scholarly journals Effect of resveratrol on the in vitro maturation of ovine (Ovis aries) oocytes and the subsequent development of handmade cloned embryos

2019 ◽  
Vol 5 (4) ◽  
Author(s):  
José Luis Martínez-Ibarra ◽  
Eugenia Adriana Espinoza-Mendoza ◽  
Raymundo Rangel-Santos ◽  
Demetrio Alonso Ambriz-García ◽  
María Del Carmen Navarro-Maldonado
Keyword(s):  
Embryo Quality ◽  
Developmental Stages ◽  
Subsequent Development ◽  
Poor Quality ◽  
Ovis Aries ◽  
Control Group ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Maturation Rate

The effect of resveratrol on the in vitro maturation (IVM) of ovine (Ovis aries) oocytes and the development of handmade cloned embryos was evaluated. The nuclear maturation and reactive oxygen species (ROS) levels in the oocytes, as well as the early development and morphological cloned embryo quality, were evaluated under different resveratrol concentrations (0, 0.5, 2 and 5 μM). After IVM, no significant difference was observed in the maturation rate of oocytes treated with 0.5 μM (81.3 %) and 2 μM (72 %) resveratrol compared to that of the control group (0 μM) (74.2 %), but the rate significantly decreased at 5 μM (56 %) (p < 0.05). When the oocyte ROS levels were determined, no significant differences among the groups were observed (p > 0.05). For cloned embryo development, the embryos obtained from the oocytes treated with 0.5 μM resveratrol showed higher (p < 0.05) compacted morula rates (10.7 %) compared to the embryos obtained from the oocytes treated with 0, 2 and 5 μM (6.2, 0 and 0 %, respectively). Regarding embryo morphological quality, the embryos from the oocytes treated with 0.5 μM resveratrol showed a lower rate of poor quality morulae (4.7 %) in comparison to those treated with 0, 2 and 5 μM (23.8, 23.3 and 33.3 %, respectively) (p < 0.05). In conclusion, resveratrol showed no significant improvement on the IVM or ROS levels in domestic ovine oocytes. However, treatment with 0.5 μM resveratrol during IVM improved embryo quality and promoted morulae compaction of Ovis aries handmade cloned embryos.Figure 3. Different developmental stages of the HMC sheep embryos cultured in the WOW system. Cleaved embryos (a-d), 8‒16 blastomere embryos (e-h), morulae (i-l) and compact morulae (m-p) (200X).

41 DEVELOPMENTAL POTENTIAL OF BUFFALO OOCYTES VITRIFIED AT THE GERMINAL VESICLE STAGE: EFFECTS OF DIFFERENT CRYOPROTECTANT COMBINATIONS AND CRYODEVICES

2016 ◽  
Vol 28 (2) ◽  
pp. 150
Author(s):  
A. S. El-Shalofy ◽  
A. R. Moawad ◽  
G. M. Darwish ◽  
S. T. Ismail ◽  
A. B. Badawy
Keyword(s):  
Solid Surface ◽  
Germinal Vesicle ◽  
Subsequent Development ◽  
The Other ◽  
Control Group ◽  
Developmental Potential ◽  
High Frequencies ◽  
Nuclear Maturation ◽  
Maturation Rate

The cryopreservation of immature oocytes would generate a readily available, nonseasonal source of female gametes for both research and reproduction. In domestic animals, the most promising results in the field of oocyte cryopreservation have been reported in cattle, and a few experiments have been conducted on buffalo. The aim of the present study was to compare the effects of different cryoprotectant combinations and different cryodevices on viability and subsequent development of buffalo oocytes vitrified at the germinal vesicle stage. Cumulus-oocyte complexes obtained at slaughter from mature buffalos were vitrified by using either straw or open pulled-straw or solid surface vitrification (SSV) in a solution composed of either 20% ethylene glycol (EG) + 20% glycerol or 20% EG + 20% dimethylsulfoxide (DMSO). Following vitrification and warming, viable oocytes were matured in vitro for 22 h. Matured oocytes were either evaluated for nuclear maturation or fertilized and cultured in vitro for 7 days. Recovery rate was significantly higher (P < 0.05) in the oocytes vitrified by straw in 20% EG + 20% glycerol (92.6%) as compared with the other groups. Percentages of viable oocytes were significantly higher (P < 0.05) in the oocytes vitrified in 20% EG + 20% DMSO using SSV (95.7%) than those in the other groups (from 80 to 88.0%). Among the vitrified groups, the highest maturation rate was achieved in SSV with 20% EG + 20% DMSO group (56.7%). This value was comparable with those in the control group (62.1%). After IVF and embryo culture, the highest cleavage and blastocyst rates were obtained in SSV with 20% EG + 20% DMSO group (35.7 and 21.4%, respectively), and these values were nearly similar to those in the control group (38.7 and 25.8%, respectively). Vitrification of oocytes by straw or open pulled-straw resulted in significantly lower (P < 0.05) blastocyst rates (2.6 and 11.5%) as compared with the control. In conclusion, buffalo oocytes vitrified at the germinal vesicle stage can be matured, fertilized, and developed in vitro and produce high frequencies of blastocyst embryos. Solid surface vitrification may be superior to straw and open pulled-straw in vitrification of immature buffalo oocytes because this technique results in higher survival and embryo development rates.

