133 VITRIFICATION CAUSES DAMAGE OF THE ANTIOXIDANT DEFENSE SYSTEM IN IVM PORCINE OOCYTES

2007 ◽  
Vol 19 (1) ◽  
pp. 184 ◽  
Author(s):  
T. Somfai ◽  
M. Ozawa ◽  
J. Noguchi ◽  
H. Kaneko ◽  
K. Ohnuma ◽  
...  
Keyword(s):  
Polar Body ◽  
Antioxidant Defense System ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Cleavage Rate ◽  
Sperm Penetration ◽  
Second Polar Body ◽  
Polar Body Extrusion

The present study investigated the ability of in vitro-matured (IVM) porcine oocytes to be fertilized in vitro after vitrification. Oocytes matured in vitro for 46 h according to Kikuchi et al. (2002 Biol. Reprod. 66, 1033–1041) were cryopreserved by solid surface vitrification (SSV; Dinnyes et al. 2000 Biol. Reprod. 63, 513–518) or subjected to the steps of SSV without cooling (toxicity control, TC). Oocyte viability was assessed 2 h after treatment by morphology and fluorescein diacetate staining. Live oocytes were in vitro-fertilized (IVF) and cultured (IVC) for 6 days according to Kikuchi et al. (2002). Fertilization and pronuclear development of oocytes were assessed 10 h after IVF by aceto-orcein staining. Cleavage and blastocyst rates were recorded during IVC. Glutathione (GSH) and hydrogen peroxide levels in oocytes were analyzed by DTNB-glutathione disulfide reductase recycling assay and 20,70-dichlorofluorescein fluorescence assay, respectively. Data were analyzed by ANOVA and paired t-test. The rate of live oocytes after SSV was lower compared to the control and the TC groups (54.4%, 100%, and 100%, respectively; P < 0.05). Sperm penetration rates of SSV oocytes were lower than those of the control group (51.9% and 67.8%, respectively; P < 0.05). Significantly fewer penetrated oocytes in the SSV group formed male pronuclei than those in the control and the TC groups (66.7%, 96.5%, and 98.5%, respectively; P < 0.05). There were no differences in second polar body extrusion and monospermy rates between the treatment groups. The cleavage rate of SSV oocytes was significantly lower than that of the control and the TC groups (13.3%, 46.6%, and 47.7%, respectively; P < 0.05). Blastocyst rates of control and TC oocytes were similar (20.7% and 23.6%, respectively), whereas only a single embryo developed to the blastocyst stage in the SSV group. GSH content of SSV oocytes was significantly lower than that of the control oocytes (7.3 pM and 10.5 pM, respectively), whereas the peroxide level was higher in SSV oocytes than in the control oocytes (59.0 and 50.5 FIU, respectively; P < 0.05). Our results reveal a cryopreservation-related drop of intracellular GSH level in oocytes, which may cause their decreased ability to form a male pronucleus and their increased sensitivity to oxidative stress. These factors might contribute to the low developmental competence of vitrified oocytes. This work was supported by a grant-in-aid for the Japanese Society for the Promotion of Science Postdoctoral Fellowship for Foreign Researchers (P05648) and the Bilateral Scientific and Technological Collaboration Grant between Hungary and Japan (TET, no. JAP-11/02).

scholarly journals Effect of Heat Stress on Developmental Competence of In Vitro Matured Oocytes of Camelus Dromedaries with Different Qualities

2020 ◽  
Vol 10 (4) ◽  
pp. 658-664
Author(s):  
G Ashour ◽  
Ashraf El-Sayed ◽  
M Khalifa ◽  
Nasser Ghanem
Keyword(s):  
Heat Stress ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Cleavage Rate ◽  
Developmental Capacity ◽  
Polar Body Extrusion

