216 PRONUCLEAR FORMATION AND DEVELOPMENTAL COMPETENCE OF MATURE PORCINE OOCYTES DERIVED FROM SMALL- AND MEDIUM-SIZED FOLLICLES

2011 ◽  
Vol 23 (1) ◽  
pp. 207
Author(s):  
C. Kohata ◽  
H. Funahashi
Keyword(s):  
Polar Body ◽  
Formation Rate ◽  
Cell Number ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Dibutyryl Camp ◽  
First Polar Body ◽  
Porcine Oocyte ◽  
Sperm Penetration ◽  
Pronuclear Formation

The maturation rate of oocytes derived from small follicles (SF) is known to be lower than that of oocytes from medium follicles (MF). The objective of this study was to assess the fertilizability and developmental competence of mature SF oocytes that were selected by the presence of the first polar body. Cumulus–oocyte complexes (COC) were aspirated from SF (1 to 2 mm in diameter) or MF (3 to 6 mm in diameter) of prepuberal ovaries. The COC were cultured in modified porcine oocyte medium supplemented with gonadotropins and dibutyryl cAMP for the first 20-h period and then in gonadotropin-free and dibutyryl cAMP-free porcine oocyte medium for another 24 h. Following IVM culture, mature oocytes with the first polar body were selected under a stereomicroscope, co-incubated with spermatozoa in a drop of modified TCM-199 containing 0.4% BSA and 5 mM caffeine for 6 h, and then incubated in porcine zygote medium-5 for 7 days. Sperm penetration, cleavage, and early development of the oocytes were examined before culture in porcine zygote medium-5 on Days 2 and 7 of culture. To analyse the fertilizability and developmental competence of oocytes from the SF and MF groups, sperm penetration, pronuclear formation, cleavage, blastocyst formation, and mean cell number in a blastocyst (as determined by fluorescence observation following Hoechst 33342 staining) were examined. Statistical analysis was performed by ANOVA with a Bonferroni-Dunn post-hoc test (P < 0.05). The percentages of oocytes in which the first polar body could be observed were 51.0 ± 4.5% and 78.5 ± 2.8% for SF- and MF-oocytes, respectively, whereas the maturation rates were 83.8 ± 4.0% and 62.8 ± 4.4% following fixation and staining. When only mature oocytes were co-cultured with sperm for 6 and 9 h, sperm penetration, monospermic penetration, and pronuclear formation were not different (P > 0.33) between mature SF- and MF-oocytes. Although there was no difference in cleavage rates between the mature SF- and MF-oocyte groups, blastocyst formation rate and mean cell number in the blastocyst were higher in mature MF-oocytes (31.0 ± 3.6% and 38.7 ± 1.9 cells, respectively) than in mature SF-oocytes (14.7 ± 3.2% and 31.2 ± 2.0 cells). From these results, we conclude that mature oocytes derived from SF have a similar fertilizability when compared with mature MF-oocytes, but the developmental competence to the blastocyst stage following IVF is significantly lower in mature SF-oocytes than in mature MF-oocytes.

44 EFFECT OF CYTOPLAST VOLUME ON FERTILIZATION AND EMBRYO DEVELOPMENT IN PORCINE M-II OOCYTES RECONSTRUCTED WITH KARYOPLASTS AND CYTOPLASTS OBTAINED BY THE 'CENTRI-FUSION' METHOD

2008 ◽  
Vol 20 (1) ◽  
pp. 102
Author(s):  
N. Maedomari ◽  
K. Kikuchi ◽  
M. Fahrudin ◽  
N. Nakai ◽  
M. Ozawa ◽  
...  
Keyword(s):  
Polar Body ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
First Polar Body ◽  
Developmental Capacity ◽  
Sperm Penetration ◽  
Pronuclear Formation ◽  
And Control

