46 VITRIFICATION AT THE GERMINAL VESICLE STAGE TRIGGERS PRECOCIOUS MEIOTIC RESUMPTION BUT DOES NOT AFFECT CYTOPLASMIC MATURATION IN CUMULUS-ENCLOSED PORCINE OOCYTES DURING IN VITRO MATURATION

2016 ◽  
Vol 28 (2) ◽  
pp. 153
Author(s):  
T. Somfai ◽  
N. T. Men ◽  
H. Kaneko ◽  
J. Noguchi ◽  
S. Haraguchi ◽  
...  
Keyword(s):  
Adenosine Triphosphate ◽  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Control Group ◽  
Control Groups ◽  
Porcine Oocyte ◽  
And Control

Previously we have reported a vitrification protocol that allows preservation of immature porcine oocytes in large numbers (Somfai et al. 2014 PLoS One 9, e97731). However, despite high survival rates, embryo development rates have remained low. The aim of our current research is to reveal factors potentially responsible for reduced developmental competence of vitrified oocytes. As a first step, we investigated the effects of vitrification at the germinal vesicle stage on subsequent nuclear progression and the normality of cytoplasmic functions during in vitro maturation (IVM). Cumulus-enclosed porcine oocytes were vitrified in microdrops, stored, and then warmed by our method (Somfai et al. 2015 Reprod. Fertil. Dev. 27, 124). Then the oocytes were subjected to IVM for 46 h in a chemically defined porcine oocyte medium. During the first 22 h of IVM, the medium was supplemented with 1 mM dibutyryl cyclic adenosine monophosphate, 10 IU mL–1 of eCG, and 10 IU mL–1 of hCG. The following 24 h of IVM was performed in porcine oocyte medium without any supplementation. We compared vitrified/warmed oocytes (vitrified group) with freshly collected immature oocytes (control group) in terms of (1) nuclear progression, (2) intracellular glutathione (GSH), and (3) adenosine triphosphate levels throughout IVM. Each experiment was replicated at least 3 times. Results were analysed by one-way ANOVA and Tukey’s multiple comparison test. A total of 510 oocytes were vitrified of which 422 (82.3%) survived. Only live oocytes were subjected to subsequent assays. Orcein staining revealed that after 22 h of IVM, a significantly higher percentage (P < 0.05) of vitrified oocytes showed germinal vesicle breakdown compared with the control group (22.0 v. 0.9%, respectively). In a similar fashion, after 30 h IVM, a significantly higher (P < 0.05) percentage of oocytes reached the metaphase-II (MII) stage in the vitrified group than in the control group (21.8 v. 0%, respectively). After 46 h of IVM, there was no difference between the vitrified and control groups in terms of the percentage of MII stage oocytes (93.9 and 86.3%, respectively). Analysis of GSH levels in oocytes by the 5,5′-dithio-bis-2-nitrobenzoic acid-glutathione disulfide reductase recycling assay showed no significant difference between the vitrified and control groups at 0 h (6.7 and 7.0 pmol, respectively), 22 h (5.5 and 5.5 pmol, respectively), and 46 h (6.9 and 7.9 pmol, respectively) of IVM. Adenosine triphosphate assay (FL-ASC; Sigma-Aldrich Co., St. Louis, MO) revealed similar adenosine triphosphate contents in the oocytes of the vitrified and control groups at 0 h (1.53 and 1.61 pmol, respectively), 22 h (1.67 and 1.70 pmol, respectively), and 46 h (1.65 and 1.83 pmol, respectively) of IVM. In conclusion, vitrification triggered precocious nuclear maturation even in the presence of dibutyryl cyclic adenosine monophosphate; however, it did not affect GSH levels and overall metabolism. This work was supported by JSPS KAKENHI (Grant Number: 26870839) and JST/JICA SATREPS.