The deleterious effect of heat stress on cumulus-oocytes complexes (COCs) competence is well recognized in different livestock species. Therefore, the present study aimed to investigate the effect of physiologically relevant heat stress on the developmental competence of camel COCs during in vitro maturation (IVM). A total of 1548 COCs were divided into six groups in this study. The groups were named K1 and K2 representing good and low-quality COCs incubated at 38.5oC for 30 hours. While K3 and k4 represent good and low-quality COCs exposed to 41oC for the first 6 hours of IVM. Finally, K5 and k6 represent the groups of good and low-quality COCs exposed to 42oC for the first 6 hours of IVM. After exposure of COCs to heat stress at 41°C and 42°C during the first 6 hours of in vitro maturation, the COCs were incubated at 38.5°C for 24 hours of IVM. The in vitro matured COCs were activated to cleave using ethanol followed by 4 mM 6-DMAP and developed embryos were cultured in vitro for 7 days post parthenogenetic activation. The results of this study indicated that heat stress at 42oC significantly decreased the Pb (polar body) extrusion rate in K4 and K6, compared to other groups. Additionally, the embryo cleavage rate was significantly lower for good and low-quality oocytes exposed to heat stress (K2, K3, K4, K5, and K6), compared to good quality COCs of the control group (K1). The cleavage rate was lower for low quality (K2; 63 ± 1.28) than good quality COCs (K1; 53 ± 1.85). The percentages of oocytes that developed to the blastocyst stage were lower for K2, K3, K4, K5, and K6 than K1. Moreover, the blastocyst rate was lower for K2 (9 ± 0.22) than K1 (15 ± 0.22). The results of this study indicated that exposure of camel oocytes to heat stress for 6 hours during in vitro maturation severely reduced extrusion of polar body, cleavage, and blastocyst rates. The low-quality camel COCs were reduced developmental capacity than good quality oocytes.

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

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.

44 EFFECT OF CYTOPLAST VOLUME ON FERTILIZATION AND EMBRYO DEVELOPMENT IN PORCINE M-II OOCYTES RECONSTRUCTED WITH KARYOPLASTS AND CYTOPLASTS OBTAINED BY THE 'CENTRI-FUSION' METHOD

2008 ◽  
Vol 20 (1) ◽  
pp. 102
Author(s):  
N. Maedomari ◽  
K. Kikuchi ◽  
M. Fahrudin ◽  
N. Nakai ◽  
M. Ozawa ◽  
...  
Keyword(s):  
Polar Body ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
First Polar Body ◽  
Developmental Capacity ◽  
Sperm Penetration ◽  
Pronuclear Formation ◽  
And Control

Metaphase-II chromosome transfer (M-II transfer) of oocytes is considered to be one of the advanced procedures to improve fertilization and developmental abilities of oocytes with poor cytoplasmic maturation. The aim of this study was to investigate the developmental capacity after IVF and IVC of porcine oocytes reconstructed from karyoplasts and cytoplasts produced by centri-fusion (Fahrudin et al. 2007 Cloning Stem Cells 9, 216–228). In brief, IVM oocytes (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041) with a visible first polar body were centrifuged at 13 000g for 9 min to stratify the cytoplasm. Then the zonae pellucidae were removed with pronase treatment. Zona-free oocytes were layered on a 300-µL discontinuous gradient of Percoll in TCM-HEPES with 5 µg mL–1 of cytochalasin B. After centrifugation at 6000g for 4 s, fragmented cytoplasms with approximately equal volumes were obtained, stained with Hoechst-33342, and classified into cytoplasm with (K; karyoplast) or without (C; cytoplast) chromosomes. One karyoplast was fused with 0, 1, 2, 3, and 4 cytoplasts (K, K + 1C, K + 2C, K + 3C, and K + 4C, respectively) by an electric stimulation with a single DC pulse (1.5 kV cm–1 for 20 µs) and cultured for 1 h. Zona-free oocytes without any reconstruction served as control oocytes. The diameters of the reconstructed and control oocytes were measured. All specimens were fertilized in vitro with frozen–thawed boar sperm, and cultured using the well of the well (WOW) system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264). Their fertilization status and developmental competence were examined. Data were analyzed by ANOVA followed by Duncan's multiple range tests. The diameter differed significantly among K to K + 4C oocytes (75.0–127.1 µm; P < 0.05), whereas the diameter of K + 2C oocytes was similar to that of the control oocytes (110.5 µm). Regardless of the cytoplast volume, sperm penetration rates (73.1–93.8%) for K to K + 4C oocytes were not significantly different compared to control oocytes (78.0%). Male pronuclear formation rates of K to K + 4C oocytes (92.3–97.1%) were also not different significantly different compared to control oocytes (96.6%). However, monospermy rates of K oocytes was significantly higher (61.6%; P < 0.05) than those of the reconstructed (K + 1C to K + 4C; 18.2–34.9%) and control oocytes (32.9%). The blastocyst formation rates in K, K + 1C, K + 2C, and K + 3C groups (0.0–9.8%; P < 0.05) were significantly lower than those in the control and K + 4C groups (17.8% and 15.3%, respectively; P < 0.05). The total cell numbers per blastocyst in K + 1C and K + 2C groups (7.5 and 8.3 cells, respectively) were significantly lower than in the control, K + 3C, and K + 4C groups (15.3–26.2 cells; P < 0.05). These results suggest that the cytoplast volume of porcine M-II transferred oocytes, produced by reconstruction from a karyoplast and cytoplast(s) and centri-fusion, is important for their ability to develop to the blastocyst stage and influences cell number.

