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

2004 ◽  
Vol 16 (2) ◽  
pp. 268 ◽  
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.

scholarly journals 313IN VITRO MATURATION AND FERTILIZATION OF OOCYTES COLLECTED FROM UNSTIMULATED MACACA NEMISTRINA OVARIES

2004 ◽  
Vol 16 (2) ◽  
pp. 276
Author(s):  
E.S. Hayes ◽  
E.C. Curnow
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Pantothenic Acid ◽  
Macaca Nemestrina ◽  
Chi Square ◽  
Maturation In Vitro ◽  
First Polar Body ◽  
Chi Square Analysis ◽  
Polar Body Extrusion

Reports describing the IVM of Macaca nemestrina (Mn) oocytes are limited (Cranfield MR et al. 1989 Zoo. Biol. (Supp. 1), 33). The use of gonadotrophins (Gnt) for IVM of non-human primate (NHP) oocytes is common but the concentrations used are often high (8–40IUmL−1) and the species of origin and biological activity of Gnt varies (Schramm RD and Paprocki AM, 2000 Hum. Reprod. 15, 2411). We have compared two different IVM systems with human Gnt on maturation and fertilization of oocytes collected from unstimulated Mn ovaries (n=6–10 animals). Oocytes were subjected to IVM in modified (minus PVA and pantothenic acid, plus 20 amino acids) HECM−10+15% FCS (Zheng P et al., 2001 Mol. Reprod. Dev. 58, 348) for a) 36h in the presence (mHECM+36, n=322) or absence (mHECM−36, n=99) of FSH and LH applied sequentially (FSH 1IUmL−1 0–24h; 10IUmL−1 FSH and LH 24–36h) or b) 24h in the presence (mHECM+24, n=119) or absence (mHECM−24, n=56) of static concentrations of Gnt (FSH and LH 1IUmL−1 0–24h; no Gnt 24–30h). Oocytes exhibiting first polar body extrusion at 36 and 30h were recorded as mature (MII) and subjected to IVF in HTF+BSA (3mg mL−1) with Mn sperm pretreated with 1.0mM caffeine and 0.1mM dbcAMP. Fertilized oocytes (pronuclei and/or 2nd polar body extrusion) were cultured in sequential culture medium for 48h, assessed for cleavage and either fixed or frozen. Proportional data (mature/total, fertilized/mature or cleaved/fertilized) were compared by chi-square analysis and are reported as percentages. Oocytes cultured in mHECM+36 and mHECM−36 exhibited similar rates of GVBD (58.7% v. 53.5%) but the percentage of MII oocytes was significantly higher (P=0.0244) in mHECM+36 (41.3%) v. mHECM−36 (28.3%). Fertilization rates were comparable between mHECM+36 (61.5%) and mHECM−36 (60.9%), whereas cleavage rates were significantly higher (P=0.0004) in mHECM+36 (74.6%) v. HECM−36 (21.4%). Oocytes cultured in mHECM+24 and mHECM−24 exhibited similar rates of GVBD (76.5% v. 62.5%) but the proportion of MII oocytes was significantly higher (P=0.0159) in mHECM+24 (55.5%) v. mHECM−24 (35.7%). Fertilization and cleavage rates were comparable between mHECM+24 (58.8% v. 63.3%) and mHECM−24 (50.0% v. 42.8%). A comparison between mHECM+36 and mHECM+24 indicated a significantly lower (P=0.0005) percentage of GV oocytes and a significantly higher (P=0.0096) percentage of MII oocytes in mHECM+24 (23.5% v. 55.5%) compared to mHECM+36 (41.3% v. 41.3%). Fertilization and cleavage rates were not significantly different between mHECM+36 and mHECM+24. Oocyte maturation and fertilization and embryo cleavage were not different for mHECM−36 and mHECM−24 (P=0.3138–0.8202). Mn oocytes exhibit high rates of Gnt-independent GVBD (52.5%–53.5%) and maturation (28.3%–35.7%) in vitro, and maturation rates were improved in Gnt supplemented maturation medium. However, reduced exposure to lower concentrations of FSH and increased exposure to lower concentrations of LH was associated with higher rates of oocyte maturation in vitro. The use of lower concentrations of FSH and LH for reduced periods may improve IVM of NHP oocytes. This work was supported by the Tissue Distribution Program of the WaNPRC (NIH grant # R00166).

