Non-vitrified Oocytes Research Articles

Effect of meiotic stages during in vitro maturation on the post thaw recovery of buffalo oocytes

The present study has been undertaken to assess the post thaw recovery of buffalo oocytes vitrified at different stages of in vitro maturation (IVM). Cumulus oocyte complexes (COCs) obtained from slaughterhouse ovaries were randomly divided into 6 different groups: control (non-vitrified oocytes were matured for 24 h in maturation medium (MM) consists of TCM-199 supplemented with 10 per cent w/v fetal calf serum (FCS) at 38±1 0 C and 5 per cent CO 2 in a humidified atmosphere, 0 h (vitrified before the onset of maturation), 6, 12, 18 and 24 h groups (vitrified at 6, 12, 18 and 24 h, respectively, after the onset of maturation). Oocytes were exposed to vitrification solution (VS) consists of 40 per cent w/v propylene glycol and 0.25 M trehalose in phosphate buffered saline (PBS) supplemented with 4 per cent w/v bovine serum albumin (BSA) for 3 min at 20-25 0 C. Oocytes in VS were loaded into 0.25 ml French mini straw with 1M sucrose solution separated by two airspace on either side of VS. The straws were sealed with hot forceps and plunged directly into liquid nitrogen (LN 2 ; -196 0 C). The straws were thawed after storage period of atleast 7 days by transferring them into a water bath at 37°C for 30 sec. The cryoprotectant was removed by exposing the oocytes to 1 M sucrose solution. Oocytes in 0, 6, 12, 18 and 24 h groups were further matured for additional 24, 18, 12, 6 and 0 h, respectively, to complete a total of 24 h maturation period.A sum of 495, 432, 457, 416 and 420 oocytes were vitrified in 0, 6, 12, 18 and 24 h groups, respectively. After thawing, 444 (89.70%), 384 (88.89%), 418(91.47%), 381 (91-59%) and 387 (92.14%) oocytes were recovered in 0, 6, 12, 18 and 24 h groups, respectively. It is evident that no significant difference was observed under different vitrification groups.

L-carnitine supplementation during vitrification of mouse germinal vesicle stage-oocytes and their subsequent in vitro maturation improves meiotic spindle configuration and mitochondrial distribution in metaphase II oocytes.

How does l-carnitine (LC) supplementation during vitrification and in vitro maturation (IVM) of germinal vesicle stage (GV)-oocytes improve the developmental competence of the resultant metaphase II (MII) oocytes? LC supplementation during both vitrification of GV-oocytes and their subsequent IVM improved nuclear maturation as well as meiotic spindle assembly and mitochondrial distribution in MII oocytes. Vitrification of GV-oocytes results in a lower success rate of blastocyst development compared with non-vitrified oocytes. LC supplementation during both vitrification and IVM of mouse GV-oocytes significantly improves embryonic development after IVF. GV-oocytes were collected from (B6.DBA)F1 and B6 mouse strains and subjected to vitrification and warming with or without 3.72 mM LC supplementation. After IVM with or without LC supplementation, the rate of nuclear maturation and the quality of MII oocytes were evaluated. At least 20 oocytes/group were examined, and each experiment was repeated at least three times. All experiments were conducted during 2013-2014. Extrusion of the first polar body in IVM oocytes was observed as an indication of nuclear maturation. Spindle assembly and chromosomal alignment were examined by immunostaining of α-tubulin and nuclear staining with 4,6-diamidino-2-phenylindole (DAPI). Mitochondrial distribution and oxidative activity were measured by staining with Mitotracker Green Fluorescence Mitochondria (Mitotracker Green FM) and chloromethyltetramethylrosamine (Mitotracker Orange CMTMRos), respectively. ATP levels were determined by using the Bioluminescent Somatic Cell Assay Kit. LC supplementation during both vitrification and IVM of GV-oocytes significantly increased the proportions of oocytes with normal MII spindles to the levels comparable with those of non-vitrified oocytes in both mouse strains. While vitrification of GV-oocytes lowered the proportions of MII oocytes with peripherally concentrated mitochondrial distribution compared with non-vitrified oocytes, LC supplementation significantly increased the proportion of such oocytes in the (B6.DBA)F1 strain. LC supplementation decreased the proportion of oocytes with mitochondrial aggregates in both vitrified and non-vitrified oocytes in the B6 strain. The oxidative activity of mitochondria was mildly decreased by vitrification and drastically increased by LC supplementation irrespective of vitrification in both mouse strains. No change was found in ATP levels irrespective of vitrification or LC supplementation. Results were considered to be statistically significant at P < 0.05 by either χ(2)- or t-test. It remains to be tested whether beneficial effect of LC supplementation during vitrification and IVM of GV-oocytes leads to fetal development and birth of healthy offspring after embryo transfer to surrogate females. This protocol has the potential to improve the quality of vitrified human oocytes and embryos during assisted reproduction treatment. Partially supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant and Mitacs Elevate Postdoctoral Fellowship, Canada.

