Grape Seed Proanthocyanidin Ameliorates FB1-Induced Meiotic Defects in Porcine Oocytes

Fumonisin B1 (FB1), as the most prevalent and toxic fumonisin, poses a health threat to humans and animals. The cytotoxicity of FB1 is closely related to oxidative stress and apoptosis. The purpose of this study is to explore whether Grape seed proanthocyanidin (GSP), a natural antioxidant, could alleviate the meiotic maturation defects of oocytes caused by FB1 exposure. Porcine cumulus oocyte complexes (COCs) were treated with 30 μM FB1 alone or cotreated with 100, 200 and 300 μM GSP during in vitro maturation for 44 h. The results show that 200 μM GSP cotreatment observably ameliorated the toxic effects of FB1 exposure, showing to be promoting first polar body extrusion and improving the subsequent cleavage rate and blastocyst development rate. Moreover, 200 μM GSP cotreatment restored cell cycle progression, reduced the proportion of aberrant spindles, improved actin distribution and protected mitochondrial function in FB1-exposed oocytes. Furthermore, reactive oxygen species (ROS) generation was significantly decreased and the mRNA levels of CAT, SOD2 and GSH-PX were obviously increased in the 200 μM GSP cotreatment group. Notably, the incidence of early apoptosis and autophagy level were also significantly decreased after GSP cotreatment and the mRNA expression levels of BAX, CASPASE3, LC3 and ATG5 were markedly decreased, whereas BCL2 and mTOR were observably increased in the oocytes after GSP cotreatment. Together, these results indicate that GSP could exert significant preventive effects on FB1-induced oocyte defects by ameliorating oxidative stress through repairing mitochondrial dysfunction.

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359 ICSI AND ACTIVATION OF OOCYTES RECOVERED FROM TRANSITIONAL AND CYCLING MARES

Reproduction Fertility and Development ◽

10.1071/rdv18n2ab359 ◽

2006 ◽

Vol 18 (2) ◽

pp. 286 ◽

Cited By ~ 3

Author(s):

T. Suh ◽

S. Purcell ◽

G. Seidel Jr

Keyword(s):

Growth Factor ◽

Polar Body ◽

Follicular Development ◽

Transitional Period ◽

Developmental Competence ◽

Blastocyst Development ◽

Cleavage Rate ◽

First Polar Body

Ovarian follicular development in mares during the transitional period before the breeding season leads to an accumulation of antral follicles of various sizes. The quality of oocytes at this stage may be compromized until the first seasonal ovulation. In this study, we evaluated the developmental competence of oocytes recovered from transitional and cyclic mares, and the effect of zygote activation after intracytoplasmic sperm injection (ICSI). A 2 × 2 × 2 factorial experiment consisting of oocytes from transitional and cyclic mares, two follicle sizes (10 to 20 and 20+ mm), and two treatments (control and activated) was conducted. Follicular oocytes of 14 mares were aspirated in March and April (transitional) and May to July (cyclic) five times per each period at 10-day intervals, without use of hCG. Oocytes aspirated from mares were matured in vitro in a defined medium similar to SOF plus FSH, LH, epidermal growth factor (EGF), insulin-like growth factor (IGF), estradiol (E2), prostaglandin (P4) and 10% FCS, for 30 ± 1 h under 5% CO2 in air at 38.5°C; oocytes with a first polar body were used for ICSI. Motile sperm from frozen-thawed semen were used for sperm injection with a piezo-driven pipet. For activation after ICSI, presumptive zygotes were cultured in G1.3 containing 0.02 µM phorbol 12-myristate 13-acetate (PMA) for 2 h, and then in 2 mM 6-dimethylaminopurine (6-DMAP) for 3 h under 6% CO2 in air at 38.5°C. Zygotes were cultured in 50 µL drops of DMEM/F12 containing 10% FCS for 9 days at 38.5°C in 5% CO2/5% O2/90% N2. Medium was replaced every 3 days. Cleavage and blastocyst rates were calculated based on non-degenerating injected oocytes. Data were analyzed by Fisher's exact test. A total of 115 and 78 oocytes were recovered from cyclic and transitional mares. Average maturation rates to MII in the respective groups were 76.5 and 65.4%, respectively (P < 0.07), and those of 10 to 20 and 20+ mm follicle groups were 70.6 and 80.0%, respectively (P > 0.05). The average cleavage rate in cyclic mares was higher than in transitional mares, and that of the activated group averaged over follicle sizes was higher than that of controls (P < 0.05; Table 1); those of 10 to 20 and 20+ mm follicle groups were not different (P < 0.05; Table 1). Blastocyst rates per oocyte within main effects were not different (P < 0.05; Table 1). Oocytes from transitional mares had lower cleavage rates than those of cyclic mares, but blastocyst development was similar. Activation of zygotes clearly improved cleavage rates of in vivo-derived immature equine oocytes after ICSI. Table 1. Main effect means of responses after ICSI

