scholarly journals 116DEVELOPMENT INTO BLASTOCYSTS OF SWAMP BUFFALO OOCYTES AFTER VITRIFICATION AND NUCLEAR TRANSFER

2004 ◽  
Vol 16 (2) ◽  
pp. 180 ◽  
Author(s):  
R. Parnpai ◽  
C. Laowtammathron ◽  
T. Terao ◽  
C. Lorthongpanich ◽  
S. Muenthaisong ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Somatic Cell ◽  
Sucrose Solution ◽  
Nuclear Transplantation ◽  
Developmental Potential ◽  
Domestic Species ◽  
Reconstructed Embryos ◽  
University Of Technology ◽  
Before And After ◽  
Swamp Buffalo

Oocyte cryopreservation in the domestic species is still at the experimental stage, but recent studies indicated that vitrification characterized by ultra-rapid cooling rate is promising for cryopreservation of bovine oocytes. In the present study, denuded buffalo oocytes were vitrified by minimum volume cooling procedure (Kuwayama and Kato, 2000, J Assist Reprod Genet 17, 477) after IVM or after IVM and enucleation, and developmental potential into blastocysts of the post-warm oocytes after somatic cell nuclear transplantation was examined. Cumulus-oocyte complexes were matured, denuded, and enucleated as described previously (Parnpai et al., 1999, Buffalo J 3, 371–384). The presumptive metaphase-II (M-II) oocytes before and after enucleation were first equilibrated in 7.5% DMSO+7.5% ethylene glycol+20% FCS in TCM199 for 10 min, and then exposed to 15% DMSO+15% ethylene glycol+0.5M sucrose+20% FCS in TCM199 for 1min. Five oocytes were placed on a Cryotop sheet (Kitazato Supply Co., Tokyo, Japan) and vitrified in liquid nitrogen. The samples were warmed in 0.5M sucrose solution for 5min, directly transferred into TCM199+20% FCS, and kept at room temperature for 1h before being used for a cloning experiment. The post-warm oocytes were fused with ear skin fibroblasts by two DC pulses (26V, 17μs) and activated with 7% ethanol for 5min and then 10μg/mL cycloheximide and 1.25μg/mL cytochalasin-D for 5h. The reconstructed embryos were cultured in mSOFaa+0.2% BSA+0.1% linoleic acid albumin for 2 days, and then co-cultured with bovine oviductal epithelial cells for an additional 5 days. Post-warm morphological survival of M-II oocytes (80%, 187/235) was similar to that of enucleated oocytes (75%, 158/212). Vitrified M-II oocytes were successfully enucleated (96%, 136/142) as were fresh control oocytes (88%, 143/162). Fusion rates of M-II oocytes vitrified before and after enucleation (81%, 94/116 and 78%, 106/136, respectively) were also similar to those of fresh oocytes (81%, 100/123). Percentages of reconstructed embryos developing into hatching blastocysts on Day 7 were 5% (5/91), 6% (6/103), and 8% (8/99) in the groups of oocytes vitrified before and after enucleation, and of fresh control oocytes, respectively (ANOVA tests were not significant different). These results indicate that swamp buffalo oocytes cryopreserved by ultra-rapid vitrification procedure can be used successfully for subsequent somatic cell nuclear transplantation. (Supported by Thailand Research Fund and R&D Fund of Suranaree University of Technology)

scholarly journals 104EFFECT OF HATCHING STATUS ON VITRIFICATION OF CLONED BOVINE BLASTOCYSTS

2004 ◽  
Vol 16 (2) ◽  
pp. 174
Author(s):  
C. Laowtammathron ◽  
T. Terao ◽  
C. Lorthongpanich ◽  
S. Muenthaisong ◽  
T. Vetchayan ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Sucrose Solution ◽  
Survival Rates ◽  
Nuclear Transplantation ◽  
Fibroblast Cells ◽  
Anova Test ◽  
University Of Technology ◽  
Vitrification Solution ◽  
Cloned Bovine

