Linolenic acid improves oocyte developmental competence and decreases apoptosis ofin vitro-produced blastocysts in goat

Zygote ◽  
2015 ◽  
Vol 24 (4) ◽  
pp. 537-548 ◽  
Author(s):  
Arash Veshkini ◽  
Ali Akbar Khadem ◽  
Abdollah Mohammadi-Sangcheshmeh ◽  
Ali Asadi Alamouti ◽  
Masoud Soleimani ◽  
...  
Keyword(s):  
Linolenic Acid ◽  
Apoptotic Cell ◽  
Cell Number ◽  
Developmental Competence ◽  
Meiotic Maturation ◽  
Blastocyst Formation ◽  
Cumulus Expansion ◽  
Embryonic Cleavage ◽  
Maturation Rate

SummaryThe effects of α-linolenic acid (ALA) on developmental competence of oocytes in goats were evaluated in this study. Initially, the level of ALA in small and large antral follicles was determined to be in a range of 0.018–0.028 mg/ml (64.6–100.6 μM, respectively).In vitromaturation was performed in the presence of various concentrations (10, 50, 100, or 200 μM) of ALA. Cumulus expansion, meiotic maturation, levels of intracellular glutathione (GSH), embryonic cleavage, blastocyst formation following parthenogenetic activation (PA) andin vitrofertilization (IVF), number of total and apoptotic cells in blastocyst, and expression ofBax, Bcl-2, and p53 genes in blastocyst cells were determined. Compared with the control, no improvement was observed in cumulus expansion in ALA-treated groups. At 50 μM concentration, ALA increased meiotic maturation rate but had no effect on GSH level. When oocytes treated with 50 μM ALA were subsequently used for PA or IVF, a higher rate of blastocyst formation was observed, and these embryos had a higher total cell number and a lower apoptotic cell number. Expression analyses of genes in blastocysts revealed lesser transcript abundances forBaxgene, and higher transcript abundances forBcl-2gene in 50 μM ALA group. Expression ofp53gene was also less observed in ALA-treated blastocysts. Our results show that ALA treatment at 50 μM duringin vitromaturation (IVM) had a beneficial effect on maturation of goat oocytes and this, in turn, stimulated embryonic development and regulated apoptotic gene expression.

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.

scholarly journals 152EFFECT OF REPLACEMENT OF PYRUVATE/LACTATE IN CULTURE MEDIUM WITH GLUCOSE ON PREIMPLANTATION DEVELOPMENT OF PORCINE EMBRYOS IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 198
Author(s):  
N.W.K. Karja ◽  
S. Medvedev ◽  
D. Fuchimoto ◽  
A. Onishi ◽  
M. Iwamoto ◽  
...  
Keyword(s):  
Energy Source ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Culture Period ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Energy Substrates ◽  
Anova Test

Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) reported that replacement of pyruvate and lactate with glucose, as energy substrates, at 48h of culture in IVC medium enhanced the quality of IVP porcine blastocysts. However, the exact time during early cleavage stages when the utilization of glucose as an energy source is optimal has not yet been determined. The purpose of this study was to examine the effects of glucose supplementation at different times of culture on the developmental competence of IVP porcine embryos. Porcine cumulus-oocytes complexes were matured in modified NCSU-37 solution and fertilized in vitro according to Kikuchi et al. All cultures were performed at 38.5°C, 5% O2, 5% CO2, and 90% N2. In experiment 1, after being fertilized (Day 0), putative zygotes (1158 in 6 trials) were cultured in NCSU-37 supplemented with 0.4% BSA, 0.17mM sodium pyruvate, and 2.73mM sodium lactate (IVC-pyr/lac). Embryos (30–50 in each group) were then transferred into NCSU-37 supplemented with 0.4% BSA and 5.55mM D-glucose (IVC-glu) at 24, 48, 72, 96, or 118h of culture. As control groups, putative zygotes (391) were cultured in IVC-pyr/lac or IVC-glu for the whole culture period. In experiment 2, after being fertilized, putative zygotes (543 in 4 trials, 30–50 in each group) were cultured in IVC-pyr/lac, and then were transferred into IVC-glu at 48h, 53h, 58h, or 63h of culture, because glycolytic activity of in vitro-derived porcine embryos was reported to increase around the 8-cell stage, and some embryos develop to that stage before 72h of culture in experiment 1. All embryos were cultured for 6 days, and then development to the blastocyst stage and number of cells per blastocyst were assessed. When IVF embryos were cultured in IVC pyr/lac for 24h or 48h and subsequently in IVC-glu until day 6 in experiment 1, the rates of blastocyst formation were significantly higher (P<0.05, ANOVA test) than those of embryos cultured in IVC-pyr/lac for the whole culture period (24.4% and 23.0% v. 14.5%, respectively). However, when IVC pyr/lac was replaced with IVC-glu, there were no significant differences between the energy source replacement groups and the glucose-only group in terms of the proportions of cleavage, development to the blastocyst stage and mean cell number per blastocyst (P>0.05, ANOVA test) (15.2%–24.4%, and 16.8%, respectively). Replacement of pyruvate and lactate with glucose at 58h of culture in experiment 2 significantly enhanced the rate of blastocyst formation (P<0.05, ANOVA test) but not the mean cell number compared with zygotes in which the replacement was done at 48, 53, and 63h of culture (31.3% v. 20.6%, 20.8%, and 21.1%, respectively) (P<0.05, ANOVA test). In conclusion, replacement of pyruvate and lactate with glucose as energy substrates was optimal at 58h of culture for the in vitro development of pig embryos to the blastocyst stage.

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).

Depletion of BIRC6 leads to retarded bovine early embryonic development and blastocyst formation in vitro

2010 ◽  
Vol 22 (3) ◽  
pp. 564 ◽  
Author(s):  
Dessie Salilew-Wondim ◽  
Micheal Hölker ◽  
Franca Rings ◽  
Chirawath Phatsara ◽  
Abdollah Mohammadi-Sangcheshmeh ◽  
...  
Keyword(s):  
Embryo Development ◽  
Preimplantation Embryo ◽  
Apoptotic Cell ◽  
Cell Number ◽  
Blastocyst Formation ◽  
Caspase 9 ◽  
Embryo Survival ◽  
Double Stranded Rna ◽  
Preimplantation Embryo Development

Baculoviral inhibitors of apoptosis repeat-containing 6 (BIRC6) is believed to inhibit apoptosis by targeting key cell-death proteins. To understand its involvement during bovine preimplantation embryo development, two consecutive experiments were conducted by targeted knockdown of its mRNA and protein using RNA interference. In Experiment 1, the effect of BIRC6 knockdown during the early stages of preimplantation embryo development was assessed by injecting zygotes with long double-stranded RNA (ldsRNA) and short hairpin RNA (shRNA) against BIRC6 mRNA followed by in vitro culturing until 96 h post insemination (hpi). The results showed that in RNA-injected zygote groups, reduced levels of BIRC6 mRNA and protein were accompanied by an increase (P < 0.05) in the proportion of 2- and 4-cell and uncleaved embryos and a corresponding decrease (P < 0.05) in the number of 8-cell embryos. In Experiment 2, the effect of BIRC6 knockdown on blastocyst formation, blastocyst total cell number and the extent of apoptosis was investigated. Consequently, zygotes injected with ldsRNA and shRNA resulted in lower (P < 0.05) blastocyst formation and total blastocyst cell number. Moreover, the apoptotic cell ratio, CASPASE 3 and 7 activity, BAX to BCL-2 ratio and levels of SMAC and CASPASE 9 were higher in blastocysts derived from the ldsRNA and shRNA groups, suggesting increased apoptosis in those blastocysts. The results of this study reveal the importance of BIRC6 expression for embryo survival during bovine preimplantation embryo development. However, whether BIRC6 is essential for implantation and fetal development during bovine pregnancy needs further research.