scholarly journals Use of Melatonin in the In Vitro Production of Bovine Embryos

2020 ◽  
Vol 21 ◽  
Author(s):  
Alan da Silva LIRA ◽  
Ricardo de Macedo CHAVES ◽  
Felipe de Jesus MORAES JUNIOR ◽  
Sergio Henrique COSTA JUNIOR ◽  
Brenda Karine Lima do AMARAL ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Control Group ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Maturation Medium ◽  
Significant Difference ◽  
Maturation Rate ◽  
Vitro Production

ABSTRACT We aimed to assess the effects of melatonin in the in vitro production of bovine embryos. Our experiment was conducted at the Laboratório de Reprodução Animal of the Universidade Estadual do Maranhão. The cumulus-oocyte complexes (COCs) were distributed among treatments at concentrations of 0, 10-1, 10-3 and 10-5 µMol/L melatonin. Our experiment was further divided into two: the first was to assess the effect of different concentrations of melatonin (treatments) on the maturation rate of COCs, and the second was to assess the effects of melatonin treatments on the in vitro production of bovine embryos. The results from the first experiment demonstrated no significant difference between the in vitro maturation rate of the cultivated COCs in treatments with melatonin. In the second experiment, however, melatonin treatments yielded statistically higher cleavage, morula and blastocyst rates in the 10-5 µM group (52.9%, 52.9%, and 35.3%, respectively), and lower rates in the 10-1 µM group (19.5%, 19.5% and 7.8%, respectively), compared to the others. The control group (no melatonin) and the 10-3 µM group showed similar results. We concluded that supplementation of melatonin in the in vitro maturation medium resulted in no improvement in the oocyte maturation rate, but in the in vitro production of embryos at different concentrations, the 10-5 µM group displayed better results, but with no improvement in the variables (P < 0.05).

Vitrification of in vitro matured oocytes of Mangalica and Large White pigs

2008 ◽  
Vol 56 (3) ◽  
pp. 399-410 ◽  
Author(s):  
Erika Varga ◽  
Erzsébet Gajdócsi ◽  
Brigitta Petz Makkosné ◽  
Ildikó Salamon ◽  
Ágnes Bali Papp
Keyword(s):  
Embryonic Development ◽  
Developmental Competence ◽  
Large White ◽  
Normal Morphology ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Maturation Rate ◽  
Morula Stage ◽  
Potential Tool

The breeding of Mangalica, a native pig breed in Hungary, had been started in 1833, but this pig breed almost became extinct in Hungary in the past decades. In 1991, the number of sows was only 200. Although in these days the existing Mangalica population consists of more than 6000 animals representing different colour variations, the preservation of this traditional pig breed is still very important. Vitrification is a potential tool for the preservation of gametes and embryos of these animals. The aim of this study was to investigate the effects of vitrification on the developmental competence of Mangalica (M) and Large White (LW) oocytes following fertilisation. The oocytes were vitrified by the Open Pulled Straw (OPS) method using different concentrations of ethylene glycol and dimethyl sulphoxide as cryoprotectants. After rehydration the oocytes underwent in vitro fertilisation; the resultant zygotes were then cultured in vitro for four days to assess embryonic development. In the first experiment, in vitro maturation of M and LW oocytes was compared. No significant difference was observed in the nuclear maturation rate of LW and M oocytes. In the second experiment, the sensitivity of oocytes to vitrification was examined by evaluating oocyte morphology after thawing. A higher percentage of LW oocytes showed normal morphology compared to M oocytes, indicating that Mangalica oocytes are more sensitive to cryoprotectants than Large White oocytes. After warming and in vitro fertilisation, more than 50% of the oocytes started embryonic development and by the end of the incubation period morula stage embryos had developed in both groups. The results show that the OPS vitrification technique is well suited to preserve Mangalica oocytes and from these oocytes morula embryos can be produced.

