70 ONE-STEP CRYOPROTECTANT DILUTION FOLLOWING VITRIFICATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 182
Author(s):  
R. Morató ◽  
T. Mogas
Keyword(s):  
Embryo Transfer ◽  
Statistical Significance ◽  
Survival Rates ◽  
Blastocyst Stage ◽  
Slow Freezing ◽  
Embryo Cryopreservation ◽  
Dilution Method ◽  
Bovine Embryo ◽  
One Step

Although slow freezing continues to be the most widely used technique of cryopreservation for bovine in vivo- and in vitro-produced embryos, vitrification has been tested in different species with good results, especially when dealing with in vitro-produced embryos. Vitrification represents a minor expense in time and equipment associated with cryopreservation compared with conventional slow freezing. However, vitrification, which is the most common method for human embryo cryopreservation, has not been widely adopted by embryo-transfer practitioners for commercial use in cattle. In general, vitrification requires gradual cryoprotectant dilution in a laboratory setting, and it is difficult to perform in the field. The objective of this study was to develop a one-step dilution method suitable for one-step bovine embryo transfer using the cryotop vitrification method. Embryos produced in vitro by standard procedures were vitrified at the blastocyst stage at Day 7 post-insemination in a mixture of 15% ethylene glycol + 15% dimethyl sulfoxide + 0.5 M sucrose using cryotop devices. Embryos were randomly assigned to 1 of 3 warming methods: (1) W3: warming was carried out following the cryotop method (1 M sucrose for 1 min, 0.5 M sucrose for 3 min, and 0 M sucrose for 6 min); (2) W1/0.5: embryos were warmed directly in 0.5 M sucrose for 3 min; and (3) W1/0: embryos were warmed directly in 0 M sucrose for 5 min. Survival rates were assessed in terms of blastocyst re-expansion, hatching, and hatched status at 3 and 24 h after warming. Data were analyzed using the statistical analysis systems package (SAS, v9.1). Data from at least 3 replicates were collected. Comparisons of vitrified–warmed blastocyst survival rates between groups were performed using the chi-squared test. The level of statistical significance was set at P < 0.05. When embryo survival was evaluated at 3 h postwarming, embryos warmed using the 3-step dilution protocol and those warmed directly in 0.5 M sucrose showed higher percentages of survival (W3: 89.8%, n = 98; W1/0.5: 87.5%, n = 64; P < 0.05) than those blastocysts that were warmed directly in 0 M sucrose (W1/0: 66.4%, n = 146). However, similar rates irrespective of the warming procedure were observed at 24 h postwarming (W3: 85.7%, W1/0.5: 88.2%, W1/0: 70.5%). Warmed in vitro-produced embryos exposed to W3 (47.6%) and W1/0.5 (35.6%) achieved higher percentages of embryos developing to the hatched blastocyst stage after 24 h of culture than those embryos warmed in W1/0 (20.4%; P < 0.05). Our results indicate that direct warming and dilution of cyotop-vitrified embryos in 0.5 M sucrose for 3 min may enable one-step bovine embryo transfer without requirement of a microscope or other laboratory equipment, simplifying the embryo-transfer procedure of vitrified embryos on farm at the same level of complexity as carrying out AI. Support came from Spanish MEC (RZ2010-00015-0-00; AGL2010-19069) and Generalitat de Catalunya (2009 SGR 621).

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.

