scholarly journals Development of a reliable in vitro maturation system for zebrafish oocytes

Reproduction ◽  
2008 ◽  
Vol 135 (3) ◽  
pp. 285-292 ◽  
Author(s):  
Shinsuke Seki ◽  
Toshimitsu Kouya ◽  
Ryoma Tsuchiya ◽  
Delgado M Valdez ◽  
Bo Jin ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Saline Solution ◽  
Stage Iii ◽  
Hatching Rate ◽  
Cleavage Rate ◽  
Immature Oocytes ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

In zebrafish oocytes, it has been reported that a 60 or 75% Leibovitz L-15 medium or simple balanced saline solution containing 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) is effective for nuclear maturation. However, most of the oocytes that matured under these conditions were not fertilized and did not hatch. Thus, thesein vitromaturation methods could not support the cytoplasmic maturation of zebrafish oocytes. Therefore, we tried to develop a reliablein vitromaturation method for zebrafish oocytes, which supports their ability to be fertilized and to develop till hatching. When zebrafish oocytes at stage III were cultured in 50–100% Leibovitz L-15 medium supplemented with DHP, the highest rates of cleavage (24%) and hatching (12%) were obtained from oocytes matured in 90% Leibovitz L-15 medium. When we examined the suitable pH (7.5–9.5) of the 90% medium, higher rates of cleavage (45%) and hatching (33%) were obtained in oocytes matured at pH 9.0 than at pH 7.5, 8.5, or 9.5 (cleavage rate, 16–29%; hatching rate, 8–21%). In oocytes matured in 90% Leibovitz L-15 medium at pH 9.0, high rates of cleavage (70%) and hatching (63%) were obtained when oocytes were cultured for 270 min with 0.5 mg/ml BSA. Thus, 90% Leibovitz L-15 medium at pH 9.0 containing 0.5 mg/ml BSA was effective for normal maturation of zebrafish oocytes. This method will become a powerful tool for understanding the mechanism ofin vitromaturation in zebrafish oocytes and for the practical use of immature oocytes.

scholarly journals Effect of Alpha-Tocopherol Supplementation in TCM199 Medium on In Vitro Maturation and Cleavage of Buffalo Oocytes

2020 ◽  
Vol 15 (04) ◽  
pp. 66-70
Author(s):  
Vijayalakshmi . ◽  
Shrikant Kulkarni ◽  
KB Sathisha ◽  
HM Yathish ◽  
MH Girish ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Culture Medium ◽  
Alpha Tocopherol ◽  
Cleavage Rate ◽  
Nuclear Maturation ◽  
Maturation Rate ◽  
The Mean ◽  
Cytoplasmic Maturation ◽  
T1 And T2

An experiment was conducted to assess the effect of supplementing α-tocopherol at two different concentrations to the in vitro maturation (IVM) medium (TCM 199 culture medium) on in vitro maturation and cleavage of buffalo oocytes. Ovaries were collected from local slaughter house. Oocytes were collected by aspiration method and were matured in IVM medium (T0, control), IVM medium supplemented with α-tocopherol @ 10 μg/ml (T1) and IVM medium supplemented with α-tocopherol @ 20 μg/ml (T2). The mean cytoplasmic maturation rate was 82.39 ± 0.81, 88.81 ± 1.08, 81.67 ± 1.82 % and nuclear maturation rate was 62.50 ± 8.33, 87.50 ± 5.59, 66.66 ± 7.68 % in T0, T1 and T2 groups, respectively. The fertilized oocytes reaching 2-cell and 4-cell stages of cleavage for T0, T1 and T2 groups were 14.92 ± 1.52, 32.39 ± 1.01 and 16.39 ± 1.25 %, respectively. Significantly (p less than 0.05) higher level of cytoplasmic maturation rate, nuclear maturation rate and cleavage rate was observed in IVM medium supplemented with α-tocopherol @10 μg/ml than other two groups.