168 EFFECT OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS DURING OOCYTE IN VITRO MATURATION ON BOVINE EMBRYOS (Gyr×HOLSTEIN) QUALITY

2016 ◽  
Vol 28 (2) ◽  
pp. 214
Author(s):  
G. R. Leal ◽  
C. A. S. Monteiro ◽  
H. F. R. A. Saraiva ◽  
A. J. R. Camargo ◽  
P. M. S. Rosa ◽  
...  
Keyword(s):  
Lipid Content ◽  
In Vitro Maturation ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Control Group ◽  
Bovine Embryos ◽  
Tropical Conditions ◽  
Significant Difference ◽  
Number Of Cells

In vitro embryo production (IVP) is an important tool for cattle breeding. Brazilian dairy systems are based on Gyr × Holstein crossbreds, which integrates adaptability to tropical conditions and milk production. Quality determines the oocyte proportion that will develop to blastocyst stage, and although the lipid content is important in oocyte development, a high concentration in embryos is associated with cryotolerance reduction, making this a relevant issue for IVP systems. The in vitro maturation system (IVM) simulated physiological oocyte maturation (SPOM) mimics the physiological maturation events by using cyclic adenosine monophosphate (cAMP) modulators, which promote the increase of oocyte competence. Among the modulators, Forskolin has lipolytic properties. The aim of this study was to evaluate the effect of the SPOM system (Albuz 2010 Hum. Reprod. 25, 12) on bovine embryos (Gyr × Holstein) regarding their total number of cells (TNC) and lipid content. Oocytes were obtained by ovum pick-up from Gyr cows in 5 replications. After selection, they were randomly divided into 2 groups: SPOM (S) and control (C). The IVM lasted 24 h for group C (TCM 199 medium without FBS) in culture oven at 38.5°C, 5% CO2 in atmospheric air and high humidity. In the SPOM system, oocytes were in pre-IVM [TCM 199 medium + 100 µM Forskolin + 500 µM 3-isobutyl-1-methylxanthine (IBMX)] for 2 h and followed for extended IVM (TCM 199 medium + 20 µM cilostamide) for 28 h under the same conditions as control group. After IVM, oocytes were fertilised with semen from a single Holstein bull that was prepared by Percoll gradient method in Fert-TALP medium (Bioklone® Animal Reproduction, São Paulo, Brazil) for 22 h and transfered to culture droplets, where they remained for 7 days (n = 10–13 per group). The lipid content analysis was performed by staining with Oil red and the stained area fraction of each embryo was measured using software ImageJ (NIH, Bethesda, MD, USA). The TNC was measured after being stained with Hoechst 33342 and results were analysed by Student's t-test in Instat GraphPad program, with a 5% significance level. There was no significant difference (P > 0.05) between embryos from both groups on TNC (group S: 88.9 ± 28.0A; group C: 101.6 ± 29.1a) and lipid content (group S: 0.93 ± 12:18A; group C: ±0.15 to 0.96) analysis. Some studies have shown there is a beneficial effect on embryo quality when using this system; however, our results demonstrated that there was no effect on total number of cells using our conditions. Some authors have also demonstrated a reduction in embryo lipid content using Forskolin during in vitro culture. Our results suggest that the time of Forskolin exposure was not enough to ensure lipolytic action on the structures produced from oocytes (Gyr) treated in pre-IVM. It was concluded that the SPOM system had no effect on TNC and lipid content of Gyr/Holstein embryos. Financial support from FAPERJ and CAPES is acknowledged.

306 IN VITRO PENETRATION OF SWINE OOCYTES MATURED IN TCM-199 WITH ADDED DIBUTYRYL CYCLIC ADENOSINE MONOPHOSPHATE AND FOLLICULAR FLUID

2007 ◽  
Vol 19 (1) ◽  
pp. 268
Author(s):  
A. B. Nascimento ◽  
M. G. Marques ◽  
A. R. de S. Coutinho ◽  
M. N. Tavares ◽  
M. E. O. D'Avila Assumpção ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Penetration Rate ◽  
Early Period ◽  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Control Group ◽  
In Vitro Production