47 A New Device and Method for Successful Vitrification of In Vitro-Produced Ovine Embryos

2018 ◽  
Vol 30 (1) ◽  
pp. 163
Author(s):  
S. Ledda ◽  
J. M. Kelly ◽  
S. K. Walker ◽  
Y. Natan ◽  
A. Arav
Keyword(s):  
Survival Rate ◽  
Embryo Transfer ◽  
Blastocyst Stage ◽  
Embryo Cryopreservation ◽  
Control Group ◽  
Hatching Rate ◽  
High Survival ◽  
Embryo Survival ◽  
New Device

To advance the use of embryo vitrification technology in veterinary practice, we developed a system in which embryo vitrification, warming, and dilution can be performed within a straw. An in-straw embryo cryopreservation method reduces the need for equipment and technical skills and can facilitate direct embryo transfer to the uterus. This study proposes the use of a new device named “Sarah” that is designed to permit all in-straw embryo cryopreservation procedures. Ovine in vitro-produced (IVP) embryos were vitrified at either early blastocyst stage (EB, n = 65, 6 days post-IVF) or fully expanded blastocyst stage (FB, n = 168, 7 days post-IVF). The vitrification procedure using Sarah constituted a 0.25-mL straw with a capsule having 50-µm pores inserted at one end. Embryos at each stage (EB and FB) were divided into 2 subgroups and vitrified by 1 of 2 methods: (1) multi-step (MS) group-a straw containing 2 embryos was sequentially loaded vertically into 1.5-mL tubes containing 6 different vitrification solutions: 10, 20, 40, 60, 80, or 100% ES (with 100% ES being 7.5% DMSO +7.5% EG + 20% FCS in TCM-199; 90 s each step) followed by 30 s each in 75 and 100% VS (100% VS being 18% DMSO +18% EG + 0.5 M trehalose + BSA in TCM-199); and (2) two-step (TS) group-the straw (2 embryos/straw) was loaded with 100% of ES (5 min), followed by 100% VS solution for 30 s. For both methods, at the end of the preparation steps, the straws were plunged directly into liquid N2. Non-vitrified embryos were maintained in in vitro culture as a control group (n = 102). The warming procedure consisted of placing the straws directly into 5-mL tubes containing 100, 50, 25% WS (WS = 1 M sucrose in TCM-199+ 20% FCS) at 38.6°C (for first solution) and at room temperature for all the rest (5 min each), before being placed into the holding medium. Embryos were recovered from the straws, incubated at 38.6 C in 5% CO2 in air in TCM 199 + 5% FCS, and evaluated for blastocoel re-expansion, embryo survival, and hatching rate at 2, 14, 48 h post-warming. Blastocyst re-expansion (2 h) after warming increased as the developmental stage progressed and was not affected by the vitrification method. In fact, it was significantly (P < 0.05) higher for FB vitrified in the MS and TS methods (77.90% and 71.25%, respectively) compared with the EB method (62.5% and 48.50%, respectively). At 24 h, survival rate of vitrified FB was significantly higher (P < 0.05) in the MS system (95.35%) compared with those in TS (86.25%). Survival rates of FB embryos for both methods (MS and TS) were significantly higher (P < 0.001) than EB embryos vitrified in MS (56.25%) and TS (56.55) methods. After 48 h of culture, the hatching rate for FB vitrified in the MS system (87.21%) was comparable with TS (77.5%) and control (85.3%) groups but significantly higher (P < 0.001) than vitrified EB in MS (43.75%) and TS (36.36%). In conclusion, we showed that a high survival rate of IVP embryos can be achieved by this new in-straw vitrification and warming device (“Sarah”), with hatching rates in vitro comparable with that of control fresh embryos. This method has the potential for use in direct embryo transfer in field conditions.

22 Vitrification of bovine embryo using antifreeze polyamino acid

2019 ◽  
Vol 31 (1) ◽  
pp. 137
Author(s):  
T. Fujikawa ◽  
Y. Gen ◽  
S.-H. Hyon ◽  
C. Kubota
Keyword(s):  
Ethylene Glycol ◽  
Survival Rates ◽  
Basal Medium ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Chi Square ◽  
Embryo Survival ◽  
Polyamino Acid

