379 EFFECT OF SUPPLEMENTARY INOSITOL 1,4,5-TRIPHOSPHATE INJECTION ON FERTILIZATION AND EMBRYO DEVELOPMENT AFTER BOVINE ICSI

2007 ◽  
Vol 19 (1) ◽  
pp. 305
Author(s):  
T. K. Suh ◽  
G.E. Seidel, Jr
Keyword(s):  
Embryo Development ◽  
Polar Body ◽  
Formation Rate ◽  
Cortical Granule ◽  
Blastocyst Development ◽  
Exact Test ◽  
Injection Process ◽  
First Polar Body ◽  
Membrane Bound

During fertilization in mammals, sperm membrane-bound phospholipase C zeta induces breakdown of ooplasmic membrane-bound phosphatidylinositol-4,5-bisphosphate (PIP2), which leads to the production of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The IP3 induces intracellular Ca2+ oscillations ([Ca2+]i), which trigger inactivation of maturation-promoting factor (MPF) and mitogen-activated kinase (MAPK), resulting in decondensation of the sperm head, resumption of meiosis, extrusion of a second polar body, cortical granule exocytosis, formation of pronuclei (PN), and entry into the first cell cycle. In bovine ICSI, injection of a single spermatozoon into an oocyte does not consistently induce [Ca2+]i oscillations, as was observed in IVF, and this may, at least in part, explain the low rates of fertilization and embryo development. Although IP3 has been used as a powerful activator for nuclear transferred zygotes or parthenogenetic oocytes, few studies have evaluated the effect of IP3 injection on normal fertilization and embryo development after ICSI. The objective of this study was to determine the effect of injecting IP3 during bovine ICSI on fertilization and embryo development in vitro. Chemically defined media (CDM) were used throughout (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 585–596). The injection volume of IP3, which was dissolved in calcium- and magnesium-free PBS, was approximately 0.01 nL, and the total dose for injection along with a spermatozoon was about 250 �M, which was determined by the 2PN formation rate in preliminary experiments. Semen from 3 bulls was used to produce embryos in 5 replicates. Oocytes obtained from slaughterhouse ovaries were matured in vitro in CDM-M for 22–23 h under 5% CO2 in air at 38.5�C, and oocytes with a first polar body were used for ICSI. Motile sperm from frozen–thawed semen were used for sperm injection, with or without IP3 in a 50-�L drop of GMOPS medium, with a piezo-driven pipet of 7–8 �m inner diameter. After ICSI, presumptive zygotes were cultured in CDM-1 for 3 days, and then in CDM-2 for 5 days at 39�C under 5% CO2/5% O2/90% N2. Cleavage and blastocyst development were evaluated at the end of each culture period. Data were subjected to Fisher's exact test. Cleavage in control and IP3 groups was 36.4 and 50.0%, respectively (P < 0.05). Respective blastocyst rates per oocyte were 5.5 and 13.0% (P > 0.05). This study showed that injection of IP3 during the sperm injection process improved cleavage of bovine oocytes after ICSI.

341 OXYGEN TENSION AND EGF AFFECT METABOLISM OF IN VITRO-MATURED CUMULUS-ENCLOSED MOUSE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 278
Author(s):  
K. A. Preis ◽  
G. E. Seidel Jr ◽  
D. K. Gardner
Keyword(s):  
Glucose Uptake ◽  
Oxygen Tension ◽  
Embryo Development ◽  
Metabolic Activity ◽  
Lactate Production ◽  
Defined Medium ◽  
Blastocyst Development ◽  
Developmental Potential ◽  
Exact Test

