Slow-frozen Embryos Research Articles

Mitochondrial bioenergetic profiles of warmed bovine blastocysts are typically altered after cryopreservation by slow freezing and vitrification

The widespread use of cryopreserved in vitro produced (IVP) bovine embryos is limited due to their low post-warming viability compared to their ex vivo derived counterparts. Therefore, the present study aimed to analyse in detail the consequences of cryopreservation (vitrification and slow freezing) on the bioenergetic profile of the embryo and its mitochondria. To accomplish that, day 7 IVP embryos were separated in a non-cryopreserved control group (fresh, n = 120, 12 replicates) or were either slow frozen (slow frozen, n = 60, 6 replicates) or vitrified (vitrified, n = 60, 6 replicates). An in-depth analysis of the bioenergetic profiles was then performed on these 3 groups, analysing pools of 10 embryos revealing that embryo cryopreservation both via vitrification and slow freezing causes profound changes in the bioenergetic profile of bovine embryos. Noteworthy, fresh embryos demonstrate a significantly (P < 0.05) higher oxygen consumption rate (OCR) compared to vitrified and slow frozen counterparts (0.858 ± 0.039 vs. 0.635 ± 0.048 vs. 0.775 ± 0.046 pmol/min/embryo). This was found to be largely due to significantly reduced mitochondrial oxygen consumption in both vitrified and deep-frozen embryos compared to fresh counterparts (0.541 ± 0.057 vs. 0.689 ± 0.044 vs. 0.808 ± 0.025 pmol/min/embryo). Conversely, slow-frozen thawed blastocysts showed 1.8-fold (P < 0.05) higher non-mitochondrial OCR rates compared to fresh embryos. Maximum mitochondrial respiration of vitrified and slow-frozen embryos was significantly reduced by almost 1.6-fold compared to fresh embryos and the proportion of ATP-linked respiration showed significantly lower values in vitrified thawed embryos compared to fresh embryos (1.1-fold, P < 0.05). Likewise, vitrification-warming and freeze-thawing reduced reactive glycolytic capacity (1.4 fold, 1.2-fold)as well as compensatory glycolytic capacity to provide energy in response to mitochondrial deficiency (1.3-fold and 1.2-fold, P < 0.05). In conclusion, the present study has, to the best of our knowledge, identified for the first time a comprehensive overview of typical altered metabolic features of the bioenergetic profile of bovine embryos after cryopreservation, which have great potential to explain the detrimental effects of cryopreservation on embryo viability. Avoidance of these detrimental effects through technical improvements is therefore suggested to be mandatory to improve the viability of bovine embryos after cryopreservation-warming.

Assessment of a double freezing approach in the management of surplus embryos in IVF.

What pregnancy rates are achieved after transfer of cryopreserved double slow-frozen embryos in IVF cycles? Patients in whom surplus thawed cleaved embryos (day 2 or 3) were grown to the blastocyst stage, re-frozen and then re-thawed for transfer (double freezing) were included. Data were collected on all patients who had undergone the above procedure at the IVF unit of Assuta Ramat Hachayal Hospital, Tel Aviv, during a 7-year period. For each patient in the study group, the two-consecutive, matched-by-age patients treated with frozen-thawed single blastocyst transfer were selected to form a 2:1 ratio control group. All embryos were frozen using the slow freeze protocol. A total of 54 patients had 70 embryos that were re-frozen at the blastocyst stage. Twenty-eight of these blastocysts were thawed and 27 underwent transfer to 25 patients. A single embryo was transferred to 23 patients and two embryos were transferred to two patients. The survival rate of the second thawing was 96.4% (27/28). Clinical pregnancy rate was 16% (4/25) and implantation rate was 14.8% (4/27). In the study group, pregnancies were achieved in 22 out of the 25 patients using IVF treatment, indicating good receptivity of the uterus. In the control group, the implantation/pregnancy rates were significantly higher (44.2% [23/52]; P < 0.01). The transfer of twice slow-frozen and thawed embryos does not seem to be a beneficial approach in the planned management of cryopreserved surplus embryos owing to the low pregnancy rate achieved after transfer of the re-frozen blastocyst embryos.

22 Vitrification of bovine embryo using antifreeze 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&amp;lt;0.05 as significant. Survival rates were significantly greater in P11.0 at 24h post-thawing (55.7% v. 89.5%; P&amp;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&amp;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&amp;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.