44 PRODUCTION OF LIVE PIGLETS AFTER CRYOPRESERVATION OF IMMATURE PORCINE OOCYTES

Development to term of vitrified porcine follicular oocytes is reported in the present study. Immature cumulus-oocyte complexes (COC) were collected from slaughtered prepubertal gilts and were vitrified according to our method published recently (Somfai et al. 2013 J. Reprod. Dev., in press). Briefly, after pretreatment with 7.5 μg mL–1 of cytochalasin B (CB) for 30 min in modified NCSU-37 (a basic medium, BM) at 38.5°C, groups of 88 to 121 COC were equilibrated in a mixture of 2% ethylene glycol (EG), 2% propylene glycol (PG), and 7.5 μg mL–1 CB for 13 to 15 min. Then, COC were washed in vitrification solution (17.5% EG, 17.5% PG, 5% polyvinyl pyrrolidone, and 0.3 M trehalose in BM) and then dropped with 2 μL of vitrification solution onto the surface of aluminum foil floating on liquid nitrogen (LN2). Microdroplets (each containing 10–25 COC) were transferred into cryotubes. After storage in LN2 for 2 to 4 weeks, the oocytes were warmed by dropping the microdroplets directly into 2.5 mL of warming solution (0.4 M trehalose in BM) kept in a 35-mm Petri dish on a 42°C hotplate for less than 1 min. Then, the warming dish was placed on a 38°C hotplate and COC were consecutively transferred for 1-min periods into BM containing 0.2, 0.1, or 0.05 M trehalose at 38°C. The COC were matured in vitro for 44 h using porcine oocyte medium (POM) supplemented with 10% follicular fluid (Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213). Then, oocytes were denuded, and their live/dead status and nuclear maturation were determined by their morphology and the presence of the first polar body, respectively. To assess their developmental competence, vitrified and non-vitrified (control) oocytes were in vitro fertilized (IVF; Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041) and then in vitro cultured in porcine zygote medium-5 (PZM-5; Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213). Blastocyst rates were recorded on Days 5, 6, and 7 of culture (Day 0 = the day of IVF). The experiment was replicated 4 times. Data were analysed with 1-way ANOVA and the Tukey test. The results revealed that 86.4% (364/424) of oocytes survived after vitrification, which was significantly lower (P &lt; 0.05) than that of controls [100% (326/326)]. Live oocytes in vitrified and control groups did not differ statistically in terms of nuclear maturation (63.9 v. 65.3%). Blastocyst rates of surviving vitrified oocytes were significantly lower compared with controls on Days 5 (2.4 v. 12.7%), 6 (4.8 v. 17.6%), and 7 (5.6 v. 18.4%). To test their ability to develop to term, 16 and 27 blastocysts on Day 5 developing from vitrified COC were transferred into 2 recipients. Both recipients became pregnant and farrowed a total of 10 live piglets (4 and 6 piglets, respectively). These data demonstrate that large groups of immature porcine oocytes could be cryopreserved by this method showing high survival and maturation rates. Furthermore, despite a low rate of blastocyst development, transfer of Day-5 blastocysts generated from vitrified oocytes resulted in piglet production for the first time in the world. Partially supported by JSPS and HAS under the Japan-Hungary Research Cooperative Program.

A novel method for human oocyte vitrification with a closed device using super-cooled air

Human oocytes have been successfully vitrified with open systems that involve direct contact with liquid nitrogen. Concerns have been raised regarding the safety and sterility of this methodology. It is therefore highly desirable to use a device that does not utilize direct contact with liquid nitrogen during vitrification and storage. It is thought, however, that the high cooling rate afforded by direct liquid nitrogen contact is essential for human oocyte vitrification. Human oocytes were collected from donors and vitrified/warmed for transfer into recipients. Following oocyte warming; fertilization, embryo development, implantation, biochemical pregnancy and clinical pregnancy rates were recorded. The results were compared retrospectively to data from non-vitrified oocytes within the same time period. Almost 600 oocytes from 53 donors were vitrified using a newly developed vitrification medium specifically for oocytes (RapidVit and RapidWarm Oocyte: Vitrolife AB) and a closed vitrification device that uses a straw with super-cooled air (Rapid-i™: Vitrolife AB). Oocytes were collected from 53 oocyte donors (mean age of 24.6 ± 4.0) and vitrified/warmed. The survival and fertilization rates were 94 % and 76 %, respectively. A mean number of 1.8 ± 0.5 embryos were transferred on day 5/6 per recipient (mean age 40.4 ± 4.6). The implantation rate was 34.4 % (33/96). The biochemical and clinical pregnancy rates were 65 % (33/51) and 51 % (26/51), respectively. The ongoing (>12 weeks) pregnancy rate was 49 % with 5 healthy live births reported so far. These results were not statistically different from outcomes with non-vitrified oocytes. This study demonstrates that, despite lower cooling rates, human oocytes can be vitrified without direct contact with liquid nitrogen in a closed device (Rapid-i™). A closed system that uses super-cooled air to vitrify removes the potential concerns regarding contamination from liquid nitrogen.

In vitro blastocyst development of post-thaw vitrified bovine oocytes

Aim: To evaluate the developmental competence of post-thaw vitrified bovine cumulus-oocyte complexes (COCs) in vitro. Materials and Methods: A total of 129 COCs were cryopreserved using vitrification solution comprising of 15% ethylene glycol (EG) + 15% dimethyl sulfoxide (DMSO) + 0.6 M sucrose in medium TCM-199 with 10% FBS. Immediately, within a minute they are plunged into liquid nitrogen using 0.25 ml straws. Thawing was made with a step wise dilution method. Postthaw normal vitrified and non-vitrified oocytes were subjected to in vitro maturation and in vitro fertilization. Results: Post-thaw survival percentage of vitrified oocytes was 88.37% and maturation performance of vitrified oocytes on the basis of cumulus expansion was 81.58% as compared to non-vitrified control 93.85%. The in vitro fertilization performance of vitrified oocytes was 49.46% as compared to the non-vitrified ones (63.11%). Similarly, blastocyst formation of vitrified oocytes was 21.74% as compared to 32.47% in non-vitrified oocytes. Conclusion: Vitrification of immature bovine oocytes using 7.5% EG + 7.5% DMSO for equilibration and 15% EG +15% DMSO + 0.6 M sucrose as vitrification solution yielded better in vitro fertilization and blastocyst formation rate.