Bovine blastocysts produced by nuclear transplantation have mechanical slits in their zonae pellucidae, and therefore initiate hatching earlier than the non-manipulated embryos. The present study was undertaken to examine whether the hatching stage of cloned blastocysts is among the factors influencing their survival after vitrification and warming. Cloned bovine blastocysts were produced by using adult ear fibroblast cells as reported previously (Parnpai et al., 2002, Theriogenology 57, 443), except that fused couplets were co-cultured with bovine oviductal epithelial cells in mSOFaa medium supplemented with 0.1% linoleic acid-albumin (LAA)+0.2% BSA (Hochi et al., 1999, Theriogenology 52, 497–504). Hatching blastocysts harvested on Day 7 were classified into one of three groups according to the ratio of extruding embryonic diameter from zona (D2) to embryonic diameter inside the zona (D1); category-A: D2/D1=0.01–0.70; category-B: D2/D1=0.71–1.00; category-C: D2/D1=1.01–1.70. The blastocysts were first exposed to 10% DMSO+10% ethylene glycol in TCM199+20% FCS for 2min, and then equilibrated in 20% DMSO+20% ethylene glycol+0.5M sucrose with or without 10% Ficoll in TCM199+20% FCS for 30s. One to three blastocysts were placed on a Cryotop sheet (Kitazato Supply Co., Tokyo, Japan) and vitrified in liquid nitrogen. The samples were warmed in 0.5M sucrose solution for 2min and transferred into TCM199+20% FCS in five steps (5min per step). The post-warm survival of the blastocysts was assessed by in vitro culture for 24h. When Ficoll-free vitrification solution was used, post-warm survival rate of the category-A blastocysts (77%, 23/30) was not significantly different (ANOVA test) from those of category-B and category-C blastocysts (74%, 20/27; and 80%, 24/30; respectively). Inclusion of 10% Ficoll in the vitrification solution did not improve (ANOVA test) the post-warm survival rates of cloned blastocysts (category-A: 65%, 22/34; category-B: 54%, 15/28; category-C: 59%, 19/32). Groups of fresh nonsurgical embryos, vitrified with or without Ficoll, yielded 66.7% (4/6), 66.7% (2/3) and 40.0% (2/5), respectively, of recipients pregnant at 48 days of gestation. In conclusion, cloned bovine blastocysts, regardless of their hatching stages, were relatively resistant to cryopreservation by vitrification. (Supported by Thailand Research Fund and R&D Fund of Suranaree University of Technology.)

65 CLONED EMBRYOS FROM HORSE SOMATIC CELLS AND ENUCLEATED BOVINE AND OVINE OOCYTES AND THEIR DEVELOPMENTAL COMPETENCE

2008 ◽  
Vol 20 (1) ◽  
pp. 113
Author(s):  
H. M. Zhou ◽  
B. S. Li ◽  
L. J. Zhang
Keyword(s):  
Somatic Cell ◽  
Somatic Cells ◽  
Calf Serum ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
Electrical Pulses ◽  
Mongolian Horse

The objective of this study was to investigate the reprogramming potential of equine somatic cell donor nuclei in either bovine or ovine recipient oocyte cytoplasmic environments. Heterogeneous embryos were reconstructed by somatic cell nuclear transfer (NT). The percentage of fusion and developmental competence, assessed by rates of cleavage and morula and blastocyst formation, were determined. Skin fibroblast cells, obtained from the ear of an adult female Mongolian horse, were dissociated using 0.25% trypsin and cultured in vitro in a humidified atmosphere of 5% CO2 in air at 37°C. Donor somatic cells were serum-starved before NT and used between passages 4 and 6. Bovine and ovine oocytes derived from slaughterhouse ovaries were matured in vitro for 17–19 and 22–24 h, respectively, in a humidified atmosphere of 5% CO2 in air at 38.5°C, before they were enucleated and used as recipient cytoplasts. The fibroblasts were injected under the zona pellucida of the cytoplasts and electrically fused by 2 DC electrical pulses of 1.58 kV cm–1 for 10 μs, with an interval of 0.13 s. The reconstructed embryos were then activated with 5 μm ionomycin in H-M199 for 5 min and then in 2 mm 6-DMAP for 4 h. The equine-bovine and equine-ovine reconstructed embryos were co-cultured, respectively, with bovine and ovine cumulus cells in synthetic oviduct fluid supplemented with amino acids (SOFaa) and 10% fetal calf serum (FCS) for 168 h. The data were analyzed with ANOVA and differences among the groups were evaluated with t-test. The results of the percentages of fusion, cleavage, and development to morula (8 to 64 cells) and blastocyst stages of equine-bovine and equine-ovine heterogeneous embryos are shown in Table 1. This study demonstrates that heterogeneous embryos can undergo early embryonic divisions and that reprogramming of equine fibroblast nuclei can be initiated in foreign cytoplasts. It appears that embryos reconstructed with equine somatic nuclei and ovine cytoplasts have a higher developmental potential than those using bovine cytoplasts. Table 1. Developmental competence of equine-bovine and equine-ovine reconstructed embryos