84 Effect of Embryo Stage and Cryopreservation Method on Pregnancy Rates Obtained Following the Transfer of In Vivo-Derived Ovine Embryos on Small-Scale Farms in Thailand

Assisted reproductive technologies including superovulation, laparoscopic AI (LAI), and embryo transfer (ET) are important tools for genetic improvement in the sheep industry. The present study aimed to determine the effects of embryo stage and cryopreservation method on field trial outcomes of embryo transfer on small-scale farms in Thailand. Black Dorper ewes (n = 16) were used as donors and mixed breed ewes (n = 21) were used as recipients. Donors were superovulated as previously described (Tríbulo et al. 2012 Theriogenology 77, 1679-1685, 10.1016/j.theriogenology.2011.12.013) and inseminated by LAI within 22 to 24 h after standing heat (Day 0). Donors females were flushed on Day 2 to recover 2- to 8-cell embryos (n = 8) or on Day 6 to recover blastocyst-stage embryos (n = 8). Recovered embryos were randomly cryopreserved by either slow freezing or vitrification methods. Into 21 recipients was inserted an intravaginal device impregnated with CIDR® that was left in place for 10 days. Those received 300 IU of pregnant mare serum gonadotropin (PMSG) on Day 9 and randomly assigned to receive embryos on either Day 2 or 6 after oestrus. Two- to 8-cell embryos were thawed and transferred into the ipsilateral oviduct (n = 1-5 embryos/recipient) on Day 2. Five recipients received 15 slow-frozen embryos and 5 received 13 vitrified embryos. Blastocyst stage embryos were thawed and transferred into the ipsilateral uterine horn (n = 1-4 embryos/recipient) on Day 6. Five recipients received 11 slow-frozen embryos and 4 received 7 embryos. Pregnancy diagnosis was determined by ultrasonography 45 days after embryo transfer. Pregnancy rate was calculated as the proportion of ewes with at least one pregnancy out of the total number of ewes that received embryos. Chi-squared analysis was used to determine the effects of embryo freezing technique and embryo stage on pregnancy rate (SAS 9.2, SAS Institute Inc., Cary, NC, USA). All donor ewes responded to the superovulation program (donor had 3 to 6 corpora lutea). The mean number of viable embryos recovered was 4.3 ± 2.4 and 3.1 ± 3.7 for Day 2 and Day 6, respectively. Nineteen of the 21 recipient ewes responded to the synchronization program and received embryos in the study. There was no effect (P &gt; 0.05) of embryo stage (5/10 = 50% v. 3/9 = 33.3% for 2- to 8-cell v. blastocyst, respectively) or cryopreservation method (4/10 = 40% v. 4/9 = 44.4% for slow-freezing and vitrification, respectively) on pregnancy rate following embryo transfer. Results of the present study suggest that similar pregnancy rates following embryo transfer in sheep under tropical conditions in Thailand can be obtained using either 2- to 8-cell embryos or blastocyst-stage embryos and with embryos that have been cryopreserved by slow-freezing or vitrification. Further research with larger numbers of animals is necessary to confirm the preliminary results of the present study.

206 Effect of L-Ascorbic Acid, Polydatin, and In-Straw Rehydration of Slow-Frozen In Vitro-Produced Bovine Embryos

In vitro-produced (IVP) bovine embryos have poor cryotolerance due to high lipid content and reactive oxygen species levels that hinder post-thaw survival. We hypothesised that in-straw rehydration of slow-frozen embryos with sucrose and the addition of the antioxidant polydatin and l-ascorbic acid would increase post-thaw survival. The IVP embryos (n = 116) were generated in 7 replicates by aspirating oocytes from 2- to 8-mm follicles of abattoir ovaries, matured for 23 h, fertilized with semen from 1 of 3 bulls using standard procedures, and cultured in SCF1 medium for 7 days (Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130). Stage 7 embryos were slow-frozen using 1 of 4 protocols in a 2 × 2 factorial design: embryos were equilibrated in conventional slow-freezing media for 20 min [1.5 M ethylene glycol (EG) and 0.5 M sucrose] with 1 mm l-ascorbic acid or 1 μM polydatin, and loaded in the straw adjacent to columns of freezing medium or 0.75 M EG and 0.6 M sucrose and then seeded at −6°C, cooled at 0.5°C min−1, and plunged at –32°C. Embryos were thawed in air for 10 s followed by 30 s in 32 to 35°C water bath. Once straw columns were disrupted, embryos were allowed to equilibrate for 5 min. Subsequently, embryos were washed and placed back in culture and re-expansion was assessed at 24 and 48 h. Data (Table 1) were analysed by ANOVA with means separated by Tukey’s HSD. Results indicate that there was no main effect between the 2 antioxidants or the use of rehydration columns (P &lt; 0.05); however, there was higher (P &lt; 0.05) re-expansion for embryos frozen with polydatin and with rehydration column than embryos frozen with l-ascorbic acid and no rehydration column. This suggests that polydatin coupled with in-straw rehydration (0.75 M EG and 0.6 M sucrose) may improve post-thaw survival of IVP bovine embryos. Table 1.Post-thaw re-expansion rates of embryos exposed to antioxidants and in-straw rehydration (± SEM)