Carboxylated poly-l-lysine (CPLL) is an ampholytic polymer compound and a polyamino acid with a known functional resemblance to antifreeze proteins. We previously reported that CPLL is an effective cryoprotectant for bovine cells, sperm, and slow-frozen embryos. In this study, we investigated CPLL as a cryoprotectant for vitrified bovine embryos. We developed bovine embryos in vitro and vitrified them at the blastocyst stage. Embryos were equilibrated (3min) and vitrified (1min). Vitrified embryos were cryopreserved in LN (Cryotop® device; Kitazato Corp., Tokyo, Japan) for at least 1 week, thawed with a 0.3M sucrose warming solution, and then cultured in a basal medium (Gibco® medium 199, Grand Island, NY, USA; supplemented with 100µM 2-mercaptoethanol, 10% fetal bovine serum, and antibiotics) at 38.5°C in a humidified atmosphere (5% CO2, 5% O2, 90% N2). We evaluated the embryos morphologically for survival and hatched rate at 0, 24, 48, and 72h post-thawing. In control, the equilibration solution (ES) consisted of 7.5% (vol/vol) dimethyl sulfoxide (DMSO) and 7.5% (vol/vol) ethylene glycol, and the vitrification solution (VS) consisted of 16.5% (vol/vol) DMSO and 16.5% (vol/vol) ethylene glycol and 0.5M sucrose. In this study, CPLL was added to ES and VS at various concentrations instead of DMSO. The CPLL was added at 16.5, 11.0, 5.5, and 2.2% (wt/vol) to VS; respectively, these solutions were named P16.5, P11.0, P5.5, and P2.2. The ES was used 45% CPLL of VS each. Embryos underwent the above procedure concurrently, with testing replicated at least 3 times. We evaluated 88, 34, 38, 44, and 28 embryos with each solution (control, P16.5, P11.0, P5.5, and P2.2, respectively). Results were analysed statistically with a chi-square test and residual analysis, regarding P&lt;0.05 as significant. Survival rates were significantly greater in P11.0 at 24h post-thawing (55.7% v. 89.5%; P&lt;0.05) and in P11.0 and P5.5 at 48h post-thawing (47.7% v. 78.9% and 47.7% v. 79.5%, respectively; P&lt;0.05) relative to controls but showed no significant differences at 0h post-thawing. Hatched rates were significantly greater in P11.0 and P5.5 through 72h post-thawing relative to controls (44.7% v. 22.7% and 52.3% v. 22.7%, respectively; P&lt;0.05). The CPLL improved post-thawing embryo survival and hatched rates when applied during vitrification, thus demonstrating cryoprotective effectiveness. We conclude that CPLL acts as a low-toxicity cryoprotectant for vitrified bovine embryos, and our results are consistent with previous reports of protective CPLL effects for cells and cell membranes.

110 THE USE OF FORSKOLIN AND ITS EFFECT ON IN VITRO-PRODUCED BRAHMAN-SIRED EMBRYOS SUBMITTED TO SLOW COOL FREEZING OR VITRIFICATION

2010 ◽  
Vol 22 (1) ◽  
pp. 214 ◽  
Author(s):  
J. H. Pryor ◽  
J. A. Trant ◽  
C. B. Ponchirolli-Schneider ◽  
C. R. Looney ◽  
C. R. Long ◽  
...  
Keyword(s):  
Statistical Significance ◽  
Survival Rates ◽  
Slow Cool ◽  
Blastocyst Stage ◽  
Developmental Differences ◽  
Treatment Groups ◽  
Student’S T ◽  
Blastocyst Hatching ◽  
Hatching Rates