Metaphase-II chromosome transfer (M-II transfer) of oocytes is considered to be one of the advanced procedures to improve fertilization and developmental abilities of oocytes with poor cytoplasmic maturation. The aim of this study was to investigate the developmental capacity after IVF and IVC of porcine oocytes reconstructed from karyoplasts and cytoplasts produced by centri-fusion (Fahrudin et al. 2007 Cloning Stem Cells 9, 216–228). In brief, IVM oocytes (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041) with a visible first polar body were centrifuged at 13 000g for 9 min to stratify the cytoplasm. Then the zonae pellucidae were removed with pronase treatment. Zona-free oocytes were layered on a 300-µL discontinuous gradient of Percoll in TCM-HEPES with 5 µg mL–1 of cytochalasin B. After centrifugation at 6000g for 4 s, fragmented cytoplasms with approximately equal volumes were obtained, stained with Hoechst-33342, and classified into cytoplasm with (K; karyoplast) or without (C; cytoplast) chromosomes. One karyoplast was fused with 0, 1, 2, 3, and 4 cytoplasts (K, K + 1C, K + 2C, K + 3C, and K + 4C, respectively) by an electric stimulation with a single DC pulse (1.5 kV cm–1 for 20 µs) and cultured for 1 h. Zona-free oocytes without any reconstruction served as control oocytes. The diameters of the reconstructed and control oocytes were measured. All specimens were fertilized in vitro with frozen–thawed boar sperm, and cultured using the well of the well (WOW) system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264). Their fertilization status and developmental competence were examined. Data were analyzed by ANOVA followed by Duncan's multiple range tests. The diameter differed significantly among K to K + 4C oocytes (75.0–127.1 µm; P < 0.05), whereas the diameter of K + 2C oocytes was similar to that of the control oocytes (110.5 µm). Regardless of the cytoplast volume, sperm penetration rates (73.1–93.8%) for K to K + 4C oocytes were not significantly different compared to control oocytes (78.0%). Male pronuclear formation rates of K to K + 4C oocytes (92.3–97.1%) were also not different significantly different compared to control oocytes (96.6%). However, monospermy rates of K oocytes was significantly higher (61.6%; P < 0.05) than those of the reconstructed (K + 1C to K + 4C; 18.2–34.9%) and control oocytes (32.9%). The blastocyst formation rates in K, K + 1C, K + 2C, and K + 3C groups (0.0–9.8%; P < 0.05) were significantly lower than those in the control and K + 4C groups (17.8% and 15.3%, respectively; P < 0.05). The total cell numbers per blastocyst in K + 1C and K + 2C groups (7.5 and 8.3 cells, respectively) were significantly lower than in the control, K + 3C, and K + 4C groups (15.3–26.2 cells; P < 0.05). These results suggest that the cytoplast volume of porcine M-II transferred oocytes, produced by reconstruction from a karyoplast and cytoplast(s) and centri-fusion, is important for their ability to develop to the blastocyst stage and influences cell number.

348 CO-CULTURE OF PORCINE OOCYTES - CUMULUS COMPLEXES DERIVED FROM SMALL FOLLICLE WITH CUMULUS-CELL MASSES FROM MIDDLE FOLLICLE IMPROVES MEIOTIC MATURATION OF THE OOCYTES

2010 ◽  
Vol 22 (1) ◽  
pp. 330
Author(s):  
R. Matsunaga ◽  
H. Funahashi
Keyword(s):  
Early Development ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Meiotic Maturation ◽  
Protein Profiles ◽  
Blastocyst Formation ◽  
Maturation Rate ◽  
Sperm Penetration ◽  
Functional Peptide ◽  
Pronuclear Formation