Appendix F: Quinn's Advantage Embryo Freeze Kit.

Despite a large focus on the use of vitrification to cryopreserve embryos in recent years, there are still arguments for the use of slow freezing for the cleavage-stage embryo. Having said this, there are lessons to be learned from the process of vitrification that could be applied to slow freezing to improve post-thaw survival and ultimately clinical pregnancy rates. Specifically, increasing the concentration of sucrose in the freezing solution from 0.1 to 0.2M and subsequently increasing the sucrose concentrations in thawing solutions could prove beneficial. The use of vitrification warming solutions in the thawing of slow-frozen embryos may also be an option that not only improves survival but also streamlines product purchasing and protocols within the laboratory.

The freezing method of cleavage stage embryos has no impact on the weight of the newborns.

The aim of this study was to study the effect of the embryo freezing method on the birth weight of newborns from frozen embryo transfer (FET) cycles, and the pregnancy results of cleavage stage embryos cryopreserved by slow freezing or vitrification. This is a retrospective cohort study undertaken in a University Hospital IVF unit using concurrently both the slow-freezing and the vitrification techniques. All frozen-thawed and vitrified-warmed day 2 and day 3 embryo transfers during the time period from 1 April 2009 to 31 November 2013 were included in the study. There was no statistically significant weight difference between newborns from vitrified or slow-frozen embryos (3588 vs 3670 g). A higher post-thaw viability rate was achieved after cryopreservation by the vitrification technique compared to the slow-freezing protocol (83.4 vs 61.4%). The miscarriage rate was lower in the vitrification group (15.7 vs 29.0%). The live birth rates were similar (19.5 vs 19.1%) in the slow-freezing and vitrification groups, respectively. Among vitrified embryos, 7.4 embryos needed to be thawed to produce one delivery; in the slow-freezing group, that number was 11.9. The freezing method has no impact on the weight of the newborn. With lower post-thaw survival rates and higher miscarriage rates, the slow-freezing cryopreservation protocol is inferior to the vitrification technique.

Cytoskeletal alterations in different developmental stages of in vivo cryopreserved preimplantation murine embryos.

BackgroundThis study aimed to investigate the effects of vitrification and slow freezing on actin, tubulin, and nuclei of in vivo preimplantation murine embryos at various developmental stages using a Confocal Laser Scanning Microscope (CLSM).Material/MethodsFifty female mice, aged 4–6 weeks, were used in this study. Animals were superovulated, cohabitated overnight, and sacrificed. Fallopian tubes were excised and flushed. Embryos at the 2-cell stage were collected and cultured to obtain 4- and 8-cell stages before being cryopreserved using vitrification and slow freezing. Fixed embryos were stained with fluorescence-labelled antibodies against actin and tubulin, as well as DAPI for staining the nucleus. Labelled embryos were scanned using CLSM and images were analyzed with Q-Win software V3.ResultsThe fluorescence intensity of both vitrified and slow-frozen embryos was significantly lower for tubulin, actin, and nucleus as compared to non-cryopreserved embryos (p<0.001). Intensities of tubulin, actin, and nucleus in each stage were also decreased in vitrified and slow-frozen groups as compared to non-cryopreserved embryos.ConclusionsCryopreservation of mouse embryos by slow freezing had a more detrimental effect on the actin, tubulin, and nucleus structure of the embryos compared to vitrification. Vitrification is therefore superior to slow freezing in terms of embryonic cryotolerance.

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

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 &gt; 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 &lt; 0.05). Vitrified embryos (143.5 ± 12.7) had higher (P &lt; 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.