61 EFFECT OF ROOM TEMPERATURE HOLDING PROCEDURE ON ABILITY OF OOCYTES TO MATURE AND DEVELOP IN VITRO AFTER EQUINE SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 136 ◽  
Author(s):  
K. Song ◽  
J. Lee ◽  
J. Park ◽  
W. Lee ◽  
Y. Chun ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Room Temperature ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Maturation Rate ◽  
Vitro Development

In Korea, it takes time to transport the ovaries of mares to the laboratory because horses are slaughtered only on Jeju island. Also, initiation of in vitro maturation (IVM) may be a little more delayed because of the oocyte collection by scraping of the follicular wall. It was reported that holding procedure of equine oocytes before IVM did not affect the developmental competence after intracytoplasmic sperm injection (Choi et al. 2006 Theriogenology 66, 955–963). The aims of present study were 1) to investigate the meiotic competence of equine oocytes held before IVM according to the type of oocytes, and 2) to examine the in vitro development after somatic cell nuclear transfer (SCNT). Cumulus–oocyte complexes (COCs) were recovered by scraping and washing of the follicular wall with Dulbecco’s modified Eagle medium (D-MEM) supplemented with 0.05% PVA, and classified as compact (Cp) or expended (Ex) depending on the expansion of cumulus or granulosa cells. 2 types of IVM procedures were compared: 1) COCs were matured immediately in IVM medium (TCM-199 supplemented with 5 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS) at 38.5°C under 5% CO2 in air for 24 to 27 h, and then held in holding medium (40% TCM-199 with Earle’s salts, 40% TCM-199 with Hanks’ salts, and 20% FBS) at room temperature for 6 to 7 h (control); or 2) COCs were initially held in holding medium for 6 to 7 h, and then matured in IVM medium for 24 to 27 h (holding). For SCNT, matured oocytes (pooled) were enucleated and electrically fused with equine skin fibroblasts (2.25 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 2 mM 6-DMAP, and cultured in D-MEM supplemented with 10% FBS and 50 ng mL–1 EGF at 38.5°C under 5% CO2, 5% O2, and 90% N2 for 7 to 9 days. Cleavage and blastocyst formation were evaluated on Days 2 and 8, respectively. All analyses were performed using SAS (version 9.1; SAS Institute Inc., Cary, NC, USA). 4 replicates were conducted from May to June 2010. In Ex oocytes, the maturation rate of the holding group (71.4%; 10/16) was not different from that of the control (65.6%; 44/73), and the rate of degenerated oocytes (4.8%; 1/16) in the holding group was not different from that in the control (5.6%; 5/73). However, in Cp oocytes, the degeneration rate of the holding group (65.0%; 31/49) was higher (P < 0.001) than that of the control (28.4%; 23/83), and the maturation rate of the holding group (20.6%; 12/49) was slightly lower (P = 0.07) than that of the control (46.0%; 38/83). After SCNT, the cleavage rate of the holding group (66.7%; 8/9) was not different from that of the control (60.8%; 14/25), and the rates of blastocyst formation of the control and the holding group were 8.1% (2/25) and 16.7% (2/9), respectively. Although the holding procedure may influence to the degeneration of Cp oocytes, it is considered that the developmental competence of equine oocytes held before IVM is not affected after SCNT.

Effect of antioxidant ascorbic acid on in vitro maturation of Caprine oocytes under normal and elevated temperatures

2018 ◽  
Author(s):  
S.B. Khanday ◽  
J.A. Ahmed ◽  
N. Nashiruddullah ◽  
U. Sharma and D. Chakraborty
Keyword(s):  
Ascorbic Acid ◽  
Heat Stress ◽  
In Vitro Maturation ◽  
Elevated Temperatures ◽  
Developmental Competence ◽  
Cumulus Expansion ◽  
Nuclear Maturation ◽  
Maturation Rate ◽  
Metaphase Stage

The aim of the present study was to assess the effect of ascorbic acid on in vitro maturation of caprine oocytes under normal and elevated temperatures. Goat ovaries were collected at slaughter and both A and B grade cumulus-oocyte-complexes (COCs) were aspirated out and were matured in vitro under normal (38.5°C) and elevated temperatures (41°C). On the basis of cumulus expansion and nuclear maturation, the maturation competencewere compared with and without ascorbic acid supplementation (100 µM). Heat stress significantly (P£ 0.01) reduced cumulus expansion, maturation rate and lowered metaphase stage II of nuclear maturation. Ascorbic acid improved developmental competence of oocytes during heat stress (41 °C) and ascorbic acid supplemented COCs demonstrated significantly (P£ 0.05) higher maturation rates when compared to non-supplemented groups.

