160 In vitro maturation of pre-pubertal goat oocytes and their development after chemical activation

Experiments were conducted to study in vitro maturation of pre-pubertal goat oocytes and their developmental potential after chemical activation. Cumulus-oocyte complexes (n=1170) collected from the ovaries of pre-pubertal goats slaughtered at a local abattoir were matured in TCM-199 supplemented with 0.15mg mL−1 l-glutamine, 0.25mM sodium pyruvate, 0.1mM l-cysteine, 20ng mL−1 epidermal growth factor, 10mg mL−1 FSH, 10mg mL−1 LH, 1μg mL−1 oestradiol and 10% FCS for 24h at 39°C under 5% CO2 in humidified air. In Experiment 1, matured oocytes were activated (r=6) with either 5mM ionomycin (n=85) or 7% ethanol (n=91) followed by culture in 6-DMAP for 4h. All the activated oocytes were then cultured in KSOM supplemented with 3mg mL−1 BSA and were fixed and stained with Hoechst 33342 after 18h of culture to evaluate their activation status. In Experiment 2, oocytes activated with 5mM ionomycin and 6-DMAP were cultured for 7 days (r=6) in 1 of the 4 different culture media [Charles Rosenkrans medium (CR-1), modified TCM-199, KSOM and SOF] to study their developmental potential. All media were supplemented with 3.0mg mL−1 BSA for the first 3 days and 10% FCS for the subsequent 4 days. Of these pre-pubertal oocytes, 59% reached metaphase II stage, and 83% of these oocytes were classified as activated in the group using ionomycin in comparison with 69% in the group using ethanol as an activating agent (P<0.05). No difference was observed in the cleavage rate of activated oocytes cultured in any of the 4 culture media (65.7v. 55.0v. 61.0v. 56.2%, respectively). However, the development to blastocyst stage was observed in only KSOM (16%) and SOF (5%) media. In conclusion, the present study demonstrates that pre-pubertal goat oocytes can mature in vitro and can be activated with 5mM ionomycin, and KSOM, and to a lesser extent SOF, supports development to the blastocyst stage. We plan to use these oocytes as a cytoplast source for interspecies somatic cell NT; however, before that, more studies are needed to evaluate their requirements in culture media to enhance their development to the blastocyst stage.

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130 PREDICTION OF PARTHENOGENETIC DEVELOPMENTAL POTENTIAL BY POLAR BODY EXTRUSION AND FIRST CLEAVAGE ON IN VITRO MATURATION AND DEVELOPMENT OF PORCINE FOLLICULAR OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab130 ◽

2008 ◽

Vol 20 (1) ◽

pp. 145

Author(s):

H. J. Kim ◽

S. R. Cho ◽

C. Y. Choe ◽

S. H. Choi ◽

D. S. Son ◽

...

Keyword(s):