60 VITRIFICATION AND CONVENTIONAL CRYOPRESERVATION OF EQUINE EMBRYOS

Vitrification and conventional cryopreservation are effective methods of preserving equine embryos smaller than 300 μm in diameter. This study was designed to compare pregnancy rates using these methods to cryopreserve embryos of similar size. Sport horse mares approximately 2–20 years old were flushed nonsurgically between Days 6.5 and 7 post-ovulation with 2 L of lactated-Ringers solution (Braun, Melsungen, Germany). Thirty-one embryos were collected, washed 4 times with 1 mL of ViGro® holding medium (Bioniche Animal Health, Bogart, GA), graded for quality, measured for diameter, and blocked into 2 groups (&lt;200 μm, 200 to 300 μm). Embryos were either vitrified with a commercial equine vitrification kit (Bioniche Animal Health) according to the manufacturer’s instructions in 0.25-mL straws or subjected to a slow cooling method. For vitrification, embryos were sequentially transferred to 2 wells containing 0.5 mL of 2 Syngro®-based vitrification solutions (VS1 and VS2) and held for 5 min each. Embryos were incubated in a third vitrification solution (VS3) for 45 s during which time they were loaded into straws. Straws were held in liquid nitrogen-cooled air for 1 min before submersion in liquid nitrogen. For slow freezing, embryos were consecutively placed into 0.5 mL of the following Syngro®-based solutions for 5 min each: 1.8 m glycerol, 1.8 m glycerol + 1.8 m ethylene glycol (EG), and 0.9 m glycerol + 0.9 m EG + 0.5 m galactose. Embryos were loaded into 0.25-mL straws, placed in a chamber pre-cooled to –6°C, and held for 10 min. Straws were seeded after 2 min. The temperature was lowered to –32°C at a rate of 0.5°C min–1. Embryos were then plunged into liquid nitrogen within 3 min of reaching –32°C. For warming vitrified embryos, straws were held in air for 10 s followed by submersion into a 35°C water bath for 20 s. Straws were flicked 5 times to mix the diluent solution with the VS3-containing embryos, which were transferred within 7 min of being thawed. For thawing conventionally frozen embryos, straws were held in air for 10 s followed by submersion into a 35°C water bath for 30 s. Contents of the straw were immediately expelled into a Petri dish, and the embryos were transferred immediately to 0.5 mL of 1.2 m glycerol +1.2 m EG + 0.5 m galactose and held for 5 min. This was followed by a 5-min incubation in 0.5 mL of each of the following solutions: 0.6 m glycerol + 0.6 m EG + 0.5 m galactose, 0.25 m glycerol + 0.25 m EG + 0.5 m galactose, and 0.5 m galactose. After exposure to the last solution, embryos were transferred to Syngro®, loaded into a straw, and immediately transferred into 2-year-old virgin recipients 6 days after ovulation as detected by rectal palpation and ultrasonography. Of the 21 embryos &lt;200 μm collected, 11 were vitrified and 10 frozen slowly. Three of these 11 vitrified embryos and 7 of 10 slow-frozen embryos resulted in Day 16 pregnancies (27 and 70%, respectively). None of the embryos &gt;200 μm resulted in pregnancies in either the vitrification (n = 5) or slow-freeze treatments (n = 4).

P-206: Non-immersion vitrification - a viable alternative to slow freezing of blastocysts