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Antioxidant β-cryptoxanthin enhances porcine oocyte maturation and subsequent embryo development in vitro

Reproduction Fertility and Development ◽

10.1071/rd17444 ◽

2018 ◽

Vol 30 (9) ◽

pp. 1204 ◽

Cited By ~ 4

Author(s):

Yun-Gwi Park ◽

Seung-Eun Lee ◽

Yeo-Jin Son ◽

Sang-Gi Jeong ◽

Min-Young Shin ◽

...

Keyword(s):

Oxidative Stress ◽

Polar Body ◽

Formation Rate ◽

Cumulus Cells ◽

Control Group ◽

Mrna Levels ◽

Cleavage Rate ◽

Antioxidant Genes ◽

Developmental Potential

Oxidative stress is partly responsible for the poor quality of IVM oocytes. The present study investigated the effects of the antioxidant β-cryptoxanthin on the IVM of porcine oocytes and the in vitro development of the ensuing embryos. Oocytes were matured in IVM medium containing different concentrations of β-cryptoxanthin (0, 0.1, 1, 10 or 100 μM). Treatment with 1 µM β-cryptoxanthin (Group 1B) improved polar body extrusion and the expression of maturation-related genes in cumulus cells and oocytes compared with control. In addition, levels of reactive oxygen species decreased significantly in Group 1B, whereas there were significant increases in glutathione levels and expression of the antioxidant genes superoxide dismutase 1 and peroxiredoxin 5 in this group. After parthenogenetic activation, although the cleavage rate did not differ between the control and 1B groups, the blastocyst formation rate was higher in the latter. Moreover, the total number of cells per blastocyst and relative mRNA levels of pluripotency marker and antioxidant genes were significantly higher in the 1B compared with control group. These results demonstrate that β-cryptoxanthin decreases oxidative stress in porcine oocytes and improves their quality and developmental potential.

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In vitro production of bovine blastocyst with oocyte collected from abattoir ovary

Bangladesh Journal of Animal Science ◽

10.3329/bjas.v45i1.27485 ◽

2016 ◽

Vol 45 (1) ◽

pp. 31-35

Author(s):

GK Deb ◽

SR Dey ◽

TN Nahar ◽

MYA Khan ◽

MM Rahman

Keyword(s):

Polar Body ◽

Cumulus Cell ◽

Morphological Characteristics ◽

Bovine Embryo ◽

In Vitro Production ◽

Blastocyst Development ◽

Cleavage Rate ◽

Development Rates ◽

First Polar Body

This study was designed to adopt in vitro embryo production (IVP) protocol using abattoir ovary. Ovaries were collected from local abattoir; cumulus-oocyte-complexes (COCs) were aspirated from 3 to 8 mm diameter follicles using a 10 ml disposable syringe attached with a 21G needle. The COCs were selected based on morphological characteristics and selected COCs were transferred into in vitro maturation (IVM) medium for 22 to 24 hrs. The matured COC were fertilized in vitro (IVF) using fresh semen capacitated through incubation with heparin sodium salt (20 ?g/ml). After IVF, the presumed denuded zygotes were cultured in in vitro culture medium-I (IVC-I) for 3 days. After 3 days, the ?8 cell embryos were transferred into IVC-II medium until day 8 of embryonic development (day 0: day of IVF). Cleavage and blastocyst development rates were evaluated at day 3 and day 7. The maturation rates of COC were examined through detection of first polar body and cumulus cell expansion. Results showed that 74.16 ± 5.49% of the total immature COCs were matured as detected by the presence of first polar body. The diameter of matured COC was 2.21 times higher than that of the immature COC. Moreover, about 64.30 ± 6.71% COC showed full expansion of their cumulus cell. The cleavage rate of presumed zygotes was 62.05 ± 7.07%. Among the cleaved embryos, 26.67 ± 11.78%, 10.84 ± 6.13%, 22.51 ± 9.57% and 39.98 ± 5.25% embryos were at 2-, 4-, 8- and 16 to 32-cell embryonic stages, respectively at day 3. Blastocyst development rates were 14.95 ± 4.39%. This study inferred that the BLRI adopted culture system supports development of bovine embryo in vitro.Bang. J. Anim. Sci. 2016. 45 (1): 31-35

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44 PRODUCTION OF LIVE PIGLETS AFTER CRYOPRESERVATION OF IMMATURE PORCINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab44 ◽

2014 ◽

Vol 26 (1) ◽

pp. 136

Author(s):

T. Somfai ◽

K. Kikuchi ◽

K. Yoshioka ◽

F. Tanihara ◽

H. Kaneko ◽

...