24 BUFFALO (BUBALUS BUBALIS) SOMATIC CELL NUCLEAR TRANSFER EMBRYOS PRODUCED FROM FROZEN–THAWED SEMEN-DERIVED SOMATIC CELLS: EFFECT OF TRICHOSTATIN A ON THE IN VITRO AND IN VIVO DEVELOPMENTAL POTENTIAL, QUALITY, AND EPIGENETIC STATUS

2015 ◽  
Vol 27 (1) ◽  
pp. 104
Author(s):  
N. L. Selokar ◽  
M. Saini ◽  
H. Agrawal ◽  
P. Palta ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Somatic Cells ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
Frozen Semen ◽  
Significant Difference

Cryopreservation of semen allows preservation of somatic cells, which can be used for the production of progeny through somatic cell nuclear transfer (SCNT). This approach could enable restoration of valuable high-genetic-merit progeny-tested bulls, which may be dead but the cryopreserved semen is available. We have successfully produced a live buffalo calf by SCNT using somatic cells isolated from >10 year old frozen semen (Selokar et al. 2014 PLoS One 9, e90755). However, the calf survived only for 12 h, which indicates faulty reprogramming of these cells. The present study was, therefore, carried out to study the effect of treatment with trichostatin A (TSA), an epigenetic modifier, on reprogramming of these cells. Production of cloned embryos and determination of quality and level of epigenetic markers in blastocysts were performed according to the methods described previously (Selokar et al. 2014 PLoS One 9, e90755). To examine the effects of TSA (0, 50, and 75 nM), 10 separate experiments were performed on 125, 175, and 207 reconstructed embryos, respectively. The percentage data were analysed using SYSTAT 12.0 (SPSS Inc., Chicago, IL, USA) after arcsine transformation. Differences between means were analysed by one-way ANOVA followed by Fisher's least significant difference test for significance at P < 0.05. When the reconstructed buffalo embryos produced by hand-made clones were treated with 0, 50, or 75 nM TSA post-electrofusion for 10 h, the cleavage percentage (100.0 ± 0, 94.5 ± 2.3, and 96.1 ± 1.2, respectively) and blastocyst percentage (50.6 ± 2.3, 48.4 ± 2.7, and 48.1 ± 2.6, respectively), total cell number (274.9 ± 17.4, 289.1 ± 30.1, and 317.0 ± 24.2, respectively), and apoptotic index (3.4 ± 0.9, 4.5 ± 1.4, and 5.6 ± 0.7, respectively) in Day 8 blastocysts were not significantly different among different groups. The TSA treatment increased (P < 0.05) the global level of H4K5ac but not that of H3K18a in embryos treated with 50 or 75 nM TSA compared with that in controls. In contrast, the level of H3K27me3 was significantly lower (P < 0.05) in cloned embryos treated with 75 nM TSA than in embryos treated with 50 nM TSA or controls. The ultimate test of the reprogramming potential of any donor cell type is its ability to produce live offspring. To examine the in vivo developmental potential of the 0, 50, or 75 nM TSA treated embryos, we transferred Day 8 blastocysts, 2 each to 5, 6, and 5 recipients, respectively, which resulted in 2 pregnancies from 75 nM TSA treated embryos. However, one pregnancy was aborted in the first trimester and the other in the third trimester. In conclusion, TSA treatment of reconstructed embryos produced from semen-derived somatic cells alters their epigenetic status but does not improve the live birth rate. We are currently optimizing an effective strategy to improve the cloning efficiency of semen-derived somatic cells.

