230 EVALUATION OF APOPTOSIS IN IN VITRO-PRODUCED BOVINE EMBRYOS MATURED WITH FORSKOLIN

2013 ◽  
Vol 25 (1) ◽  
pp. 263
Author(s):  
D. M. Paschoal ◽  
M. J. Sudano ◽  
R. R. D. Maziero ◽  
M. D. Guastali ◽  
L. F. Crocomo ◽  
...  
Keyword(s):  
Linear Models ◽  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
High Dose ◽  
Bovine Embryos ◽  
Nuclear Maturation ◽  
Tunel Reaction ◽  
Level Of Significance

The maintenance of oocytes in the germinal vesicle stage for a few hours could result in more competent oocytes for use in biotechnology. Related to this, forskolin (Sigma-Aldrich, St. Louis, MO, USA) is an efficient inhibitor of nuclear maturation because of its ability to increase the levels of intracellular cyclic adenosine monophosphate. This study aimed to show whether the use of forskolin would be able to inhibit maturation in bovine oocytes, producing a higher rate of in vitro embryos. Nellore oocytes from a slaughterhouse (n = 960) were matured in TCM-199 with Earle’s salt + 10% FCS, FSH, and LH, in a 5% CO2 atmosphere. To delay meiosis, the oocytes were maintained for 6 h in medium with forskolin at 3 different concentrations, 0. 1 mM (n = 240), 0.05 mM (n = 240), and 0.025 mM (n = 240), whereas untreated oocytes acted as controls (n = 240). The oocytes were then cultured for 18 h in agent-free medium to resume meiosis, completing 24 h of maturation. After 24 h (Day 0) of maturation, oocytes were fertilized in human tubal fluid (HTF, Irvine, New Zealand) under the same conditions as described above. Semen was selected through Percoll gradient, and the concentration was adjusted to 2 × 106 sperm mL–1. The presumed zygotes were cultured in 90-µL droplets of SOFaa + 0.6% BSA + 2.5% FCS in a 5% CO2, 5% O2, and 90% N2 atmosphere until Day 7, when blastocysts were evaluated. Apoptosis in blastocysts was accessed through TUNEL reaction. Data were analysed by ANOVA, followed by Tukey’s test using the general linear models procedure (PROC GLM) of SAS (SAS Institute Inc., Cary, NC, USA). The level of significance adopted was 5%. No statistical differences were observed in blastocyst production rate (n = 297): control (n = 88): 36.7% ± 3.7; 0.1 mM forskolin (n = 61): 25.1% ± 3.7; 0.05 mM forskolin (n = 70): 29.2% ± 3.7; 0.025 mM forskolin (n = 78): 32.6% ± 3.7 (P > 0.05). However, when we analysed the apoptosis rates, differences were found among groups: control: 6.0% ± 6.3a; 0.1 mM forskolin: 33.4% ± 6.3b; 0.05 mM forskolin: 27.2% ± 6.3ab; 0.025 mM forskolin: 10.0% ± 6.3ab (P < 0.05). Although there was no difference in blastocyst rate, the TUNEL technique allowed us to identify that a high dose of forskolin was detrimental for in vitro-produced bovine embryos. FAPESP: 10/50410-2.

161 USE OF FORSKOLIN TO DELAY MEIOSIS AND PRODUCE IN VITRO BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 194
Author(s):  
D. Paschoal ◽  
R. Maziero ◽  
M. Sudano ◽  
M. Guastali ◽  
L. Vergara ◽  
...  
Keyword(s):  
Rate Control ◽  
Germinal Vesicle ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Bovine Embryos ◽  
Hoechst 33342 ◽  
Germinal Vesicle Breakdown ◽  
Tunel Reaction ◽  
Level Of Significance ◽  
Bovine Oocytes

