Hepes-buffered TCM-199+10% steer serum +50microg/mL gentamycin. The IVP embryos were cryopreserved by a two-step vitrification method at 24 degrees C. After warming, the embryos were cultured in vitro for 72h. The vitrification of morulae and blastocysts in 25% EG+25% DMSO with an exposure time of 2 and 4min, respectively, resulted in a better hatching rate than other combinations. The hatching rate of morulae vitrified in 25% EG+25% G, 25% EG+25% DMSO, and blastocysts vitrified in 40% EG, 25% EG+25% DMSO were negatively correlated with exposure time. However, the hatching rate of blastocysts vitrified in 25% EG+25% G was positively correlated with exposure time. The study demonstrated that the post-thaw in vitro development of IVP buffalo embryos was affected by the vitrification medium composition and exposure time.  相似文献   

18.
The aim of this study was to determine whether vitrification is an effective method when used for Japanese Black Cattle (Wagyu) in vivo‐derived embryos, collected following a superovulation treatment and embryo transfer (MOET) programme. In vivo‐derived morula and blastocysts collected on day 7 after artificial insemination, were vitrified using a modified droplet vitrification (MDV) procedure and subsequently warmed for transfer (ET) into synchronized recipients. Fresh embryos, and embryos cryopreserved using a standardized slow freezing procedure (direct thaw/direct transfer, DT) served as ET controls. Two different follicle‐stimulating hormone (FSH) sources, Folltropin® Canada (FSH BAH, 24 donors) and a brand prepared by the Chinese Academy of Science (FSH CAS, 16 donors), were compared in a series of superovulation outcomes following well‐established FSH administration protocols. Following data analysis, the total number of ovulations recorded at the time of embryo flushing (10.5 vs 8.5; p = 0.28) and the total number of transferable embryos (6.2 vs 5.1; p = 0.52) were similar between the two FSH sources. ET for MDV (39.7%, n = 78), DT (35.2%, n = 71) and fresh controls (47.1%, n = 34) resulted in similar pregnancy rates (p > 0.05). When MDV was used, a higher pregnancy rate (42.6%) resulted from the transfer of vitrified morulae, when compared to the DT counterparts (24.3%), (p = 0.05). Transfer of vitrified morulae resulted also in higher pregnancy rate, when compared to the transfer of vitrified blastocysts (42.6% vs. 29.4%; p < 0.05). Transfer of DT blastocysts resulted in higher pregnancy rate than morulae, similarly cryopreserved (47.1% vs. 24.3%, p < 0.05). In conclusion, MDV is an effective alternative methodology for cryopreservation of in vivo‐derived embryos. This study gives also indication that, compared to vitrified blastocysts, MDV of morula stage embryos results in higher pregnancy rates following warming and transfer into synchronized recipients.  相似文献   

19.
This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2–4-cell embryos, 8–16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages.  相似文献   

20.
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.  相似文献   

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