During the in vitro maturation (IVM) of pig oocytes, a large variation in the nuclear morphology of the germinal vesicle stage is observed. Thus, some oocytes can start meiosis earlier than others. A reversible alternative to inhibit meiotic resumption is the use of dibutyryl cyclic adenosine monophosphate (dbcAMP) in the early period of IVM, which may synchronize the oocytes to a specific germinal vesicle stage and improve early embryonic development. This study investigated the effects of additional dbcAMP and porcine follicular fluid (PFF) on monospermic and polyspermic penetration rates after IVF. Oocytes from prepuberal females were selected under a stereomicroscope, and those with uniform ooplasm and surrounded by several layers of compact cumulus cells were divided into 2 groups: T1 (control) group: TCM-199 supplemented with polyvinyl alcohol (0.1%), FSH (0.5 �g mL-1), LH (0.5 �g mL-1), epidermal growth factor (10 ng mL-1), pyruvate (0.9 mM), d-glucose (3.05 mM), cysteine (0.1 mg mL-1), and gentamycin (50 �g mL-1); and T2 group: T1 with the addition of 25% PFF and 1 mM dbcAMP. Both the T1 and T2 groups were IVM for 42 to 46 h, with FSH, LH, and dbcAMP used only in the first 22 h. At the end of the maturation period, cumulus cells were chemically removed; the oocytes were washed 3 times in IVF medium (modified Tyrode&apos;s buffered medium, mTBM) and placed in petri dishes containing 50 &micro;L of the same medium. The sperm-rich fraction was collected from 2 boars by digital pressure with a gloved hand, extended in Beltsville thawing solution, and incubated for 24 h at 17&deg;C. It was then centrifuged at 1200g for 3 min and standardized for 1 &times; 105 spermatozoa mL&minus;1. Oocytes were co-incubated with the sperm for 6 h in mTBM at 38.5&deg;C and 5&percnt; CO2. After insemination, oocytes were cultured in porcine zygote medium-3 (80 &micro;L), covered with paraffin oil, for 18 h. The presumptive zygotes were fixed and stained with 1&percnt; orcein in 45&percnt; acetic acid and evaluated under phase-contrast microscopy at a 400&times; magnification. Differences among groups were determined by one-way ANOVA. In the T1 group, the penetration rate was 39.3 &plusmn; 9.6&percnt; (162/412), and no difference was observed in comparison with the T2 group, 29.5 &plusmn; 4.9&percnt; (113/383). The monospermic penetration rate was 31.5 &plusmn; 6&percnt; (51/162) in the T1 group and differed from that in the T2 group, 71.7 &plusmn; 3.3&percnt; (81/113). Moreover, the polyspermic penetration was significantly higher in the T1 group, 68.5 &plusmn; 6&percnt; (111/162), compared with the T2 group, 28.3 &plusmn; 3.3&percnt; (32/113). These data suggest that the IVM with TCM-199 with added dbcAMP &plus; PFF can improve in vitro production of swine embryos and decrease polyspermic penetration.

scholarly journals The Accuracy and Reliability of Tooth Shade Selection Using Different Instrumental Techniques: An In Vitro Study

Sensors ◽  
2021 ◽  
Vol 21 (22) ◽  
pp. 7490
Author(s):  
Nattapong Sirintawat ◽  
Tanyaporn Leelaratrungruang ◽  
Pongsakorn Poovarodom ◽  
Sirichai Kiattavorncharoen ◽  
Parinya Amornsettachai
Keyword(s):  
Intraclass Correlation ◽  
In Vitro Study ◽  
Control Group ◽  
Intraoral Scanner ◽  
Control Groups ◽  
B Values ◽  
Cie Lab ◽  
And Control ◽  
Shade Selection

This study aimed to investigate and compare the reliability and accuracy of tooth shade selection in the model using 30 milled crowns via five methods: (1) digital single-lens reflex (DSLR) camera with twin flash (TF) and polarized filter (DSLR + TF), (2) DSLR camera with a ring flash (RF) and polarized filter (DSLR + RF), (3) smartphone camera with light corrector and polarized filter (SMART), (4) intraoral scanner (IOS), and (5) spectrophotometer (SPEC). These methods were compared with the control group or manufacturer’s shade. The CIE Lab values (L, a, and b values) were obtained from five of the methods to indicate the color of the tooth. Adobe Photoshop was used to generate CIE Lab values from the digital photographs. The reliability was calculated from the intraclass correlation based on two repetitions. The accuracy was calculated from; (a) ΔE calculated by the formula comparing each method to the control group, (b) study and control groups were analyzed by using the Kruskal–Wallis test, and (c) the relationship between study and control groups were calculated using Spearman’s correlation. The reliability of the intraclass correlation of L, a, and b values obtained from the five methods showed satisfactory correlations ranging from 0.732–0.996, 0.887–0.994, and 0.884–0.999, respectively. The ΔE from all groups had statistically significant differences when compared to the border of clinical acceptance (ΔE = 6.8). The ΔE from DSLR + TF, DSLR + RF, SMART, and SPEC were higher than clinical acceptance (ΔE > 6.8), whereas the ΔE from IOS was 5.96 and all of the L, a, and b values were not statistically significantly different from the manufacturer’s shade (p < 0.01). The ΔE of the DSLR + RF group showed the least accuracy (ΔE = 19.98), whereas the ∆E of DSLR + TF, SMART, and SPEC showed similar accuracy ∆E (ΔE = 10.90, 10.57, and 11.57, respectively). The DSLR camera combined with a ring flash system and polarized filter provided the least accuracy. The intraoral scanner provided the highest accuracy. However, tooth shade selection deserves the combination of various techniques and a professional learning curve to establish the most accurate outcome.