scholarly journals 117 CYTOPLASMIC FACTORS INFLUENCE DEVELOPMENTAL POTENTIAL OF SAMP1/Yit MOUSE EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 209
Author(s):  
J. Otsuka ◽  
H. Funabashi ◽  
T. Kono
Keyword(s):  
Blastocyst Stage ◽  
Development Rate ◽  
Mouse Embryos ◽  
The Other ◽  
Nuclear Transplantation ◽  
Chi Square ◽  
Developmental Potential ◽  
Reconstructed Embryos

Nuclear transplantation is an efficient means to investigate nucleo-cytoplasmic interactions of mammalian embryos during early development. A recent study has shown that the developmental potential of embryos is affected by the type of cytoplasm. The SAMP1/Yit mouse, an inbred strain that develops spontaneous chronic ileitis resembling Crohn's disease (Matsumoto S 1999 Bioscience Microflora 18, 1–9), has poor reproductive performance, and the developmental ability of embryos is low (unpublished data). Therefore we need to enhance productivity of the SAMP1/Yit mouse. Recently it was reported that cytoplasm of F1 mouse egg supported the development of embryos which have low developmental ability (Muggleton-Harris A et al. 1982 Nature 299, 460–462). In the present study, we examined the influences of the nucleus and cytoplasm on the development of reconstructed embryos in vitro and in vivo, using reciprocal nuclear transplantation between SAMP1/Yit and B6P1F1 (C57BL/6J × SAMP1/Yit) mouse embryos. We evaluated the developmental ability of reconstructed embryos by the development rate into blastocysts in vitro and by the rate of offspring after transfer of blastocysts to recipient mice. Pronuclear transplantation was carried out as reported previously (McGrath J and Solter D 1983 Science 220, 1300–1302). Briefly, karyoplasts from one-cell SAMP1/Yit embryos were introduced into enucleated B6P1F1 zygotes (SAMP1/B6P1F1) and fused by addition of inactivated HVJ (2700 U L−1). The other group of reconstructed embryos (B6P1F1/SAMP1) was manipulated similarly. After fusion, reconstructed embryos were cultured in drops of KSOM medium for 120 h at 37°C in 5% CO2 in humidified air. Some reconstructed and control (unmanipulated) embryos that developed to the blastocyst stage were transferred to the uteri of recipient mice. Data were compared using chi-square test; differences were considered significant at P < 0.01. The development rate of [SAMP1/B6P1F1] embryos to the blastocyst stage was significantly (P < 0.01) higher (75.0%) than that of SAMP1/Yit controls (39.1%). The rate of offspring in [SAMP1/B6P1F1] was also significantly (P < 0.01) higher (47.5%) than that of SAMP1/Yit controls (22.1%). On the other hand, [B6P1F1/SAMP1] embryos showed low developmental potential compared to B6P1F1 control embryos. These results indicate that the source of the cytoplasm strongly influences the development of reconstructed embryos containing SAMP1/Yit karyoplasts. Table 1.

Zebularine significantly improves the preimplantation development of ovine somatic cell nuclear transfer embryos

2019 ◽  
Vol 31 (2) ◽  
pp. 357 ◽  
Author(s):  
Hui Cao ◽  
Jun Li ◽  
Wenlong Su ◽  
Junjie Li ◽  
Zhigang Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cumulus Cells ◽  
Control Group ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
Donor Cells ◽  
Pluripotency Genes

Aberrant DNA methylation reduces the developmental competence of mammalian somatic cell nuclear transfer (SCNT) embryos. Thus, hypomethylation-associated drugs are beneficial for improving reprogramming efficiency. Therefore, in the present study we investigated the effect of zebularine, a relatively novel DNA methyltransferase inhibitor, on the developmental potential of ovine SCNT embryos. First, reduced overall DNA methylation patterns and gene-specific DNA methylation levels at the promoter regions of pluripotency genes (octamer-binding transcription factor 4 (Oct4), SRY (sex determining region Y)-box 2 (Sox2) and Nanog) were found in zebularine-treated cumulus cells. In addition, the DNA methylation levels in SCNT embryos derived from zebularine-treated cumulus cells were significantly reduced at the 2-, 4-, 8-cell, and blastocyst stages compared with their corresponding controls (P&lt;0.05). The blastocyst rate was significantly improved in SCNT embryos reconstructed by the cumulus donor cells treated with 5nM zebularine for 12h compared with the control group (25.4±1.6 vs 11.8±1.7%, P&lt;0.05). Moreover, the abundance of Oct4 and Sox2 mRNA was significantly increased during the preimplantation stages after zebularine treatment (P&lt;0.05). In conclusion, the results indicate that, in an ovine model, zebularine decreases overall DNA methylation levels in donor cumulus cells and reconstructed embryos, downregulates the DNA methylation profile in the promoter region of pluripotency genes in donor cells and ultimately elevates the expression of pluripotency genes in the reconstructed embryos, which can lead to improved development of SCNT embryos.

