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

2010 ◽  
Vol 22 (1) ◽  
pp. 220
Author(s):  
X. L. Zhou ◽  
A. Al Naib ◽  
D. W. Sun ◽  
P. Lonergan
Keyword(s):  
Subsequent Development ◽  
Cumulus Cells ◽  
Theoretical Modelling ◽  
Blastocyst Development ◽  
Oocyte Vitrification ◽  
Permeability Characteristics ◽  
Survival And Development ◽  
Blastocyst Rate ◽  
Improve Rate ◽  
Bovine Oocytes

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 < 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 < 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 < 0.05) than nonvitrified control oocytes. In Experiment 3, the presence of ice-blockers did not improve rate or blastocyst development (P < 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.

131 BOVINE OOCYTE VITRIFICATION IS IMPROVED BY INCREASED PYRUVATE CONCENTRATION AND THE INCLUSION OF HYALURONAN IN THE VITRIFICATION MEDIUM

2007 ◽  
Vol 19 (1) ◽  
pp. 183
Author(s):  
C. B. Sheehan ◽  
M. G. Larman ◽  
D. K. Gardner
Keyword(s):  
Bovine Oocyte ◽  
Blastocyst Development ◽  
Oocyte Vitrification ◽  
Embryo Survival ◽  
Ongoing Research ◽  
Exact Test ◽  
Base Medium ◽  
Intracellular Ice Formation ◽  
Survival And Development ◽  
Bovine Oocytes

Bovine oocytes have limited cryo-tolerance and are susceptible to damage partially caused by intracellular ice formation and reactive oxygen species. The ability to routinely cryopreserve bovine oocytes has remained elusive, with high rates of degeneration upon warming or fertilization failure. Hyaluronan has been shown to increase bovine blastocyst survival after vitrification, and the addition of an antioxidant improves mouse embryo survival. The aim of this study was, therefore, to determine if supplementation of the base medium with hyaluronan and increasing the pyruvate concentration would improve the success of bovine oocyte vitrification. Bovine oocytes were recovered by aspiration of abattoir ovaries. Oocytes were matured for 21 h in a defined system using G-MAT supplemented with recombinant albumin (2.5 mg mL-1) and epidermal growth factor (EGF; 100 ng mL-1). All oocytes were partially denuded using hyaluronidase and divided into 3 groups: Control (non-vitrified, n = 789), G-MOPSTM (Vitrolife Sweden AB, Kungsbacka, Sweden; n = 697), and G-MOPS supplemented with 0.125 mg mL-1 hyaluronan with a 1.5-fold pyruvate increase (designated G-VIT; n = 697). Oocytes were soaked in either G-MOPS or G-VIT for 45 min prior to being vitrified. G-MOPS or G-VIT was then used as the base medium for all vitrification and warming steps. The vitrification procedure was carried out at 37�C using the Cryoloop. Oocytes were incubated in 8% dimethyl sulfoxide (DMSO) and ethylene glycol (EG) for 1 min and 16% DMSO and EG (with 0.65 M sucrose and 10 mg mL-1 Ficol, MW 400 000) for under 30 s, before loading onto the Cryoloop and submerging into liquid nitrogen. Warming was performed using 0.25 M sucrose for 1 min, 0.125 M sucrose for 5 min, and no sucrose for at least 10 min. All oocytes (within 1 h of warming) were then fertilized for 18 h and their survival assessed before being washed and moved to G1.3 (with 8 mg mL-1 BSA). Embryos were again washed, cleavage rates assessed, and embryos were moved to G2.3 (with BSA) on the morning of Day 4 post-fertilization. Blastocyst formation was assessed at 144 h. Differences between treatments were assessed by Fisher&apos;s exact test. Survival, cleavage, and blastocyst development in the G-VIT and G-MOPS groups were 76.9 vs. 58.2% (P &lt; 0.001), 48.5 vs. 41.6% (P &lt; 0.05), and 15.4 vs. 4.1% (P &lt; 0.001), respectively. The cleavage and blastocyst development rates of control oocytes were higher than for both vitrification groups (61.7 and 26.7%, respectively; P &lt; 0.001). Survival and development of vitrified oocytes improved with the addition of hyaluronan and increased pyruvate concentration. Ongoing research into the role of macromolecules and antioxidants should further increase the success of oocyte cryopreservation. This study was supported by a grant from Vitrolife.