Slow freezing of human blastocysts is a widely used technique in ART but is often associated with a high rate of cell lysis leading to decreased embryo viability. It is time consuming and also has high equipment costs. In contrast, vitrification reduces the risk of ice crystal formation hence reducing cell lysis, has shown promising implantation rates in many species including human, is rapid and relatively inexpensive. In this ongoing study we compare the preliminary clinical results using the Cryologic Vitrification Method 1 (CVM 1) with historical data using a standard slow freezing procedure. Oocytes were fertilised and cultured in the Sydney IVF suite of sequential media (Cook IVF, Brisbane) for up to 144 hours. Day 5/6 supernumerary blastocysts produced prior to January 2006 were frozen using a programmed control rate freezer (Planer, UK) in Sydney IVF blastocyst freezing media. Slow freeze embryos were passaged through a series of ambient temperature solutions containing glycerol (9%) and sucrose (0.2M) in the final solution, transferred into embryo freeze straws, placed into a programmed freezer and then seeded manually. From January 2006 all blastocysts were vitrified In Sydney IVF Vitrification Media using the CVM 1 kit (Cryologic, Australia). Embryos were passaged through a series of vitrification solutions at 37°C, with the final solution containing trehalose (0.68 M), ethylene glycol (16%) and DMSO (16%) . Within 30 to 40 seconds in this final vitrification solution blastocysts were loaded in a 2-3 μl drop onto a fine plastic hook at the end of a fibreplug. This microdrop was vitrified by placing it onto the surface of the liquid nitrogen pre-cooled metal vitrification block and were then stored in sealable plastic straws. The blastocysts did not have direct contact with liquid nitrogen at any stage. Straws containing the slow freeze embryos were thawed in air for 30 seconds then in a water bath (30°C) for 30 seconds and then were transferred into a series of thawing solutions containing varying concentrations of sucrose at ambient temperature. Vitrified blastocysts were thawed by plunging the hook containing the vitrified embryo directly into the warming solution at 37°C. Historically, most pregnancies (55%) obtained using our slow freezing protocol were from embryos that had 90 -100% cell survival. Our 2005 data indicates that 41% (432/1047) of slow frozen embryos were in this survival band. Preliminary 2006 data indicates that a higher embryo survival rate of 79% (19/24) of vitrified embryos had cell survival of 90% or greater, (p < 0.005). To date we have obtained 10 +βhCG pregnancies from 18 embryo transfers (56%, 1.1 embryos per transfer). Comparative data from slow frozen embryos in 2005 gave a +βhCG pregnancy rate of 34% (222/658), transferring on average1.3 embryos, (p=0.095). These results indicate that non-immersion vitrification of human blastocysts could provide a safe and viable alternative to current slow freezing protocols. High post-thaw survival and pregnancy rates are necessary to encourage an increase in the proportion of cases where single embryo transfer (SET) is the preferred protocol to avoid multiple pregnancies [Fertil. Steril. 2005;84: 1620].

Effect of estrous cow serum during bovine embryo culture on blastocyst development and cryotolerance after slow freezing or vitrification

The present study investigated the effect of estrous cow serum (ECS) during culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. Embryos were derived from in vitro maturation (IVM) and in vitro fertilization (IVF) of abbatoir-derived oocytes. At Day 3, embryos were cultured in three different media: Charles Ronsenkrans medium + amino acids (CR1aa; without bovine serum albumin (BSA)) + 5% estrous cow serum (CR1-ECS), CR1aa + 3 mg/mL BSA (CR1-BSA) or CR1aa + 5% ECS + 3 mg/mL BSA (CR1-ECS-BSA). At 7.5 d post-insemination (PI), blastocyst yield and quality were evaluated; blastocysts and expanded blastocysts from each media were cryopreserved by Open Pulled Straw (OPS) vitrification method or slow freezing (1.5 M ethylene glycol, EM). Total blastocyst yield did not differ among CR1-ECS, CR1-BSA and CR1-ECS-BSA (30.9, 33.1 and 32.9%, respectively, P < 0.05). Embryo survival (hatching rate) was higher in vitrified versus slow-frozen embryos (43% versus 12%, respectively, P < 0.01), and in embryos cultured in CR1-BSA (40.3%) compared with those cultured in serum-containing media (CR1-ECS, 21.5% and CR1-ECS-BSA, 19.8%; P < 0.01). In conclusion: (a) it was possible to produce in vitro bovine embryos in serum-free culture medium without affecting blastocyst yield and quality; (b) serum-free medium produced the best quality embryos (in terms of post-cryopreservation survival); and (c) vitrification yielded the highest post-cryopreservation survival rates, regardless of the presence of serum in the culture medium.