102 EFFECT OF DEMECOLCINE PRE-TREATMENT ON VIABILITY, TIMING OF FIRST POLAR BODY EXTRUSION, SPINDLE CONFIGURATION, AND SUBSEQUENT DEVELOPMENT OF OVINE OOCYTES VITRIFIED AT GERMINAL VESICLE STAGE

2010 ◽  
Vol 22 (1) ◽  
pp. 210 ◽  
Author(s):  
A. R. Moawad ◽  
J. Zhu ◽  
I. Choi ◽  
K. H. S. Campbell
Keyword(s):  
Polar Body ◽  
Germinal Vesicle ◽  
Subsequent Development ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
First Polar Body ◽  
Polar Body Extrusion ◽  
And Control

Oocyte cryopreservation is a potentially valuable way of preserving female germ cells. However, to date the reported developmental competence of cryopreserved oocytes is low. The objectives of this study were to investigate the effects of demecolcine pretreatment on viability, timing of the first polar body extrusion (PBI), spindle, chromatin organization, and in vitro embryo development of ovine vitrified germinal vesicle (GV) oocytes after in vitro fertilization (IVF) and parthenogenetic activation. Cumulus-oocyte complexes (COC) aspirated from ovine ovaries collected at slaughter were selected and randomly divided into 3 groups: (1) untreated (in vitro matured, IVM) as a control, (2) vitrified (Moawad AR et al. 2009 Reprod. Fertil. Dev. 21, 135 abst), and (3) deme + vitrified (oocytes were incubated with 0.1 μg mL-1 demecolcine for 20 min before vitrification). After vitrification COC were thawed and matured in vitro for 24 h. Following IVM, oocytes from 3 groups were subsequently subjected to (1) immunostaining, (2) IVF, or (3) activation. Presumptive zygotes were cultured in vitro in SOF media for 7 days. Data were analyzed using chisquare and t-test. No significant differences (P > 0.05) were observed in survival rates between deme + vitrified (90.8%, 324/357) and vitrified (87.2%, 211/242). However, the numbers of oocytes with PBI in two vitrified groups at 18 h (20.4 and 8.5 v. 47.1%) and 24 h post IVM (51 and 43.2 v. 88.5%) were significantly lower (P < 0.01) than those in the control. Percentage of normal spindle and chromatin configuration in the two vitrified groups also significantly decreased (P < 0.05) compared with those in the control (42.5 and 41.8 v. 76.5%), whereas missing spindle in the 2 vitrified groups significantly increased (P < 0.001) compared with the controls (47.5 and 32.7 v. 3.9%). Following IVF (pi), cleavage rates at 24.48 hpi and morula development (5 days pi) were significantly lower (P < 0.001) in deme + vitrified (6.1, 43.1, and 28.5%) and vitrified groups (3.3, 30.1, and 22.9%) than control (50.4, 82.4, and 46.4%). Blastocyst development in deme + vitrified (9.8%) and control (33.6%) was significantly higher (P < 0.01) than in vitrified group (1.3%). Hatched blastocysts were observed only in deme + vitrified and control groups (4.9 v. 12.8%). In addition, post activation (pa) cleavage rates in deme + vitrified (10.3 v. 40.7%) and control (52.5 v. 76.7%) at 24 and 48 hpa were significantly higher (P < 0.05) than those in the vitrified group. Blastocyst development in deme + vitrified (4.8%) was higher than that in the vitrified group (1.8%), but not significant (P > 0.05); however, these values were still significantly lower (P < 0.001) than those in the control (24.2%). No significant differences were observed in total cell numbers per blastocyst between all the groups. Taken together, these results suggest that pretreatment of oocytes with demecolcine before vitrification could improve the developmental competence of ovine vitrified-thawed GV-stage oocytes. A. R. Moawad was supported by the Egyptian government.