In vitro maturation of immature oocytes results in limited success in both clinical and research laboratories. Although reduced oxygen concentration is beneficial to embryo development, the optimal concentration for oocyte maturation has yet to be determined. The objective of this study was to determine whether oxygen tension (20% or 5% O2) affects oocyte physiology. Additionally, the effect of epidermal growth factor (EGF) in maturation medium on oocyte metabolic activity and subsequent embryo development was determined. Cumulus–oocyte complexes (COCs; n = 231) were collected from 28-day-old unprimed F1 (C57BL/6 × CBA/ca) mice. COCs were individually matured in defined medium at 37°C in 6% CO2 in one of four groups (Table 1). For the metabolism study, COCs were further divided into two groups: individual maturation in a 2-µL drop of medium for 16 h (n = 131); or individual maturation in 5-μL for 12 h and then placed in a 0.5-μL drop of medium for 4 h (n = 100), the time of greatest metabolic activity of the COC. At 17 h of maturation, COCs were individually fertilized, and zygotes were individually cultured until 96 h, at which time blastocyst development was assessed. Metabolic profiles were analyzed by ANOVA, and blastocyst rates were analyzed by Fisher's exact test. Maturation rates and blastocyst development were not different between groups. However, at 12–16 h of maturation, metabolism of COCs was affected by both oxygen tension and EGF (Table 1). Concerning metabolism over the entire course of maturation, glucose uptake and lactate production were higher in COCs in 5% O2 + 100 ng EGF (P < 0.05) than in the remaining three groups. There was no difference between 5% O2 and 20% O2 + 100 ng EGF, but 20% O2 caused less glucose uptake and lactate production than did the other three treatment groups (P < 0.05). Results of this study are the first to show that oxygen tension alters COC metabolism: COCs matured under 5% O2 were more active metabolically than COCs matured under 20% O2. The effect of oxygen tension is to some extent moderated by the presence of EGF, as metabolic activity of COCs matured under 20% O2 + 100 ng EGF was closer to that of COCs matured under 5% O2 conditions. Although blastocyst rates were similar across the four groups, embryos derived from oocytes matured in different oxygen tensions may exhibit different developmental potential. In conclusion, results of this study have implications for the improvement of maturation conditions in both clinical and research laboratories. Table 1. Carbohydrate metabolism of individual COCs at 12–16 h of maturation

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.

53 EFFECT OF THE NUCLEAR REPROGRAMMING TIME ON IN VITRO DEVELOPMENT OF EQUINE AGGREGATED CLONED EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 174
Author(s):  
R. Olivera ◽  
C. Alvarez ◽  
I. Stumpo ◽  
G. Vichera
Keyword(s):  
Embryo Development ◽  
Polar Body ◽  
Nuclear Reprogramming ◽  
Chi Square ◽  
In Vitro Development ◽  
First Polar Body ◽  
Group 3 ◽  
Group 2 ◽  
Vitro Development

The time allowed for nuclear reprogramming is considered an essential factor for the efficiency of cloning and has not been evaluated in equine aggregated cloned embryos. The aim of our work was to assess the effect of different timing of activation stimulus after fusion of adult equine fibroblast cells to enucleated equine oocytes on embryo development and embryo quality. We processed a total of 1874 equine ovaries, recovering 3948 oocytes, of which 1914 (48.5%) had extruded the first polar body after 24 h of maturation. Oocyte collection, maturation, and the NT procedure were performed as described by Lagutina et al. (2007 Theriogenology 67, 90–98). Reconstructed oocytes (RO) were activated at 3 different times after cell fusion: (1) 1 h, (2) 1.5 h, and (3) 2 h. Activation was performed using 8.7 µM ionomycin for 4 min, followed by a 4-h culture in a combination of 1 mM DMAP and 5 mg mL–1 of cycloheximide. The RO were cultured in the well of the well system, aggregating 3 RO per well. The RO were cultured in DMEM-F12 with 5% fetal bovine serum (FBS) and antibiotics. Cleavage (48 h after activation), blastocyst, and expanded blastocyst rates (8–9 days) were assessed. In vitro development was compared using the chi-square test (P < 0.05). A total of 1608 RO were cultured. Cleavage was significantly lower in group 3 with respect to the other 2 groups [(1): 396/450, 88%; (2): 540/639, 84.5%; (3): 365/519, 70.3%]. There were no significant differences in blastocyst rates within the 3 groups considering the number of total RO [(1): 19/450, 4.2%; (2): 23/639, 3.6%; (3): 15/519, 2.9%] or aggregated RO per well [(1): 12.7%; (2): 10.8%; (3): 8.7%]. However, the rate of blastocyst expansion was higher (P < 0.05) in group 2 than in group 3 [(1): 17/19, 89.5%; (2): 23/23, 100%; (3): 11/15, 73.3%]. In conclusion, the timing of nuclear reprogramming did not affect blastocyst rates but affected cleavage rates and blastocyst quality. This indicates that 1 h before activation stimulus is enough for embryo development of equine aggregated cloned embryos.