Keyword(s):

Polar Body ◽

Petri Dish ◽

Developmental Competence ◽

Basic Medium ◽

High Survival ◽

Blastocyst Development ◽

Nuclear Maturation ◽

First Polar Body ◽

Vitrification Solution

Development to term of vitrified porcine follicular oocytes is reported in the present study. Immature cumulus-oocyte complexes (COC) were collected from slaughtered prepubertal gilts and were vitrified according to our method published recently (Somfai et al. 2013 J. Reprod. Dev., in press). Briefly, after pretreatment with 7.5 μg mL–1 of cytochalasin B (CB) for 30 min in modified NCSU-37 (a basic medium, BM) at 38.5°C, groups of 88 to 121 COC were equilibrated in a mixture of 2% ethylene glycol (EG), 2% propylene glycol (PG), and 7.5 μg mL–1 CB for 13 to 15 min. Then, COC were washed in vitrification solution (17.5% EG, 17.5% PG, 5% polyvinyl pyrrolidone, and 0.3 M trehalose in BM) and then dropped with 2 μL of vitrification solution onto the surface of aluminum foil floating on liquid nitrogen (LN2). Microdroplets (each containing 10–25 COC) were transferred into cryotubes. After storage in LN2 for 2 to 4 weeks, the oocytes were warmed by dropping the microdroplets directly into 2.5 mL of warming solution (0.4 M trehalose in BM) kept in a 35-mm Petri dish on a 42°C hotplate for less than 1 min. Then, the warming dish was placed on a 38°C hotplate and COC were consecutively transferred for 1-min periods into BM containing 0.2, 0.1, or 0.05 M trehalose at 38°C. The COC were matured in vitro for 44 h using porcine oocyte medium (POM) supplemented with 10% follicular fluid (Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213). Then, oocytes were denuded, and their live/dead status and nuclear maturation were determined by their morphology and the presence of the first polar body, respectively. To assess their developmental competence, vitrified and non-vitrified (control) oocytes were in vitro fertilized (IVF; Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041) and then in vitro cultured in porcine zygote medium-5 (PZM-5; Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213). Blastocyst rates were recorded on Days 5, 6, and 7 of culture (Day 0 = the day of IVF). The experiment was replicated 4 times. Data were analysed with 1-way ANOVA and the Tukey test. The results revealed that 86.4% (364/424) of oocytes survived after vitrification, which was significantly lower (P < 0.05) than that of controls [100% (326/326)]. Live oocytes in vitrified and control groups did not differ statistically in terms of nuclear maturation (63.9 v. 65.3%). Blastocyst rates of surviving vitrified oocytes were significantly lower compared with controls on Days 5 (2.4 v. 12.7%), 6 (4.8 v. 17.6%), and 7 (5.6 v. 18.4%). To test their ability to develop to term, 16 and 27 blastocysts on Day 5 developing from vitrified COC were transferred into 2 recipients. Both recipients became pregnant and farrowed a total of 10 live piglets (4 and 6 piglets, respectively). These data demonstrate that large groups of immature porcine oocytes could be cryopreserved by this method showing high survival and maturation rates. Furthermore, despite a low rate of blastocyst development, transfer of Day-5 blastocysts generated from vitrified oocytes resulted in piglet production for the first time in the world. Partially supported by JSPS and HAS under the Japan-Hungary Research Cooperative Program.

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Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa

Reproduction ◽

10.1530/rep.0.1230455 ◽

2002 ◽

pp. 455-465 ◽

Cited By ~ 74

Author(s):

YH Choi ◽

CC Love ◽

LB Love ◽

DD Varner ◽

S Brinsko ◽

...

Keyword(s):

Intracytoplasmic Sperm Injection ◽

Polar Body ◽

Cumulus Cells ◽

Fertilization Rate ◽

Developmental Competence ◽

Cleavage Rate ◽

First Polar Body ◽

Bovine Oocytes

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.