Influence of hyaluronic acid synthesis and cumulus mucification on bovine oocyte in vitro maturation, fertilisation and embryo development

2007 ◽  
Vol 19 (3) ◽  
pp. 488 ◽  
Author(s):  
Cynthia Gutnisky ◽  
Gabriel C. Dalvit ◽  
Laura N. Pintos ◽  
Jeremy G. Thompson ◽  
Martha T. Beconi ◽  
...  
Keyword(s):  
Hyaluronic Acid ◽  
Glucose Uptake ◽  
Acrosome Reaction ◽  
Cumulus Cell ◽  
Acid Synthesis ◽  
Cell Number ◽  
Meiotic Maturation ◽  
Blastocyst Development ◽  
Cumulus Expansion

During cumulus–oocyte complex (COC) maturation, cumulus expansion involves the deposition of mucoelastic compounds, especially hyaluronic acid, synthesised from glucose via the hexosamine biosynthesis pathway. The aim of the present study was to determine the effects of uridine monophosphate (UMP) and 6-diazo-5-oxo-l-norleucine (DON), inhibitors of hyaluronic acid synthesis, during bovine oocyte in vitro maturation (IVM) on cumulus expansion, glucose uptake, protein synthesis, cumulus cell number, meiotic maturation, cleavage rate and subsequent embryo development. A further aim of the study was to examine the effect of hyaluronic acid on sperm capacitation and acrosome reaction in relation to the capacity of COCs to be fertilised in vitro. A low correlation between glucose uptake and degree of cumulus expansion was observed. Total and partial inhibition of cumulus expansion was observed with DON and UMP, respectively, and was accompanied by a decrease in glucose uptake with DON. Total protein content and cumulus cell number per COC increased during IVM, but was unaffected by the presence of DON or UMP, as was oocyte meiotic maturation. Rates of cleavage and blastocyst development decreased in oocytes matured with DON and UMP, although this inhibition was reversed when the in vitro fertilisation (IVF) medium contained heparin. Hyaluronic acid induced capacitation and the acrosome reaction, and in IVF medium prevented the inhibition of cleavage and blastocyst development by DON in a similar fashion to heparin. Hyaluronic acid synthesis during cumulus mucification contributes to the penetration and fertilisation of bovine oocytes, most likely by facilitating the processes of capacitation and acrosome reaction. Mucification during IVM is independent of cumulus cell proliferation, COC protein content, oocyte meiotic maturation and subsequent developmental competence once fertilised.

Effect of oocyte-secreted factors on porcine in vitro maturation, cumulus expansion and developmental competence of parthenotes

Zygote ◽  
2011 ◽  
Vol 20 (2) ◽  
pp. 135-145 ◽  
Author(s):  
Ma. Ninia L. Gomez ◽  
Jung Taek Kang ◽  
Ok Jae Koo ◽  
Su Jin Kim ◽  
Dae Kee Kwon ◽  
...  
Keyword(s):  
Cell Function ◽  
Cell Number ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Cumulus Expansion ◽  
Treatment Groups ◽  
Secreted Factors ◽  
Significant Difference