Decreased Expression of CD9 in Bovine Oocytes After Cryopreservation and the Relationship to Fertilization Capacity

This study was conducted to investigate the effect of vitrification of bovine metaphase-II (MII) oocytes on CD9 expression and fertilization capacity. Surviving vitrified/warmed oocytes were used to detect CD9 distribution (fluorescence microscopy), CD9 mRNA (qRT-PCR), and CD9 protein expression (Western blot), and to analyze in vitro fertilization rates (number of sperm bound to or that penetrated the oocytes) after removing the zona pellucida. Fresh oocytes acted as control. The experimental results showed that the vitrification/warming procedures significantly decreased CD9 expression at the mRNA and protein levels, and changed the CD9 distribution pattern in bovine oocytes. After fertilization in vitro, the average number of sperm binding and penetration of vitrified oocytes were significantly lower than those of the non-vitrified oocytes. In conclusion, vitrification of bovine oocytes caused a decrease in CD9 expression at the mRNA and protein levels, and an alteration of CD9 distribution pattern, which may have resulted in lowered fertilization capacity.

Supplementation of maturation medium with L-carnitine improves cryo-tolerance of bovine in vitro matured oocytes

The objective was to determine the effects of adding L-carnitine (an enhancer of lipid metabolism) during IVM, on cryotolerance and developmental competence of bovine oocytes. Oocytes matured in the absence (control) or presence (0.6 mg/mL) of L-carnitine were subjected to IVF and embryo culture after Cryotop vitrification or nonvitrification at the metaphase stage of the second meiotic cell division. Cleavage and blastocyst formation rates, and inner cell mass and trophectoderm cell numbers were determined. Also, ATP content in IVM oocytes was measured and intracellular lipid droplets were observed (Nile red staining and confocal microscopy). L-carnitine had no significant effect on the rate of matured oocytes. Vitrification reduced (P < 0.05) mean (±SEM) rates of live oocytes both in control (80.6 ± 1.9%) and L-carnitine groups (82.7 ± 5.1%) compared with nonvitrified oocytes (100%). After IVF, cleavage rates of vitrified control and L-carnitine groups (56.5 ± 3.9% and 62.8 ± 5.1%, respectively) were significantly lower than those in nonvitrified control and L-carnitine groups (83.9 ± 4.2% and 84.3 ± 1.3%). After vitrification, blastocyst formation rate in the L-carnitine group (54.4 ± 5.2%) was significantly higher compared with the control (34.9 ± 4.4%), and did not significantly differ from those in nonvitrified control and L-carnitine groups (52.1 ± 4.2% and 52.8 ± 3.0%). The numbers and ratio of inner cell mass and trophectoderm cells in blastocysts did not differ significantly among groups. The ATP content in L-carnitine–treated oocytes tended to be higher compared with the control. Vitrification did not reduce ATP content in oocytes, irrespective of L-carnitine treatment. Treatment with L-carnitine dislocated lipid droplets from the peripheral area to the inner cytoplasm. In conclusion, L-carnitine supplementation during IVM redistributed lipid droplets in oocytes; if they survived vitrification, their developmental competence was similar to that of nonvitrified oocytes.