The maintenance of oocytes in germinal vesicle (GV) stage for a few hours could result in more competent oocytes for use in biotechnology. This study aimed to show if the use of forskolin is able to inhibit and reverse the maturation in bovine oocytes, producing a higher rate of in vitro embryos without apoptosis rates. Eight replicates in total were performed. Nellore oocytes were matured in TCM-199 and to delay meiosis, the oocytes (n = 584) were maintained for 6 h in medium in presence of 0.025, 0.05, or 0.1 mM Forskolin. Then, the oocytes were cultured for 18 h in agent-free medium to resume meiosis. After resumption of meiosis, the oocytes (n = 336) were stained with Hoechst 33342 to evaluate the state of the nucleus: germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII), or degenerated or unidentified (D/U). Then (Day 0) oocytes were fertilized in human tubal fluid (Irvine, New Zealand) and the presumed zygotes were culture in SOFaa + 0.6% BSA + 2.5% FCS until Day 7, when the blastocyst (n = 177) rate was evaluated. Apoptosis in blastocysts was assessed by terminal deoxynucleotidyl transferase uracil nick-end labeling (TUNEL) reaction. Data were analysed by ANOVA, followed by Tukey test using the general linear model (PROC GLM) of SAS (SAS Institute Inc., Cary, NC, USA). The level of significance adopted was 5%. There were no statistical differences in state of the nucleus, only in MI (Control = GV: 0.0, GVBD: 0.8, MI: 8.3a, MII: 67.7, D/U: 7.3; F 0.025 mM = GV: 2.8, GVBD: 0.7, MI: 20.8ab, MII: 67.7, D/U: 8.9; F 0.05 mM = GV: 0.0, GVBD: 4.4, MI: 15.8ab, MII: 65.9, D/U: 13.7; and F 0.1 mM = GV: 0.0, GVBD: 1.0, MI: 34.1b, MII: 50.2, D/U: 14.6; P < 0.05). There were no statistical differences in blastocyst rate (Control: 36.7, F 0.025 mM: 32.6, F 0.05 mM: 29.2 and F 0.1 mM: 25.1 – P > 0.05). But when we analysed the apoptosis rate, differences were found among groups: (Control: 12.1a, F 0.025 mM: 12.9a, F 0.05 mM: 13.5a and F 0.1 mM: 30b; P < 0.05). Although Forskolin was able to inhibit meiosis and produce embryos at the same rates as controls, the higher dosage of this drug damaged the embryos. The authors acknowledge FAPESP 10/50410-2 for support.

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.

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.

268 CYCLIC GUANOSINE MONOPHOSPHATE INHIBITS PHOSPHODIESTERASE 3 ACTIVITY IN PORCINE OOCYTES

2013 ◽  
Vol 25 (1) ◽  
pp. 282 ◽  
Author(s):  
F. G. Zaffalon ◽  
C. Guimmelette ◽  
C. L. V. Leal ◽  
F. J. Richard
Keyword(s):  
Critical Role ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cyclic Guanosine Monophosphate ◽  
Guanosine Monophosphate ◽  
Intracellular Camp ◽  
Activity Levels ◽  
Nuclear Maturation ◽  
Serum Gonadotropin

The level of cyclic adenosine monophosphate (cAMP) within oocytes has been shown to play a critical role in maintaining meiotic arrest. High levels of intracellular cAMP prevent spontaneous oocyte maturation in vitro, whereas a decrease in oocyte cAMP is associated with the resumption of meiosis. Another cyclic nucleotide that also was recently proposed as being involved in meiotic resumption is cyclic guanosine monophosphate (cGMP), which could be regulating phosphodiesterase (PDE) 3 activity. The aim of this study was to determine whether cGMP inhibits cAMP-PDE activity in porcine oocytes. With the method described previously by Sasseville et al. (2006 BMC Dev. Biol. 6, 47), PDE activity was measured in groups of 10 oocytes cultured in the absence (control) or presence of different concentrations of cGMP (1, 3, 10, 30, 100, 300, 1000, and 3000 nM) or with the PDE3 inhibitor cilostamide (10 µM). Before assaying PDE activity, the cumulus–oocyte complexes (COC) were matured in vitro for 24 h in the presence of pregnant mare serum gonadotropin (5 IU) and hCG (5 IU) at 38.5°C in 5% CO2. The COC were homogenised in a hypotonic buffer. Data were analysed using one-way ANOVA followed by Duncan’s post-hoc test. Differences with P < 0.05 were considered significant. Results showed that 300, 1000, and 3000 nM cGMP inhibited PDE3 activity (7.9, 5.1, and 4.1 fmol min–1 per COC; P < 0.05) at levels below the controls (13.2 fmol min–1 per COC) and were similar to the activity observed in the presence of (2.4 fmol min–1 per COC; P > 0.05). The other concentrations tested were similar to activity levels seen in the control (1 to 100 nM; 12.2, 11.3, 10.8, 11.5, and 10.4; P > 0.05). In conclusion, the results support the concept that increasing concentrations of cGMP inhibit PDE activity, suggesting the inhibition of the predominant form of cAMP-PDE present in porcine oocytes, PDE3. These results support the hypothesis that cGMP inhibits PDE activity in porcine oocytes. Further work is needed to determine the role this plays in maintaining high cAMP levels and inhibiting oocyte nuclear maturation. Financial support from FGZ FAPESP 2010/20188-6 and 2010/18023-9 is acknowledged.