scholarly journals Effect of storage tube material and resveratrol during liquid storage of matured bovine oocytes on subsequent development

2017 ◽  
Vol 65 (4) ◽  
pp. 546-555
Author(s):  
Tayita Suttirojpattana ◽  
Tamás Somfai ◽  
Satoko Matoba ◽  
Takashi Nagai ◽  
Rangsun Parnpai ◽  
...  
Keyword(s):  
Embryo Development ◽  
Subsequent Development ◽  
Control Group ◽  
Tube Material ◽  
Blastocyst Development ◽  
Liquid Storage ◽  
Glass Tubes ◽  
Bovine Oocytes

This study determined the optimum storage vessel and the effects of resveratrol for the storage of in vitro matured (IVM) bovine oocytes. After IVM, the oocytes were kept in a Hepes-buffered medium at 25 °C for 20 h in different containers including Eppendorf tubes (ET) made of polypropylene (PP) and polystyrene (PS), and tissue culture tubes (TCT) made of PP, PS, and glass. Then oocytes were subjected to IVF and subsequent in vitro embryo development was compared among the groups and to that of a control group without storage. The percentage of blastocyst development in the control group was significantly higher than in the stored groups (P < 0.05). Among oocytes stored in TCT, the percentage of blastocyst development of oocytes stored in glass TCT was significantly higher than that of oocytes stored in PP and PS TCT (P < 0.05); however, it did not differ from that of oocytes stored in ET. The quality of blastocysts did not differ among the control and stored groups. Embryo development was not affected when 0.1, 1 or 10 μM resveratrol was added to the medium during oocyte storage. In conclusion, glass tubes were optimal for oocyte storage and resveratrol did not improve the development of stored oocytes.

scholarly journals A Method for Chronological Intravital Imaging of Bovine Oocytes during In Vitro Maturation

2008 ◽  
Vol 14 (6) ◽  
pp. 549-560 ◽  
Author(s):  
Morten R. Petersen ◽  
Michael Hansen ◽  
Birthe Avery ◽  
Ingrid B. Bøgh
Keyword(s):  
Embryonic Development ◽  
Oocyte Maturation ◽  
Laser Scanning ◽  
Subsequent Development ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Laser Scanning Microscopy ◽  
Intravital Imaging ◽  
Bovine Oocytes

AbstractOocyte maturation is known to affect the chances for successful fertilization, embryonic development, establishment of pregnancy and delivery of a live, healthy, and viable offspring. Two-photon laser scanning microscopy (TPLSM) has previously been used to evaluate early embryonic development without a detectable impairment of subsequent development, but has never been applied to assess mammalian oocytes throughout in vitro maturation (IVM). Visualization of structures within live oocytes during IVM, followed by fertilization and embryo culture, may improve the understanding of oocyte maturation. To visualize structures within bovine oocytes using TPLSM, it is necessary to remove the cumulus cells that normally surround the oocyte during maturation. Repeated visualization of structures within the same oocyte is possible, if movement of the oocyte can be avoided. In this article, we describe the development of a method for repeated intravital imaging of denuded bovine oocytes using an upright TPLSM equipped with a specially constructed incubator. Oocytes were stained with Hoechst 33258, and the nuclear structures were evaluated. Oocyte fertilization rate was not affected by TPLSM exposure, but the developmental capacity of the denuded oocytes was significantly reduced. This is, to our knowledge, the first article describing repeated intravital imaging during mammalian oocyte maturation using TPLSM.

scholarly journals 67 PREMATURE CHROMOSOME CONDENSATION IS NOT ESSENTIAL FOR BOVINE SOMATIC NUCLEAR REPROGRAMMING

2005 ◽  
Vol 17 (2) ◽  
pp. 183
Author(s):  
L.Y. Sung ◽  
F. Du ◽  
B.S. Jeong ◽  
C.C. Chang ◽  
J. Xu ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Subsequent Development ◽  
Donor Cell ◽  
Cumulus Cells ◽  
Chromosome Condensation ◽  
Nuclear Reprogramming ◽  
Premature Chromosome Condensation ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Cloned Bovine