328 IN VITRO MATURATION OF OOCYTES FROM Canindé GOATS SUBMITTED TO DIFFERENT HORMONAL TREATMENTS FOR OVARIAN STIMULATION

2010 ◽  
Vol 22 (1) ◽  
pp. 320
Author(s):  
K. C. Almeida ◽  
A. F. Pereira ◽  
A. S. Alcântara Neto ◽  
S. R. G. Avelar ◽  
F. C. Sousa ◽  
...  
Keyword(s):  
Ovarian Stimulation ◽  
Polar Body ◽  
Hormonal Treatment ◽  
Oocyte Quality ◽  
Exact Test ◽  
Gentamicin Sulfate ◽  
First Polar Body ◽  
The One ◽  
Hormonal Treatments

Oocyte IVM is a long process during which oocytes acquire their ability to support the stages of development in a stepwise manner, ultimately reaching activation of the embryonic genome. The overall success of this process can be affected by factors such as hormonal treatment for ovarian stimulation. Thus, the current study aims to evaluate the possible effects of the ovarian stimulatory protocols on the goat oocyte quality and IVM rate. Adult and cyclic Canindé goats were heat-synchronized by means of intravaginal sponges impregnated with 60 mg medroxyprogesterone acetate (MAP, Progespon, Syntex, Buenos Aires, Argentina) inserted for 11 days coupled with a luteolytic injection of 50 μg cloprostenol (Ciosin, Coopers, São Paulo, Brazil) in the 8th day of treatment. The ovarian stimulation was carried out using one of the following protocols: a) standard multi-doses (MD) with 120 mg pFSH (Folltropin-V, Vetrepharm, Canada) distributed in five injections (30/30; 20/20; 20 mg) at 12 h intervals (n = 18); b) three- doses (TD) with 120 mg pFSH administered in three injections (60; 40; 20 mg) at 24 h intervals (n = 17); c) one shot (OD) of 70 mg pFSH plus 200 IU of eCG (Novormon, Syntex) administered 36 h before sponge removal (n = 17). In MD andTD groups, the pFSH injections started in Day 8 of progestagen treatment. The follicles were aspirated just after the sponge removal using laparoscopic oocyte recovery (LOR). This procedure was performed with a 22-gauge needle and a vacuum pump at 30 mmHg. The collection medium was TCM-199 supplemented with HEPES (10 mM), heparin (20 IU mL-1), and gentamicin sulfate (40 μg mL-1). COCs were classified as grade I, II, III, or IV based on visual criteria (Baldassarre H et al. 2003 Theriogenology 56, 831-839). Good quality oocytes (grade I and II) were incubated in TCM-199 supplemented with cysteamine (100 μM), EGF (10 ng mL-1) and gentamicin sulfate (40 μgm L-1) at 38.5°C in a humidified atmosphere with 5% CO2 in air for 24 h. Oocyte maturation was assessed by the visualization of first polar body under inverted microscope. Data were expressed as percentages and analyzed using the Fischer’s exact test. No statistical differences among hormonal treatments (P > 0.05) were observed for the percentage of the good quality oocytes, with 70.4 ± 3.0% of COCs graded in I and II. The IVM rate inTD (31.4%) was statistically lower than MD (31.4% v. 46.5%, P = 0.04) group. However, no significant differences (P = 0.89) were observed between OD (45.2%) and MD groups. Thus, current results indicate that oocyte production for IVM can be facilitated using ovarian stimulation with the one shot FSH/eCG regime without affecting meiotic competence. In summary, OD and MD treatments can be used for oocyte IVM in an embryo production programme in Canindé goats. This study was supported by the following Brazilian agencies: FINEP, CNPq, FUNCAP, and CAPES.