It is known that maturation rate of oocytes derived from small follicles (SF) is lower than that of oocytes from middle follicles (MF). Since it has been reported that cumulus cells have important role during oocytes maturation, the ability of SF oocytes to complete the meiotic maturation may be affected by additional cumulus-cell mass. The present study was undertaken to examine the effects of co-culture of oocyte-cumulus complexes (OCCs) derived from SF with additional cumulus-cell masses on in vitro maturation and developmental competence of the oocytes. OCCs were aspirated from small (SF; 1-2 mm in diameter) or middle follicles (MF; 3-6 mm in diameter) of prepuberal ovaries. OCCs were cultured in porcine oocyte medium (POM; Research Institute for the Functional Peptide, Yamagata, Japan) supplemented with gonadotropins and dbcAMP for a first 20-h period and then in gonadotropin-free and dbcAMP-free POM for another 24 h. Culture medium was collected after the first 20-h culture and the end of IVM, and analyzed for the protein profiles. Following IVM, some oocytes were co-incubated with spermatozoa in a drop of modified Medium199 containing 0.4% BSA and 5 mM caffeine for 8 h and then incubated in PZM5 (Research Institute for the Functional Peptide, Yamagata, Japan) for 6 days. Sperm penetration, cleavage, and the early development of the oocytes were examined before culture in PZM5 or Day 2 and Day 6 of culture, respectively. OCCs derived from SF were co-cultured with cumulus-cell masses derived from SF or MF during IVM (SFO-SFC and SFO-MFC groups, respectively). Some OCCs derived from SF or MF were cultured for IVM without additional cumulus-cell masses (SFO and MFO, respectively). After culture, meiotic maturation of the oocytes was examined. To analyze the developmental competence of oocytes of SF, MF, and SFO-MFC groups, sperm penetration, pronuclear formation, cleavage, and blastocyst formation were examined. Protein profiles in the IVM media were examined by 10% SDS-PAGE. Statistical analysis was performed by ANOVA with a Bonferroni-Dunn post hoc test (significance, P ≤ 0.05). After culture for IVM, the diameters of SFO and SFO-MFC were not different from that of MFO (113.3-114.5 μm). The maturation rate of SFO-MFC oocytes (75.5 ± 6.2%) was higher than SFO (52.2 ± 2.8%) and comparable with the rate of MFO oocytes (83.2 ± 6.3%), while there was not significant difference between the mature rate of SFO+SFC oocytes (63.6 ± 4.0%) and SFO oocytes. There were no significant differences between groups in sperm penetration, pronuclear formation, and cleavage. Blastocyst formation of SF oocytes was not improved by co-culture with MF cumulus-cell masses. Certain band was detected only in MF medium of collected at 20 h (24.5 kD). From these results, we conclude that secretions from cumulus-cell masses derived from MF well improve the meiotic progress of oocytes derived from SF, but not the early development following IVF.

Effects of milrinone and butyrolactone-I on porcine oocyte meiotic progression and developmental competence

2006 ◽  
Vol 18 (3) ◽  
pp. 309 ◽  
Author(s):  
Christopher G. Grupen ◽  
Maggie Fung ◽  
David T. Armstrong
Keyword(s):  
Formation Rate ◽  
Inhibitory Effect ◽  
Cell Number ◽  
Developmental Competence ◽  
In Vitro Fertilisation ◽  
Cleavage Rate ◽  
Meiotic Progression ◽  
Porcine Oocyte ◽  
Type 3

Inappropriate coordination of oocyte nuclear and cytoplasmic maturation is thought to contribute to the poor efficiency of embryo production in vitro. With the aim of improving this coordination, the effects of milrinone, an inhibitor of type 3 phosphodiesterases, and butyrolactone-I, a selective inhibitor of cdc2 kinases, on porcine oocyte maturation were investigated. Oocytes recovered from slaughterhouse-derived ovaries of prepubertal animals were treated with the inhibitors for 24 h. At concentrations of 50 and 250 μm, milrinone reversibly inhibited meiotic progression in 57% and 71% of oocytes, respectively. The presence or absence of milrinone in the medium used to wash oocytes for 30 min did not alter the inhibitory effect of the 24 h treatment. At concentrations of 25 and 50 μm, butyrolactone-I inhibited meiotic progression in 61% and 66% of oocytes, respectively, but the effect was not fully reversible in the absence of follicle-stimulating hormone (FSH). The presence of FSH during the butyrolactone-I treatment period increased the ability of oocytes to subsequently complete meiosis at 44 h without changing the inhibitory effect at 24 h. Following in vitro fertilisation at 44 and 50 h, treatment with butyrolactone-I and milrinone, alone or in combination, did not alter embryo cleavage rate, blastocyst formation rate or blastocyst cell number. Despite the different actions of milrinone and butyrolactone-I, the present study demonstrates that these reagents inhibit meiotic progression to a similar extent in the presence of FSH while maintaining developmental competence in porcine oocytes.