The objective of this study was to determine the effect of donor nuclear exposure in MII oocyte cytoplasm on nuclear reprogramming events and subsequent development of cloned embryos in cattle. Somatic nuclear transfer (NT) was performed by electrofusion of the enucleated MII oocytes with cultured cumulus cells by ovum pickup. Donor cell-cytoplast pairs were fused by applying two direct current pulses at 2 kV/cm for 10 μs. Fused NT embryos were randomly divided in to Treatment A (immediate activation) and Treatment B (delayed activation, 4-h exposure in MII cytoplast before activation). In both treatment groups, the activation protocol was identical and consisted of incubation in cycloheximide (10 μg/mL) plus cytochalasin D (2.5 μg/mL) in M199 + 7.5% FBS for 1 h, followed by culture in cycloheximide (10 μg/mL) for an additional 4 h. Reconstructed embryos in both groups were subsequently cultured in CR1aa in 5% CO2, 5% O2, and 90% N2 at 39°C. Samples from both treatments were fixed at 0, 1, 2, 4, 6, 12, 18, 24, 30, 36, and 44 h after fusion. All fixed samples were double stained for tubulin and DNA, and observed with a laser-scanning confocal microscope for changes in nuclei and microtubules. The experiment was replicated three times. Cleavage rate and blastocyst rate were recorded and analyzed by Studen's t-test (SPSS 11.0, Chicago, IL). The staining revealed an absence of premature chromosome condensation (PCC) in all embryos in Treatment A. However, delayed activation (Treatment B) resulted in a high incidence of PCC, probably due to high levels of MPF in the MII cytoplasts. Chromosome condensation was observed in Treatment B at 4 h (82%, n = 17), 6 h (80%, n = 10), 12 h (36%, n = 25), 18 h (71%, n = 24), 24 h (50%, n = 16) and 30 h (6%, n = 18) after fusion. Subsequent culture results of these cloned embryos (Table 1) indicated that there were significantly higher cleavage rates and blastocyst development in Treatment A than in Treatment B. This study clearly demonstrated that PCC is not essential to support bovine cloned blastocyst development. Direct exposure of donor nuclei in a MII cytoplast enviroment for a very short time was sufficient for nuclear reprogramming. Table 1. Effect of donor nuclear exposure duration in MII cytoplasm on the development potential of cloned bovine embryos

Cryopreservation of equine oocytes: looking into the crystal ball

2020 ◽  
Vol 32 (5) ◽  
pp. 453 ◽  
Author(s):  
Tine De Coster ◽  
Daniel Angel Velez ◽  
Ann Van Soom ◽  
Henri Woelders ◽  
Katrien Smits
Keyword(s):  
Cumulus Cells ◽  
Short Exposure ◽  
Biological Factors ◽  
Blastocyst Development ◽  
Oocyte Vitrification ◽  
Crystal Ball ◽  
Immature Oocytes ◽  
Cryoprotective Agents ◽  
The Past ◽  
Chromosome Alignment

Invitro embryo production has evolved rapidly in the horse over the past decade, but blastocyst rates from vitrified equine oocytes remain quite poor and further research is needed to warrant application. Oocyte vitrification is affected by several technical and biological factors. In the horse, short exposure of immature oocytes to the combination of permeating and non-permeating cryoprotective agents has been associated with the best results so far. High cooling and warming rates are also crucial and can be obtained by using minimal volumes and open cryodevices. Vitrification of invivo-matured oocytes has yielded better results, but is less practical. The presence of the corona radiata seems to partially protect those factors that are necessary for the construction of the normal spindle and for chromosome alignment, but multiple layers of cumulus cells may impair permeation of cryoprotective agents. In addition to the spindle, the oolemma and mitochondria are also particularly sensitive to vitrification damage, which should be minimised in future vitrification procedures. This review presents promising protocols and novel strategies in equine oocyte vitrification, with a focus on blastocyst development and foal production as most reliable outcome parameters.

scholarly journals Role of cumulus cells during vitrification and fertilization of mature bovine oocytes: Effects on survival, fertilization, and blastocyst development

2016 ◽  
Vol 86 (2) ◽  
pp. 635-641 ◽  
Author(s):  
N. Ortiz-Escribano ◽  
K. Smits ◽  
S. Piepers ◽  
E. Van den Abbeel ◽  
H. Woelders ◽  
...  
Keyword(s):  
Cumulus Cells ◽  
Blastocyst Development ◽  
Bovine Oocytes