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.

scholarly journals Effects of Stem Cell Factor/c-Kit Signaling on In Vitro Maturation of Porcine Oocytes and Subsequent Developmental Competence After Fertilization

2021 ◽  
Vol 8 ◽  
Author(s):  
Eunhye Kim ◽  
Lian Cai ◽  
Sang-Hwan Hyun
Keyword(s):  
Stem Cell ◽  
Mrna Expression ◽  
Stem Cell Factor ◽  
Polar Body ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Control Group ◽  
Secreted Factors ◽  
Polar Body Extrusion

Stem cell factor (SCF), also known as c-Kit ligand, plays an important role in the proliferation of primordial germ cells and the survival of oocytes during follicular development. The aim of this study was to investigate the effect of SCF/c-Kit signaling on in vitro maturation (IVM) of porcine oocytes by analyzing nuclear and cytoplasmic maturation, oocyte size, cumulus cell expansion, and developmental competence to the blastocyst stage. Moreover, mRNA expression patterns of porcine cumulus cells and oocytes were evaluated using qRT-PCR. Following 42 h of IVM, 10 and 50 ng/mL SCF-treated groups exhibited significantly (P &lt; 0.05) increased polar body extrusion rates and intracellular glutathione levels compared with the control group. The cumulus expansion index significantly (P &lt; 0.05) increased in all SCF-treated groups compared with the control samples. mRNA levels of the proapoptotic gene Bax and apoptosis-related cysteine peptidase Caspase3 were lower in SCF-treated cumulus cells than in the control group. Notably, the diameter of oocytes after IVM, the mRNA expression of well-known oocyte-secreted factors (GDF9 and BMP15), and an oocyte-specific protein essential for ovulation and oocyte health (YBX2) were significantly (P &lt; 0.05) higher in SCF-treated than in non-treated oocytes. Inhibition of c-Kit during porcine IVM using ACK2, an antagonistic blocker of c-Kit, significantly (P &lt; 0.05) decreased the polar body extrusion rate compared with the control, as well as blastocyst formation rate compared with the 10 ng/mL SCF-treated group. In conclusion, the effect of SCF/c-Kit-mediated signaling during porcine IVM could be ascribed to the reduced expression of apoptosis-related genes and higher expression of oocyte-specific/secreted factors.

188 HAPLOID ACTIVATION OF BOVINE OOCYTES WITH IONOMYCIN AND SINGLE OR COMBINED ACTIVATING AGENTS

2016 ◽  
Vol 28 (2) ◽  
pp. 225 ◽  
Author(s):  
M. Suvá ◽  
N. G. Canel ◽  
D. F. Salamone
Keyword(s):  
Embryo Development ◽  
Polar Body ◽  
Reproductive Technologies ◽  
Mitogen Activated Protein Kinase ◽  
Cleavage Rate ◽  
Extrusion Rate ◽  
Second Polar Body ◽  
Polar Body Extrusion ◽  
Bovine Oocytes