In Vitro Maturation ◽

Polar Body ◽

Blastocyst Stage ◽

Developmental Competence ◽

Cleavage Rate ◽

Developmental Potential ◽

Non Invasive ◽

First Polar Body ◽

Polar Body Extrusion

The objective of this study was to examine the selection effects of in vitro matured porcine follicular oocytes with polar body extrusion and early cleavage as a non-invasive marker to know the developmental competence in advance. Porcine oocytes matured for 48 h and then examined for polar body extrusion. The examined oocytes were matured for an additional 16–18 h, activated with 7% ethanol, and cultured in 5 µg mL–1 cytochalasin B for 5 h for diploid formation. The treated oocytes were examined for cleavage after 48 h and continued culturing for 5 days. Each treatment was replicated by 3–4 times. Oocytes of 21.9% (70/320) were discarded in morphological selection, and 32.1% (167/520) oocytes were discarded by failure of first polar body extrusion. The selected oocytes were matured and activated, and after 48 h, the cleavage rate was examined. In morphologically selected oocytes, 15.8% (30/190) were not cleaved, 52.6% (100/190) were normally cleaved (consisted of 2–7 cells), and 31.6% (60/190) were hyper-cleaved (consisted of 8 cells or more) at 48 h after activation. However, in the first polar body extruded oocytes, 7.1% (18/253) were not cleaved, 73.1% (185/253) were normally cleaved, and 19.8% (50/253) were hyper-cleaved. From the morphologically selected oocytes, 16.7% (10/60) were developed up to blastocyst stage from those in which cleavage selection was not performed and 31.7% (19/60) from those in which cleavage selection was performed. From the polar body extruded oocytes, 39.0% (39/100) were developed up to blastocyst stage from those in which cleavage selection was not performed and 49.0% (49/100) from those in which cleavage selection was performed. Cleavage was examined within 12 h interval after activation (0 = time of activation) up to 48 h. At 0–12, 12–24, 24–36, and 36–48 h intervals, 4.1% (9/220), 68.6% (151/220), 19.1% (42/220), and 2.3% (5/220) oocytes were cleaved, respectively, and 5.9% (13/220) oocytes were not cleaved at 48 h after activation. The cleaved embryos in each interval were cultured and developed up to blastocyst with 0 (0/9), 39.1 (59/151), 9.5 (4/42), and 0% (0/5), respectively. This result suggests that the polar body extruded and cleaved at 12–36 h embryo has higher developmental potential than the others.

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324SPHINGOSINE-1-PHOSPHATE PROTECTS CULTURED BOVINE OOCYTES FROM PHYSIOLOGICALLY RELEVANT THERMAL STRESS

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab324 ◽

2004 ◽

Vol 16 (2) ◽

pp. 282 ◽

Cited By ~ 1

Author(s):

Z. Roth ◽

P.J. Hansen

Keyword(s):

Thermal Stress ◽

Heat Shock ◽

Blastocyst Stage ◽

Caspase Activity ◽

Blastocyst Development ◽

Cleavage Rate ◽

Developmental Potential ◽

Oocyte Competence ◽

Cell Death Pathway

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that can block the sphingomyelin cell-death pathway by suppressing ceramide-induced apoptosis. The present study was performed to test whether S1P protects oocytes from heat shock during in vitro maturation. Cumulus-oocyte complexes obtained by slicing follicles were placed in maturation medium with or without 50nM S1P and cultured at 38.5°C (CON) or 41°C (41C) for the first 12h of maturation. Incubation during the last 10h of maturation (22-h total maturation time), fertilization, and embryonic development were performed at 38.5°C and 5% (v/v) CO2. Blastocyst development was recorded at 8 days post-insemination (dpi) and activity of group II caspases in 8-day blastocysts was determined using a fluoroprobe, PhiPhiLux-G1D2 (OncoImmunin, Gaithersburg, MD, USA). Data were analysed by least-squares ANOVA with the GLM procedure of SAS. Percentage data were subjected to arcsin transformation before analysis. Exposure of oocytes to thermal stress during the first 12h of maturation reduced cleavage rate (P<0.01) and the number of oocytes developing to the blastocyst stage (P<0.04). There was a temperature x S1P interaction for cleavage rate (P<0.03) because S1P blocked effects of thermal stress on cleavage rate. Without S1P, the percentage of oocytes that cleaved by 3 dpi were 83.6±2.7% and 65.8±2.7% for CON and 41C, respectively. In the presence of S1P, percent cleavage was 86.7±2.7% and 83.9±2.7% for CON and 41C, respectively. There was a trend (P=0.06) for a temperature x S1P interaction for percent oocytes developing to blastocyst stage because S1P blocked effects of heat shock on development. Without S1P, the percentages of oocytes that developed to the blastocyst stage were 28.7±3.0% and 15.2±3.0% for CON and 41C, respectively. In the presence of S1P, percent blastocysts were 24.3±3.4% and 23.9±3.0% for CON and 41C, respectively. When development was expressed as percentage of cleaved embryos, however, there were no effects of temperature, S1P, or temperature x S1P on percent development to the blastocyst stage. Blastocyst caspase activity was not affected by temperature or S1P. In summary, exposure to physiologically relevant thermal stress during the first 12h of maturation has a deleterious effect on oocyte competence and this effect can be reduced by S1P. The fact that heat shock reduced the percentage of oocytes but not the percentage of cleaved embryos that became blastocysts suggests that oocytes that survive effects of heat shock and cleave have normal potential to develop to the blastocyst stage. Moreover, since heat shock did not affect caspase activity, it is likely that blastocysts from heat-shocked oocytes have normal developmental potential, at least as determined by caspase activity. Support: BARD FI-330-2002 and USDA Grants 2002-35203-12664 and 2001-52101-11318.