Platelet Alpha-2 Adrenergic Receptor Binding and Plasma Free 3-Methoxy-4-hydroxyphenyl-ethylene Glycol in Depressed Patients before and after Treatment with Mianserin

1992 ◽  
Vol 25 (1) ◽  
pp. 14-19 ◽  
Author(s):  
Motohisa Kaneko ◽  
Tomoyuki Kanno ◽  
Kyoichi Honda ◽  
Hirobumi Mashiko ◽  
Nobuyoshi Oosuga ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Receptor Binding ◽  
Adrenergic Receptor ◽  
Depressed Patients ◽  
Before And After

Efficient reactivation of Xenopus erythrocyte nuclei in Xenopus egg extracts

1995 ◽  
Vol 108 (6) ◽  
pp. 2187-2196 ◽  
Author(s):  
L.J. Wangh ◽  
D. DeGrace ◽  
J.A. Sanchez ◽  
A. Gold ◽  
Y. Yeghiazarians ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Somatic Cell ◽  
Nuclear Transplantation ◽  
Optimal Time ◽  
Genome Replication ◽  
Cell Nuclei ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Rapid genome replication is one of the hallmarks of the frog embryonic cell cycle. We report here that complete reactivation of quiescent somatic cell nuclei in Xenopus egg extracts depends on prior restructuring of the nuclear substrate and prior preparation of cytoplasmic extract with the highest capacity to initiate and sustain DNA synthesis. Nuclei from mature erythrocytes swell, replicate their DNA efficiently, and enter mitosis in frozen/thawed extracts prepared from activated Xenopus eggs, provided the nuclei are first treated with trypsin, heparin, and an extract prepared from unactivated, meiotically arrested, eggs. Optimal replicating extracts are prepared from large batches of unfertilized eggs that are synchronously activated into the cell cycle for 28 minutes (at 20 degrees C). Because the Xenopus cell cycle progresses so rapidly, extracts prepared just a few minutes before or after this time have substantially lower DNA synthetic capacities. At the optimal time and temperature, eggs have just reached the G1/S boundary of the first cell cycle. This fact was revealed by injecting and replicating an SV40 plasmid in intact unfertilized eggs as described previously. We estimate that under optimal conditions approximately 6.14 × 10(9) base pairs of DNA/per nucleus are synthesized in 30–40 minutes, a rate that rivals that observed in the zygotic nucleus. The findings reported here are one step in our long term effort to develop a new in vitro/in vivo approach to nuclear transplantation. Nuclear transplantation in amphibian embryos has been used to establish that the genomes of many types of differentiated somatic cells are pluripotent. But very few such nuclei have ever developed into advanced tadpoles or adult frogs, probably because somatic nuclei injected directly into activated eggs fail to reactivate quickly enough to avoid being damaged during first mitosis. We have already shown that unfertilized eggs can be injected prior to activation of the first cell cycle. Future experiments will reveal whether in vitro reactivated somatic cell nuclei transplanted into such eggs reliably reach advanced stages of development.

scholarly journals e-Learning readiness amongst nursing students at the Durban University of Technology

2017 ◽  
Vol 22 ◽  
pp. 300-306 ◽  
Author(s):  
Marilynne Coopasami ◽  
Stephen Knight ◽  
Mari Pete
Keyword(s):  
Nursing Education ◽  
Nursing Students ◽  
Traditional Learning ◽  
First Year ◽  
Technological Readiness ◽  
Technological Culture ◽  
University Of Technology ◽  
Before And After ◽  
Quasi Experimental ◽  
E Learning

e-Learning and other innovative open learning multimedia modalities of delivering education are being introduced to enhance learning opportunities and facilitate student access to and success in education. This article reports on a study that assessed students' readiness to make the shift from traditional learning to the technological culture of e-Learning at a university in Durban. A quasi-experimental study design was employed to assess such readiness in first year nursing students before and after an appropriate educational intervention. A modified Chapnick Readiness Score was used to measure their psychological, equipment and technological readiness for the change in learning method. It was found that, while students' psychological readiness for e-Learning was high, they lacked technological and equipment readiness. Although e-Learning could be used in nursing education, technological and equipment readiness require attention before it can be implemented effectively in this institution. Fortunately, these technical aspects are easier to resolve than improving psychological readiness.