239 USE OF CYCLIC ADENOSINE MONOPHOSPHATE MODULATORS IN IN VITRO PRODUCTION OF BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 209
Author(s):  
T. Fanti ◽  
N. M. Ortega ◽  
R. Garaguso ◽  
M. J. Franco ◽  
C. Herrera ◽  
...  
Keyword(s):  
Phosphodiesterase Inhibitor ◽  
Basal Medium ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Oocyte Competence

In vitro embryo production systems (IVP) try to emulate and enhance molecular events that occur in in vivo reproductive systems in order to increase, not only the number of embryos generated, but also their quality. Despite advances, IVP processes are still inefficient compared with in vivo systems. Several studies have attributed this deficiency to a lack of oocyte competence due to spontaneous premature resumption of meiotic maturation in the oocyte following the removal from its follicular environment. Therefore, our objective was to increase oocyte competence avoiding premature resumption of meiosis by using cyclic adenosine monophosphate modulators. Cumulus-oocyte complexes (COC) were obtained from ovaries of slaughterhouses, washed, and randomly allocated in 2 culture systems. Oocytes in the control group (IVM) were cultured for a period of 24 h in basal medium TCM-199 with EGF (1 µg mL–1) supplemented with rhFSH (25 mIU mL–1). Oocytes in the biphasic in vitro maturation (b-IVM) group were cultured for 2 h in a basal medium supplemented with a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 500 µM), and an activator of adenylate cyclase (forskolin, 100 µM). Subsequently, COC were washed and cultured in basal medium supplemented with cilostamide (20 µM) and rhFSH (25 mIU mL–1) for 24 h. Maturation rates were analysed and IVF was performed with a dose of 1 × 106 sperm cells mL–1 in IVF-SOF medium. The presumptive zygotes were cultured in continuous-single-culture medium (Irvine) supplemented with 8 mg mL–1 of BSA until they reached the blastocyst stage. No significant differences in maturation, cleavage, and cryotolerance were observed between b-IVM and IVM groups (P > 0.05; Table 1). This study showed that b-IVM produced a significant increase in IVP compared with the control (IVM) at Days 7 and 8 (P < 0.01). Blastocyst hatching rate was significant (P < 0.05) for both treatment and day of analysis. The b-IVM group yielded an increase of 10 and 7.5% at Days 7 and 8, respectively, of IVP. The biphasic maturation showed an improvement in quality regarding the control group, in the timing analysis of production, and hatching percentages, and these results show that the use of cyclic adenosine monophosphate modulators in the oocyte maturation process enhances oocyte competence, which is reflected in increased productivity and embryo quality. We propose this treatment as an alternative to the standard protocols currently used in IVP of bovine embryos. Table 1.Effect of treatment on maturation, cleavage, and cryotolerance

159 EFFECT OF 3-ISOBUTYL-1-METHYLXANTHINE (IBMX) ON THE GERMINAL VESICLE STAGE OF PORCINE OOCYTES DURING OOCYTE COLLECTION IN VITRO

2014 ◽  
Vol 26 (1) ◽  
pp. 193
Author(s):  
R. Appeltant ◽  
J. Beek ◽  
D. Maes ◽  
A. Van Soom
Keyword(s):  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Guanosine Monophosphate ◽  
Developmental Competence ◽  
Meiotic Arrest ◽  
Oocyte Collection