scholarly journals 301EFFECT OF THE MII-AGE AND ACTIVATION PROTOCOL ON THE PARTHENOGENETIC DEVELOPMENT OF PORCINE OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 270
Author(s):  
I. Lagutina ◽  
G. Lazzari ◽  
C. Galli
Keyword(s):  
Cytochalasin B ◽  
Average Rate ◽  
Polar Body ◽  
Cell Number ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Average Cell ◽  
Electric Impulse ◽  
Polar Body Extrusion

The completion of porcine oocyte nuclear maturation (MII) in vitro, characterized by the time of polar body extrusion, starts at about 32h of maturation and lasts more than 12h. This leads to the simultaneous presence in the population of matured oocytes with differing abilities to be activated. We investigated age-dependent changes in pig oocyte maturation, activation and development in SOFaa in response to electric impulse (EL) in the presence of cytochalasin B (CB) and EL in combination with cycloheximide and cytochalasin B (EL+CHX+CB). Oocytes were matured in TCM 199 with 10% FCS, cysteine, LH, FSH (Pergovet, Serono, Geneva, Switzerland) for 36h and then decumulated. Matured oocytes were activated at 40 and 44h by double pulse of 30μs DC 1, 5kVcm−1 and cultured in 5μgmL−1 CB for 4h or by EL followed by incubation in 10μgmL−1 CHX+5μgmL−1 CB for 4h. According to the MII-age before activation oocytes were divided into 2 age classes: 3–7 and 7–11h after polar body extrusion. Embryos were cultured in SOFaa in 5% CO2, 5% O2 at 38.5°C. The rates of cleavage, blastocyst formation and cell number of BL on Day 7 (BLD7) were recorded. Our results showed that the average rate of maturation at 44h was 72% (n=1377). About 50% and 87% of oocytes, that eventually matured, extruded the polar body at 37 and 40h, respectively. The average cell number of BLD7 developed in SOFaa was 80±36 (n=52) and was not affected by activation protocol. Seventy-nine and 27% of BL had more than 50 and 100 cells per BL, respectively. Porcine oocytes activated by EL acquired their developmental competence gradually, achieving the highest rates of cleavage and blastocyst formation 7h after polar body extrusion. By contrast, oocytes activated by EL+CHX+CB showed their maximal developmental competence earlier (3–7h group). In conclusion, we demonstrate that electric impulse in combination with CHX+CB treatment permits earlier efficient activation of porcine oocytes (3–7h after polar body extrusion).

167 DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES DERIVED FROM SMALL- OR MEDIUM-SIZED FOLLICLES AND DENUDED OF CUMULUS CELLS BEFORE AND DURING IN VITRO MATURATION

2017 ◽  
Vol 29 (1) ◽  
pp. 192
Author(s):  
P. Ferré ◽  
K. X. Nguyen ◽  
T. Wakai ◽  
H. Funahashi
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
First Polar Body