The objective of this study was to further test the lipolytic effect of 10 μM forskolin on developmental differences between bovine in vitro produced (IVP) embryos submitted to slow cool (SC) freezing or vitrification (VT). Previously reported (phase I) IVP embryo hatching rates for control embryos (62%) were no different than 10 μM forskolin (67%; Pryoretal et al. 2009 Reprod. Fertil. Dev. 21, 163). For phase II: on Day 6 post-fertilization (IVF = Day 0), 207 Brahman-sired viable embryos were evenly divided and cultured for 24 h in G2.5 medium (Vitrolife, Englewood, CO, USA) with or devoid of 10 μL forskolin (Sigma, St. Louis, MO, USA). On Day 7, compact morula (CM, n = 31), blastocyst (BL, n = 76), and expanded BL (XBL, n = 100) were washed in Vigro Holding Plus medium (Bioniche, Pullman, WA, USA) and randomly allocated to 4 treatment groups; control SC (CSC; no treatment, n = 52), 10 μM forskolin SC (FSC; n = 55), control VT (CVT; n = 49), and 10 μM forskolin VT (FVT; n = 51). All embryos were packaged in sterile 0.25-mL plastic straws. The SC embryos were submitted to Vigro Ethylene Glycol Freeze Plus medium (Bioniche) for 5 min before freezing at 0.5°C/min from -6°C to -32°C and plunging in LN2. Embryos were vitrified using a bovine VT kit (Bioniche): VS1, 3 min; VS2, 45 s in 15 μL; diluent, in straw with VS2 separated by air columns, vitrified in LN2 vapor 1 cm from liquid for 1 to 15 min before plunging. The SC embryos were air thawed 5 s and placed in 30°C H2O bath for 10 s. The VT straws were air warmed 10 s and then in 35°C H2O for 20 s prior to shaking them down to mix columns. All embryos were cultured in G2.5 for 24-h survival and 48-h hatching rates. All percentage data were transformed using arcsin square root function prior to analysis, and means were compared for statistical significance using Student’s t. For mean survival rates, FSC was different than CSC but showed no difference between FVT and CVT (81.7 ± 0.09, 42.6 ± 0.09, 59.4 ± 0.09, 49.0 ± 0.10, respectively (P < 0.01). There were no statistical differences for hatching rates for combined embryo stages (58.2 ± 0.10, 37.8 ± 0.10, 34.4 ± 0.10, 28.1 ± 0.11 for FSC, FVT, CSC, and CVT, respectively; P > 0.07). However, when comparing hatching rates of only the blastocyst stage embryos (n = 176), FSC was superior to CSC and CVT but not different than FVT (74.8 ± 0.11, 29.5 ± 0.11, 29.1 ± 0.11, 48.5 ± 0.11, respectively; P < 0.01). In conclusion, FSC yielded significantly higher survival and blastocyst hatching rates than CSC, but there were no differences between CVT and FVT for survival and FVT for blastocyst hatching rates. These results indicate that the addition of 10 μM forskolin to culture 24 h prior to freezing 7 d IVP Brahman-sired embryos can increase survival and blastocyst hatching rates. The authors acknowledge support from the American Brahman Breeders Association.

24 Survival rates of vitrified biopsied bovine in vitro-produced blastocysts using the VitTrans device

2019 ◽  
Vol 31 (1) ◽  
pp. 138
Author(s):  
N. González ◽  
J. Scherzer ◽  
M. Reichenbach ◽  
C. Otzdorff ◽  
H. Zerbe
Keyword(s):  
Ethylene Glycol ◽  
Dimethyl Sulfoxide ◽  
Embryo Transfer ◽  
Zona Pellucida ◽  
Survival Rates ◽  
Petri Dish ◽  
Blastocyst Stage ◽  
Control Group ◽  
Embryo Survival