82 EVALUATION OF CELL DEATH IN CRYOPRESERVED MOUSE EMBRYOS

Cryopreservation of mammalian embryos is an important tool for the application of reproductive biotechnology. Recent evidence indicates that apoptosis may be important in determining the viability of cryopreserved embryos. Our goal was to detect apoptosis and characterize and quantify the embryonic cell death caused by cryopreservation. Mouse morulae were collected, selected, and separated into three groups: fresh, slow-freezing, and vitrification. In the slow-freezing procedure, embryos were exposed to 10% ethylene glycol (EG) for 10 min. After loading, the straws were placed into methanol at −7°C for 5 min, seeded and after 5 min cooled at 0.5°C/minute. After 10 minutes at −31°C, straws were plunged into and stored in liquid nitrogen. Slow-frozen straws were thawed in air for 10 s, and then immersed in a 25°C water bath for 20 s. Embryos were vitrified by exposing them to 10% and 20% EG for 5 min followed by 40% EG + 18% Ficoll + 10% sucrose (EFS) for 30 s and the 0.25-mL straws then plunged into and stored in liquid nitrogen. The vitrified straws were warmed by immersing them in 25°C water for 20 s. Cell membrane integrity was assessed by Hoechst and propidium iodide double staining (H/PI). Fresh and thawed embryos were scored (following IETS recommendations) and then fixed after 30 min in PBS + 10% FCS. Morphology and apoptosis were assessed with Haematoxylin-Eosin staining (HE) and by electron microscopy (MET). The number of Grade I embryos recovered after thawing was higher for slow-frozen embryos (61.5%) than vitrified embryos (29.5%). H/PI detected more membrane permeability in the vitrified embryos (69.7%), than in the slow-frozen (48.4%) or non-frozen (13.8%) groups (P &lt; 0,05, Wilcoxon's test). Nuclear evaluation by HE revealed that vitrification and slow-freezing induced pyknosis and chromatin condensation. Mitotic pattern was observed in the fresh and slow-frozen group, but not in vitrification group suggesting that the embryos were either not randomly allocated to the groups or not-treated and fixed at the same age, or that vitrification changed the nuclear status of the embryos. HE staining revealed weakly staining cytoplasm and degenerated cells in the vitrification group (indicating oncosis), while in the slow-frozen group the presence of cytoplasmic condensation and eosinophilic structures indicated apoptosis. The ultrastructure examination confirmed the HE observations. In conclusion, the results demonstrated that staining with HE allows detection of oncosis and apoptosis in cryopreserved embryos. According to these data, vitrification caused more cellular injuries than slow-freezing, and oncosis was the predominant injury. It is important to point that specific molecular apoptosis tests must be performed to confirm these results. This work was supported by FAPESP 04/01252-4.

Vitrification of in vitro cultured rabbit morulae

In the present work, we attempt to establish an efficient vitrification procedure for 32-cell rabbit embryos obtained in vitro. In experiment 1, both the effect of the composition of the vitrification solutions and the cryoprotectant addition (either in one or two steps) were studied. For one-step addition, straws with embryos in the final vitrification solution (total time 60 s) were plunged into liquid nitrogen. For two-step addition, previously embryos were 2 min pre-equilibrated in 0.5 ml of (1:1) PBS plus 20% FCS: vitrification solution without sucrose. Different solutions of cryoprotectants were compared: 25 vol.% ethylene glycol supplemented with 0.25 M sucrose (25EG+S) and 20% ethylene glycol plus 20% dimethyl sulfoxide, alone (20EG+20DMSO−S) or supplemented with 0.25 M sucrose (20EG+20DMSO+S). Six percent (30/487) of the total of 32-cell embryos obtained by in vitro culture in each experimental session was slow-frozen by a classical method as a technical efficiency control. Only 30% slow-frozen embryos reached blastocyst stage. Significant differences in embryo development were detected between the one-step (25EG+S) and two-step (25EG+S) groups and the one-step (20EG−20DMSO+S) and two-step (20EG−20DMSO−S) groups (0–6% versus 36–50%, respectively). Consequently, in the following experiments only these two vitrification procedures were used. In experiment 2, we attempted to substitute the use of PBS by HEPES-buffered Ham’s F-10 (h-CM) in all cryoprotective solutions or media. When h-CM was used, a significant reduction in the in vitro embryo development was observed when the HEPES-buffered groups were compared with one-step (20EG−20DMSO+S) group in s-PBS (35–45% versus 73%). In experiment 3, the one-step (20EG+20DMSO+S) and two-step (20EG+20DMSO−S) procedures were assayed using two FCS levels (20 and 40%) in the PBS-based media. Relative to in vitro development, the highest rates were reached with one step (20EG−20DMSO+S), using PBS plus 20% FCS, which was different from two steps (20EG−20DMSO−S), regardless of percentage of FCS in the PBS-based media (81% versus 41–45%; P<0.05). In conclusion, we propose either the one step (20EG−20DMSO+S) or two steps (20EG−20DMSO−S) prepared in PBS plus 20% serum for use in future works.