372 SPERM ASTER FORMATION AND BLASTOCYST DEVELOPMENT OF IN VIVO-MATURED BOVINE OOCYTES FERTILIZED BY INTRACYTOPLASMIC SPERM INJECTION

2007 ◽  
Vol 19 (1) ◽  
pp. 301 ◽  
Author(s):  
T. Horiuchi ◽  
M. Takenaka ◽  
C. Kani ◽  
C. Emuta ◽  
Y. Ogata ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Blastocyst Development ◽  
Chi Square ◽  
First Polar Body ◽  
Bovine Oocytes ◽  
Sperm Aster

In cattle, activation treatment after intracytoplasmic sperm injection (ICSI) is required to improve cleavage and blastocyst rates (Horiuchi et al. 2002 Theriogenology 57, 1013–1024). The reason why the exogenous activation treatment in bovine ICSI is needed to promote cleavage and blastocyst development is not clear. The objective of this study was to examine the effect of activation treatment on sperm aster formation, cleavage, and blastocyst development of in vivo- and in vitro-matured bovine oocytes following ICSI. In vivo-matured oocytes were collected using transvaginal devices under ultrasound guide at about 29 h after GnRH injection from Japanese Black cows superstimulated with a total 19 mg FSH (Antrin�; Denka Pharmaceutical Co., Kanagawa, Japan) divided into twice daily over 3 days, and treated with 750 �g cloprostenol (Estramate�; Sumitomo Chemical Co., Tokyo, Japan). In a total of 8 aspiration sessions, 131 oocytes were collected; of 116 oocytes with expanded cumulus cells, 84 (72%) had a first polar body and were used for ICSI. On the other hand, in vitro-matured bovine oocytes were prepared by culturing immature follicular oocytes derived from abattoir ovaries. Bull spermatozoa, immobilized by scoring their tails, were injected into in vivo- or in vitro-matured oocytes. At 4 h after ICSI, the oocytes were treated with or without 7% ethanol for 5 min for activation. The injected oocytes were fixed at 8 h after ICSI, and sperm aster formation was examined by using specific antibodies and immunofluorescence microscopy. Data were analyzed by the chi-square test in all experiments. The rate of sperm aster formation in in vivo-matured oocytes was similar regardless of activation treatment (71% vs. 65%), but the rate in in vitro-matured oocytes was significantly (P &lt; 0.05) higher in the group receiving activation treatment than in the non-activation group (57% vs. 19%). Cleavage (88% vs. 88%) and blastocyst rates (59% vs. 47%) of in vivo-matured oocytes after ICSI were also similar, regardless of activation treatment, but cleavage (72% and 20%) and blastocyst rates (19% and 7%) of in vitro-matured oocytes were significantly (P &lt; 0.05) higher in the group receiving activation treatment than in the non-activation group. Moreover, the blastocyst rate of in vivo-matured oocytes was significantly (P &lt; 0.05) higher than the rate in in vitro-matured oocytes. These results show that activation treatment after ICSI of in vivo-matured bovine oocytes is not necessary for cleavage and blastocyst development, and suggest that the necessity of activation treatment in bovine ICSI has relevance to in vitro maturation of bovine oocytes.

26 Effect of different cryoprotectant concentrations on vitrification of invitro-matured bovine oocytes in paper containers

2021 ◽  
Vol 33 (2) ◽  
pp. 120
Author(s):  
T. A. Oliveira ◽  
L. F. L. Correia ◽  
L. Schmaltz ◽  
Y. Locatelli ◽  
V. J. F. Freitas ◽  
...  
Keyword(s):  
Cumulus Cells ◽  
Final Concentration ◽  
Control Group ◽  
Cleavage Rate ◽  
Oocyte Vitrification ◽  
Low Concentrations ◽  
Paper Device ◽  
Fetal Bovine ◽  
Post Hoc ◽  
Bovine Oocytes