CPP-ACP: Effect on Dental Plaque Acidity after Water Rinsing Following Topical Fluoride Therapy

2017 ◽  
Vol 41 (1) ◽  
pp. 22-26 ◽  
Author(s):  
Mazhari Fatemeh ◽  
Sharifi Marjan ◽  
Noorollahian Homa ◽  
Sharifi Mahsa
Keyword(s):  
Dental Plaque ◽  
Sucrose Solution ◽  
Inhibitory Effect ◽  
Control Groups ◽  
Treatment Protocols ◽  
Challenge Study ◽  
Plaque Ph ◽  
Before And After ◽  
Water Rinsing ◽  
Topical Fluoride

Objectives: There is some evidence that water rinsing immediately after topical fluoride therapy has the potential to reduce the effectiveness of fluoride. The aim was to determine if covering fluoridated teeth with a layer of mousse containing CPP-ACP could prevent the adverse effect of rinsing on fluoride and consequently its buffering effect on dental plaque pH during cariogenic challenge. Study design: This randomized, controlled, crossover, in situ study was conducted on 25 participants. The participants were subjected to acidulated phosphate fluoride (APF) application followed by five treatment protocols: (1) water rinsing after 30 minutes (APF-30) or (2) immediate water rinsing (APF-0); (3) using CPP-ACP immediately before water rinsing (F-CPP-ACP); and two control groups: (4) no fluoride therapy (No-F) and (5) using CPP-ACP and immediate water rinsing (CPP-ACP-0). After 48 hours, teeth were rinsed with 10% sucrose solution and plaque pH was measured before and after 5, 10, 15, 20 and 30 minutes. Results: The least pH changes, the lowest pH drop, and the quickest pH recovery were found in the APF-30 and F-CPP-ACP groups. APF-0 ranked in the middle and the highest values were in the control groups. Conclusions: The results show that in the case using CPP-ACP on fluoridated teeth, water rinsing immediately after topical fluoride therapy did not seem to influence the inhibitory effect of fluoride on plaque acidity.

scholarly journals Recent Progress in Cryopreservation of Bovine Oocytes

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
In-Sul Hwang ◽  
Shinichi Hochi
Keyword(s):  
Lipid Droplets ◽  
Coiled Coil ◽  
High Sensitivity ◽  
Short Term ◽  
Rock Inhibitor ◽  
Developmental Potential ◽  
Domestic Species ◽  
Cytoplasmic Lipid Droplets ◽  
Recovery Culture ◽  
Bovine Oocytes

Principle of oocyte cryoinjury is first overviewed and then research history of cryopreservation using bovine oocytes is summarized for the last two decades with a few special references to recent progresses. Various types of cryodevices have been developed to accelerate the cooling rate and applied to the oocytes from large domestic species enriched with cytoplasmic lipid droplets. Two recent approaches include the qualitative improvement of IVM oocytes prior to the vitrification and the short-term recovery culture of vitrified-warmed oocytes prior to the subsequent IVF. Supplementation of L-carnitine to IVM medium of bovine oocytes has been reported to reduce the amount of cytoplasmic lipid droplets and improve the cryotolerance of the oocytes, but it is still controversial whether the positive effect of L-carnitine is reproducible. Incidence of multiple aster formation, a possible cause for low developmental potential of vitrified-warmed bovine oocytes, was inhibited by a short-term culture of the postwarm oocytes in the presence of Rho-associated coiled-coil kinase (ROCK) inhibitor. Use of an antioxidantα-tocopherol, instead of the ROCK inhibitor, also supported the revivability of the postwarm bovine oocytes. Further improvements of the vitrification procedure, combined with pre- and postvitrification chemical treatment, would overcome the high sensitivity of bovine oocytes to cryopreservation.

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