This experiment was undertaken to assess the meiotic and developmental competences of oocytes derived from different sized follicles and denuded of cumulus cells 0, 20, and 44 h after the start of culture for in vitro maturation (IVM). Groups of 60 oocyte-cumulus complexes from small- (SF; <3 mm) and medium-sized follicles (MF; 3–6 mm) were cultured for IVM in porcine oocyte medium with 50 μM β-mercaptoethanol supplemented with 1 mM dibutyryl-cyclic adenosine monophosphate, 10 IU mL−1 of eCG, and 10 IU mL−1 of hCG for 20 h at 39°C and 5% CO2 in air. Then, after washing, they continued culture in fresh β-mercaptoethanol without dibutyryl-cyclic adenosine monophosphate and gonadotropins under the same conditions for another 24 h. At 0, 20, and 44 h of IVM, cumulus cells were removed with 0.1% (wt/vol) hyaluronidase and the denuded oocytes continued IVM culture following the protocol. Mature oocytes with the first polar body were selected, parthenogenetically activated with a single electrical pulse (DC: 1.2 kV/cm, 30 µs), incubated with 4% (wt/vol) BSA and 5 μM cytochalasin B for 4 h, and cultured in porcine zygote medium for 5 days. Cleavage and blastocyst formation rates were observed on Day 2 and 5, respectively. Blastocysts were stained with 4’,6-diamidino-2-phenylindole for cell count assessment. The experiment was replicated 5 times and analysed with a 1- or 2-way ANOVA. If P < 0.05 in ANOVA, a Tukey multiple comparisons test was performed. Regardless of the time of cumulus cell removal, oocytes from MF had significantly higher in rates of maturation, cleavage, and blastocyst rates, as compared with those from SF, whereas there were no significant differences in the cell number of blastocysts between SF and MF (32 v. 34 cells, respectively). When oocytes were denuded before IVM culture, rates of oocyte maturation (37.6% in SF and 50.8% in MF), and blastocyst formation (2.7% in SF and 27.3% in MF) were significantly lower than controls (51.2% in SF and 76% in MF; 25.8% in SF and 48.5% in MF, respectively). When oocytes were denuded 20 h after the start of IVM, oocyte maturation rates were significantly increased (64.1% in SF and 82.5% in MF) as compared with controls, whereas no significant differences were observed in cleavage and blastocyst formation rates in comparison with controls. These results conclude that removing cumulus cells from oocyte-cumulus complexes 20 h after the start of IVM improves the meiotic competence of oocytes derived from both SF and MF, without any reduction of developmental competence of the oocytes following parthenogenetical activation.

scholarly journals Diploid porcine parthenotes produced by inhibition of first polar body extrusion during in vitro maturation of follicular oocytes

Reproduction ◽  
2006 ◽  
Vol 132 (4) ◽  
pp. 559-570 ◽  
Author(s):  
Tamás Somfai ◽  
Manabu Ozawa ◽  
Junko Noguchi ◽  
Hiroyuki Kaneko ◽  
Katsuhiko Ohnuma ◽  
...  
Keyword(s):  
Polar Body ◽  
Formation Rate ◽  
Metaphase Plate ◽  
Chromosome Analysis ◽  
Blastocyst Stage ◽  
Control Group ◽  
Blastocyst Formation ◽  
First Polar Body ◽  
Polar Body Extrusion

We investigated nuclear progression and in vitro embryonic development after parthenogenetic activation of porcine oocytes exposed to cytochalasin B (CB) during in vitro maturation (IVM). Nuclear progression was similar in control oocytes and oocytes matured in the presence of 1 μg/ml CB (IVM-CB group) by 37 h IVM; at this time the proportion of oocytes that had reached or passed through the anaphase-I stage did not differ significantly between the IVM-CB and the control groups (61.3 and 69.9% respectively; P < 0.05). After IVM for 37 h, no polar body extrusion was observed in the IVM-CB group. In these oocytes, the two lumps of homologous chromosomes remained in the ooplasm after their segregation and turned into two irregular sets of condensed chromosomes. By 41 h IVM, the double sets of chromosomes had reunited in 89.5% IVM-CB oocytes and formed a single large metaphase plate, whereas 68.8% of the control oocytes had reached the metaphase-II stage by this time. When IVM-CB oocytes cultured for 46 h were stimulated with an electrical pulse and subsequently cultured for 8 h without CB, 39.0% of them extruded a polar body and 82.9% of them had a female pronucleus. Chromosome analysis revealed that the majority of oocytes that extruded a polar body were diploid in both the control and the IVM-CB groups. However, the incidence of polyploidy in the IVM-CB group was higher than that in the control group (P < 0.05). In vitro development of diploid parthenotes in the control and the IVM-CB groups was similar in terms of blastocyst formation rates (45.8 and 42.8% respectively), number of blastomeres (39.9 and 44.4 respectively), the percentage of dead cells (4.3 and 2.9% respectively), and the frequency of apoptotic cells (7.3 and 6.3% respectively). Tetraploid embryos had a lower blastocyst formation rate (25.5%) and number of cells (26.2); however, the proportion of apoptotic nuclei (7.0%) was similar to that in diploid parthenotes. These results suggest that the proportion of homozygous and heterozygous genes does not affect in vitro embryo development to the blastocyst stage.