29 The effect of vitrification at the immature stage on DNA methylation in porcine oocytes and its relevance to subsequent embryo development

2021 ◽  
Vol 33 (2) ◽  
pp. 122
Author(s):  
T. Somfai ◽  
N. T. Hiep ◽  
K. Kikuchi ◽  
Y. Hirao
Keyword(s):  
Dna Methylation ◽  
Embryo Development ◽  
Polar Body ◽  
Immature Stage ◽  
Negative Control ◽  
Control Group ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Significant Difference ◽  
Polar Body Extrusion

Oocyte vitrification is an important approach for invitro gene banking of female germplasm; however, in pigs, it hampers embryo development. In cattle, vitrification at the MII stage was reported to alter epigenetic status in oocytes and even in subsequently developing embryos (Chen et al. 2016 Theriogenology 86, 868-878). The present study investigated the effect of vitrification at the immature stage of porcine oocytes on DNA methylation status and its relevance to subsequent embryo development. Immature cumulus–oocyte complexes were vitrified in microdrops and warmed (vitrified group) or treated with cryoprotectant agents (17.5% ethylene glycol + 17.5% propylene glycol, CPA group) by our method (Appeltant et al. 2018 Cryobiology 85, 87-94). Then they were subjected to IVM, parthenogenetic activation (PA), and embryo culture. From each batch, a group of oocytes was processed without treatment (control group). Oocyte survival and polar body extrusion were recorded after IVM. Cleavage and blastocyst developmental rates were recorded on Day 2 and 6 of culture, respectively (Day 0=PA). In each replication, DNA methylation was assayed in representative oocytes at the MII stage after IVM and in embryos at the 2- to 4-cell stage on Day 2 by immunostaining with 5-methylcytosine (5mC). Relative fluorescent intensity of 5mC in the chromatin was compared among groups. The experiment was replicated 3 times. Data were analysed by ANOVA. After IVM, there was no significant difference among the control, CPA, and vitrified groups in terms of the percentage of live oocytes (99.3, 96.4, and 94.0%, respectively) or polar body extrusion (88.6, 86.9, and 79.6%, respectively). After PA of oocytes with a polar body, there was no difference between the control and CPA groups in the percentage of cleavage (84.1 and 80.7%, respectively) or blastocyst development of cleaved embryos (63.3 and 79.3%, respectively). However, in the vitrified group, cleavage and blastocyst development rates (46.6 and 33.5%, respectively) were reduced (P&lt;0.05) compared with the other groups. The 5mC fluorescence in the DNA of oocytes at the MII stage in the CPA and vitrified groups were similar and significantly lower than that in the control group (0.88±0.02, 0.87±0.001, and 1.0±0.02, respectively) but higher than that in the negative control processed without primary antibody (0.33±0.02). In the embryos at the 2- to 4-cell stage, 5mC fluorescence was not significantly different among the control, CPA, and vitrified groups (1.0±0.1, 0.99±0.1, and 0.96±0.1, respectively) but was significantly higher than that of the negative control (0.36±0.04). In conclusion, CPA treatment reduced DNA methylation levels in oocytes. However, it was restored during early embryo development and did not affect blastocyst development. The results suggest that reduced DNA methylation in vitrified oocytes is caused by CPA but it may not be responsible for their reduced ability to develop to blastocysts.

scholarly journals Effect of Melatonin on the In Vitro Maturation of Porcine Oocytes, Development of Parthenogenetically Activated Embryos, and Expression of Genes Related to the Oocyte Developmental Capability

Animals ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 209 ◽  
Author(s):  
Ling Yang ◽  
Qingkai Wang ◽  
Maosheng Cui ◽  
Qianjun Li ◽  
Shuqin Mu ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Polar Body ◽  
Melatonin Receptors ◽  
Melatonin Treatment ◽  
In Vitro Development ◽  
First Polar Body ◽  
Mitochondrial Distribution ◽  
Polar Body Extrusion ◽  
Vitro Development