75 ADDITION OF HYALURONAN TO A VITRIFICATION SOLUTION FOR IMMATURE BOVINE OOCYTES SELECTED BY BRILLIANT CRESYL BLUE

An experiment was designed to evaluate the effect of brilliant cresyl blue (BCB) selection of immature oocytes and the addition of sodium hyaluronate (HA) to the vitrification solution on survival rates of bovine oocytes vitrified using solid-phase vitrification. Bovine cumulus–oocyte complexes (COC; n = 716) obtained from slaughterhouse ovaries were used in 6 replicates. Cumulus–oocyte complexes were washed in tissue culture medium 199 (TCM-199) and randomly allocated to 2 groups to be exposed to BCB stain (Sigma Chemical Company, St. Louis, MO, USA) for 90 min as described by Alm et al. (2005 Theriogenology 63, 2194–2205) or (control) maintained in Vigro holding medium (Bioniche Animal Health, Belleville, Canada) for 90 min (n = 220). Cumulus–oocyte complexes in the BCB group were selected based on their response to BCB as BCB+ (colored, n = 248) or BCB– (colorless, n = 248), whereas those in the control group were selected morphologically as described by Rodríguez-González et al. (2002 Theriogenology 57, 1397–1409). Oocytes from both BCB groups and 100 oocytes in the control group were vitrified by solid-phase vitrification as previously described by Rodriguez et al. (2012 Reprod. Fertil. Dev. 24, 132). The remaining 120 oocytes in the control group were not vitrified and were matured, fertilized, and cultured in vitro (in SOFaa in a controlled atmosphere) for 7 days. Vitrified oocytes were exposed to 10% ethylene glycol for 10 min, and 20% ethylene glycol + 0.2-M trehalose for 30 s, and then were subdivided to be exposed to 30% ethylene glycol + 0.5-M trehalose with or without 0.1 mg mL–1 HA (MAP 5, Bioniche Animal Health). Vitrified oocytes were stored in liquid nitrogen for at least one week and then placed directly into a 0.5-M sucrose solution (in TCM 199) at 37°C for 5 min, 0.25 M of sucrose for another 5 min, and finally TCM-199 and matured, fertilized, and cultured. Development rates (i.e. proportion of blastocysts) were examined on Day 7 after fertilization. Proportional data were first transformed by square root and then analyzed by ANOVA to detect the effect of replicate, type of oocyte (BCB+, BCB–, controls), and vitrified with or without HA or not vitrified as main effects, using the software Infostat (UNC, Argentina, 2010). There was a significant effect of oocyte type on blastocyst rate (P &lt; 0.01) following vitrification (BCB+, 6.4 ± 0.4%. v. BCB–, 1.6 ± 0.6%). Control oocytes (not exposed to BCB) resulted in 3.0 ± 2.0% blastocysts following vitrification, which was lower to that obtained with the BCB+ oocytes. Vitrification also influenced development rates (3.0 ± 2.0 v. 32.0 ± 1.3%) for blastocysts produced from vitrified v. nonvitrified oocytes, respectively (P &lt; 0.01). Furthermore, the use of HA in the vitrification solutions did not have a significant effect on development rates (4.7 ± 0.9 v. 3.3 ± 0.9%, for blastocysts obtained from vitrified oocytes with or without HA, respectively). In conclusion, the selection of oocytes by BCB increased the in vitro development rates of vitrified immature oocytes, whereas the use of HA in the vitrification solution did not improve the survival rates of vitrified oocytes.

Achieving oocyte survival and stable spindles after vitrification using closed pulled straws regardless of zona status

ObjectivesEmbryo cryopreservation is a well-established technique at in vitro fertilization centers but the best protocol for vitrification of oocytes and zona manipulation remains inconclusive. This study aimed to determine if the closed pulled straws (CPS) method and zona drilling for vitrification provide a higher survival rate for thawed oocytes. Materials and MethodsFemale MF1 mice were superovulated by injection of gonadotropins. Cumulus-oocyte complexes were derived from excised fallopian tubes and the oocytes were divided into the following three groups: (1) one-hole zona drilling by laser, (n = 40); (2) intact zona (n = 48); and (3) zona digestion by pronase (n = 43). The control group consisted of 40 nonvitrified oocytes. After thawing, surviving oocytes were stained for spindles and chromosomes after 1-hour and 3-hour incubations, and compared to controls. ResultsThere were no significant differences in the survival rates among Groups 1 (34/40, 85%), 2 (34/48, 71%), and 3 (30/43, 70%), but there were significant differences compared with the control oocytes (100%). After 1-hour and 3-hour incubations, vitrified oocytes in the three groups did not have significantly fewer normal spindles than the controls (1 hour: Group 1, 70.5%; Group 2, 64.7%; and Group 3, 66.6% vs. control 90%). There was also no significant difference in the percentage of oocytes with a normal spindle shape between the 1-hour and 3-hour recovery times (3 hours: Group 1, 88.2%; Group 2, 88.2%; and Group 3, 80%; control, 95%). ConclusionsThe zona pellucidum with CPS method has no effect on spindle injury and oocyte survival. Sufficient culture time for the recovery of the meiotic spindle is imperative for fertilization of vitrified oocytes. A CPS vitrified oocyte has the advantages of high survival and preserved good spindles.

Morphological survival and subsequent in vitro maturation of denuded and cumulus compact Bubaline oocytes cryopreserved by ultra rapid cooling

Culturable grade oocytes (n=380) recovered by aspiration of surface follicles from buffalo ovaries (n=97) were either mechanically denuded (DN) or kept cumulus compact (CC) and were vitrified in Dulbecco’s phosphate buffered saline + 0.4% sucrose, 0.4% bovine serum albumin and 6 M concentrations of either ethylene glycol (EG) or propylene glycol (PG). Oocytes were randomly allocated to four groups of vitrification (EGCC, EGDN, PGCC and PGDN) and cryostorage for 7-10 days in liquid nitrogen. They were then warmed to record morphological survival and morphologically normal oocytes were matured in vitro along with fresh oocytes (control) for 24 h in TCM-199 containing hormones (LH + FSH + estradiol) at 38.5 0C and 5% CO2 in humidified air in a CO2 incubator. The arcsine transformed data of the proportion of morphologic survival of oocytes and in vitro maturation of oocytes was compared by DNMR-test. The morphologically normal oocytes were significantly higher (P&lt;0.05) for cumulus compact oocytes compared with denuded oocytes for both cryoprotectants EG and PG. The in vitro maturation was significantly higher (P&lt;0.05) for non-vitrified oocytes (control) compared to vitrified oocytes. Significantly higher (P&lt;0.05) proportion of cumulus compact oocytes matured in vitro compared to denuded oocytes for both cryoprotectants EG and PG. The differences between the cryoprotectants were non-significant. It was concluded that cryo-damage to the oocytes during vitrification can be minimized by the presence of cumulus cells with the oocyte, whereas the two cryoprotectants EG and PG are equally effective in preventing cryodamage to oocytes.