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MicroRNAs from Extracellular Vesicles Secreted by Bovine Embryos as Early Biomarkers of Developmental Competence

International Journal of Molecular Sciences ◽

10.3390/ijms21238888 ◽

2020 ◽

Vol 21 (23) ◽

pp. 8888

Author(s):

Bárbara Melo-Baez ◽

Yat S. Wong ◽

Constanza J. Aguilera ◽

Joel Cabezas ◽

Ana C. F. Mançanares ◽

...

Keyword(s):

Extracellular Vesicles ◽

Fatty Acid Biosynthesis ◽

Culture Media ◽

Blastocyst Stage ◽

Developmental Competence ◽

Target Cells ◽

Bovine Embryos ◽

Developmental Potential ◽

Maternal Communication

During early development, embryos secrete extracellular vesicles (EVs) that participate in embryo–maternal communication. Among other molecules, EVs carry microRNAs (miRNAs) that interfere with gene expression in target cells; miRNAs participate in embryo–maternal communication. Embryo selection based on secreted miRNAs may have an impact on bovine breeding programs. This research aimed to evaluate the size, concentration, and miRNA content of EVs secreted by bovine embryos with different developmental potential, during the compaction period (days 3.5–5). Individual culture media from in vitro–produced embryos were collected at day 5, while embryos were further cultured and classified at day 7, as G1 (conditioned-culture media by embryos arrested in the 8–16-cells stage) and G2 (conditioned-culture media by embryos that reached blastocyst stages at day 7). Collected nanoparticles from embryo conditioned culture media were cataloged as EVs by their morphology and the presence of classical molecular markers. Size and concentration of EVs from G1 were higher than EVs secreted by G2. We identified 95 miRNAs; bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were upregulated in G1, whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were downregulated. The most significant upregulated pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The characteristics of EVs secreted by bovine embryos during the compaction period vary according to embryo competence. Embryos that reach the blastocyst stage secrete fewer and smaller vesicles. Furthermore, the loading of specific miRNAs into the EVs depends on embryo developmental competence.

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Development of prepubertal goat oocytes after their in vitro maturation and chemical activation

Zygote ◽

10.1017/s0967199420000313 ◽

2020 ◽

Vol 28 (6) ◽

pp. 447-452

Author(s):

Seungbum Hong ◽

Binoy S. Vettical ◽

Nisar Ahmad Wani

Keyword(s):