36 The effects of E-64 on the developmental competence of porcine oocytes vitrified at the germinal vesicle stage

2019 ◽  
Vol 31 (1) ◽  
pp. 144
Author(s):  
T. Somfai ◽  
H. T. Nguyen ◽  
N. T. Men ◽  
T. Q. Dang-Nguyen ◽  
H. Kaneko ◽  
...  
Keyword(s):  
Survival Rates ◽  
Germinal Vesicle ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Immature Stage ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Embryo Production ◽  
Cell Numbers

Previous studies reported the activation of the apoptotic cascade by vitrification in mature porcine oocytes (Vallorani et al. 2012 Anim. Reprod. Sci. 135, 68-74) and that the cathepsin B inhibitor E-64 improved developmental competence of bovine oocytes via an antiapoptotic effect (Balboula et al. 2013 Reproduction 146, 407-417). The present study was carried out to test whether E-64 affected the developmental competency of porcine oocytes vitrified at the germinal vesicle stage. Cumulus-enclosed porcine oocytes were vitrified in microdrops and warmed by our method (Somfai et al. 2015 J. Reprod. Dev. 61, 571-579). Then, the oocytes were subjected to in vitro maturation (IVM) for 46h in a chemically defined porcine oocyte medium supplemented with 10ng mL−1 of epidermal growth factor, 10IU mL−1 of eCG, and 10IU mL−1 of hCG and during the first 22h of IVM with 1mM dibutyryl cyclic adenosine monophosphate. Then, cumulus-oocyte complexes were fertilized in vitro and presumptive zygotes were cultured in 50-µL drops of porcine zygote medium-3 for 7 days in 6-well dishes covered by paraffin oil in an atmosphere of 5% CO2, 5% O2, and 90% N2 at 39°C. On Day 5 (Day 0=IVF), the porcine zygote medium-3 was supplemented with 10% (vol/vol) FCS. The effects of 1.0μM of E-64 supplementation during IVM of non-vitrified and vitrified cumulus-oocyte complexes were investigated in a 2×2 factorial design. Survival rates after IVM, cleavage rates on Day 2, blastocyst rates, and total cell numbers in blastocysts on Day 7 were compared among groups. The experiment was replicated 5 times. Results were analysed by ANOVA and Tukey’s multiple comparison test. The percentages of live oocytes were statistically similar when oocytes were matured in the absence or presence of E-64 both in non-vitrified (99.2% v. 99.6%, respectively) and vitrified (94.3% v. 90.8%, respectively) groups. Similarly, IVM without or with E-64 supplementation had no effect on subsequent cleavage and blastocyst development rates in non-vitrified (67.4% v. 71.2% and 38.7% v. 43.2%, respectively) and vitrified (46.8% v. 48.8% and 14.6% v. 22.8%, respectively) oocytes. Irrespective of E-64 treatment, all survival and developmental rates in the vitrified groups were significantly lower (P&lt;0.05) compared with those of their non-vitrified counterparts except for the blastocyst development rate in the E-64-treated vitrified group, which did not differ significantly from those of the non-vitrified groups with or without E-64 treatment. There was no statistical difference in mean blastocyst cell numbers among the groups, ranging between 86.5±15.8 and 118±10.6. In conclusion, E-64 treatment had no effect on embryo production rates, which suggests that in our system, cathepsin-mediated apoptosis during IVM might not be the factor to limit embryo production using either fresh oocytes or those vitrified at the immature stage. This work was supported by JST/JICA SATREPS.

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.

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.

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