Successful pregnancy following transfer of feline embryos derived from vitrified immature cat oocytes using ‘stepwise’ cryoprotectant exposure technique

Oocyte cryopreservation is the desired tool for the ‘long-term’ storage of female genetic potential especially for endangered/valuable species. This study aims at examining the ability of different cryoprotectant (CPA) and CPA exposure techniques to protect immature feline oocytes against cryoinjury during vitrification. Immature oocytes were submitted to different CPA exposure techniques: 1) 2-step DMSO, 2) 4-step DMSO, 3) 2-step EG, 4) 4-step EG, 5) 2-step EG plus DMSO and 6) 4-step EG plus DMSO. Non-CPA treated, non-vitrified oocytes served as controls. The oocytes were then submitted either to in vitro maturation (Experiment 1, n = 334) or to vitrification/warming (Experiment 2, n = 440). The stage of nuclear maturation was subsequently determined. In Experiment 3, the vitrified immature oocytes (n = 254) were matured and fertilized in vitro, and their developmental competence was assessed. A total of 424 embryos derived from vitrified immature oocytes were transferred into the oviduct of 6 recipient queens (Experiment 4). Vitrification reduced significantly the meiotic and developmental competence of immature cat oocytes compared with the non-vitrified controls. The EG alone or a combination of EG and DMSO yielded higher maturation rates than DMSO, irrespective of the CPA equilibration techniques used. The 4-step EG vitrification resulted in the highest maturation rate (37.6%) but cleavage and blastocyst rates were significantly lower than the non-vitrified controls (24.8% and 30.2% vs 62.5% and 49.3%, respectively). Pregnancy was established in recipients receiving embryos derived from non-vitrified and vitrified/warmed immature oocytes. It is concluded that the stepwise CPA exposure technique can be successfully applied for vitrification of immature cat oocytes, in terms of in vitro development but it is likely to affect in utero development.

74 MECHANICAL DELIPIDATION IMPROVES CRYOSURVIVAL AND IN VITRO DEVELOPMENT OF VITRIFIED CAT OOCYTES

Although considerable progress has been made in the development of successful methods for cryopreservation of embryos, oocytes are much less cryotolerant. There appears to be an inverse relationship between cryosurvival and intracellular lipid levels. For example, cat oocytes, which appear microscopically as coffee-coloured, nearly opaque spheres due to their high lipid content, are extremely sensitive to cryopreservation. Oocyte delipidation thus represents a potential approach to improving cryosurvival. The objectives of the present study were to examine 1) the effects of calcium (Ca2+, 0 v. 10 nM), FBS (0 v. 10%), and cytochalasin B (CB, 7.5 v. 20.0 μg mL–1) during mechanical delipidation by high-speed centrifugation on in vitro development of IVM cat oocytes, and 2) the influence of centrifugation, degree of lipid polarization (partial v. full), and co-culture with cat fetal fibroblasts (CFF) on in vitro development of vitrified IVM cat oocytes. In Experiment 1, oocytes were randomly allocated to each centrifugation medium and centrifuged at 12 000 × g for 20 min. Oocytes were then fertilized with epididymal sperm (motile sperm mL–1) and cultured until Day 8 (Pope et al. 2006 Theriogenology 66, 59–71). In Experiment 2, oocytes were centrifuged with the optimal centrifugation medium obtained in experiment 1, allocated to each treatment and vitrified in a solution of 15% DMSO, 15% ethylene glycol, and 18% sucrose (2008 Reprod. Fertil. Dev. 20, 188). Liquified oocytes were fertilized and cultured until Day 8. In both experiments, cleavage and degeneration rates were determined on Day 2 and blastocyst development on Day 8. Data were analysed by 2-way ANOVA and chi-square tests. In Experiment 1, of 939 oocytes that were centrifuged and fertilized, 40% of those treated in 0 nM Ca2+ cleaved and 22% developed into blastocysts, v. 33 and 6%, respectively, in 10 nM Ca2+ (P &lt; 0.05). The respective cleavage and degeneration frequencies for oocytes treated in 10% FBS were 43 and 19% v. 19 and 3% in 0% FBS (P &lt; 0.05). Cleavage and blastocyst development after treatment with 7.5 and 20.0 μg mL–1 CB were 36 and 15% v. 42 and 22%, respectively. In Experiment 2, 493 oocytes were vitrified/liquified and fertilized. The degeneration, cleavage, and blastocyst rates of non-centrifuged oocytes were 49, 21, and 0% v. 31 (P &lt; 0.05), 38 (P &lt; 0.05), and 7%, respectively, of centrifuged oocytes. Of centrifuged oocytes with partially extruded lipids, 34% degenerated, 34% cleaved, and 4% developed into blastocysts v. 29, 42, and 10%, respectively, of oocytes with fully extruded lipids. Degeneration, cleavage and blastocyst rates of co-cultured v. control oocytes were 18, 36, and 10%, v. 26 (P &lt; 0.05), 34, and 3%, respectively. In summary, cryotolerance of domestic cat oocytes to vitrification was 1) affected by their lipid content, and 2) improved by mechanical reduction of intracellular lipids. When oocytes were fully delipidated in Ca2+-free medium containing 10% FBS and 20.0 μg mL–1 CB before vitrification and co-cultured after IVF with CFF, blastocyst development was similar to that of control, non-vitrified oocytes.