Selection of Rattus norvegicus oocytes for in vitro maturation by brilliant cresyl blue staining

Zygote ◽  
2011 ◽  
Vol 21 (3) ◽  
pp. 238-245 ◽  
Author(s):  
Diego Duarte Alcoba ◽  
Bianca Letícia da Rosa Braga ◽  
Nathallie Louise Sandi-Monroy ◽  
Letícia Auler Proença ◽  
Rui Fernando Felix Lopes ◽  
...  
Keyword(s):  
Rattus Norvegicus ◽  
Blue Staining ◽  
Control Group ◽  
Brilliant Cresyl Blue ◽  
Cresyl Blue ◽  
Nuclear Maturation ◽  
Effective Evaluation ◽  
Maturation Rate ◽  
Selection Of

SummaryThe objective of this work was to evaluate the rate of meiosis resumption and nuclear maturation of rat (Rattus norvegicus) oocytes selected for in vitro maturation (IVM) after staining of cumulus–oocyte complexes (COCs) with blue cresyl brilliant (BCB) using different protocols: exposure for 30, 60 or 90 min at 26 μM BCB (Experiment 1), and exposure for 60 min at 13, 20 or 26 μM BCB (Experiment 2). In Experiment 1, the selection of oocytes exposed to BCB for 60 min was found to be the most suitable, as meiosis resumption rates in the BCB+ group (n = 35/61; 57.37%) were the closest to the observed in the control (not exposed) group (n = 70/90; 77.77%) and statistically higher than the values observed for the BCB− group (n = 3/41; 7.32%). Additionally, the more effective evaluation of diagnostic tests (sensitivity and negative predictive value 100%) was observed in COCs exposed for 60 min. In Experiment 2, the 13 μM BCB+ group presented rates of meiosis resumption (n = 57/72; 72.22%) similar to the control group (n = 87/105; 82.86%) and higher than other concentration groups. However, this results of the analysis between BCB− oocytes was also higher in the 13 μM BCB group (n = 28/91; 30.78%) when compared with BCB− COCs exposed to 20 μM (n = 3/62; 4.84%) or 26 μM (n = 3/61; 4.92%) BCB. The nuclear maturation rate in the 13 μM BCB group was similar between BCB+ or BCB− oocytes. The 20 μM BCB group had a lower rate of nuclear maturation of BCB− oocytes than other groups. Thus, our best results in the selection of Rattus norvegicus oocytes by staining with BCB were obtained using the concentration of 13 μM and 20 μM, and an incubation period of 60 min.

scholarly journals Expression of miR-302 in human embryo derived from in-vitro matured oocyte

2019 ◽  
Author(s):  
Parvin Dorfeshan ◽  
Marefat Ghaffari Novin ◽  
Mohammad Salehi ◽  
Fatane Farifteh
Keyword(s):  
Human Embryo ◽  
Embryo Quality ◽  
Molecular Study ◽  
Quality Score ◽  
Control Group ◽  
Polar Bodies ◽  
Significant Difference ◽  
Negative Effect

Background: The expression of miR-302 over the period of early embryogenesis could possibly regulate the maternal transcript clearance. Zygotic transcription activation is mostly related to maternal messages degradation. Objective: In this study, the effects of in-vitro maturation technique (IVM) on the expression of miR-302 in human embryo produced from immature and mature human oocytes (matured in vitro and in vivo, before sperm exposure) obtained from females under gonadotrophin therapy were evaluated for assisted reproduction. Materials and Methods: Immature oocytes were cultured in vitro. The injection of oocytes-producing polar bodies was given using fresh sperm. Then, the embryo quality score was assessed in the IVM group compared with the control group. In both the groups, embryos with normal morphology were included in the molecular study. Only one blastomere was removed from three-day embryos and then the embryos were frozen. The expression of miR-302 in embryos was measured through quantitative realtime polymerase chain reaction. Results: Our data showed a significant reduction of miR-302 expression in the IVM group vs. the control group (p = 0.02). The embryo quality score showed a significant difference between the two groups (p = 0.01). Conclusion: The present study demonstrated that the IVM process had a negative effect on the expression level of miR-302 in human pre-implantation embryos. Considering the major role of expression miR-302, a reduced potential in miR-302 expression could be related to a decrease in the early embryonic development.