Culture Media ◽

Chemical Activation ◽

Control Treatment ◽

Developmental Potential ◽

Embryo Culture Medium ◽

Significant Difference ◽

Maturation Rate ◽

Further Development ◽

Oviductal Fluid

SummaryExperiments were conducted to study in vitro maturation of prepubertal goat oocytes and their developmental potential after chemical activation. In Experiment 1, cumulus–oocytes complexes collected from the ovaries of prepubertal goats slaughtered at a local abattoir were matured in vitro in TCM-199-based medium supplemented with 10 µg/ml luteinizing hormone (LH) (treatment 1) or 10 µg/ml LH + 0.1 mM l-cysteine (treatment 2). In Experiment 2, mature oocytes were activated with either 5 µM ionomycin or 7% ethanol. After 18 h, some oocytes were randomly fixed and stained to evaluate their chromatin status, while others were cultured in embryo culture medium to study their further development. In Experiment 3, oocytes activated with 5 µM ionomycin were cultured for 7 days in one of the four different culture media [Charles Rosenkrans medium (CR-1), TCM-199, potassium simplex optimization medium (KSOM) and synthetic oviductal fluid (SOF)] to study their developmental potential. The maturation rate in control, treatment 1, and treatment 2 media did not differ from each other (P > 0.05). However, the lowest degeneration of oocytes was observed in treatment 3 (P < 0.05) when compared with the other two groups. The proportion of activated oocytes was higher, while non-activated oocytes were lower in ionomycin group when compared with the group activated with ethanol (P < 0.05). The proportions of oocytes cleaved were 65.7, 56.8, 61.0 and 54.4% in CR-1, TCM-199, KSOM and SOF medium, respectively, with no significant difference. However, further development of cleaved oocytes was better in KSOM followed by SOF.

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Generation of parthenogenetic goat blastocysts: effects of different activation methods and culture media

Zygote ◽

10.1017/s0967199413000580 ◽

2014 ◽

Vol 23 (3) ◽

pp. 327-335 ◽

Cited By ~ 5

Author(s):

Hruda Nanda Malik ◽

Dinesh Kumar Singhal ◽

Shrabani Saugandhika ◽

Amit Dubey ◽

Ayan Mukherjee ◽

...

Keyword(s):

Culture Media ◽

Combined Treatment ◽

Blastocyst Stage ◽

Developmental Competence ◽

Cleavage Rate ◽

Cell Stage ◽

Quantitative Expression ◽

Pattern Of Variation ◽

Better Than

SummaryThe present study was carried out to investigate the effects of different activation methods and culture media on the in vitro development of parthenogenetic goat blastocysts. Calcium (Ca2+) ionophore, ethanol or a combination of the two, used as activating reagents, and embryo development medium (EDM), modified Charles Rosenkrans (mCR2a) medium and research vitro cleave (RVCL) medium were used to evaluate the developmental competence of goat blastocysts. Quantitative expression of apoptosis, stress and developmental competence-related genes were analysed in different stages of embryos. In RVCL medium, the cleavage rate of Ca2+ ionophore-treated oocytes (79.61 ± 0.86) was significantly (P < 0.05) higher than in ethanol (74.90 ± 1.51) or in the combination of both Ca2+ ionophore and ethanol. In mCR2a or EDM, hatched blastocyst production rate of Ca2+ ionophore-treated oocytes (8.33 ± 1.44) was significantly higher than in ethanol (6.46 ± 0.11) or in the combined treatment (6.70 ± 0.24). In ethanol, the cleavage, blastocyst and hatched blastocyst production rates in RVCL medium (74.90 ± 1.51, 18.30 ± 1.52 and 8.24 ± 0.15, respectively) were significantly higher than in EDM (67.81 ± 3.21, 14.59 ± 0.27 and 5.59 ± 0.42) or mCR2a medium (65.09 ± 1.57, 15.36 ± 0.52 and 6.46 ± 0.11). The expression of BAX, Oct-4 and GlUT1 transcripts increased gradually from 2-cell stage to blastocyst-stage embryos, whereas the transcript levels of Bcl-2 and MnSOD were significantly lower in blastocysts. In addition, different activation methods and culture media had little effect on the pattern of variation and relative abundance of the above genes in different stages of parthenogenetic activated goat embryos. In conclusion, Ca2+ ionophore as the activating agent, and RVCL as the culture medium are better than other tested options for development of parthenogenetic activated goat blastocysts.

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314EFFECT OF MACROMOLECULE SUPPLEMENTATION DURING IN VITRO MATURATION ON THE DEVELOPMENTAL COMPETENCE OF GOAT OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab314 ◽

2004 ◽

Vol 16 (2) ◽

pp. 276

Author(s):

J.R. Herrick ◽

E. Behboodi ◽

E. Memili ◽

S. Blash ◽

Y Echelard ◽

...