242 USE OF BRAIN-DERIVED NEUROTROPHIC FACTOR IN IN VITRO PREMATURATION OF BOVINE OOCYTES SUBJECTED TO PARTHENOGENETIC ACTIVATION

2008 ◽  
Vol 20 (1) ◽  
pp. 200
Author(s):  
T. H. C. De Bem ◽  
R. Rochetti ◽  
P. R. L. Pires ◽  
F. F. Bressan ◽  
P. R. Adona ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Polar Body ◽  
Hatching Rate ◽  
Blastocyst Development ◽  
Embryo Production ◽  
Parthenogenetic Activation ◽  
Bovine Oocytes ◽  
Hatching Rates

Prematuration provides an additional time for oocyte capacitation and maturation in an attempt to improve in vitro embryo production (IVP) rates and allows media supplementation during this period for IVP. The aim of this study was to use brain-derived neurotropic factor (BDNF) in prematuration to improve maturation of bovine oocytes subjected to parthenogenetic activation and cultured with different media. Oocytes were subjected to prematuration in TCM-199 medium supplemented with 10 µm butyrolactone I, 2.0 mm pyruvate, and 10 µg mL–1 gentamicin for 24 h in the absence of BDNF (control) or in the presence of 10 ng mL–1 BDNF (BD). Oocytes were then in vitro-matured (IVM) in TCM-199 medium supplemented with 10% FCS, 0.5 µg mL–1 FSH, 5.0 µg mL–1 LH, 2.0 mm pyruvate, and 10 µg mL–1 gentamicin at 38.5�C under 5% CO2 in air. After 19 h oocytes were denuded using hyaluronidase and vortexing for 3 min for the 1st polar body (1PB) selection. Those which extruded the 1PB were maintained in IVM until 26 h, when parthenogenetic activation was performed (5 min in 5 µm ionomycin, followed by 3 h in 2 mm 6-DMAP). Activated oocytes were then transferred to in vitro culture (IVC) for embryo development evaluation. Embryos from both groups were cultured in SOF medium with 2.5% FCS, 0.05 g mL–1 BSA, 0.2 mm pyruvate, and 10 mg mL–1 gentamicin. Cleavage rates on the second day of in vitro culture (D2), embryo production at Days 7 and 8 (D7 and D8), and hatching rate at Day 8 were evaluated. Data regarding 1PB extrusion, cleavage, blastocyst development on D7 and D8, and blastocyst D8 hatching rates of three replicates were analyzed by chi-square test at 5% significance using the BIOESTATS 4.0 software. Control and BD, respectively, did not show differences (P > 0.05) regarding 1PB extrusion (n = 164, 63.81%, and n = 175, 66.79%) or cleavage (n = 117, 71.34%, and n = 138, 78.86%). However, for control and BD, respectively, blastocyst development on D7 (n = 63, 38.41%, and n = 89, 50.86%), D8 (n = 63, 38.41%, and n = 91, 52.00%), and hatching on D8 (n = 22, 34.92%, and n = 39, 43.82%) were all significantly higher for BD when compared with control (P < 0.05). In conclusion, BDNF during prematuration improved in vitro embryo development by increasing blastocyst and hatching rates of parthenogenetic embryos.

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 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&gt;0.05 ); but a significant difference was found in blastocyst development rates (P&lt;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.

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

2006 ◽  
Vol 18 (2) ◽  
pp. 286 ◽  
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

scholarly journals Diploid porcine parthenotes produced by inhibition of first polar body extrusion during in vitro maturation of follicular oocytes

Reproduction ◽  
2006 ◽  
Vol 132 (4) ◽  
pp. 559-570 ◽  
Author(s):  
Tamás Somfai ◽  
Manabu Ozawa ◽  
Junko Noguchi ◽  
Hiroyuki Kaneko ◽  
Katsuhiko Ohnuma ◽  
...  
Keyword(s):  
Polar Body ◽  
Formation Rate ◽  
Metaphase Plate ◽  
Chromosome Analysis ◽  
Blastocyst Stage ◽  
Control Group ◽  
Blastocyst Formation ◽  
First Polar Body ◽  
Polar Body Extrusion