In breeding programs, the application of a vitrification method suitable for direct transfer of biopsied embryos can increase the genetic improvement of cattle and help reduce the costs of embryo transfer. The aim of this study was to determine the in vitro survival of biopsied vitrified blastocysts using the new VitTrans device (Morató and Mogas 2014 Cryobiology 68, 288-293), a 1-step in-straw warming system. Immature bovine oocytes were in vitro matured, fertilized, and cultured to the blastocyst stage. A total of 110 grade 1 blastocysts (IETS codes 6 and 7) were randomly allocated to 2 groups: (1) biopsy (n=49) and (2) without biopsy, or control (n=61). Blastocysts were biopsied using a microblade mounted on a micromanipulator. A small portion of the trophoblast, approximately 15%, was cut off and a significant part of the zona pellucida was sliced away. Both groups were then vitrified using the VitTrans device. For vitrification, all blastocysts were exposed to an equilibration medium with 7.5% ethylene glycol+7.5% dimethyl sulfoxide in holding medium (HM) consisting of TCM-199 with 20% FCS, moved into a drop with 16.5% ethylene glycol+16.5% dimethyl sulfoxide+0.5M sucrose in HM, and then placed in a microdroplet on the VitTrans. The VitTrans was plunged into LN and covered with a 0.5-mL straw. For warming, the protective cover was removed from the VitTrans while still submerged in LN. Subsequently, a new 0.5-mL plastic embryo transfer straw was placed on the VitTrans while flushing the warming solution (0.3mL of 0.5M sucrose in HM at 45°C) with a syringe through the lumen of the device. By entering the warming solution into the VitTrans device, the embryo is flushed inside the plastic straw. The straw containing the embryo can then be readily used for transfer after the VitTrans is removed. To recover the embryo in the laboratory, the content of the straw was put into a Petri dish and blastocysts were placed in the culture medium and incubated at 38.5°C in 5% CO2 and 5% O2 in air. Morphology and re-expansion were evaluated 24h post-warming. The embryo survival rate was defined as the ratio of blastocysts that were able to re-expand with regards to the total number of warmed blastocysts. Due to the attachment of embryos inside the straw, a total of 18 embryos were lost during recovery (12 from the biopsied group and 6 from the nonbiopsied group). The ratio of re-expanded blastocysts from the recovered embryos was 40% in the biopsy group and 61% in the control group. In conclusion, vitrification using the VitTrans device showed good results with intact embryos compared with biopsied embryos. In addition, biopsied embryos had a tendency to adhere to the inside of the straw, which is probably due to the damage or loss of the zona pellucida. Additional research is required to minimize the loss of embryos.

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.

43 NEW METHOD FOR ONE-STEP WARMING/IN-STRAW CRYOPROTECTANT DILUTION FOR IN VITRO-PRODUCED BOVINE BLASTOCYSTS AFTER VITRIFICATION WITH THE CRYOLOGIC VITRIFICATION METHOD

2015 ◽  
Vol 27 (1) ◽  
pp. 114
Author(s):  
J. N. Caamaño ◽  
E. Gómez ◽  
B. Trigal ◽  
M. Muñoz ◽  
S. Carrocera ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Survival Rates ◽  
Field Conditions ◽  
Control Group ◽  
Embryo Survival ◽  
Grant Support ◽  
One Step ◽  
The One ◽  
New System

Vitrification is considered an alternative to slow-rate freezing to cryopreserve in vitro-produced (IVP) bovine embryos. However, the use of vitrified IVP embryos for embryo transfer under field conditions is difficult because of the requirements of the current thawing protocols. The objective of this study was to develop a simple one-step warming/in-straw cryoprotectant dilution procedure for IVP bovine blastocysts that were vitrified using the cryologic vitrification method. In this study, 109 Day-7 IVP blastocysts were subjected to vitrification using the conventional fibreplugs (groups of 5 embryos were loaded in 3 mL of vitrification medium). Warming was performed in one-step in MS1 (0.25 M sucrose in BV = TCM 199-Hepes + 20% FCS) either using a 4-well plate for 5 min (control group) or in a new system that allowed in-straw cryoprotectant dilution designed to avoid losses of embryos and to maintain the temperature required during this procedure. This new system is composed of an adaptor with a wider opening that is coupled to the French straw and a heated metal chamber to protect and keep the straw at 41°C. Warmed embryos were washed and subsequently cultured in mSOFaaci + 6 gL–1 BSA + 10% FCS for 48 h. Re-expansion (at 2, 24, and 48 h) and hatching rates (at 24 and 48 h) were recorded. Data were analysed by ANOVA and are presented as LSM ± standard error. Embryo survival rates of embryos warmed by the one-step warming/in-straw cryoprotectant dilution procedure did not differ from the control group (see Table 1). These results suggest that the cryologic vitrification method combined with our warming system for in-straw cryoprotectant dilution may be used for direct embryo transfer under field conditions. Table 1.Embryo survival rates of in vitro-produced embryos vitrified by the cryologic vitrification method and warmed by the new one-step warming/in-straw cryoprotectant dilution procedure This study received grant support: INIA-RTA 2011–0090 and FEDER. M. Muñoz was supported by grant MICINN-RYC08-03454, and B. Trigal by a grant from Cajastur. The authors are members of the COST Action FA1201 Epiconcept.