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298RED DEER (CERVUS ELAPHUS) PARTHENOGENETIC BLASTOCYSTS PRODUCED USING IONOMYCIN/6DMAP ACTIVATION

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab298 ◽

2004 ◽

Vol 16 (2) ◽

pp. 268 ◽

Cited By ~ 2

Author(s):

S.E. Beaumont ◽

D.K. Berg ◽

G.W. Asher

Keyword(s):

Red Deer ◽

Polar Body ◽

Negative Control ◽

Blastocyst Development ◽

Cell Counts ◽

First Polar Body ◽

Significant Difference ◽

Chi Square Analysis ◽

Polar Body Extrusion

Successful activation of red deer oocytes is a necessary prerequisite for the cloning of red deer individuals with desirable genetic characteristics. To investigate this, an established biphasic protocol used for oocyte activation in sheep was investigated for suitability. The method chosen was 5μM Ionomycin for 5min followed by 2mM 6DMAP for 3h ( Loi P et al., 1998 Biol. Reprod. 58, 1177–1187). The medium used during activation and subsequent culture was Deer Synthetic Oviduct Fluid, which has been shown to support routine in vitro fertilization and blastocyst development (15%) of in vitro-matured red deer oocytes (DSOF, Berg D et al., 2003 Theriogenology 59, 189–205). Red deer abattoir-derived COCs were matured in vitro for 22h before random allocation across 3 treatment groups comprising a standard IVF group, the activation group and a negative control group exposed to medium only. Activation treatment oocytes were stripped of cumulus by vortexing in 0.1% hyaluronidase before selecting for first polar body extrusion. First-step activation was performed in medium comprising HEPES-buffered IVF-DSOF containing 4mM Ca2+. Second-step activation used 3mM Ca2+ early DSOF under 7% O2, 5% CO2, and 88% N2 at 38.5°C. Standard IVF was conducted at 23h post-IVM using 4mM Ca2+ IVF-DSOF and 0.5×106mL−1 final sperm concentration. Following activation and IVF, oocytes were washed 3 times in HEPES DSOF before culture for 7 days in sequential DSOF with late DSOF on Day 4 containing 1.5mM Ca2+. Cleavage was assessed 24h after activation, and all blastocysts were fixed for cell counts. Four replicates of each treatment were performed. Cleavage and blastocyst rates were examined by chi-square analysis and cell numbers by ANOVA. First polar body extrusion rate was 84%. Cleavage was similar between the activation treatment and IVF (P>0.05 ); but a significant difference was found in blastocyst development rates (P<0.05) with the Ionomycin and 6DMAP protocol being superior to the IVF treatment. Exposure to high Ca2+ media alone resulted in only 5% of the negative control oocytes cleaving to 2 cells. Results show that Ionomycin and 6DMAP are effective in activating red deer oocytes and DSOF is a suitable medium to produce parthenogenetic blastocysts.

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Resveratrol Hinders Postovulatory Aging by Modulating Oxidative Stress in Porcine Oocytes

Molecules ◽

10.3390/molecules26216346 ◽

2021 ◽

Vol 26 (21) ◽

pp. 6346

Author(s):

Benazir Abbasi ◽

Yan Dong ◽

Rong Rui

Keyword(s):

Oxidative Stress ◽

Polar Body ◽

Treated Group ◽

Control Group ◽

Relative Level ◽

Antioxidant Genes ◽

First Polar Body ◽

Postovulatory Aging ◽

Relative Mrna Expression

Postovulatory aging of the mammalian oocytes causes deterioration of oocytes through several factors including oxidative stress. Keeping that in mind, we aimed to investigate the potential of a well-known antioxidant, resveratrol (RV), to evaluate the adverse effects of postovulatory aging in porcine oocytes. After in vitro maturation (IVM), a group of (25–30) oocytes (in three replicates) were exposed to 0, 1, 2, and 4 μmol/L of RV, respectively. The results revealed that the first polar body (PB1) extrusion rate of the oocytes significantly increased when the RV concentration reached up to 2 μmol/L (p < 0.05). Considering optimum RV concentration of 2 μmol/L, the potential of RV was evaluated in oocytes aged for 24 and 48 h. We used fluorescence microscopy to detect the relative level of reactive oxygen species (ROS), while GHS contents were measured through the enzymatic method. Our results revealed that aged groups (24 h and 48 h) treated with RV (2 μmol/L) showed higher (p < 0.05) ROS fluorescence intensity than the control group, but lower (p < 0.05) than untreated aged groups. The GSH content in untreated aged groups (24 h and 48 h) was lower (p < 0.05) than RV-treated groups, but both groups showed higher levels than the control. Similarly, the relative expression of the genes involved in antioxidant activity (CAT, GPXGSH-Px, and SOD1) in RV-treated groups was lower (p < 0.05) as compared to the control group but higher than that of untreated aged groups. Moreover, the relative mRNA expression of caspase-3 and Bax in RV-treated groups was higher (p < 0.05) than the control group but lower than untreated groups. Furthermore, the expression of Bcl-2 in the RV-treated group was significantly lower than control but higher than untreated aged groups. Taken together, our findings revealed that the RV can increase the expression of antioxidant genes by decreasing the level of ROS, and its potent antiapoptotic effects resisted against the decline in mitochondrial membrane potential in aged oocytes.