scholarly journals The effect of follicle size and cryoprotectants on nuclear maturation and early embryonic development of vitrified - thawed Awassi sheep oocytes (Ovis aries)

2021 ◽  
Vol 91 (5) ◽  
pp. 483-493
Author(s):  
Omar Mardenli ◽  
◽  
Mahdi S. Mohammad Al-Kerwi ◽  
Ahmad Y. Alolo
Keyword(s):  
Embryo Quality ◽  
Germinal Vesicle ◽  
Ovis Aries ◽  
Control Group ◽  
Three Solutions ◽  
Germinal Vesicle Breakdown ◽  
Nuclear Maturation ◽  
Awassi Sheep ◽  
Morula Stage ◽  
Follicle Size

In this study, two experiments were conducted to study the effect of both the follicle size and the cryoprotectants dimethyl sulfoxide (DMSO) and ethylene glycol (EG) on the main phases of nuclear maturation (Experiment I), cleavage stages and embryo quality (Experiment II) of Awassi sheep oocytes. Follicles were classified into two groups: small follicles (SF) (1-2 mm) and large follicles (LF) (> 2 mm). Oocytes were vitrified in three solutions: A (30% DMSO), B (30% EG) and C (15% DMSO and 15% EG). In Experiment I, the resulting vitrified-thawed oocytes in solution C achieved the best rates after the control group (fresh), respectively as the rates of maturation, germinal vesicle (GV), metaphase II(M-II), arrest, and lyses were 85.71% (P = 0.04), 8.33% (P = 0.02), 72.92% (P = 0.04); LF group, 15.25% (P = 0.04), and 5.08% (P = 0.04); SF group, respectively. In Experiment II, the same group of oocytes achieved the best rates after the control group, as the rates of fertilization, cleavage, 2-16 cell, Type3, blastocyst, and Type1 embryos were 63.28% (P = 0.001), 57.46% (P = 0.001), 40.38% (P = 0.04), 38.46% (P = 0.04); LF group, 30.00% (P = 0.01), and SF group 36.67% (P = 0.001), respectively, while the vitrified-thawed oocytes in A solution (SF group) reached the highest rate of Type 2 embryo quality (58.06%; P = 0.01). No significant differences were noticed in the germinal vesicle breakdown (GVBD), metaphase I (M-I) and morula stage. Vitrification of oocytes obtained from follicles with a diameter of more than 2 mm in a cocktail solution of DMSO (15%) and EG (15%) led to a significant increase in the yield and quality of the resulting sheep embryos.

scholarly journals Impairment on nuclear maturation rate in oocytes from cows naturally infected by bovine herpesvirus 1 (BoHV-1)

2018 ◽  
Vol 38 (12) ◽  
pp. 2207-2212
Author(s):  
Vívian R.A. Mendes ◽  
Eduardo P. Costa ◽  
Vanessa L.D. Queiroz ◽  
Abelardo Silva Júnior ◽  
Saullo V.P. Alves ◽  
...  
Keyword(s):  
Bovine Herpesvirus ◽  
Cumulus Cells ◽  
Control Group ◽  
Viral Dna ◽  
Bovine Herpesvirus 1 ◽  
Nuclear Maturation ◽  
Maturation Rate ◽  
Developmental Capacity ◽  
Herpesvirus 1