A great challenge for successful oocyte vitrification is the development of a low-cytotoxic cryoprotectant solution in a safe device allowing ultra-rapid cooling. This study compared different concentrations of cryoprotectants for bovine IVM-oocyte vitrification in a safe paper container device on oocyte survival and cleavage rates. Abattoir ovaries were obtained and cumulus–oocyte complexes (COCs) were recovered by aspirating follicles of 3 to 6mm in diameter. A total of 470 COCs with homogeneous cytoplasm oocytes, surrounded by several layers of cumulus cells were selected, in 5 replicates. Groups of ∼50 COCs were matured in 500µL of semi-defined IVM medium for 22h at 38.8°C in a humidified atmosphere with 5% CO2. After IVM, COCs were allocated to 1 of 3 groups of 20 to 30 COCs, differing only in final concentration of cryoprotectants. A nonvitrified control group (CG) was also tested, totalling 4 groups. Before vitrification, each group was transferred to 500µL of TCM-199 HEPES with 20% fetal bovine serum (FBS) (Base medium, BM) for 5min at 34°C, and COCs were partially denuded by gentle pipetting. Vitrification followed a 3-step protocol at room temperature and groups of 4 to 5 COCs were transferred to BM solution drops containing (1) 5% ethylene glycol (EG) + 5% dimethyl sulfoxide (DMSO) for 30 s; (2) 10% EG + 10% DMSO + 0.25M sucrose for 30 s; and (3) vitrification solution (VS), according to each group: high (HG), 20% EG + 20% DMSO + 0.5M sucrose; medium (MG), 15% EG + 15% DMSO + 0.5M sucrose; or low (LG), 10% EG + 10% DMSO + 0.5M sucrose for 30s. Afterwards, COCs were loaded in &lt;1µL of solution and placed in a homemade paper container device, and immediately plunged in liquid nitrogen. Warming was performed placing the paper container in 3mL of 1M sucrose in BM for 2min. After warming, a 3-step protocol was conducted and COCs were transferred to (1) 500µL of 0.5M sucrose in BM for 2 min; (2) 500µL of 0.25M sucrose for 2 min; (3) 500µL of BM for 2min. Then, COCs from each group were transferred to 250µL of semi-defined IVF medium. Motile sperm were recovered by Percoll washing from one bull and added to IVF medium (Day 0) at final concentration of 106 sperm mL−1 for 18h. At Day 1, all presumptive zygotes were cultured in 25µL of SOF medium with 5% FBS under mineral oil at 38.8°C with 5% CO2 and 5% O2. Normal data were subjected to ANOVA and post hoc Tukey test. Cleavage rate was recorded at Day 2 after IVF. Oocyte survival rate was similar (P&gt;0.05) among vitrified groups (HG, 80%; MG, 86%; LG,87%). Cleavage rate differed (P&lt;0.05) in all vitrified groups compared with control (CG, 82%; HG, 10%; MG, 16%; LG, 16%). Although no difference (P&gt;0.05) was observed among vitrified groups, MG and LG showed a slightly increased oocyte survival and cleavage rates compared with HG. In conclusion, the use of either medium or low concentrations of cryoprotectants may be a less toxic alternative for vitrification of IVM bovine oocytes on paper device. This research was funded by CAPES/COFECUB (#88881.142966/2017-01).

Methodological approaches for vitrification of bovine oocytes

Zygote ◽  
2020 ◽  
pp. 1-11
Author(s):  
Linda Dujíčková ◽  
Alexander V. Makarevich ◽  
Lucia Olexiková ◽  
Elena Kubovičová ◽  
František Strejček
Keyword(s):  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Oocyte Vitrification ◽  
Factors Affecting ◽  
Oocyte Stage ◽  
Follicle Size ◽  
Oocyte Donors ◽  
Methodological Approaches ◽  
Bovine Oocytes

Summary Numerous factors affect vitrification success and post-thaw development of oocytes after in vitro fertilization. Therefore, elaboration of an optimal methodology ensuring higher cryotolerance of oocytes and subsequent blastocyst yield is still of great interest. This paper describes and evaluates critical factors affecting the success of oocyte vitrification. In particular, an appropriate oocyte stage such as maturation status (germinal vesicle stage, metaphase II stage), presence/absence of cumulus cells before vitrification, and the effect of follicle size, as well as different culture systems and media for in vitro production of embryos, the types and concentrations of cryoprotectants, and cooling and warming rates at vitrification are considered. Special attention is paid to various cryocarriers used for low-volume vitrification, which ensures safe storage of oocytes/embryos in liquid nitrogen and their successful post-thaw recovery. At the end, we focussed on how age of oocyte donors (heifers, cows) influences post-thaw development. This review summarizes results of recently published studies describing different methodologies of cryopreservation and post-thaw oocyte development with the main focus on vitrification of bovine oocytes.

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