Haploid activation of bovine oocytes is important for reproductive technologies such as intracytoplasmic sperm injection (ICSI) or somatic cell nuclear transfer (SCNT). Nevertheless, it is still a highly inefficient procedure. The aim of this work was to combine different activation drugs, known to have different targets along the activation cascade, to find a more effective activation protocol. Cumulus-oocyte complexes (COC) were aspirated from slaughtered ovaries and in vitro-matured (IVM) for 22 h. Oocytes were activated with 5 µM ionomycin (IO) for 4 min and then randomly allocated into 1 of the following treatments: 50 µM roscovitine (ROSC), 10 µg mL–1 cycloheximide (CHX), ROSC and 10 µM PD0325901 (ROSC/PD), or CHX and PD (CHX/PD) for 5 h; 15 µM dehydroleucodine (DHL) or DHL and ROSC (DHL/ROSC) for 3 h; DHL and CHX for 3 h followed by 2 h with CHX; 5-min exposure to 7% ethanol 4 h post-IO (ET); or ET followed by ROSC (ET-ROSC). Controls were IO followed by 3 h of exposure to 1.9 mM 6-DMAP with or without a previous 3-h culture in TCM-199 (3 h in DMAP and DMAP, respectively). Embryos were cultured in SOF medium. Pronuclear formation (PN) and second polar body extrusion (2PB) were assessed by 5 µg mL–1 propidium iodide oocyte staining, 17 h after IO. Activation was defined as the presence of at least 1 PN, and 2PB extrusion rate was calculated regardless of the nuclear stage. Data were analysed by Fisher’s Test (P < 0.05). Activation (Table 1) was similar in all groups, with the exception of ROSC/PD and ET-ROSC that were the highest and DHL that was the lowest. Although ROSC or CHX seemed to improve 2PB rate when combined with DHL, cleavage decreased significantly, suggesting DHL itself, or its combination with these drugs, negatively affects embryo development. Group ET showed activation rates comparable to other treatments, but it was not reflected on cleavage, suggesting that ET induces PN formation but it might be inefficient to trigger embryo development. Nevertheless, this observation was not made for ET-ROSC, as it showed a higher cleavage rate than ET and ROSC alone. The mitogen-activated protein kinase (MAPK) inhibitor PD showed different effects when combined with ROSC or CHX, despite that they both act on the mammary fat pad (MPF). In ROSC/PD, a slight improvement was observed on activation and cleavage rates compared with ROSC. Group CHX/PD resulted in a slightly higher 2PB percentage, but a lower activation percentage that derived in a significantly lower cleavage than CHX. In conclusion, ROSC and CHX were the most effective single treatments for haploid activation. Moreover, some combined treatments, namely DHL/ROSC and DHL/CHX, proved to be as effective or better at 2PB extrusion rate, which is the defining feature in haploid activation. Table 1.Activation, second polar body extrusion (2PB) and cleavage of bovine oocytes activated with ionomycin followed by single or combined activating agents1

39 THE EFFECTS OF RESVERATROL DURING IN VITRO MATURATION ON THE DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES VITRIFIED AT THE IMMATURE STAGE

2017 ◽  
Vol 29 (1) ◽  
pp. 127 ◽  
Author(s):  
E. C. S. Santos ◽  
T. Somfai ◽  
R. Appeltant ◽  
T. Q. Dang-Nguyen ◽  
H. Kaneko ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Polar Body ◽  
Developmental Rate ◽  
Immature Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Dibutyryl Camp ◽  
Cleavage Rate ◽  
Nuclear Maturation