We investigated nuclear progression and in vitro embryonic development after parthenogenetic activation of porcine oocytes exposed to cytochalasin B (CB) during in vitro maturation (IVM). Nuclear progression was similar in control oocytes and oocytes matured in the presence of 1 μg/ml CB (IVM-CB group) by 37 h IVM; at this time the proportion of oocytes that had reached or passed through the anaphase-I stage did not differ significantly between the IVM-CB and the control groups (61.3 and 69.9% respectively; P < 0.05). After IVM for 37 h, no polar body extrusion was observed in the IVM-CB group. In these oocytes, the two lumps of homologous chromosomes remained in the ooplasm after their segregation and turned into two irregular sets of condensed chromosomes. By 41 h IVM, the double sets of chromosomes had reunited in 89.5% IVM-CB oocytes and formed a single large metaphase plate, whereas 68.8% of the control oocytes had reached the metaphase-II stage by this time. When IVM-CB oocytes cultured for 46 h were stimulated with an electrical pulse and subsequently cultured for 8 h without CB, 39.0% of them extruded a polar body and 82.9% of them had a female pronucleus. Chromosome analysis revealed that the majority of oocytes that extruded a polar body were diploid in both the control and the IVM-CB groups. However, the incidence of polyploidy in the IVM-CB group was higher than that in the control group (P < 0.05). In vitro development of diploid parthenotes in the control and the IVM-CB groups was similar in terms of blastocyst formation rates (45.8 and 42.8% respectively), number of blastomeres (39.9 and 44.4 respectively), the percentage of dead cells (4.3 and 2.9% respectively), and the frequency of apoptotic cells (7.3 and 6.3% respectively). Tetraploid embryos had a lower blastocyst formation rate (25.5%) and number of cells (26.2); however, the proportion of apoptotic nuclei (7.0%) was similar to that in diploid parthenotes. These results suggest that the proportion of homozygous and heterozygous genes does not affect in vitro embryo development to the blastocyst stage.

372 SPERM ASTER FORMATION AND BLASTOCYST DEVELOPMENT OF IN VIVO-MATURED BOVINE OOCYTES FERTILIZED BY INTRACYTOPLASMIC SPERM INJECTION

2007 ◽  
Vol 19 (1) ◽  
pp. 301 ◽  
Author(s):  
T. Horiuchi ◽  
M. Takenaka ◽  
C. Kani ◽  
C. Emuta ◽  
Y. Ogata ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Blastocyst Development ◽  
Chi Square ◽  
First Polar Body ◽  
Bovine Oocytes ◽  
Sperm Aster

In cattle, activation treatment after intracytoplasmic sperm injection (ICSI) is required to improve cleavage and blastocyst rates (Horiuchi et al. 2002 Theriogenology 57, 1013–1024). The reason why the exogenous activation treatment in bovine ICSI is needed to promote cleavage and blastocyst development is not clear. The objective of this study was to examine the effect of activation treatment on sperm aster formation, cleavage, and blastocyst development of in vivo- and in vitro-matured bovine oocytes following ICSI. In vivo-matured oocytes were collected using transvaginal devices under ultrasound guide at about 29 h after GnRH injection from Japanese Black cows superstimulated with a total 19 mg FSH (Antrin�; Denka Pharmaceutical Co., Kanagawa, Japan) divided into twice daily over 3 days, and treated with 750 �g cloprostenol (Estramate�; Sumitomo Chemical Co., Tokyo, Japan). In a total of 8 aspiration sessions, 131 oocytes were collected; of 116 oocytes with expanded cumulus cells, 84 (72%) had a first polar body and were used for ICSI. On the other hand, in vitro-matured bovine oocytes were prepared by culturing immature follicular oocytes derived from abattoir ovaries. Bull spermatozoa, immobilized by scoring their tails, were injected into in vivo- or in vitro-matured oocytes. At 4 h after ICSI, the oocytes were treated with or without 7% ethanol for 5 min for activation. The injected oocytes were fixed at 8 h after ICSI, and sperm aster formation was examined by using specific antibodies and immunofluorescence microscopy. Data were analyzed by the chi-square test in all experiments. The rate of sperm aster formation in in vivo-matured oocytes was similar regardless of activation treatment (71% vs. 65%), but the rate in in vitro-matured oocytes was significantly (P &lt; 0.05) higher in the group receiving activation treatment than in the non-activation group (57% vs. 19%). Cleavage (88% vs. 88%) and blastocyst rates (59% vs. 47%) of in vivo-matured oocytes after ICSI were also similar, regardless of activation treatment, but cleavage (72% and 20%) and blastocyst rates (19% and 7%) of in vitro-matured oocytes were significantly (P &lt; 0.05) higher in the group receiving activation treatment than in the non-activation group. Moreover, the blastocyst rate of in vivo-matured oocytes was significantly (P &lt; 0.05) higher than the rate in in vitro-matured oocytes. These results show that activation treatment after ICSI of in vivo-matured bovine oocytes is not necessary for cleavage and blastocyst development, and suggest that the necessity of activation treatment in bovine ICSI has relevance to in vitro maturation of bovine oocytes.

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