When using modern maturation conditions for in vitro maturation, pig oocytes yield ~20% blastocysts only. One problem is that cumulus cells, which are normally connected with the immature oocyte by cellular projections penetrating through the zona pellucida and with the oolemma via gap junctions, are prematurely losing these connections after the cumulus–oocyte complex is removed from the follicle. The oocyte possesses a type 3 phosphodiesterase, which degrades 3′,5′-cyclic adenosine monophosphate (cAMP), and this activity is inhibited by supply of 3′,5′-cyclic guanosine monophosphate (cGMP) to the oocyte via the cumulus cells. Consequently, cAMP levels, which are typically high during early stages of oocyte maturation in vivo, decrease, leading to spontaneous nuclear maturation and oocytes of low developmental competence. Therefore, the maintenance of these cumulus-oocyte connections is important to keep cAMP high and the oocyte under meiotic arrest. One way to prevent this drop in cAMP is using N6, 2′-o-dibutyryladenosine 3′,5′-cyclic monophosphate sodium (dbcAMP) that causes an arrest at germinal vesicle (GV) stage II (Funahashi et al. 1997 Biol. Reprod. 57, 49–53). Another option is collecting the oocytes in a medium containing the phoshodiesterase inhibitor, IBMX. The present study investigated the influence of IBMX on the progression of the GV of the oocyte after collection, just before the start of the maturation procedure. The GV stage was defined according to Sun et al. (2004 Mol. Reprod. Dev. 69, 228–234). In parallel with the findings on dbcAMP, we hypothesised an arrest at GV II by the presence of IBMX during collection. One group of oocytes were collected in HEPES-buffered TALP without IBMX (n = 375) and another group in the same medium containing 0.5 mM IBMX (n = 586). An average incubation time of 140 min was applied in both groups, and 3 replicates were performed. The proportions of oocytes before or at GV II and beyond GV II were compared in both groups using logistic regression analysis. The proportion of oocytes was included as dependent variable and group (IBMX addition or not) as independent variable. Replicate was also included in the model. The proportion of oocytes before or at GV II was not statistically significant between the group without and the group with IBMX (59.2 v. 58.7% respectively; P > 0.05). In conclusion, the use of IBMX during oocyte collection did not influence the state of the germinal vesicle of the oocyte during collection, indicating that IBMX did not cause a meiotic arrest in the oocytes during collecting in vitro.

312 EFFECTS OF TRANSFORMING GROWTH FACTOR ALPHA AND INSULIN-LIKE GROWTH FACTOR 1 SUPPLEMENTATION ON IN VITRO MATURATION OF CANINE OOCYTES

2015 ◽  
Vol 27 (1) ◽  
pp. 245
Author(s):  
A. Sato ◽  
B. Sarentonglaga ◽  
K. Ogata ◽  
M. Yamaguchi ◽  
A. Hara ◽  
...  
Keyword(s):  
Growth Factor ◽  
Synergistic Effect ◽  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Transforming Growth Factor ◽  
Germinal Vesicle ◽  
Control Group ◽  
Insulin Like Growth Factor ◽  
Treatment Groups