Keyword(s):

Amino Acids ◽

In Vitro Maturation ◽

Mixed Model ◽

Linear Mixed Model ◽

Blastocyst Stage ◽

Developmental Competence ◽

Similar Proportion ◽

Developmental Potential ◽

Cell Counts

In vitro maturation of goat oocytes has traditionally involved the use of serum or BSA. However, these products introduce variability and complicate evaluation of the effects of other medium components. The objective of this study was to examine the effects of citrate and hyaluronate in the absence or presence of BSA during IVM on the developmental competence of goat oocytes. Abattoir-derived, cumulus-oocyte complexes (COC) were matured for 20–22h (6.0% CO2 in air, 38.7°C) in modified SOF medium (1.5mM glucose, 3.0mM L-lactate, 0.1mM pyruvate, 1.0mM glutamine, 0.1mM taurine) supplemented with 1×MEM nonessential amino acids, 0.5×MEM essential amino acids, 1×MEM vitamins, 0.1mM cysteamine, 5μg mL−1 insulin, 5μgmL−1 transferrin, 5ng mL−1 selenium, 50ngmL−1 EGF, 0.01U mL−1 LH and FSH, and 50μgmL−1 gentamicin. Treatments were: (1) 1mgmL−1 PVA (protein-free, defined); (2) 4mgmL−1 BSA (semi-defined); (3) 0.5mM citrate and 0.5mgmL−1 hylauronate (C+H, defined); and (4) 0.5mM citrate and 0.5mgmL−1 hylauronate with 4mgmL−1 BSA (C+H+BSA, semi-defined). At the end of IVM, COC were transferred to modified Brackett and Oliphant’s medium with 7.7mM Ca-(l)-lactate and 20% FCS for IVF. Frozen-thawed sperm were processed through a 45%:90% Percoll gradient and added to IVF drops (50μL) containing COC at a final concentration of 14–15×106 spermmL−1. Gametes were coincubated in the presence of heparin (25μgmL−1) for 22–24h in 7% CO2 in air at 38.7°C. After coincubation, cumulus cells were removed and zygotes were cultured (6% CO2, 5% O2, 89% N2, 38.7°C) in G1 v.3 for 3 days followed by 4 days in G2 v.3. Cleavage was evaluated when embryos were moved to G2, and development to the blastocyst stage was assessed at the end of culture. All blastocysts were fixed and stained with Hoechst 33342 for total cell counts. Analysis of variance was performed using the general linear mixed model macro of SAS. Means are presented ±SEM and probability values P<0.05 were considered significant. The use of BSA did not improve (P>0.05) the developmental potential of goat oocytes (Table 1). Furthermore, a similar proportion (P>0.05) of oocytes developed to the blastocyst and hatching blastocyst stage after maturation under defined conditions compared to oocytes matured with BSA. In conclusion, developmentally competent goat oocytes can be produced by IVM under defined conditions. Table 1 Development of goat oocytes following IVM with different macromolecules.

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79 EFFECTS OF EPIDERMAL GROWTH FACTOR, β-MERCAPTOETHANOL, GLUCOSE, OXYGEN CONCENTRATION, AND FIBROBLAST SUBCULTURE ON THE DEVELOPMENT OF PORCINE NT EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab79 ◽

2007 ◽

Vol 19 (1) ◽

pp. 157

Author(s):

H. B. Seok ◽

J. H. Quan ◽

S. K. Kim

Keyword(s):