336 MEIOTIC DEVELOPMENT AND CORTICAL GRANULES DISTRIBUTION IN CANINE OOCYTES DURING IN VITRO MATURATION

2010 ◽  
Vol 22 (1) ◽  
pp. 324 ◽  
Author(s):  
M. De los Reyes ◽  
D. Luna ◽  
J. Palomino
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Fluorescein Isothiocyanate ◽  
Cumulus Cells ◽  
Homogeneous Distribution ◽  
Cortical Granules ◽  
Dapi Staining ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

Low development of IVM canine oocytes could be in part attributed to an impaired cytoplasmic maturation. In mammalian oocytes, migration and the redistribution of cortical granules (CGs) around the periphery of the oocyte contribute to the inhibition of polyspermy and it is an important criterion to evaluate cytoplasmic maturation. The state of nuclear maturation and the distribution of CGs were evaluated in canine oocytes cultured for different periods in order to compare the synchrony of nuclear and cytoplasmic maturation during in vitro maturation. Bitch ovaries at different stages of the estrous cycle were obtained following ovariectomy. COCs with compact cumulus cells showing a homogeneous cytoplasm were selected for experiments. Thirty-six COCs were processed at immature stage, placed in PBS medium until evaluation. A total of 275 COCs were matured in vitro for 48, 72, and 96 h in TCM-199 with Earle’s salt supplemented with 25 mM Hepes, 10% FCS, 0.25 mM pyruvate, 10 IU mL-1 of hCG, 300 IU mL-1 penicillin, and 20 mg mL-1 streptomycin, at 38.5°C and 5% CO2. At each culture period, the oocytes were stained with Lens culinaris agglutinin (LCA), labeled with fluorescein isothiocyanate, and the CGs distributions were examined under a fluorescent microscope. The nuclear status of the denuded oocytes was determined by DAPI staining under a fluorescence microscope. For each treatment, at least four replicates were performed and the data was analyzed by ANOVA using Tukey’s test to determine the differences P < 0.05. Three types of CGs distribution were distinguished during canine oocyte maturation: (1) homogeneous distribution throughout the cytoplasm including the cortex; (2) heterogeneous (clusters) within the cytoplasm and (3) densely distributed beneath the oolemma. Nuclear stages were classified as immature or germinal vesicle (GV) stage; resumption of meiosis or germinal vesicle break down (GVBD); metaphase I to telophase I (MI toTel I); and mature or second metaphase (MII). The distribution patterns of GCs were different (P < 0.05) among oocytes cultured for different periods and the nuclear maturation status also differed between oocytes cultured for different intervals (P < 0.05). Most (>84%) of the immature oocytes at GV showed a uniform distribution of CGs throughout the cytoplasm. At 48 h of culture, CGs distribution was mainly Type 2 (25%) and 3 (61%) and the oocytes were at GVBD (33%) and MI-Tel I (33%) stages. Most nuclei of the type 3 oocytes were in the MI (40%) and MII (11%) stages, corresponding to those oocytes matured for 72 (88%) or 96 h (71%). These results indicate that canine oocytes migrate to the cortex during IVM and this process is not finished before 72 h of culture. In addition, although the re-distribution of the CGs occurred in parallel with nuclear maturation, the oocytes cannot always proceed to the MII stage; however, in such oocytes the CGs are distributed beneath the oolemma. Supported by Grant FONDECYT 1080618.

scholarly journals Porcine follicular fluid derived from > 8 mm sized follicles improves oocyte maturation and embryo development during in vitro maturation of pigs

Zygote ◽  
2020 ◽  
pp. 1-6
Author(s):  
Ji-Eun Park ◽  
Sang-Hee Lee ◽  
Yong Hwangbo ◽  
Choon-Keun Park
Keyword(s):  
Embryonic Development ◽  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Developmental Competence ◽  
Control Group ◽  
Cumulus Expansion ◽  
Treatment Groups ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation

Summary The aim of the present study was to investigate the effects of porcine follicular fluid (pFF) from large-sized (LFF; >8 mm in diameter) and medium-sized (MFF; 3–6 mm in diameter) follicles on the maturation and developmental competence of porcine oocytes. Cumulus–oocyte complexes (COCs) were collected from follicles 3–6 mm in diameter. The collected COCs were incubated for 22 h with LFF or MFF (in vitro maturation (IVM)-I stage) and were incubated subsequently for 22 h with LFF or MFF (IVM-II stage). Cumulus expansion was confirmed after the IVM-I stage and nuclear maturation was evaluated after the IVM-II stage. Intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured and embryonic development was evaluated. Relative cumulus expansion and GSH levels were higher in the LFF group compared with in the MFF group after the IVM-I stage (P < 0.05). After the IVM-II stage, the numbers of oocytes in metaphase-II were increased in the LFF group and GSH content was higher in all of the LFF treatment groups compared with in the MFF treatment groups during both IVM stages (P < 0.05). ROS levels were reduced by LFF treatment regardless of IVM stage (P < 0.05). Blastocyst formation and the total numbers of cells in blastocysts were increased in all LFF treatment groups compared with the control group (P < 0.05). These results suggested that pFF from large follicles at the IVM stage could improve nucleic and cytoplasmic maturation status and further embryonic development through reducing ROS levels and enhancing responsiveness to gonadotropins.

202 CHARACTERIZATION OF BOVINE OOCYTE CYTOPLASMIC MATURATION WITH COMMON IN VITRO MATURATION PROTOCOLS

2016 ◽  
Vol 28 (2) ◽  
pp. 232
Author(s):  
B. A. Foster ◽  
F. A. Diaz ◽  
P. T. Hardin ◽  
E. J. Gutierrez ◽  
K. R. Bondioli
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
In Vitro Maturation ◽  
Transvaginal Ultrasound ◽  
Bovine Oocyte ◽  
Hoechst 33342 ◽  
Nuclear Maturation ◽  
Mitochondrial Distribution ◽  
Cytoplasmic Maturation

Modulators of 3′-5′-cyclic adenosine monophosphate have been extensively researched to delay nuclear maturation in in vitro maturation (IVM) systems to improve synchronization of nuclear and cytoplasmic maturation. However while normal maturation for many organelles has been characterised, there is a lack of information on how modulators affect cytoplasmic maturation. The goal of this study was to identify the effect of different components of bovine oocyte maturation systems on 3 aspects of cytoplasmic maturation. Bovine oocytes were collected from mixed breed beef cattle using transvaginal ultrasound guided oocyte aspiration. Oocytes were assigned to 1 of 4 treatments; staining immediately after collection (n = 249) or after 24 h of IVM (n = 270), 2 h of pre-IVM in Forskolin and 3-isobutyl-1-methylxanthine (IBMX; n = 254), or 2 h of pre-IVM followed by IVM (n = 259). Following treatment, half of the recovered oocytes were stained with Hoechst 33342 to determine nuclear maturation status, and Calcein AM for gap junction status. The other half were stained with Hoechst 33342, Mitotracker deep red to identify mitochondria distribution patterns and Alexa Fluor 488 conjugated phalloidin for F actin microfilament distribution. Organelle patterns were coded and statistically analysed using linear models to determine if treatment had an effect on the indicators of cytoplasmic maturation or their agreement with nuclear maturation. Results indicated that there was a high degree of variability in both cytoplasmic and nuclear maturation of oocytes irrespective of treatment group, with many oocytes exhibiting aberrant patterns in both mitochondrial and microfilament distribution. Gap junctions were classified as open (immature), partially open or closed (mature), based on the strength of Calcein fluorescence within the ooplasm. Both treatment and nuclear maturation had a significant effect on gap junction status (P < 0.001) with gap junctions tending to close as oocytes matured, while treatment in pre-IVM maintained open gap junctions, even as meiosis progressed. Mitochondria were classified as peripheral (immature), diffuse, central (mature) or too sparse to accurately classify. There was an unexpectedly high proportion of oocytes with few mitochondria (17%), suggesting an incomplete growth phase before collection. There was no correlation between meiotic stage and mitochondrial distribution (P = 0.73), with the majority of oocytes having diffuse mitochondrial distribution. As normal maturation proceeds, microfilaments aggregate and migrate peripherally. However, neither microfilament aggregation nor redistribution were correlated with nuclear maturation (P = 0.6 and P = 0.11 respectively) or mitochondrial distribution (P = 0.33 and P = 0.06 respectively). Overall, results show that while pre-IVM maintains open gap junctions, the system studied here is not sufficient for improving correlation between cytoplasmic and nuclear maturation. Many deviations from normal cytoplasmic maturation are seen with IVM and these irregularities are maintained with prematuration in Forskolin and IBMX.