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The Role of Ca2 + in Maturation and Reprogramming of Bovine Oocytes: A System Study of Low-Calcium Model

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.746237 ◽

2021 ◽

Vol 9 ◽

Author(s):

Lin Meng ◽

Hongmei Hu ◽

Zhiqiang Liu ◽

Luyao Zhang ◽

Qingrui Zhuan ◽

...

Keyword(s):

Oxidative Stress ◽

Polar Body ◽

Rna Seq ◽

Low Calcium ◽

Maturation Medium ◽

First Polar Body ◽

Significant Difference ◽

Bovine Oocytes

[Ca2+]i is essential for mammalian oocyte maturation and early embryonic development, as those processes are Ca2+ dependent. In the present study, we investigated the effect of [Ca2+]i on in vitro maturation and reprogramming of oocytes in a lower calcium model of oocyte at metaphase II (MII) stage, which was established by adding cell-permeant Ca2+ chelator BAPTA-AM to the maturation medium. Results showed that the extrusion of the first polar body (PB1) was delayed, and oocyte cytoplasmic maturation, including mitochondrial and endoplasmic reticulum distribution, was impaired in lower calcium model. The low-calcium-model oocytes presented a poor developmental phenotype of somatic cell nuclear transfer (SCNT) embryos at the beginning of activation of zygotic genome. At the same time, oxidative stress and apoptosis were observed in the low-calcium-model oocytes; subsequently, an RNA-seq analysis of the lower-calcium-model oocytes screened 24 genes responsible for the poor oocyte reprogramming, and six genes (ID1, SOX2, DPPA3, ASF1A, MSL3, and KDM6B) were identified by quantitative PCR. Analyzing the expression of these genes is helpful to elucidate the mechanisms of [Ca2+]i regulating oocyte reprogramming. The most significant difference gene in this enriched item was ID1. Our results showed that the low calcium might give rise to oxidative stress and apoptosis, resulting in impaired maturation of bovine oocytes and possibly affecting subsequent reprogramming ability through the reduction of ID1.

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Luteolin Orchestrates Porcine Oocyte Meiotic Progression by Maintaining Organelle Dynamics Under Oxidative Stress

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.689826 ◽

2021 ◽

Vol 9 ◽

Author(s):

Soo-Hyun Park ◽

Pil-Soo Jeong ◽

Ye Eun Joo ◽

Hyo-Gu Kang ◽

Min Ju Kim ◽

...

Keyword(s):

Oxidative Stress ◽

Oocyte Maturation ◽

Polar Body ◽

Blastocyst Formation ◽

Cortical Granules ◽

Expression Levels ◽

Meiotic Progression ◽

First Polar Body ◽

Underlying Mechanisms

Increasing evidence has demonstrated that oxidative stress impairs oocyte maturation, but the underlying mechanisms remain largely unknown. Here, for the first time, we examined the antioxidant role of luteolin in meiotic progression and the underlying mechanisms. Supplementation of 5 μM luteolin increased the rates of first polar body extrusion and blastocyst formation after parthenogenetic activation, and the expression levels of oocyte competence (BMP15 and GDF9)-, mitogen-activated protein kinase (MOS)-, and maturation promoting factor (CDK1 and Cyclin B)-related genes were also improved. Luteolin supplementation decreased intracellular reactive oxygen species levels and increased the expression levels of oxidative stress-related genes (SOD1, SOD2, and CAT). Interestingly, luteolin alleviated defects in cell organelles, including actin filaments, the spindle, mitochondria, the endoplasmic reticulum, and cortical granules, caused by H2O2 exposure. Moreover, luteolin significantly improved the developmental competence of in vitro-fertilized embryos in terms of the cleavage rate, blastocyst formation rate, cell number, cellular survival rate, and gene expression and markedly restored the competencies decreased by H2O2 treatment. These findings revealed that luteolin supplementation during in vitro maturation improves porcine meiotic progression and subsequent embryonic development by protecting various organelle dynamics against oxidative stress, potentially increasing our understanding of the underlying mechanisms governing the relationship between oxidative stress and the meiotic events required for successful oocyte maturation.

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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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