Melatonin treatment can improve quality and in vitro development of porcine oocytes, but the mechanism of improving quality and developmental competence is not fully understood. In this study, porcine cumulus–oocyte complexes were cultured in TCM199 medium with non-treated (control), 10−5 M luzindole (melatonin receptor antagonist), 10−5 M melatonin, and melatonin + luzindole during in vitro maturation, and parthenogenetically activated (PA) embryos were treated with nothing (control), or 10−5 M melatonin. Cumulus oophorus expansion, oocyte survival rate, first polar body extrusion rate, mitochondrial distribution, and intracellular levels of reactive oxygen species (ROS) and glutathione of oocytes, and cleavage rate and blastocyst rate of the PA embryos were assessed. In addition, expression of growth differentiation factor 9 (GDF9), tumor protein p53 (P53), BCL2 associated X protein (BAX), catalase (CAT), and bone morphogenetic protein 15 (BMP15) were analyzed by real-time quantitative PCR. The results revealed that melatonin treatment not only improved the first polar body extrusion rate and cumulus expansion of oocytes via melatonin receptors, but also enhanced the rates of cleavage and blastocyst formation of PA embryos. Additionally, melatonin treatment significantly increased intraooplasmic level of glutathione independently of melatonin receptors. Furthermore, melatonin supplementation not only significantly enhanced mitochondrial distribution and relative abundances of BMP15 and CAT mRNA, but also decreased intracellular level of ROS and relative abundances of P53 and BAX mRNA of the oocytes. In conclusion, melatonin enhanced the quality and in vitro development of porcine oocytes, which may be related to antioxidant and anti-apoptotic mechanisms.

111 SLOW FREEZING AND VITRIFICATION OF IN VITRO-MATURED DOMESTIC CAT OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 173 ◽  
Author(s):  
J. Braun ◽  
C. Otzdorff ◽  
T. Tsujioka ◽  
S. Hochi
Keyword(s):  
Polar Body ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Slow Freezing ◽  
Developmental Potential ◽  
Freezing Medium ◽  
First Polar Body ◽  
Blastocyst Rate ◽  
Chi Square Analysis

The effects of slow freezing or vitrification as well as exposure to the cryoprotective media without cooling and warming of in vitro-matured domestic cat oocytes on the in vitro development to the blastocyst stage was investigated. Cumulus–oocyte complexes were matured for 24 h in TCM-199 supplemented with 3 mg mL−1 BSA, 1 µg mL−1 estradiol, 0.1 IU mL−1 FSH, and 0.0063 IU mL−1 LH. Denuded oocytes with a detectable first polar body were inseminated with 2 × 106 cells mL−1 cauda epididymal spermatozoa for 22 h in TALP solution. Presumptive zygotes were cultured in modified SOF medium at 38.5°C in 5% CO2 in air. For slow freezing, oocytes were equilibrated for 20 min at ambient temperatures in PBS with 20% FCS containing either 1.5 M ethylene glycol (EG) + 0.2 M sucrose or 1.5 M EG + 0.2 M trehalose. Oocytes were loaded into 0.25-mL straws, cooled to −7°C at 2°C min, held for 5 min, seeded, cooled down to −30°C at 0.3°C min, and finally plunged into liquid nitrogen. The straws were thawed for 5 s at room temperature and for 30 s in a waterbath at 30°C. Oocytes were washed 3 times before insemination. In vitro-matured oocytes were exposed to the cryoprotective media for 30 min before they were inseminated and then they were cultured for 7 days. For vitrification (Hochi et al. 2004 Theriogenology 61, 267–275), a minimum-volume cooling procedure using Cryotop (Kitazato Supply Co., Tokyo, Japan) as a cryodevice was applied. No blastocysts could be obtained after slow freezing with a cryoprotective medium containing 0.2 M sucrose. Simple exposure to the same freezing medium after in vitro maturation without cryopreservation resulted in a blastocyst rate of 7.9% (control oocytes, 10.7%; not significant (NS); chi-square analysis). Use of trehalose as an extracellular cryoprotectant resulted in the harvest of one blastocyst (0.6%) after slow freezing. Exposure to the same cryoprotective medium resulted in a blastocyst rate of 10.0% (fresh control, 10.9%; NS). After exposure of in vitro-matured oocytes to the vitrification solution, a blastocyst rate of 16.0% was observed (8/50), which was not statistically different from the blastocyst rate in fresh control oocytes (16.3%; 15/92). No blastocysts could be obtained after vitrification (0/64). The results (Table 1) demonstrate that there is no obvious toxic effect of the cryoprotectants employed here for slow freezing or vitrification on the in vitro-matured oocytes, but the developmental potential of cryopreserved oocytes to the blastocyst stage is severely impaired. Table 1. Effect of slow freezing or exposure to freezing medium of matured cat oocytes on the development to the blastocyst stage in vitro