scholarly journals Development to Blastocyst Stage of Pig Oocytes Matured, Fertilized and Electroactivated In Vitro

2002 ◽  
Vol 45 (6) ◽  
pp. 547-556
Author(s):  
N. R. Mtango ◽  
M. D. Varisanga ◽  
D. Y. Juan ◽  
P. Wongrisekeao ◽  
T. Suzuki
Keyword(s):  
In Vitro Maturation ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Activation Rates ◽  
Oviduct Fluid

Abstract. This study was designed 1) to determine the effectiveness of two in vitro maturation (IVM) media (tissue culture medium [TCM] and modified synthetic oviduct fluid supplemented with amino acids [mSOFaa]), 2) to compare the effects of two in vitro fertilization (IVF) media (modified Tris-buffered medium [mTBM] and mSOFaa) on the developmental competence of pig oocytes, and 3) to test the activation ability of IVM pig oocytes matured in TCM or mSOFaa, electroactivated and cultured in mSOFaa. The nuclear maturation rates were similar between IVM media (91.0 % vs. 89.0 %). A similar result was obtained when the activation rates were 54.2 % in TCM and 56.0 % in mSOFaa, and the blastocyst rates were 7.9 % and 6.1 %, respectively. There was no significant difference between mSOFaa and mTBM in the percentage of embryos with two pronuclei 33.2 % vs. 13.8 % or polypronuclei 5.3 % vs. 13.4 %. The cleavage rate was the same in both media. The medium mSOFaa gave a significantly higher (P< 0.05) blastocyst rate than mTBM (12.7 % vs. 3.9 %). We concluded that mSOFaa can enhance in vitro maturation, fertilization and culture of pig oocytes.

scholarly journals Oxygen tension and oocyte density during in vitro maturation affect the in vitro fertilization of bovine oocytes

2015 ◽  
Vol 36 (6Supl2) ◽  
pp. 4277
Author(s):  
Angelo Bertani Giotto ◽  
Daniela Dos Santos Brum ◽  
Francielli Weber Santos ◽  
Antonio Carlos Galarça Guimarães ◽  
Cibele Garcia Moreira Gonçalves ◽  
...  
Keyword(s):  
Antioxidant Activity ◽  
In Vitro Fertilization ◽  
Oxygen Tension ◽  
In Vitro Maturation ◽  
Ros Production ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Cytoplasmic Maturation ◽  
Bovine Oocytes

Oocyte maturation is the key factor affecting the fertilization and embryonic development. Factors such as oocyte density and oxygen tension can directly influence the IMV. Thus, the objective of this study was to evaluate the effect of the association of oxygen tensions (5% or 20%) with different oocyte densities (1:10?l or 1:20?l) in the in vitro maturation (IVM) of bovine oocytes on maturation and fertilization rates, ROS production and antioxidant activity. Three experiments were performed with bovine oocytes that were obtained from slaughterhouse ovaries. After selection, the oocytes were randomly distributed in four treatments: 1:10/5%; 1:10/20%; 1:20/5%and 1:20/20% for each experiment. In experiment I, nuclear maturation status and cytoplasmic maturation were evaluated through detection of the first polar body by immunofluorescence and the mitochondrial reorganization assay. In experiment II, ROS production and antioxidant activity were analyzed in oocytes and IVM medium after 24 h of maturation through detection of ROS, reduced glutathione (GSH) and Superoxide dismutase activity by spectrofluorimetric methods. In experiment III, fertilization was evaluated through pronucleus formation, sperm penetration with or without decondensation and polyspermy rates by immunofluorescence. In experiment I, the nuclear maturation and cytoplasmic maturation were similar among treatments (P>0.05). In experiment II, reactive oxygen species in oocytes were elevated in treatments with low oxygen tension which was independent of oocyte density (P<0.05). Additionally, ROS levels in IVM medium were higher in treatments with high oocyte density by volume of medium, which was independent of oxygen tension (P<0.05). In Experiment III, the fertilization and penetration rates were higher in the treatment with 20% oxygen tension and high oocyte density (P<0.05). Furthermore, a high incidence of polyspermy was observed in groups with high oxygen tension and low oocyte density (P<0.05). In conclusion, the results of this study indicate an interaction between oxygen tension and oocyte density, which increases ROS production in certain associations and subsequently influences the rates of in vitro fertilization of bovine oocytes. The improved rates of IVF were obtained when IVM was conducted using 20% oxygen tension and high oocyte density (1:20 ul).