Effects of vitrification solutions and equilibration times on the morphology of cynomolgus ovarian tissues

This study assessed the effects of vitrification solutions and equilibration times on morphology of cynomolgus ovarian tissues. Ovarian cortical sections (0.1-0.2 cm thickness) of seven cynomolgus monkeys were randomly allocated to either a control group or one of six vitrification groups. Ovarian tissue sections were vitrified ultra-rapidly by placing them directly into liquid nitrogen using two different vitrification solutions (VSEGP: 5.64 mol/l ethylene glycol+5% (w/v) polyvinylpyrrolidone+0.5 mol/l sucrose; and VSED: 3.22 mol/l ethylene glycol+2.56 mol/l dimethylsulphoxide+0.5 mol/l sucrose) after three different exposure times (5-20 min). After warming, follicle morphology was analysed using light and transmission electron microscopy. The proportion of morphologically normal follicles vitrified using VSED after a 5-min exposure was lower (P<0.05) than those vitrified by other conditions. The proportion of normally structured mitochondria in oocytes of preantral follicles vitrified after a 5-min exposure to VSED (56%) was lower (P<0.01) than those vitrified by other conditions (78-88%). Following tissue vitrification with VSED, the surface ratio of lysosome was increased compared with non-vitrified oocytes (1.64% versus 1.11%; P<0.05). These results indicate that VSEGP can support the morphology of vitrified preantral follicles and oocytes.

123 BOVINE OOCYTE VITRIFICATION USING THE CRYOTOP METHOD: EFFECT OF CUMULUS CELLS AND VITRIFICATION PROTOCOL ON SURVIVAL AND SUBSEQUENT DEVELOPMENT

The ability to successfully cryopreserve mammalian oocytes has numerous practical and economic ethical benefits that may positively affect animal breeding programs and assisted conception in humans. However, oocyte survival and development following cryopreservation remain poor. The aim of the present study was (1) to evaluate the effect of the presence of cumulus cells on the outcome of vitrification of immature (GV) or mature (MII) oocytes, (2) to compare empirical and theoretical vitrification protocol, and (3) to assess the effect of adding ice blockers to vitrification media on survival and development competence of bovine oocytes following vitrification using the Cryotop method. Bovine oocytes were collected from the ovaries of slaughtered cross-bred beef heifers. In Experiment 1, cumulus-enclosed and partially denuded GV and MII oocytes were vitrified in 15% EG + 15% DMSO + 0.5 M sucrose in 2 steps. In Experiment 2, GV oocytes were vitrified as above or using theoretical modelling based on permeability and osmotic tolerance characteristics (Wang et al. Reprod. Fertil. Dev. 21 141) in 30% EG +11.4% trehalose in 3 steps or 40% EG + 11.4% trehalose in 4 steps. In Experiment 3, GV oocytes were vitrified in media supplemented or not with 1 of 2 ice blockers (21st Century Medicine, Fontana, CA, USA) 1% X-1000, 1% Z-1000, or both in 3 steps. The survival, cleavage, and blastocyst rate of cumulus-enclosed oocytes was significantly higher (ANOVA) than those of partially denuded oocytes when vitrified at GV (93.8% v. 81.3%, 65.8% v. 47.3%, 11.3% v. 4.0%, respectively, P &lt; 0.05). However, no significant effect of cumulus cover was detected between the two groups when vitrified at MII (93.0% v. 91.8%, 35.2% v. 36.8%, 5.0% v. 4.4%, respectively). Furthermore, cumulus-enclosed oocytes vitrified at the GV stage exhibited a significantly higher development competence than those vitrified at MII (P &lt; 0.05). In Experiment 2, there were no significant differences in the survival, cleavage, and blastocyst rates among 3 protocols (86.0% v. 92.8% v. 91.2%, 44.8% v. 54.4% v. 45.6%, 5.0% v. 5.4% v. 4.0%, respectively). However, cleavage and blastocyst rate were significantly lower (P &lt; 0.05) than nonvitrified control oocytes. In Experiment 3, the presence of ice-blockers did not improve rate or blastocyst development (P &lt; 0.05). In conclusion, cumulus-enclosed GV bovine oocytes survived vitrification and subsequently developed at higher rates than MII oocytes. Theoretical analysis of permeability characteristics and tolerance limits alone may not be sufficient to improve vitrification protocols.

Improved parthenogenetic development of vitrified-warmed bovine oocytes activated with 9% ethanol plus 6-DMAP

The objective was to compare various activation protocols on developmental potential of vitrified bovine oocytes. Bovine oocytes matured in vitro for 23 h were vitrified with EDFSF30 in open pulled straws. After warming, they were cultured in vitro for 1 h, followed by parthenogenetic activation. Vitrified-warmed oocytes had a morphologically normal rate similar to that of controls (nonvitrified oocytes cultured in vitro for 24 h; 98.6% vs. 100%, P > 0.05). When vitrified-warmed oocytes were first activated with 7% ethanol for 5 min and then incubated in 6-dimethylaminopurin (6-DMAP) for 4 h, cleavage and blastocyst rates were 41.2% and 23.2%, respectively, which were lower than those of controls (77.5% and 42.0%, P < 0.05). Subsequently, we varied the ethanol concentration to increase the effectiveness of parthenogenetic activation. When either 5%, 6%, 7%, 8%, 9%, 10%, or 11% ethanol alone (for 5 min) or in combination with 6-DMAP (4 h) was used to activate vitrified-warmed oocytes, cleavage rates ranged from 22.3% to 61.1% and blastocyst rates ranged from 1.1% to 30.6%. These rates were optimized when oocytes were treated with 9% ethanol plus 6-DMAP; this was verified in experiments evaluating other activation protocols with 9% ethanol, calcium ionophore A23187, or ionomycin alone, or in combination with DMAP or cycloheximide (CHX). In conclusion, the oocyte activation protocol affected developmental capacity of vitrified bovine oocytes; 9% ethanol (5 min) followed by 6-DMAP (4 h) promoted optimal parthenogenetic activation.