Although in vitro maturation (IVM) of oocytes has been successfully established for many species, the efficiency of IVM in canine oocytes is still very low. As growth factors have been shown to promote oocyte maturation in some species, we investigated whether use of transforming growth factor α (TGF-a) and insulin-like growth factor 1 (IGF-1) might overcome the difficulties of achieving meiotic maturation in cultured canine cumulus-oocyte complexes (COC). Ovaries were obtained from bitches at 6 months to 7 years of age by ovariohysterectomy and were sliced repeatedly to release COC. In the first experiment, the COC were cultured at 38.8°C for 48 h in 5% CO2 in air in medium 199 supplemented with either TGF-a (0, 1, 10, or 100 ng mL–1) or IGF-1 (0, 0.5, 5, 10, or 50 µg mL–1). In the second experiment, the synergistic effect of TGF-a and IGF-1 was investigated by culturing COC in medium 199 supplemented with both TGF-a (0, 1, 10, or 100 ng mL–1) and IGF-1 (0, 0.5, 5, 10, or 50 µg mL–1). At the end of the culture period, the oocytes were denuded of cumulus cells by pipetting with a fine bore glass pipette; the denuded oocytes were then fixed in Carnoy's solution and stained with Hoechst 33342. The nuclear configuration and chromatin morphology of the oocytes were evaluated under confocal laser scanning microscopy. The cells were assigned to 1 of the following meiotic stages: germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), or metaphase II (MII). Data were analysed by ANOVA with Fisher's PLSD test. In experiment 1, no significant difference were observed in the rates of cells maturing to the MI and MII stages, but that in the 10 ng mL–1 of TGF-a group (56.3%) were larger than in the other treatment groups (38.8–51.0%). The frequencies of MII stage cells in the 5, 10, and 50 µg mL–1 of IGF-1 treatment groups (9.8, 13.3, and 12.2%, respectively) were significantly higher than in the 0.5 µg mL–1 of IGF-1 group and the control group (5.3 and 2.2%, respectively). In experiment 2, the frequency of MI and MII cells in the control, 1 ng mL–1 of TGF-a plus 0.5 µg mL–1 of IGF-1, 10 ng mL–1 of TGF-a plus 5 µg mL–1 of IGF-1, 10 ng mL–1 of TGF-a plus 10 µg mL–1 of IGF-1, and 100 ng mL–1 of TGF-a plus 50 µg mL–1 of IGF-1 group were 44.1, 36.1, 63.5, 70.8, and 50.8%, respectively. The frequency of MII cells in the control group and the same treatment groups were 2.8, 7.2, 10.4, 15.3, and 10.8%, respectively. Both frequencies in the 10 ng mL–1 of TGF-a plus 10 µg mL–1 of IGF-1 group were significantly higher than in the control group. The TGF-a may act in a paracrine fashion on the surrounding granulosa cells, and IGF-1 may play multiple roles in cellular metabolism, proliferation, growth, and differentiation in canine oocyte maturation, as has been reported for many other species. In conclusion, these results demonstrate that a synergistic effect between TGF-a and IGF-1 produces an increased rate of in vitro maturation to the MI and MII stages in canine oocytes.

scholarly journals Use of oocytes selected by brilliant cresyl blue staining enhances rabbit cloned embryo development in vitro

Zygote ◽  
2019 ◽  
Vol 27 (3) ◽  
pp. 166-172 ◽  
Author(s):  
Linying Jia ◽  
Bo Ding ◽  
Chong Shen ◽  
Shiwei Luo ◽  
Yanru Zhang ◽  
...  
Keyword(s):  
Cell Mass ◽  
Developmental Competence ◽  
Blue Staining ◽  
Control Group ◽  
Control Groups ◽  
Brilliant Cresyl Blue ◽  
Positive Group ◽  
Cresyl Blue ◽  
And Control

SummaryRabbits play an important role in people’s lives due to their high nutritional value and high-quality hair that can be used as raw material for textiles. Furthermore, rabbits are an important animal model for human disease, as genome-edited animals are particularly valuable for studying gene functions and pathogenesis. Somatic cell nuclear transfer (SCNT) is an important technique for producing genome-edited animals and it has great value in saving endangered species and in clone stem cell therapy. However, the low efficiency of SCNT limits its application, with the selection of suitable rabbit oocytes being crucial to its success. In the present study, we collected oocytes from ovarian follicles and stained them with 26 μM brilliant cresyl blue (BCB). We then matured the oocytes in vitro and used them for SCNT. Comparison of the BCB-positive oocytes with BCB-negative oocytes and the control group showed that the BCB-positive group had a significantly higher maturation rate (81.4% vs. 48.9% and 65.3% for the negative and control groups, respectively), cleavage rate (86.6% vs. 67.9% and 77.9%), blastocyst rate (30.5% vs. 12.8% and 19.6%), total number of blastocysts (90±7.5 vs. 65.3±6.3 and 67.5±5.7), and inner cell mass (ICM)/ trophectoderm (TE) index (42.3±4.2 vs. 30.2±2.1 and 33.9±5.1) (P<0.05). The BCB-positive group had a significantly lower apoptosis index (2.1±0.6 vs. 8.2±0.9 and 6.7±1.1 for the negative and control groups, respectively) (P<0.05). These findings demonstrate that BCB-positive oocytes have a higher maturation ability and developmental competence in vitro, indicating that BCB staining is a reliable method for selecting oocytes to enhance the efficiency of SCNT.