Growth Factor ◽

Epidermal Growth Factor ◽

In Vitro Maturation ◽

Developmental Rate ◽

Blastocyst Stage ◽

First Polar Body ◽

Fetal Fibroblasts ◽

O2 Concentration ◽

Epidermal Growth

The purpose of this study was to investigate in vitro maturation rate of oocytes cultured in maturation medium supplemented with epidermal growth factor (EGF), β-mercaptoethanol (ME), and glucose, and the further development of NT embryos under various conditions. The basic media used for oocyte maturation were NCSU-23 and PZM-3 supplemented with 0.1 mg mL-1 cysteine, 10% (v/v) porcine follicular fluid (pFF), 10 �g mL-1 FSH, 10 �g mL-1 LH, 20 ng mL-1 EGF, and 25 �M ME. Porcine ovaries were collected at a local slaughterhouse, and donor cells from a 35-day-old fetus were dissociated, resuspended, and cultured for 6–8 days in DMEM supplemented with 10% (v/v) FBS, penicillin G (75 �g mL-1), streptomycin (50 �g mL-1), 1 mM sodium pyruvate, and 1% (v/v) nonessential amino acids. The first polar body and adjacent cytoplasm were enucleated by a micropipette in HEPES-buffered NCSU-23 supplemented with 4 mg mL-1 BSA and 7.5 �g mL-1 cytochalasin B. Couplets were equilibrated with 0.3 M mannitol solution and transferred to a chamber containing 2 electrodes with a pulse of 2.1 kV cm-1 for 30 �s. When the embryos were cultured in NCSU-23 or PZM-3 supplemented with or without 20 ng mL-1 EGF for 144 h, the development rates to the blastocyst stage were 12.0 � 1.3%, 9.6 � 1.9%, 10.9 � 2.1%, and 9.1 � 2.3%, respectively. When the embryos were cultured in NCSU-23 or PZM-3 supplemented with or without 25 �M ME for 144 h, the rates to blastocyst stage were 9.6 � 1.7%, 7.3 � 2.3%, 11.9 � 1.8%, and 7.4 � 2.1%, respectively. The developmental rate to the blastocyst stage of NT embryos cultured in PZM-3 supplemented with ME was significantly higher than when cultured without ME supplementation (P < 0.05). When the embryos were cultured in NCSU-23 or PZM-3 supplemented with or without 1.5 mM glucose for 144 h, the rates to blastocyst stage were 9.4 � 2.2%, 6.8 � 2.7%, 10.9 � 2.4%, and 8.9 � 2.6%, respectively. The developmental rate to the blastocyst stage of NT embryos cultured in NCSU-23 and PZM-3 supplemented with glucose was higher than when cultured without glucose supplementation. When NT embryos were cultured in NUSU-23 and PZM-3 at 5% and 20% O2 concentration, the rates were 11.1 � 1.8%, 9.8 � 1.4%, 12.5 � 1.6%, and 10.9 � 1.5%, respectively. The developmental rate to the blastocyst stage of NT embryos cultured in both NCSU-23 and PZM-3 at 5% O2 concentration was higher than when cultured at 20% O2 concentration. When fetal fibroblasts were cultured in NCSU-23 and PZM-3, the fusion rate of less than 10 passages was higher than for those of 11–15 passages. In conclusion, the present study indicates that EGF and glucose have beneficial effects on the in vitro maturation of oocytes, and ME improves the developmental ability of NT embryos. Furthermore, the developmental rate in subcultured fibroblast cells was improved when reconstruction was made with less than 10 passages.

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In vitro production of cattle-water buffalo (Bos taurus - Bubalus bubalis) hybrid embryos

Zygote ◽

10.1017/s0967199402002216 ◽

2002 ◽

Vol 10 (2) ◽

pp. 155-162 ◽

Cited By ~ 13

Author(s):

H.P.S. Kochhar ◽

K.B.C. Appa Rao ◽

A.M. Luciano ◽

S.M. Totey ◽

F. Gandolfi ◽

...

Keyword(s):