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.

106 PREGNANCY RATE AFTER EMBRYO TRANSFER OF IN VIVO-PRODUCED OVINE EMBRYOS CRYOPRESERVED BY SLOW FREEZING OR VITRIFICATION

2010 ◽  
Vol 22 (1) ◽  
pp. 212
Author(s):  
N. Mucci ◽  
F. Hozbor ◽  
G. G. Kaiser ◽  
E. Sanchez ◽  
R. H. Alberio
Keyword(s):  
Ethylene Glycol ◽  
Pregnancy Rate ◽  
Liquid Nitrogen ◽  
Embryo Transfer ◽  
Slow Freezing ◽  
Embryo Cryopreservation ◽  
Chi Square ◽  
Chi Square Test

Although slow freezing is the method of choice to cryopreserve in vivo-produced ovine embryos, vitrification has became an alternative procedure mostly developed for in vitro-produced bovine embryos. The aim of this work was to compare pregnancy rates after cryopreservation of in vivo-produced ovine embryos with slow freezing or open pulled straw (OPS) vitrification method. Ewes were synchronized using intravaginal sponges containing 60 mg of medroxyprogesterone acetate for 14 d. Superovulation was performed using a total dose of 176 IU of ovine FSH (Ovagen), in 6 decreasing doses (i.m.) from Day 12 to 14 of treatment (Day 0 = sponge placing). Ewes were hand mated with 2 rams of proven fertility. Embryos were recovered 6 days after estrous detection by surgical procedure, evaluated under stereomicroscope, and randomly assigned to the cryopreservation treatments. Slow freezing was performed in D-PBS supplemented with 1.78 M ethylene glycol, 0.1 M sucrose, 4 mg mL-1 of BSA, and 20% serum. Embryos were loaded into 0.25-mL plastic straws and placed into a -7°C methanol bath chamber. After seeding embryos were cooled to -35°C at a rate of 0.5°C/min and then stored in liquid nitrogen. Thawing was performed by placing the straws in a 30°C water bath for 30 sec. Vitrification was performed by using the OPS method (Vajta et al. 1998) with minor modifications. Embryos were incubated in D-PBS supplemented with 1.78 M ethylene glycol, 1.3 M DMSO for 3 min and then transferred for 25 s in vitrification solution of D-PBS with 3.56 M ethylene glycol, 2.6 M DMSO, and 0.5 M sucrose, loaded in a 1 mL drop in the OPS, and immediately submerged into and stored in liquid nitrogen. Warming was performed in D-PBS plus 0.25 M sucrose for 5 min and then into D-PBS plus 0.15 M sucrose for another 5 min. Before embryo transfer, the presence of corpus luteum (CL) was detected by laparoscopic examination. One embryo per recipient was surgically transferred in the apical extreme of the uterine horn ipsilateral to the CL. Pregnancies were determined by ultrasonography 41 days after embryo transfer. Data were analyzed using the chi-square test. We found 47.8% pregnancy rate using slow freezing (11/23) and 43.5% pregnancy rate using OPS vitrification (10/23). Statistical differences were not detected (P = 0.09). We conclude that vitrification by OPS system, with minor modifications, is a suitable procedure for in vivo-produced ovine embryo cryopreservation.

Sign in / Sign up

Export Citation Format

Share Document