scholarly journals Identification and characterization of an oocyte factor required for sperm decondensation in pig

Reproduction ◽  
2014 ◽  
Vol 148 (4) ◽  
pp. 367-375 ◽  
Author(s):  
Jingyu Li ◽  
Yanjun Huan ◽  
Bingteng Xie ◽  
Jiaqiang Wang ◽  
Yanhua Zhao ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Polar Body ◽  
Developmental Competence ◽  
First Polar Body ◽  
Specific Factors ◽  
Pronuclear Formation ◽  
Protein Disulfide ◽  
Sperm Decondensation

Mammalian oocytes possess factors to support fertilization and embryonic development, but knowledge on these oocyte-specific factors is limited. In the current study, we demonstrated that porcine oocytes with the first polar body collected at 33 h ofin vitromaturation sustain IVF with higher sperm decondensation and pronuclear formation rates and supportin vitrodevelopment with higher cleavage and blastocyst rates, compared with those collected at 42 h (P<0.05). Proteomic analysis performed to clarify the mechanisms underlying the differences in developmental competence between oocytes collected at 33 and 42 h led to the identification of 18 differentially expressed proteins, among which protein disulfide isomerase associated 3 (PDIA3) was selected for further study. Inhibition of maternal PDIA3 via antibody injection disrupted sperm decondensation; conversely, overexpression of PDIA3 in oocytes improved sperm decondensation. In addition, sperm decondensation failure in PDIA3 antibody-injected oocytes was rescued by dithiothreitol, a commonly used disulfide bond reducer. Our results collectively report that maternal PDIA3 plays a crucial role in sperm decondensation by reducing protamine disulfide bonds in porcine oocytes, supporting its utility as a potential tool for oocyte selection in assisted reproduction techniques.

scholarly journals 303PARTHENOGENETIC DEVELOPMENT OF PIG OOCYTES BY CHEMICAL ACTIVATION

2004 ◽  
Vol 16 (2) ◽  
pp. 271
Author(s):  
C.S. Park ◽  
D.I. Jin ◽  
M.Y. Kim ◽  
Y.J. Chang ◽  
Y.J. Yi
Keyword(s):  
Cytochalasin B ◽  
Polar Body ◽  
Chemical Activation ◽  
Formation Rate ◽  
Penicillin G ◽  
Oocyte Activation ◽  
Blastocyst Formation ◽  
Parthenogenetic Development ◽  
First Polar Body

Efficient activation is essential for the success of animal cloning by nuclear transfer. The aim of this study was to investigate the effects of chemical activation agents on parthenogenetic development of pig oocytes matured in vitro. The medium used for oocyte maturation was TCM-199 supplemented with 26.19mM sodium bicarbonate, 0.9mM sodium pyruvate, 10μgmL−1 insulin, 2μgmL−1 vitamin B12, 25mM HEPES, 10μgmL−1 bovine apotransferrin, 150μM cysteamine, 10IUmL−1 PMSG, 10IUmL−1 hCG, 10ngmL−1 EGF, 0.4% BSA, 75μgmL−1 sodium penicillin G, 50μgmL−1 streptomycin sulfate and 10% pFF. After about 22h of maturation, oocytes were cultured without cysteamine and hormones for 22h at 38.5°C, 5% CO2 in air. Cumulus-free oocytes showing first polar body were selected for activation. Oocytes were activated as follows. First, all oocytes were activated with 25mM HEPES buffered NCSU-23 medium containing 8% ethanol for 10min. After that, in treatment 1, oocytes were incubated in the NCSU-23 medium supplemented with 7.5μgmL−1 cytochalasin B for 3h. In treatment 2, oocytes were incubated in the NCSU-23 medium supplemented with 10μgmL−1 cycloheximide for 3h. In treatment 3, oocytes were incubated in the NCSU-23 medium supplemented with 7.5μgmL−1 cytochalasin B for 1.5h, and then were incubated in the NCSU-23 medium supplemented with 10μgmL−1 cycloheximide for 1.5h. In treatment 4, oocytes were incubated in the NCSU-23 medium supplemented with 7.5μgmL−1 cytochalasin B plus 10μgmL−1 cycloheximide for 3h. Following activation, oocytes were transferred into 500μL NCSU-23 culture medium containing 0.4% BSA for further culture for 20 and 144h. Activated oocytes were fixed and stained for evaluation of activation rate, cleaved oocytes, blastocyst formation rate and cell numbers per blastocyst. Data were analysed by ANOVA and Duncan’s multiple range test using the SAS program. The rate of oocyte activation was higher in treatment 4 (62.1%) than in treatment 1, 2 and 3 (52.0, 49.6 and 58.0%, respectively). The percentage of cleaved oocytes was lower in treatment 1 and 2 (56.9 and 55.2%) than in treatment 3 and 4 (68.8 and 68.5%). The rate of blastocyst formation from the cleaved oocytes was higher in treatment 3 and 4 (19.8 and 22.0%) than in treatment 1 and 2 (12.1 and 11.7%). Mean cells per blastocyst were lowest in treatment 2 (21.2±0.9) compared to treatment 1, 3 and 4 (27.3±2.2, 30.4±3.8 and 30.9±3.4, respectively). In conclusion, cytochalasin B combined with cycloheximide was more efficient for parthenogenetic development of pig oocytes matured in vitro.