Water Buffalo ◽

Bos Taurus ◽

Inner Cell Mass ◽

Cell Mass ◽

Bubalus Bubalis ◽

Blastocyst Stage ◽

In Vitro Production ◽

Cleavage Rate ◽

Developmental Potential

Interspecific hybrid embryos are useful models for the study of maternal-fetal interactions, transmission pattern of species-specific markers and parental contributions to growth and developmental potential of pre-attachment embryos. In an attempt to investigate the possibility of producing hybrid embryos of domestic cattle (Bos taurus) and water buffalo (Bubalus bubalis), cattle oocytes were exposed to buffalo sperm and buffalo oocytes were exposed to cattle sperm and the cleavage rate and the post-fertilisation features of hybrid embryos up to the blastocyst stage were compared with those of buffalo and cattle embryos. The cleavage rate in buffalo oocytes exposed to cattle sperm was low (40.8%), with only 8.8% of these hybrid embryos reaching the blastocyst stage. Cattle oocytes exposed to buffalo sperm showed 86.3% cleavage, while 25.9% of these attained the blastocyst stage. The speed of development of both types of hybrids was intermediate between that of cattle and buffalo embryos, with hatching occurring on day 7.5 in hybrid embryos, day 8-9 in cattle and day 7 in buffalo. The proportions of cells contributing to the trophectoderm and the inner cell mass were closer to those of the maternal species in both types of hybrid embryos. Our results indicate that cattle-water buffalo hybrid embryos produced using interspecies gametes are capable of developing to advanced blastocyst stages and that their in vitro fate, and developmental potential, are influenced by the origin of the oocyte.

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

Archives Animal Breeding ◽

10.5194/aab-45-547-2002 ◽

2002 ◽

Vol 45 (6) ◽

pp. 547-556

Author(s):

N. R. Mtango ◽

M. D. Varisanga ◽

D. Y. Juan ◽

P. Wongrisekeao ◽

T. Suzuki

Keyword(s):

In Vitro Maturation ◽

Blastocyst Stage ◽

Developmental Competence ◽

Cleavage Rate ◽

Vitro Fertilization ◽

Nuclear Maturation ◽

Significant Difference ◽

Activation Rates ◽

Oviduct Fluid

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

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RESPON PENGGUNAAN BERBAGAI BAHAN AKTIVATOR PADA AKTIVASI PARTENOGENESIS OOSIT KAMBING HASIL IVM

SAINSTIS ◽

10.18860/sains.v0i0.1860 ◽

2012 ◽

Author(s):

Kholifah Holil, Eva Ari Wahyuni, Hari Soepriandono Gatot Ciptadi

Keyword(s):

In Vitro Maturation ◽

Fetal Bovine Serum ◽

Reproductive Technologies ◽

Cleavage Rate ◽

Parthenogenetic Activation ◽

Sperm Extract ◽

Fetal Bovine ◽

Humidified Air

Parthenogenetic activation is one method that can be used to determine the quality of IVM oocytes results before further use to other reproductive technologies (IVF and transfer core). In parthenogenetic activation can be used various activators such as ethanol, Ca Ionophore, and Crude Sperm Extract (CSE). Therefore, the aim of this experiment is to know the response use a variety of materials activator of parthenogenetic activation of goat oocytes IVM. The sample used in this experiment was oocytes aspirated from goat ovarian follicles taken from RPH Sukun of Malang. Oocytes were matured for 24 hr in TCM-199 supplemented with fetal bovine serum (FBS), follicle-stimulating hormone (FSH) and lutheinizing hormone (LH) at a temperature of 38,5oC and 5% CO2 in humidified air. After another 30 hours of in vitro maturation, they were then activated by various treatments. The treatment of experiment are treatment 1, activation using ethanol 7% for 7 minute, treament 2, activation using Ca Ionophore 20 µM for 7 minute. Treatment 3, activation using CSE 2,5 µg/ml for 2 hr. Based on the result of research, it is showed that activation by using 7% ethanol for 7 minutes is able to produce cleavage rate of 70.40%. Activation by Ca Ionophore 20 μM for 7 minutes is able to produce cleavage rate of 52.75%. While the use of CSE activation with 2.5 ug / ml for 2 hours produces cleavage rate of 36.33%. Thus it can be concluded that the goat oocyte IVM able to respond to a variety of materials activators on parthenogenetic activation performed. The highest response given by the successive results goat IVM oocytes activated using 7% ethanol for 7 minutes, 20 μM Ca Ionophore for 7 minutes, and the CSE 2.5 microg / ml for 2 hours. Keywords: Parthenogenetic activation, goat oocytes IVM, etanol, calsium ionophore, Crude Sperm Extract (CSE).

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