ABSTRACT Bovine herpesvirus 1 (BoHV-1) is an important bovine pathogen that is responsible for causing respiratory diseases and reproductive failures. The presence of BoHV-1 in an in vitro embryo production system affects fertilization, maturation, and embryonic development. The objective of this study was to evaluate the developmental capacity of oocytes from naturally infected cows with no reproductive history. Moreover, this study investigated the presence of viral DNA in cumulus oophorus complexes (COCs). Experimental groups were differentiated by titrating the antibodies detected through seroneutralization assays, establishing three groups: seronegative animals (titer lower than 2), low titer (2 to 8), and animals with a titer above or equal to 16. COCs were obtained from 15 donors during 22 sessions of ultrasound-guided follicular aspiration. DNA was extracted from a pool of COCs obtained from all aspirations from the same donor as well as from whole blood and nested PCR reactions were performed. Only COCs with a compact layer of cumulus cells, an intact zona pellucida, and homogeneous cytoplasm were selected for in vitro culture and evaluation of nuclear maturation rate. After culturing for 24 hours, the oocytes were fixed and stained to evaluate the meiotic cell cycle stage. Oocytes that showed a chromosomal configuration in metaphase II were considered to have reached nuclear maturation. Compared with the other groups, the oocyte nuclear maturation rate in animals with a titer greater than or equal to 16 (50%) was compromised (P< 0.05). However, the viral titer did not influence the maturation rate of bovine oocytes in animals exhibiting low titration (62.2%) when compared with the control group (76.7%). Viral DNA was not observed in the blood samples but was detected in the COC pool from three seropositive donors. In view of the results obtained, we conclude that natural infections by the BoHV-1 virus can compromise the nuclear maturation rate in cows, depending on the titration levels of antibodies against the virus. Moreover, viral DNA could be present in COCs, contradicting the hypothesis that seropositive animals with no history of clinical symptomatology pose a negligible risk of transmitting BoHV-1 by COCs.

165 In vitro maturation of ovine and caprine oocytes during breeding and nonbreeding seasons

2019 ◽  
Vol 31 (1) ◽  
pp. 207
Author(s):  
M. Markle ◽  
C. K. Mak ◽  
V. Medina ◽  
C. R. F. Pinto
Keyword(s):  
Breeding Season ◽  
In Vitro Maturation ◽  
Statistical Significance ◽  
Polar Body ◽  
Cumulus Cells ◽  
Nonbreeding Season ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Maturation Rate

The current study investigated the in vitro meiotic competence of ovine and caprine oocytes that underwent nuclear maturation during the breeding and nonbreeding seasons. We hypothesised that maturation rates of ovine and caprine oocyte would be significantly lower during the nonbreeding season. Ovine (Katahdin crossbred) and caprine (mainly Spanish crossbred) ovaries were collected from a local abattoir in the southern United States. Age of the animals was not determined. Cumulus-oocyte complexes (COC) were harvested by slicing the ovaries and searching using a stereomicroscope. Oocytes with more than 3 layers of unexpanded cumulus cells and with evenly granulated cytoplasm were selected for in vitro maturation (IVM). A commercial bovine IVM media (IVF Bioscience, Falmouth, United Kingdom) was used throughout the study. After 24h of IVM, ovine and caprine oocytes were denuded and oocytes with an extruded polar body (meiotic metaphase II oocytes) were considered to have reached nuclear maturation. The seasons in this study were defined as follows: breeding season=September to April and nonbreeding season=May to July. The presence of corpus hemorrhagicum or corpus luteum in at least 70% of the ovaries indicated the breeding season for the animals. Proportions of oocytes undergoing nuclear maturation were analysed using a two-tailed Chi-squared test. Statistical significance was set at P ≤ 0.05. The ovine maturation rate was 59% (65/111) and 49% (254/519) and the caprine maturation rate was 70% (39/56) and 40% (64/162) during the breeding and nonbreeding seasons, respectively. These results show a significant difference in nuclear maturation for caprine oocytes (P&lt;0.001) during the breeding and nonbreeding seasons; however, there was no significant difference in nuclear maturation for ovine oocytes (P=0.06) during the breeding and nonbreeding seasons. High environmental temperatures during the nonbreeding season may have had detrimental effects on oocyte nuclear maturation in caprine but not in ovine oocytes. Why oocytes from these 2 species differ on how they are adversely affected by season remains to be elucidated.

scholarly journals The influence of ascorbic acid on in vitro maturation of canine oocytes

2016 ◽  
Vol 73 (2) ◽  
pp. 230
Author(s):  
Anamaria Jeni Pernes ◽  
Ileana Miclea ◽  
Marius Zahan ◽  
Vasile Miclea ◽  
Delia Orlovschi ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Meiotic Maturation ◽  
Control Group ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Oocyte Meiotic Maturation ◽  
Metaphase I