SummaryThe oocyte is known from recent studies in the mouse, cow, sheep and human to be a central regulator of follicular cell function. However, in the pig, little information is known about the regulation of cumulus expansion by oocyte-secreted factors and oocyte quality. We investigated the possible effects of oocyte-secreted factors during in vitro maturation on cumulus expansion and on porcine oocytes as judged by subsequent embryonic development after parthenogenetic activation. Cumulus–oocyte complexes (COC) from antral follicles of pig ovaries collected from a local abattoir were divided into control and treatment groups and were cultured in tissue culture medium 199 supplemented with follicle-stimulating hormone. Treatment groups consisted of increasing numbers of denuded oocytes (DO) co-cultured with COC (at ratios of COC to DO of 1:1, 1:2, 1:3, 1:4 and 1:5). After incubation for 44 h, cumulus expansion and maturation rates were assessed and oocytes were activated parthenogenetically. Cumulus expansion in the 1 COC:4 DO and 1 COC:5 DO groups was low and altered because full dispersion of the outer layer did not occur. Cell viability was not affected, as measured by the automated cell counter, but scanning electron microscopy revealed only a scanty extracellular matrix. Blastocyst rate was significantly higher in the 1 COC:4 DO (34.4%) and in the 1 COC:5 DO (34.9%) groups (p < 0.05) when compared with other groups. Maturation rate, cleavage rate and total cell number showed no significant difference between control and treatment groups. Amplification by reverse transcription polymerase chain reaction (RT-PCR) showed up-regulation of growth differentiation factor 9 (GDF9) in the cumulus cells in the 1 COC:4 DO group at 44 h. We conclude that denuded porcine oocytes could improve the maturation of COC as evidenced by increased blastocyst development in the 1 COC:4 DO, even though cumulus expansion was poor. This improvement could be a result of the GDF9 up-regulation.

275 EFFECT OF REACTIVE OXYGEN SPECIES DURING IN VITRO MATURATION ON PORCINE OOCYTE NUCLEAR MATURATION AND DEVELOPMENTAL COMPETENCE

2011 ◽  
Vol 23 (1) ◽  
pp. 235 ◽  
Author(s):  
Y. Yuan ◽  
R. Krisher
Keyword(s):  
Reactive Oxygen Species ◽  
Reactive Oxygen ◽  
Cell Number ◽  
Developmental Competence ◽  
Oxygen Species ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
Embryonic Cleavage ◽  
Porcine Oocyte

The generation of excessive reactive oxygen species (ROS) may contribute to the decreased competence of in vitro matured (IVM) oocytes. However, ROS are also generated in normal cellular metabolism and can be important regulators of cellular functions. The objective of this study was to examine the effect of ROS during IVM on porcine oocyte nuclear maturation and subsequent embryonic development. Oocytes were matured in different redox environments for 40 h in 7% CO2 in air at 38.7°C. The basic maturation medium was defined PPM supplemented with 1 mM hypoxanthine. Reactive oxygen species were generated by the hypoxanthine–xanthine oxidase (XOD) system at 3 different concentrations: XOD0 (0 mU), XOD1 (1 mU), and XOD10 (10 mU). In each XOD treatment, 2 different concentrations of cysteine (Cys) were added as an antioxidant: Cys1 (0.57 mM) and Cys2 (1.14 mM). This resulted in 6 experimental treatments in a 3 × 2 factorial design; XOD0-Cys1 was considered the control. For fertilization, gametes were co-incubated in modified Tween medium B with milk powder for 5 h and then cultured in NCSU-23 medium in 5% CO2, 10% O2 for 6 days, at which point cleavage, blastocyst development, and blastocyst cell number were determined (30–50 per treatment per replicate; 4 replicates). Data were analysed by two-way ANOVA, and differences were determined by Fisher’s least significant difference multiple-comparison test; percentage data were arcsin transformed (significance, P < 0.05). Results are shown in Table 1. Percentage of mature oocytes was not different between any XOD0 and XOD1 treatments, but maturation was decreased in both XOD10 treatments. Embryonic cleavage was also decreased in both XOD10 treatments compared with the control. Blastocyst development was decreased in XOD0-Cys2 and XOD10-Cys1 when compared with the control. Blastocyst total cell number was not different between any treatments (P > 0.05). In conclusion, 10-mU XOD during IVM resulted in decreased nuclear maturation, embryonic cleavage, and blastocyst development, possibly due to excessive ROS generated by XOD. The negative effect of high levels of XOD on blastocyst formation could be reversed by adding additional antioxidant capacity to the environment (Cys2). This result suggests that adequate ROS balance is important for oocyte quality. Interestingly, adding extra antioxidant capacity alone (XOD0-Cys2) was detrimental to blastocyst formation, possibly due to the creation of an environment that was too reduced. These results demonstrate the importance of keeping the redox environment balanced during oocyte maturation. Excessive oxidative or reducing environments both appear to be detrimental to oocyte developmental competence. Table 1.Nuclear maturation and developmental competence of oocytes matured in different redox environments