Previously, live offspring have been produced from porcine oocytes vitrified at the immature stage (Somfai et al. 2014 PLoS One 9, e97731); however, their embryo developmental rates remain low. The aim of our current research was to test the effects of resveratrol, an antioxidant and anti-apoptotic agent on the developmental competence of immature vitrified oocytes during in vitro maturation (IVM) after warming. Follicular porcine cumulus-oocyte complexes (COC) were vitrified on Cryotop® sheets (Kitazato Corp. Shizuoka, Japan) using the cryoprotectant treatment and warming method of Somfai et al. (2015 J. Reprod. Dev. 61, 571–579). After warming, the oocytes were subjected to IVM for 46 h in a chemically defined porcine oocyte medium (POM) enriched with 10 ng mL−1 epidermal growth factor, 10 IU mL−1 eCG, and 10 IU mL−1 hCG. During the first 22 h of IVM, the medium was supplemented with 1 mM dibutyryl cAMP. The following 24 h of IVM was performed in POM without dibutyryl cAMP. Vitrified/warmed COC (vitrified group) and freshly collected COC (control group) were matured either in the absence or presence of 2 µM resveratrol (RES− and RES+, respectively) throughout the entire IVM. At the end of IVM, oocytes were denuded and their survival was evaluated. Then, those with 1 polar body (PB1+) were selected for parthenogenetic activation (Day 0). Activated oocytes were cultured for 7 days in PZM-3. Survival, nuclear maturation, cleavage, and blastocyst rates were assessed. The experiment was replicated 5 times. Results were analysed by one-way ANOVA and Tukey’s multiple comparison test. Vitrification reduced the percentage of live oocytes after IVM both in RES− and RES+ groups in a similar manner (47.9 and 51.8%, respectively) compared with control RES− and RES+ groups (99.4 and 100%, respectively; P < 0.05) There was no statistical difference among groups in the percentage of PB1+ oocytes (ranging between 76.1 and 90.2%). On Day 2, the cleavage rate in vitrified RES− group was lower than those in control RES− and RES+ groups (55.9 v. 78.5% and 79.2%, respectively) whereas the vitrified RES+ group did not differ from the others (72.1%). The blastocyst developmental rate calculated from total cultured oocytes on Day 7 in vitrified RES+ group was significantly higher (P < 0.05) than that in the vitrified RES− group (26.2% v. 6.9%, respectively) and did not differ significantly from those of control RES− and RES+ groups (32.1 and 36.0%, respectively). Blastocyst rates in control RES− and RES+ groups were significantly higher (P < 0.05) than that in vitrified RES− group but did not differ from one another. In conclusion, supplementation of IVM medium with resveratrol improved the developmental competence of vitrified, but not freshly collected oocytes. This work was supported by JSPS KAKENHI (Grant Number: 26870839) and JST/JICA SATREPS. E.C.S. Santos was supported by a CNPq-Brasil fellowship.

346 BOVINE EMBRYO DEVELOPMENT AFTER IVM/IVF/IVC OF OOCYTES STORED FOR 22 H IN VARIOUS MEDIA

2007 ◽  
Vol 19 (1) ◽  
pp. 288
Author(s):  
C. Kubota ◽  
T. Kojima ◽  
T. Nagai ◽  
X. Tian ◽  
X. Yang
Keyword(s):  
Subsequent Development ◽  
Industrial Applications ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Bovine Embryo ◽  
Becton Dickinson ◽  
In Vitro Storage ◽  
Bovine Oocytes