scholarly journals 303 EFFECT OF SERUM SUPPLEMENTATION AND ESTRUS CYCLE STAGE ON IN VITRO NUCLEAR MATURATION OF CANINE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 302
Author(s):  
F.Y. Heru ◽  
H.J. Oh ◽  
M.K. Kim ◽  
J. Goo ◽  
M.S. Hossein ◽  
...  
Keyword(s):  
Luteal Phase ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Follicular Phase ◽  
Estrus Cycle ◽  
Immature Oocytes ◽  
Nuclear Maturation ◽  
Serum Supplementation ◽  
Maturation Rate

The present study investigated the effects of the estrus cycle stage and serum supplementation on nuclear maturation of canine oocytes. Ovaries were collected from a private clinic after ovariohysterectomy and classified into follicular, luteal, or anestrus stages through a combination of ovarian morphology and vaginal cytology. A total of 2214 oocytes from 196 ovaries (903 oocytes from 96 anestrus ovaries, 609 oocytes from 36 follicular ovaries, and 702 oocytes from 64 luteal ovaries) were used for experiments. The oocyte retrieval per ovary was 10, 19, and 12 for anestrus, follicular and luteal-phase ovaries, respectively. In Exp. 1, immature oocytes were cultured for 72 h in TCM-199 alone or TCM-199 supplemented with 10% canine anestrus (CAS), estrus (CES), or diestrus (CDS) serum or fetal bovine serum (FBS). In Exp. 2, immature oocytes were cultured for 72 h in TCM-199 supplemented with 0, 5, 10, or 20% CES. After staining with Hoechst 33342, chromatin state and position as well as spindle formation were evaluated to determine the stage of meiosis: germinal vesicle (GV) stage, germinal vesicle breakdown (GVBD), metaphase I (MI) stage, metaphase II (MII) stage. The experiments with anestrus and luteal-phase oocytes were repeated eight times and follicular-phase oocytes were repeated six times. Data were subjected to analysis of variance (ANOVA) and protected least significant difference (LSD) test to determine differences among experimental groups by using the Statistical Analysis System (SAS, SAS Institute, Inc., Cary, NC, USA) program. Statistical significance was determined where P value was less than 0.05. In Exp. 1, the in vitro maturation of oocytes up to MII stage was higher when oocytes were collected from ovaries in follicular phase. The maturation rate up to MII stage was 0.0 to 1.7%, 1.3 to 10.2%, and 1.0 to 3.2% for the oocytes collected from the anestrus, follicular, and luteal-phase ovaries, respectively, depending on the culture media used. In basic TCM media only, 0.0, 1.3, and 2.3% oocytes reached the MII stage for anestrus, follicular, and luteal-phase oocytes, respectively. A significantly higher rate of maturation was obtained when oocytes collected from follicular phase were cultured in TCM-199 supplemented with 10% CES (10.2%), compared to 10% CAS (4.0%), CDS (2.7%), FBS (1.3%), or the control (1.3%). In Exp. 2, supplementing with 10% CES induced the highest (P < 0.05) maturation rate to the MII stage in oocytes collected from follicular-stage ovaries (11.5%) compared to supplementing with 0% (1.0%), 5% (1.3%), or 20% CES (5.1%). Supplementing with CES (5, 10, or 20%) did not have a significant effect on nuclear maturation of canine oocytes collected from anestrus or luteal-stage ovaries. In conclusion, supplementing in vitro maturation medium with 10% CES increased nuclear maturation of canine oocytes, and canine oocytes collected from follicular-stage ovaries are the most suitable to complete nuclear maturation in vitro. This study was supported by grants from the Biogreen 21-1000520030100000.