scholarly journals 55 NUCLEUS CHANGES AND DEVELOPMENT OF PORCINE RECONSTRUCTED (NT) AND PARTHOGENETICALLY ACTIVATED (PA) EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 177
Author(s):  
N.R. Mtango ◽  
T. Kono
Keyword(s):  
Polar Body ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Normal Embryo ◽  
Basic Medium ◽  
Cell Nuclei ◽  
First Polar Body ◽  
Porcine Oocyte

Nuclear reprogramming is characterized by functional modification(s) of the transferred nucleus that allows it to direct normal embryo development with the potential to grow to term. The aim of our study was to investigate the process of nuclear changes in reconstructed and activated embryos as well as their developmental competence. All chemicals used were from Sigma Chemicals (St. Louis, MO, USA). Cumulus-oocyte complexes were aspirated from slaughterhouse ovaries of prepurbetal gilts and matured for 42 h in vitro. The cumulus cells were removed by adding in 1 mg mL −1 hyaluronidase in TLP-HEPES. For the NT experiment, oocytes with first polar body were cultured in 0.4 μg mL−1 demecolcine for 1 h. A protruding membrane was removed by micromanipulator and a single donor nucleus from fetal fibroblast was injected subzonally. Fusion was conducted immediately after transfer in 0.3 M mannitol, 0.5 mM HEPES, 0.1% PVA, and 0.1 mM MgCl2 in a fusion chamber with parallel electrodes set 1 mm apart using a singe DC pulse of 125 V mm−1 for 80 s. Activation was done 2–4 h after fusion in the same medium as fusion but with 0.1 mM CaCl2 added; embryos were cultured in 5 μg mL−1 cytochalasin B and 10 μg mL−1 cyclohexamide for 6 h. The embryos were cultured in glucose-free NCSU-37 containing 4 mg mL−1 BSA as basic medium supplemented with 0.17 mM sodium pyruvate and 2.73 mM sodium lactate from Days 0 to 2, and then in basic medium with 5.55 mM D-glucose from Days 2–6 (Kikuchi K et al. 2002 Biol. Reprod. 66, 1033–1041). Non-manipulated oocytes (PA) were electrically activated as stated above. For observing the changes of donor cells, some reconstructed oocytes were fixed 2 h after fusion, prior to activation, and some 12 h after activation in acetic acid:ethanol (1:3) and stained in 1% orcein. The activated oocytes were fixed at 12 h and stained as stated above. There were 47.5% (38/80) of reconstructed oocytes with premature chromosome condensation (PCC), and 23.7% (19/80) with nuclear swelling two hours after fusion. Pronuclear like formation 12 h after activation was 45% (27/60) and 83.3% (50/60) in NT and PA, respectively. The blastocyst rate was 8.3% (5/60) and 46% (69/150) for NT and PA, respectively. The results suggest that porcine oocyte cytoplasm can successfully reprogram somatic cell nuclei and support the development of NT embryos to the blastocyst stage.

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