The timing of IVM–IVF–IVC is restricted by the onset of oocyte maturation, and sometimes oocytes must be treated at midnight. If we could regulate the timing of IVM of oocytes without decreasing their developmental competence, the IVM–IVF–IVC system could be a more applied technology. The present study was performed to examine the effects of in vitro storage of bovine oocytes in simple media prior to maturation culture to manipulate the start of IVM. Bovine follicular fluid (bFF), Dulbecco&apos;s PBS (PBS), M199 Earle salts (M199), and Earle salts supplemented with 5 mM NaHCO3 (M199A) were used as the fundamental media, after an addition of antibiotics, for in vitro storage of bovine cumulus&ndash;oocyte complexes (COCs) collected from ovaries obtained at the slaughterhouse. The fundamental media except for bFF were supplemented with 10&percnt; fetal bovine serum (FBS) or 1 mg mL&minus;1 polyvinyl alcohol (PVA). COCs were collected from follicles (3&ndash;8 mm in diameter) and washed twice in each medium; then approximately 50 COCs were submerged in 1 mL of each medium in cryotubes (Falcon #2812, 2.5 mL; Becton Dickinson Labware, Lincoln, NJ, USA), which were stored in a container kept at 38.5&deg;C for 22 h under air-closed condition (in vitro storage: IVS). Subsequently, the stored COCs were in vitro-matured (IVM) for 22 h in M199 with 10&percnt; FBS and 20 &micro;g mL&minus;1 estradiol, fertilized (IVF), and cultured in CR1aa (IVC) for examination of their development to the blastocyst stage (Kubota et al. 1998 Mol. Reprod. Dev. 51, 281&ndash;286). Fresh oocytes without IVS were used as controls. The nuclear status of oocytes after IVS&ndash;IVM was compared to that of control oocytes by aceto-orcein stain. Their developmental rates to the blastocyst stage after IVM&ndash;IVF&ndash;IVC were compared between experimental and control groups. The experiment was repeated more than 3 times, and results were statistically analyzed using Student&apos;s t-test. When bFF and PBS supplemented with FBS or PVA were used for IVS, the rates of survived COCs after IVS and the development to the blastocyst stage after IVM&ndash;IVF&ndash;IVC (bFF (n &equals; 87): 0&percnt;, 0&percnt;; PBS/FBS (n &equals; 72): 84&percnt;, 1&percnt;; and PBS/PVA (n &equals; 81): 89&percnt;, 6&percnt;, respectively) were significantly lower than those of the control group (n &equals; 406; 97&percnt; and 29&percnt;, respectively). On the other hand, when M199A supplemented with FBS or PVA was used for IVS, the survival rate after IVS and the developmental rate to the blastocyst stage after IVS&ndash;IVM&ndash;IVF (M199A/FBS (n &equals; 97): 82&percnt;, 28&percnt;; and M199A/PVA (n &equals; 111): 98&percnt;, 31&percnt;, respectively) did not differ from those of the control group. After IVS, cumulus expansion was not seen and most of the oocyte nuclei reached the GVBD stage. These results suggest that the nuclear maturation progress of bovine oocytes can be regulated for at least 22 h in M199A without any deleterious influence on the number of oocytes surviving at an immature state after the storage and their subsequent development to the blastocyst stage after IVM&ndash;IVF&ndash;IVC. The delayed maturation allows a flexible fertilization schedule which is advantageous in research and industrial applications.

56 THE EFFECT OF VITRIFICATION FOR SHEEP OOCYTES VIABILITY

2015 ◽  
Vol 27 (1) ◽  
pp. 121 ◽  
Author(s):  
Y. M. Toishibekov ◽  
R. K. Tursunova ◽  
M. Sh. Yermekova
Keyword(s):  
Polar Body ◽  
Control Group ◽  
Polar Bodies ◽  
Maturation Medium ◽  
Chi Squared ◽  
Fetal Bovine ◽  
Second Polar Body ◽  
Cryopreservation Techniques ◽  
Humidified Air

Advances in reproduction technologies, such as in vitro maturation, IVF, and in vitro culture, stimulated research for efficient cryopreservation techniques for mammalian oocytes. It is well known that the oocyte is the largest cell of an animal's body and as such, is full of water and, in many species, fat, making it difficult to cryopreserve. The objective of this work was to study the effect of vitrification for cryopreservation of the metaphase II plate (MPII) of sheep oocytes. Ovaries from 20 ewes of Kazakh Arkharo-Merino breed were acquired after slaughter and maintained at 37°C in TCM-199. The maturation medium was TCM-199, containing 1 mM of glutamine, 10% FBS, 5 μg mL–1 FSH, 5 μg mL–1 LH, 1 μg mL–1 oestradiol, 0.3 mM sodium pyruvate, and 100 mM cysteamine. The oocytes were incubated in 400 μL of medium in 4-well dishes covered with mineral oil. The IVM conditions were 5% CO2 in humidified air at 39°C for 24 h. Then they were placed for 10 min in a media with Hoechst 33342 (3 μg mL–1) and cytochalasin B (7 μg mL–1) to facilitate the enucleation of the MPII with a minimum volume of ooplasm. The MPII plates were divided into 2 groups: the vitrification group was exposed to vitrification media containing 1.12 M ethylene glycol (ET) + 0.87 M ME2SO for 5 min and was exposed in vitrification media containing 2.24 M ET + 1.75 M ME2SO for 5 min, and then in vitrification solution containing 4.48 M ET + 40% ME2SO + 0.25 M sucrose for 30 s. Oocytes were loaded into cryoloop and plunged into liquid nitrogen (LN2). Oocytes were thawed in a 25°C water bath and then placed in TCM-199 at 20% fetal bovine serum. After 15 min of incubation the oocytes were activated for extrusion of the second polar body in 1 mg mL–1 Ca ionophore for 5 min and washed for 5 min followed by 4 h in 6-DMAP (0.12 mM) + cycloheximide (0.6 μg mL–1). After activation the MPII were washed and cultured for 20 h. The control group received the same treatment, but they were not vitrified. Differences between the experimental groups were tested using Chi-squared test. Our research showed the expulsion of the second polar body after activation was observed in more than 62.2% of the MPII that were not vitrified (control group), whereas 40.5% of vitrified plates had expulsion of polar bodies (P < 0.05). These preliminary studies showed that it is possible to vitrify MPII plates. On the other hand, the drastic reduction of the volume of the sheep oocytes might make cryopreservation possible with greater efficiency.