Cysteamine supplementation of in vitro maturation medium, in vitro culture medium or both media promotes in vitro development of buffalo (Bubalus bubalis) embryos

2008 ◽  
Vol 20 (2) ◽  
pp. 253 ◽  
Author(s):  
T. Anand ◽  
D. Kumar ◽  
M. S. Chauhan ◽  
R. S. Manik ◽  
P. Palta
Keyword(s):  
In Vitro Culture ◽  
In Vitro Maturation ◽  
Culture Medium ◽  
Bubalus Bubalis ◽  
Cell Number ◽  
Hatching Rate ◽  
Cleavage Rate ◽  
Maturation Medium ◽  
Vitro Development

The effects of supplementation of in vitro maturation (IVM) or in vitro culture (IVC) or both IVM and IVC media with cysteamine on the yield, hatching rate (HR) and total cell number (TCN) of buffalo blastocysts were examined. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to IVM and IVF. The IVM or IVC media were supplemented with 0, 50, 100 or 200 µm cysteamine. Supplementation of IVM medium with 50 µm cysteamine increased (P < 0.01) the cleavage rate and blastocyst yield without affecting the HR and TCN whereas a higher concentration of 200 µm significantly (P < 0.05) reduced the blastocyst yield but not TCN. Similar increases in blastocyst yield, without any effect on HR and TCN were observed after supplementation of the IVC medium with 100 (P < 0.01) or 50 µm (P < 0.05) cysteamine, whereas 200 µm cysteamine was ineffective. Supplementation of both IVM medium with 50 µm cysteamine and of IVC medium with 100 µm cysteamine increased the yield of blastocysts and hatched blastocyst by over 100% (P < 0.01) compared with the controls without any adverse effects on HR or TCN. The results of the present study suggest that supplementation of both IVM and IVC media improves the yield of blastocysts without compromising their health.

39 THE EFFECTS OF RESVERATROL DURING IN VITRO MATURATION ON THE DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES VITRIFIED AT THE IMMATURE STAGE

2017 ◽  
Vol 29 (1) ◽  
pp. 127 ◽  
Author(s):  
E. C. S. Santos ◽  
T. Somfai ◽  
R. Appeltant ◽  
T. Q. Dang-Nguyen ◽  
H. Kaneko ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Polar Body ◽  
Developmental Rate ◽  
Immature Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Dibutyryl Camp ◽  
Cleavage Rate ◽  
Nuclear Maturation

Previously, live offspring have been produced from porcine oocytes vitrified at the immature stage (Somfai et al. 2014 PLoS One 9, e97731); however, their embryo developmental rates remain low. The aim of our current research was to test the effects of resveratrol, an antioxidant and anti-apoptotic agent on the developmental competence of immature vitrified oocytes during in vitro maturation (IVM) after warming. Follicular porcine cumulus-oocyte complexes (COC) were vitrified on Cryotop® sheets (Kitazato Corp. Shizuoka, Japan) using the cryoprotectant treatment and warming method of Somfai et al. (2015 J. Reprod. Dev. 61, 571–579). After warming, the oocytes were subjected to IVM for 46 h in a chemically defined porcine oocyte medium (POM) enriched with 10 ng mL−1 epidermal growth factor, 10 IU mL−1 eCG, and 10 IU mL−1 hCG. During the first 22 h of IVM, the medium was supplemented with 1 mM dibutyryl cAMP. The following 24 h of IVM was performed in POM without dibutyryl cAMP. Vitrified/warmed COC (vitrified group) and freshly collected COC (control group) were matured either in the absence or presence of 2 µM resveratrol (RES− and RES+, respectively) throughout the entire IVM. At the end of IVM, oocytes were denuded and their survival was evaluated. Then, those with 1 polar body (PB1+) were selected for parthenogenetic activation (Day 0). Activated oocytes were cultured for 7 days in PZM-3. Survival, nuclear maturation, cleavage, and blastocyst rates were assessed. The experiment was replicated 5 times. Results were analysed by one-way ANOVA and Tukey’s multiple comparison test. Vitrification reduced the percentage of live oocytes after IVM both in RES− and RES+ groups in a similar manner (47.9 and 51.8%, respectively) compared with control RES− and RES+ groups (99.4 and 100%, respectively; P < 0.05) There was no statistical difference among groups in the percentage of PB1+ oocytes (ranging between 76.1 and 90.2%). On Day 2, the cleavage rate in vitrified RES− group was lower than those in control RES− and RES+ groups (55.9 v. 78.5% and 79.2%, respectively) whereas the vitrified RES+ group did not differ from the others (72.1%). The blastocyst developmental rate calculated from total cultured oocytes on Day 7 in vitrified RES+ group was significantly higher (P < 0.05) than that in the vitrified RES− group (26.2% v. 6.9%, respectively) and did not differ significantly from those of control RES− and RES+ groups (32.1 and 36.0%, respectively). Blastocyst rates in control RES− and RES+ groups were significantly higher (P < 0.05) than that in vitrified RES− group but did not differ from one another. In conclusion, supplementation of IVM medium with resveratrol improved the developmental competence of vitrified, but not freshly collected oocytes. This work was supported by JSPS KAKENHI (Grant Number: 26870839) and JST/JICA SATREPS. E.C.S. Santos was supported by a CNPq-Brasil fellowship.