29 PRODUCTION OF BOVINE CLONED EMBRYOS BY NUCLEAR TRANSFER USING VITRIFIED IMMATURE OOCYTES AS RECIPIENT CYTOPLASTS

The cryopreservation of immature oocytes is a logistic alternative to make cytoplasts available throughout the year for cloning by somatic cell nuclear transfer (SCNT). Oocyte cryopreservation will help to overcome hurdles related to oocyte availability, seasonality, or sanitary constraints. The objective of this experiment was to determine the efficiency of vitrification of bovine immature oocytes for use as cytoplasts to produce clone embryos. Cumulus–oocyte complexes (COCs) obtained from bovine ovaries by slicing from a local abattoir were selected and vitrified prior to maturation. Vitrification and warming solutions and exposure times were as previously described (Vieira AD et al. 2008 Rep. Dom. Anim. 43, 314–318) with minor modifications. Groups of 15 COCs were loaded in a 5-μL vitrification solution microdrop in beveled-cut straws (0.5 mL), which were plunged into N2L. Following warming, vitrified and control (non-vitrified) oocytes were in vitro-matured for 22 h and 17 h, respectively (Oliveira ATD et al. 2005 Theriogenology 64, 1559–1572). After maturation, cumulus cells were removed and oocytes were selected by the presence of a polar body. Embryo reconstruction by SCNT, carried out by standard micromanipulation procedures using fibroblast cells from adult origin, and in vitro culture to the blastocyst stage (Day 7) were based on our established procedures (Forell F et al. 2008 Acta Sci. Vet. 36, 141–148). Data regarding oocyte recovery following cumulus cell removal, oocyte survival after micromanipulation, and maturation, fusion, cleavage (Day 2), and blastocyst (Day 7) rates were analyzed by the chi-square test. Oocyte recovery (73.0%, n = 558/764 v. 91.4%, n = 529/579), maturation (46.8%, n = 261/558 v. 65.8%, n = 348/529) and cleavage (47.2%, n = 60/127 v. 60.2%, n = 77/128) rates were lower in the vitrified than in the non-vitrified group, respectively (P &lt; 0.05). Conversely, oocyte survival after micromanipulation (77.8% and 78.4%) and fusion (82.1% and 82.3%) and blastocyst (16.7%, 10/60 v. 23.4%, n = 18/77) rates were similar between vitrified and non-vitrified groups. However, the overall efficiency (blastocysts produced from selected COCs) was 3.4-fold lower for vitrified oocytes than controls. In conclusion, the vitrification of immature bovine oocytes was proven as a valuable procedure for the production of blastocysts by SCNT, providing that a strict selection is made following warming, being an alternative resource either for the use of large numbers of oocytes obtained from slaughterhouse ovaries or to overcome seasonal variations in oocyte supply for use in animal cloning. This work was supported by the Brazilian National Council for Scientific and Technological Development (CNPq).

Cat blastocysts produced in vitro from oocytes vitrified using the cryoloop technique and cryopreserved electroejaculated semen

Oocyte preservation is still a challenge in the cat. The aim of this study was to evaluate the efficiency of oocyte vitrification in cryoloop in the domestic cat and to assess the embryonic development after IVF with cryopreserved semen. In vitro matured cat oocytes were vitrified in cryoloop after exposure to 10% ethylene glycol (EG, 0.9 M) in hepes synthetic oviductal fluid (HSOF) for 1 min, 20% EG (1.8M) in HSOF for 1 min, and 40% EG (3.6M), 10mg/ml Ficoll 70 and 0.3M sucrose in HSOF for 20s. Warmed oocytes were fertilized in vitro with frozen-thawed semen collected by electroejaculation and presumptive zygote were cultured in vitro for 10 days. Results showed that percentage of degenerated oocytes was higher (P<0.01), while cleavage rate and morulae blastocysts rate on day 6 were significantly lower (P<0.01) for vitrified oocytes than control. Blastocyst rate on day 8 was higher (P<0.01) for control oocytes than vitrified counterparts, and also developmental ability was higher (P<0.05) for non-vitrified oocytes, while the hatched blastocyst rate on day 10 was higher (P<0.05) for vitrified oocytes than control. In conclusion cat oocytes can be vitrified in cryoloop with a fairly good survival rate, cleavage rate and embryo development until pre-implantation stage.

Use of Fetuin Before and During Vitrification of Bovine Oocytes

After vitrification of oocytes, fertilization rates and subsequent development are unsatisfactory, possibly due in part to zona hardening. Foetal calf serum (FCS) can prevent zona hardening because of its fetuin content, but FCS composition varies among batches, and may contain viruses. In this study, we therefore compared media supplemented with different sources of macromolecules, 2% bovine serum albumin (BSA), 2% BSA + 1 mg/ml fetuin and 20% FCS, for handling oocytes for 10-30 min prior to vitrification. None of the treatments resulted in developmental rates comparable with the non-vitrified controls, but FCS inclusion in pre-vitrification handling medium resulted in higher blastocyst production per oocyte (p < 0.05) (10.8%) on day 9 of culture than BSA (5.3%) or BSA + fetuin (6.4%). Blastocysts developing from oocytes from all vitrification treatments were somewhat retarded relative to those developed from non-vitrified oocytes. We also tested the use of fetuin during vitrification as well as two different exposure times with cryoprotectants, 180 and 30 s. There was no significant effect of fetuin or exposure time on rates of subsequent blastocyst production.