44 IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS GENERATED WITH BOVINE OOCYTES AND EQUINE SKIN FIBROBLASTS

2011 ◽  
Vol 23 (1) ◽  
pp. 128
Author(s):  
J. Lee ◽  
J. Park ◽  
Y. Chun ◽  
W. Lee ◽  
K. Song
Keyword(s):  
Nuclear Transfer ◽  
Polar Body ◽  
Donor Cell ◽  
Skin Fibroblasts ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Bovine Oocytes

Study for equine somatic cell nuclear transfer (SCNT) is an attractive field for research, but it has not been a major field of study because it is hard to obtain a sufficient number of ovaries and it takes a lot of time and effort for the recovery of oocytes matured in vivo by ovum pickup. It was reported that the bovine cytoplast could support the remodelling of equine donor cells (Zhou et al. 2007 Reprod. Domest. Anim. 42, 243–247). The objectives of this study are 1) to monitor the early events of equine SCNT by interspecies SCNT (isSCNT) between bovine cytoplast and equine donor cell, and 2) to investigate the developmental competence of isSCNT embryos. Bovine oocytes were recovered from the follicles of slaughtered ovaries, and matured in TCM-199 supplemented with 10 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS at 39°C under 5% CO2 in air for 22 h. Fibroblasts derived from bovine or equine skin tissues were synchronized at G0/G1 stage by contact inhibition for 72 h. After IVM, oocytes with polar body were enucleated and electrically fused with equine or bovine skin fibroblasts (1.0 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 10 μg mL–1 cycloheximide (CHX) and/or 2 mM 6-DMAP, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C under 5% CO2, 5% O2, and 90% N2 for 7 days. All analyses were performed using SAS (version 9.1; SAS Institute, Cary, NC, USA). The cleavage rate of isSCNT embryos derived from equine cell was not different (252/323, 78.7%; P = 0.94) from that of SCNT embryos derived from bovine cell (230/297, 79.2%). However, the rate of isSCNT embryos developed to over 8-cell stage was lower (3.3%; P < 0.0001) than that of bovine SCNT embryos (39.4%), and total cell number of isSCNT embryos developed to over 8-cell stage was lower (17.5, n = 12; P < 0.0001) than that (80.8, n = 110) of bovine SCNT embryos. Also, the rate of blastocyst formation of isSCNT embryos (0/323; 0.0%) was lower (P < 0.0001) than that of bovine SCNT embryos (83/297; 29.3%). Meanwhile, reconstructed oocytes for isSCNT were fixed at 8 h after activation to investigate the formation of pseudo-pronucleus (PPN) after post-activation treatment with CHX or CHX+6-DMAP. The ratio of oocytes with single PPN after treatment with CHX+6-DMAP (26/35; 74.3%) was not different (P = 0.63) from that of oocytes treated with CHX (24/36; 68.1%). Although isSCNT embryos derived from bovine cytoplast and equine donor cell could not develop to more than the 16-cell stage, it is believed that the results of this isSCNT study could be used for the preliminary data regarding the reprogramming of donor cell in equine SCNT.

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