scholarly journals 81IN VITRO MATURATION, FERTILIZATION AND DEVELOPMENT OF BOVINE IMMATURE OOCYTES CRYOPRESERVED BY VITRIFICATION WITH STEPWISE EXPOSURE USING A NYLON MESH

2004 ◽  
Vol 16 (2) ◽  
pp. 162
Author(s):  
Y. Abe ◽  
K. Hara ◽  
H. Matsumoto ◽  
H. Sasada ◽  
H. Ekwall ◽  
...  
Keyword(s):  
Structural Damage ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Single Step ◽  
Cleavage Rate ◽  
Immature Oocytes ◽  
Nylon Mesh ◽  
Transmission Electron ◽  
Alternative Material

Vitrification of bovine immature oocytes has been reported using an open pulled straw, but with limited success. In a previous report, we developed an alternative material (nylon mesh) for vitrification of large quantities of oocytes and embryos. This study was conducted to demonstrate effects of components of a cryoprotectant and a protocol of exposure for bovine immature oocytes on their subsequent in vitro maturation, fertilization and development after cryopreservation by vitrification using a nylon mesh. Bovine oocytes at the germinal vesicle stage were collected from 2–5mm follicles in ovaries, and cumulus-oocytes complexes (COCs) were randomly assigned to treatment groups. Before vitrification, COCs were exposed to the cryoprotectant, which was composed of 40% ethylene glycol, 18% ficoll and 0.3M sucrose (EFS40) or 0.3M trehalose (EFT40) by single step or stepwise exposure. Forty COCs were transferred onto a nylon mesh (0.5cm2), which was then plunged directly into liquid nitrogen. After thawing in warm medium, vitrified COCs were in vitro-matured, fertilized and cultured. After culture for in vitro maturation, the rates in the oocytes reaching to metaphase II were 64.1% and 63.1% in the stepwise exposure to EFS40 or EFT40, respectively, which was significantly higher (P&lt;0.05) than the corresponding rates after a single step (22.6% and 10.0%, respectively). There was no significant effect of the two sugars on in vitro maturation after single or step-wise equilibration. Transmission electron microscopy revealed that the cytoplasm of oocytes equilibrated in a single step had many vacuoles and broken mitochondria, while oocytes equilibrated in a step-wise manner had significantly fewer abnormalities and were similar to untreated controls. Cleavage rate of thawed oocytes after IVMFC was significantly higher after stepwise exposure to EFS40 or EFT40 than that after single step exposure (37.7% and 22.2% v. 20.8% and 0%, respectively, P&lt;0.05). Step-wise equilibration of oocytes in EFT40 was dramatically detrimental: no cleaved embryos developed to blastocysts after a single step exposure to either vitrification solution, or stepwise exposure to EFT40. However, blastocysts were obtained following stepwise exposure to EFS40 (8%). These results suggest that stepwise equilibration and vitrification on a nylon mesh minimizes structural damage to the organelles of immature oocytes and facilitates successful cryopreservation.

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