44 PRODUCTION OF LIVE PIGLETS AFTER CRYOPRESERVATION OF IMMATURE PORCINE OOCYTES

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.

scholarly journals 146COMPARISON OF EMBRYO CULTURE MEDIA FOR BLASTOCYST DEVELOPMENT AFTER INTRACYTOPLASMIC SPERM INJECTION OF IN VITRO-MATURED EQUINE OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 195
Author(s):  
Y.H. Choi ◽  
D.D. Varner ◽  
K. Hinrichs
Keyword(s):  
In Vitro Culture ◽  
Embryo Culture ◽  
Intracytoplasmic Sperm Injection ◽  
Culture Media ◽  
Polar Body ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
Significant Difference

Research on in vitro culture of equine embryos has been scant, due to failure of equine in vitro fertilization to be repeatably successful. We have recently obtained high fertilization rates of equine oocytes via intracytoplasmic sperm injection (ICSI) using a piezo drill (Choi et al., 2002 Reproduction 123, 455–465). Culture of presumptive zygotes in G1.2/2.2 medium resulted in 63% cleavage and an average of 15 cells at 4d, but only 2 to 9% blastocyst development at 7 days (Choi et al., 2003 Theriogenology 59, 1219–1229). In the present study, we evaluated the effect of two different culture media, G1.3/G2.3 v. DMEM/F-12, with or without FBS, on blastocyst development after ICSI. Oocytes were collected from slaughterhouse-derived ovaries by follicular scraping and were matured in vitro for 24h in M199 with 10% FBS and 5μUmL−1 FSH. After culture, oocytes having a polar body (198/305; 65%) were fertilized by ICSI with frozen-thawed equine sperm using a piezo drill. Presumptive zygotes were cultured in 1 of 4 media: G1.3/G2.3 (which includes 0.8% BSA) with or without 10% FBS, or in DMEM/F-12 with 0.5% BSA, with or without 10% FBS. Culture was performed in microdroplets at 5μL/zygote under oil at 38.2°C in an atmosphere of 5% CO2, 5% O2 and 90% N2 for 7.5 days. In G1.3/2.3 treatments, G1.3 media were completely refreshed at 48h, zygotes were transferred to G2.3 (with or without FBS as per the first stage) at 96h, and were completely refreshed with the same media at 144h. In DMEM/F-12 treatments, media were completely refreshed every other day. Three to 5 replicates were performed in each treatment, and data were analyzed by chi-square test. There were no significant differences in cleavage rates (59–64%) among treatments. The rate of development to blastocyst, per oocyte injected, in G1.3/G2.3/BSA (1/49, 2%) was significantly lower (P&lt;0.05) than that for the other three treatments: G1.3/2.3/BSA/FBS (9/49, 18%), DMEM/F-12/BSA (9/50, 18%), or DMEM/F-12/BSA/FBS (10/50, 20%). There was no significant difference in blastocyst development among the latter three treatments. These findings indicate that G1.3/2.3 media with BSA only do not adequately support growth of equine embryos. Development of up to 20% of injected oocytes to the blastocyst stage in G media supplemented with FBS, in DMEM/F-12/BSA or in DMEM/F-12/BSA/FBS represents the highest in vitro equine blastocyst rate in medium alone (i.e. without co-culture) yet reported. The success of DMEM/F-12 as an embryo culture medium may provide a relatively simple basis for equine in vitro culture programs. To determine whether this medium was able to support further developmental competence, we cultured equine embryos resulting from nuclear transfer of in vitro-matured oocytes in DMEM/F-12+10% FBS (without BSA). We transferred 4 resulting blastocysts to recipient mares by transcervical transfer; one pregnancy is ongoing at 230d gestation at the time of this writing. This work was supported by the Link Equine Research Endowment Fund, Texas A&amp;M University.

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

Toxins ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 841
Author(s):  
Wenhui Li ◽  
Yijing He ◽  
Hongyu Zhao ◽  
Lei Peng ◽  
Jia Li ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Polar Body ◽  
Grape Seed ◽  
Ros Generation ◽  
Mrna Levels ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
First Polar Body ◽  
Grape Seed Proanthocyanidin

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.

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

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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