scholarly journals 305DEVELOPING AN ACTIVATION PROTOCOL FOR SOMATIC CELL NUCLEAR TRANSFER (SCNT) IN THE DOMESTIC CAT

2004 ◽  
Vol 16 (2) ◽  
pp. 272 ◽  
Author(s):  
T. Shin ◽  
T. Otoi ◽  
D.C. Kraemer ◽  
M.E. Westhusin
Keyword(s):  
Cytochalasin B ◽  
Polar Body ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
Parthenogenetic Activation ◽  
Maturation Rate

In order to establish an activation protocol for somatic cloning in the domestic cat, we evaluated the developmental competence of cat embryos derived from in-vitro matured ova after parthenogenetic activation treatment. The quality of parthenogenetic embryos was assessed by D3 cleavage rates, D8 rates of blastocyst formation and total nucleus numbers in expanded/hatching blastocysts. Parthenogenetic activation treatments were as follows;; Treatment I: 3.0kVcm−1 (25μs, twice) in 0.3M mannitol containing 0.1mM CaCl2· 2H2O and 0.1mM MgSO4, administered to matured cat oocytes and followed by 10μgmL−1 cycloheximide +5μgmL−1 cytochalasin B in TCM 199-Earle’s salt supplemented with 0.3% BSA for 6–7h. Treatment II: The first electric stimulation was performed as described for treatment I except that the activation medium consisted of 0.3M mannitol containing Mg, but without Ca. Two hours later, pre-pulsed MII oocytes were electropulsed by applying 1.0kVcm−1 (50μs, twice, 5s apart) in 0.3M mannitol containing Ca and Mg for additional activation, followed by culture in 10μgmL−1 cycloheximide +5μgmL−1 cytochalasin B treatment in TCM 199-Earle’s salt supplemented with 0.3% BSA for 6–7h. Immature cat oocytes were obtained from ovaries by mincing/dissection and matured in vitro for 26–30h as previously described (Gomez et al., 2001, Therigenology, 55, 472). Only MII oocytes with a 1st polar body were utilized for the activation procedure after removal of cumulus cells with 0.1% hyaluronidase by gentle pipetting. A total of 1120 oocytes were collected and the overall maturation rate was 49.8% (551/1120). After parthenogenetic activation of the MII oocytes, the embryos were cultured in vitro as described previously (Pope et al., 2000, Theriogenology, 53, 163–174). The results are shown in Table 1. Treatment II resulted in significantly higher (P&lt;0.01) D3 cleavage rates;; however, there were no significant differences in D8 blastocyst formation and total nucleus numbers. These data suggest that an additional electric activation (Treatment II) may increase the in vitro cleavage rates compared to using a fusion and electrical stimulation simultaneously (Treatment I). In addition, we demonstrated the developmental competence of domestic cat embryos derived from in vitro maturation, activation, and culture for development to the pre-implantation stage. By using these procedures for SCNT, several pregnancies were established and a healthy cloned kitten resulted in our laboratory (Shin et al., 2002, Nature, 415, 859). Therefore, this protocol can be useful, not only for prediction of the developmental competence of domestic cat oocytes matured in vitro, but also when used with SCNT to produce cloned cats. Comparison of cleavage rates and developmental competence to blastocyst stage following parthenogenetic activation treatments in domestic cat oocytes matured in vitro

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