Production of good-quality blastocyst embryos following IVF of ovine oocytes vitrified at the germinal vesicle stage using a cryoloop

2013 ◽  
Vol 25 (8) ◽  
pp. 1204 ◽  
Author(s):  
Adel R. Moawad ◽  
Jie Zhu ◽  
Inchul Choi ◽  
Dasari Amarnath ◽  
Wenchao Chen ◽  
...  
Keyword(s):  
Germinal Vesicle ◽  
Blastocyst Stage ◽  
Control Groups ◽  
Blastocyst Development ◽  
Chromosome Configuration ◽  
Nuclear Maturation ◽  
Chromatin Configuration ◽  
And Control ◽  
First Time

The cryopreservation of immature oocytes at the germinal vesicle (GV) stage would create an easily accessible, non-seasonal source of female gametes for research and reproduction. The present study investigated the ability of ovine oocytes vitrified at the GV stage using a cryoloop to be subsequently matured, fertilised and cultured in vitro to blastocyst-stage embryos. Selected cumulus–oocyte complexes obtained from mature ewes at the time of death were randomly divided into vitrified, toxicity and control groups. Following vitrification and warming, viable oocytes were matured in vitro for 24 h. Matured oocytes were either evaluated for nuclear maturation, spindle and chromosome configuration or fertilised and cultured in vitro for 7 days. No significant differences were observed in the frequencies of IVM (oocytes at the MII stage), oocytes with normal spindle and chromatin configuration and fertilised oocytes among the three groups. Cleavage at 24 and 48 h post insemination was significantly decreased (P < 0.01) in vitrified oocytes. No significant differences were observed in the proportion of blastocyst development between vitrified and control groups (29.4% v. 45.1%, respectively). No significant differences were observed in total cell numbers, the number of apoptotic nuclei or the proportion of diploid embryos among the three groups. In conclusion, we report for the first time that ovine oocytes vitrified at the GV stage using a cryoloop have the ability to be matured, fertilised and subsequently developed in vitro to produce good-quality blastocyst embryos at frequencies comparable to those obtained using fresh oocytes.

scholarly journals Effect of crotamine, a cell-penetrating peptide, on blastocyst production and gene expression of in vitro fertilized bovine embryos

Zygote ◽  
2014 ◽  
Vol 24 (1) ◽  
pp. 48-57 ◽  
Author(s):  
Iana S. Campelo ◽  
Alexsandra F. Pereira ◽  
Agostinho S. Alcântara-Neto ◽  
Natalia G. Canel ◽  
Joanna M.G. Souza-Fabjan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Control Group ◽  
Cell Penetrating Peptide ◽  
Mrna Levels ◽  
Bovine Embryos ◽  
Control Groups ◽  
Cell Penetrating ◽  
A Cell ◽  
And Control

SummaryThe present study investigated the effects of crotamine, a cell-penetrating peptide from rattlesnake venom, at different exposure times and concentrations, on both developmental competence and gene expression (ATP1A1, AQP3, GLUT1 and GLUT3) of in vitro fertilized (IVF) bovine embryos. In Experiment 1, presumptive zygotes were exposed to 0.1 μM crotamine for 6, 12 or 24 h and control groups (vehicle and IVF) were included. In Experiment 2, presumptive zygotes were exposed to 0 (vehicle), 0.1, 1 and 10 μM crotamine for 24 h. Additionally, to visualize crotamine uptake, embryos were exposed to rhodamine B-labelled crotamine and subjected to confocal microscopy. In Experiment 1, no difference (P > 0.05) was observed among different exposure times and control groups for cleavage and blastocyst rates and total cells number per blastocyst. Within each exposure time, mRNA levels were similar (P > 0.05) in embryos cultured with or without crotamine. In Experiment 2, concentrations as high as 10 μM crotamine did not affect (P > 0.05) the blastocyst rate. Crotamine at 0.1 and 10 μM did not alter mRNA levels when compared with the control (P > 0.05). Remarkably, only 1 μM crotamine decreased both ATP1A1 and AQP3 expression levels relative to the control group (P < 0.05). Also, it was possible to visualize the intracellular localization of crotamine. These results indicate that crotamine can translocate intact IVF bovine embryos and its application in the culture medium is possible at concentrations from 0.1–10 μM for 6–24 h.

Sign in / Sign up

Export Citation Format

Share Document