Abstract It is known that L-ascorbic acid (vitamin C) can modulate many biochemical processes intracellularly or extracellularly as antioxidant. The aim of the present study was to examine the effects of media supplementation with ascorbic acid on canine oocyte meiotic maturation, viability and the cumulus cell expansion. Various concentrations of ascorbic acid supplemented in in vitro maturation (IVM) media were tested. Canine oocyte was exposed to different levels of ascorbic acid (0, 50, 150, 250, 500, 750µM). Cumulus expansion, meiotic maturation and degeneration of oocytes were assessed 72 h after in vitro culture. As results, on the group treated with 250µM ascorbic acid was a significant difference compared to the control group on nuclear maturation in stages metaphase I (MI) and metaphase II (MII) (26.98% vs. 6.00%). The groups treated with 50, 150, 250, 500µM had an increase in stage (GVBD), and a significant decrease of degenerate-undefined oocytes compared with the control (23.31%, 18.85%, 13.41% vs 40.80). Concentration 750µM had similar effect to that in the control group. The groups treated with 50, 150, 250, 500µM had an increase in meiosis resumption(GVBD), metaphase I (MI) and metaphase II (MII) with the best result in the group treated with 250 µM ascorbic acid.

Blastocyst production by in vitro maturation and development of porcine oocytes in defined media following intracytoplasmic sperm injection

Zygote ◽  
2007 ◽  
Vol 15 (2) ◽  
pp. 93-102 ◽  
Author(s):  
M. Kobayashi ◽  
S. Asakuma ◽  
Y. Fukui
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Subsequent Development ◽  
Control Group ◽  
Control Medium ◽  
Blastocyst Formation ◽  
Cell Numbers ◽  
Defined Media ◽  
Significant Difference ◽  
The Mean

SummaryThe present study was carried out to establish porcine defined IVP. In Experiments 1 and 2, we investigated the efficacy of additional 0.6 mM cystine and/or 100 µM cysteamine (Cys) to a defined TCM199 maturation medium with regard to the intracellular glutathione (GSH) concentration and the developmental competence of in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The control medium was a modified TCM199 containing 0.05% (w/v) polyvinyl alcohol (PVA). Cys and/or cystine were added to the control medium. The control group and immature oocytes (presumptive germinal vesicle oocytes; GV) were prepared for GSH assay. In Experiment 3, the efficacy of epidermal growth factor (EGF) addition to a modified porcine zygote medium (mPZM) for in vitro culture (IVC) medium was investigated on embryonic development and the mean cell number of blastocysts following ICSI. As a positive or negative control, 0.3% BSA (mPZM-3) or 0.3% PVA (mPZM-4), respectively, was added to the base medium. The defined IVC medium was supplemented with 5 or 10 ng/ml EGF. In Experiment 1, no significant difference was found in the rates of cleavage (31.4–64.3%) and blastocyst formation (6.5–22.9%) among the treatment and control groups. The mean cell numbers per blastocyst ranged from 30 to 48 among the groups without significant differences. However, in Experiment 2, the intracellular GSH concentrations in the oocytes cultured in the medium supplemented with 100 µM Cys (9.6 pmol/oocyte) or Cys + cystine (9.9 pmol/oocyte) were significantly (p < 0.05) higher than the control (2.5 pmol/oocyte) and 0.6 mM cystine (6.5 pmol/oocyte) groups, but not different from the GV group (9.0 pmol/oocyte). The GSH concentration in the cystine group was also significantly (p < 0.05) higher than that in the control group, but not different from the GV group. In Experiment 3, the rates of cleavage and blastocyst formation and the mean cell numbers of blastocysts were not significantly different among the groups. However, the addition of 5 ng/ml EGF into the mPZM-4 resulted in a significantly (p < 0.05) higher blastocyst rate per cleaved embryo than the other two defined groups (mPZM-4 + 5 ng/ml: 48.6%, mPZM-4 and mPZM-4 +10 ng/ml: 23.4% and 23.1%, respectively).The present results indicate that the addition of Cys to a defined medium for in vitro maturation (IVM) of porcine oocytes increases intracellular GSH concentration. Further addition of cystine into the IVM medium containing 100 µM Cys is not necessary and TCM199 plus Cys (100 µM) could be used as a defined IVM medium for porcine oocytes. The addition of 5 ng/ml EGF to a defined IVC medium has enhanced subsequent development after ICSI. This study shows that porcine blastocysts can be produced by defined media throughout the steps of IVP (IVM, ICSI and IVC).

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