Effect of Meiotic Stages During In Vitro Maturation on the Survival of Vitrified-Warmed Buffalo Oocytes

The present study was conducted to investigate the effect of meiotic stages during in vitro maturation (IVM) on the survival of vitrified-warmed buffalo oocytes, vitrified at different stages of IVM. Cumulus oocyte complexes obtained from slaughterhouse ovaries were randomly divided into 6 groups: control (non-vitrified, matured for 24 h at 38 +/- 1 degrees C, 5% CO2 in humidified air), and those matured for 0 h (vitrified before IVM) or 6, 12, 18 and 24 h before vitrification. Cumulus oocyte complexes were vitrified in solution consisting of 40% w/v propylene glycol and 0.25 mol/L trehalose in phosphate-buffered saline supplemented with 4% w/v bovine serum albumin. Vitrified cumulus oocyte complexes were stored at -196 degrees C (liquid nitrogen) for at least 7 days and then thawed at 37 degrees C; cryoprotectant was removed with 1 mol/L sucrose solution. Cumulus oocyte complexes in the 0, 6, 12, 18 and 24 h groups were then matured for an additional 24, 18, 12, 6 and 0 h, respectively, to complete 24 h of IVM. Among the five vitrification groups, 89-92% of cumulus oocyte complexes were recovered, after warming, of which 84-91% were morphologically normal. Overall survivability of vitrified cumulus oocyte complexes was lower (p < 0.05) than that of non-vitrified cumulus oocyte complexes (94.5%). Survival rates of cumulus oocyte complexes matured 24 h prior to vitrification (61.3%) were higher (p < 0.05) than those matured for 12 h (46.7%), 6 h (40.6%) and 0 h (37.6%). Nuclear status following 24 h IVM was assessed. A higher proportion of non-vitrified (control) oocytes (72.7%) reached metaphase II (M-II) stage in control than oocytes vitrified for 24 h (60.0%), 18 h (54.4), 12 h (42.3%), 6 h (33.3%) and 0 h (31.6%) (p < 0.05). The results suggest that length of time in maturation medium prior to vitrification influences post-thaw survivability of buffalo oocytes; longer intervals resulted in higher survival rates.

71 VITRIFICATION OF BOVINE OOCYTES IN MEDIA WITH GLYCEROL + ETHYLENE GLYCOL BEFORE AND AFTER IN VITRO MATURATION

The cryopreservation process aims to keep the cellular metabolism in a quiescent state for an indeterminate length of time. In mammals, oocyte cryopreservation success is important for the establishment of genetic banks. The objective of the present experiment was to evaluate the effect of vitrification on oocyte meiotic ability and the integrity of the metaphase plate in immature and in vitro-matured bovine oocytes. Bovine cumulus–oocytes complexes (COCs) were harvested from slaughterhouse ovaries and randomly divided into 3 groups: (G1) non-vitrified oocytes subjected to in vitro maturation, (G2) immature oocytes vitrified and then subjected to in vitro maturation after warming, and (G3) in vitro-matured oocytes subjected to vitrification. For in vitro maturation, oocytes were incubated for 22 h in 5% CO2 in air in TCM-199 with fetal calf serum, estradiol, LH, FSH, pyruvate, and gentamicin. For vitrification, the oocytes were exposed to the cryoprotectors in three steps: solution 1 containing 1.4 m glycerol in PBS for five min, and then solution 2 containing 1.4 m glycerol and 3.6 m ethylene glycol in PBS for another five min. After exposure to the second solution, the oocytes were transferred to 30-µL drops of solution 3 containing 3.4 m glycerol and 4.6 m ethylene glycol, loaded (5 oocytes per straw) in less than 1 min into 0.25-mL straws between two columns of 0.5 m galactose in PBS separated by two air bubbles, and immediately set in liquid nitrogen vapor. After 1 min of equilibration in liquid nitrogen vapor, the straws were immersed in liquid nitrogen. Warming was performed by holding the straws for 10 s in air, followed by 10 more s in a water bath at 20–22�C. The straws were then shaken 5 to 8 times to mix the bubbles (movement similar to that for a thermometer) and left horizontally for 6 to 8 min at room temperature. The rates of metaphase II and degeneration were analyzed by ANOVA followed by the Student t-test. The oocytes were stained with 100 µg mL–1 Hoechst 33342 and examined in an inverted microscope equipped with fluorescent light (UV filters 535 and 617 mm). Three different routines were realized with a total of 90 oocytes per group. The metaphase II rates in G1 (48/90, 53.3%) and G3 (42/90, 46.6%) were statistically the same (P e 0.05), but were higher (P d 0.05) than in G2 (0/90, 0%). The degeneration rates were: G1 (18/90, 20%), G2 (77/90, 85.6%), and G3 (7/90, 7.8%). The vitrification procedure damaged mainly the immature oocytes, since in the G2 the degeneration rate was higher and the oocytes were not able to resume meiosis. Meanwhile, when oocytes were vitrified after in vitro maturation, the metaphase II rate was similar to the one observed in IVM oocytes not subjected to vitrification. This indicates that the vitrification procedure performed in this experiment did not damage the structure of the metaphase II plate. However, more studies are necessary to predict the developmental potential of these in vitro-matured oocytes.