230 IN VITRO MATURATION OF OOCYTES FROM ENDANGERED DORCAS GAZELLE (GAZELLA DORCAS NEGLECTA)

2006 ◽  
Vol 18 (2) ◽  
pp. 223 ◽  
Author(s):  
E. R. S. Roldan ◽  
F. Berlinguer ◽  
S. Succu ◽  
R. Gonzalez ◽  
A. del Olmo ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Captive Breeding ◽  
Polar Body ◽  
Calf Serum ◽  
Genetic Material ◽  
Cumulus Cells ◽  
Ministry Of Education ◽  
Cell Stage ◽  
Captive Breeding Program

In vitro maturation of oocytes recovered from dead animals provides an opportunity for rescuing genetic material for biodiversity conservation. The dorcas gazelle (Gazella dorcas) is regarded by the World Conservation Union (IUCN) as ‘vulnerable’ but the subspecies G. dorcas neglecta is thought to be endangered due to excessive hunting. A captive breeding program for dorcas gazelles has been developed at the Estacion Experimental de Zonas Aridas (CSIC) in the South of Spain where efforts have so far concentrated on natural breeding and on the development of sperm cryopreservation protocols. The aim of the present study was to explore the possibility of recovering and maturing in vitro healthy oocytes from animals that die suddenly for the establishment of a program to rescue female gametes. Ovaries of a dorcas female that died unexpectedly were collected about 7 h after death of the animal. Cumulus–oocyte complexes (COCs) were recovered by slicing the ovaries. Collection and washing of COCs were performed in warmed TCM-199-HEPES with antibiotics and polyvinyl alcohol. Degenerated oocytes or those with expanded cumulus cells were removed. A total of 15 COCs were cultured in TCM-199 with 10% heat-treated fetal calf serum, 10 μg/mL ovine FSH/LH, 1 µg/mL estradiol, and 0.1 mg/mL glutamine at 38.5°C under 5% CO2/air with high humidity. After 24 h of culture, matured oocytes, as revealed by the presence of a polar body, were activated with 7% ethanol for 10 min and further incubation for 3 h. Meiotic progression and activation were evaluated by staining with Hoechst 33342 and propidium iodide (1 μg/mL each) and visualization under a fluorescence microscope. Results at the end of incubations showed that 4/15 oocytes were degenerated, 4/15 were arrested at the MI stage, and 7/15 (46.7%) progressed to the MII stage. One oocyte was found to be at the 2-cell stage but it could not be established whether this was the result of the activation method used. These results demonstrate that it is possible to recover viable oocytes several hours after death and rescue them for subsequent in vitro maturation and fertilization. More studies are needed to characterize suitable conditions for oocyte maturation, fertilization, and culture in the dorcas gazelle. This would, in turn, help in the effort to rescue biomaterials from wildlife for generating offspring. This work was supported by the Spanish Ministry of Education and Science (REN 2003–01587) and Acciones Integradas (HI20030336).

326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 270
Author(s):  
C. Hanna ◽  
C. Long ◽  
M. Westhusin ◽  
D. Kraemer
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Germinal Vesicle Breakdown ◽  
Significant Difference ◽  
Culture Duration

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

334 EFFECT OF INSULIN ON IN VITRO MATURATION OF CANINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 275
Author(s):  
H. S. Lee ◽  
Y. I. Seo ◽  
X. J. Yin ◽  
S. G. Cho ◽  
I. H. Bae ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Uv Light ◽  
Cumulus Cells ◽  
Oocyte Activation ◽  
Cell Stage ◽  
Oocyte Competence ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Oviduct Fluid

In spite of our increased knowledge of in vitro oocyte maturation techniques, the success rate of obtaining mature canine oocytes in vitro remains very low compared with that for other domestic animals. The inefficient rate of meiotic resumption of canine oocytes is probably due to both the unique reproductive cycle and inappropriate in vitro maturation (IVM) medium. In an unpublished experiment, we found that the concentration of insulin was higher in estrus bitch serum (EBS; 8833 pg/mL) than in dog follicular fluid (DFF; preovulatory follicle, 122 pg/mL), which implies its possible role in the acquisition of oocyte competence. Therefore, in the present study we investigated the effects of supplementing the IVM medium with insulin on the incidence of maturation to metaphase II. Ovaries were collected from various stages of the estrous cycle by ovariohysterectomy, and oocytes with two or more intact cumulus layers and with a diameter >110 �m were selected and used for IVM. Oocytes were cultured in modified synthetic oviduct fluid (2004 Reprod. Nutr. Dev. 44, 105-109) supplemented with 10% EBS, 20 �g/mL estradiol, and different concentrations of insulin (0, 10, 100, or 1000 ng/mL) at 38.5�C, 5% CO2 in air. After 72 h, cumulus cells were removed from around oocytes using a small glass pipette. Denuded oocytes were fixed in 3.7% paraformaldehyde supplemented with 10 �g/mL Hoechst 33342 at room temperature for 40 min. Nuclear status was observed under UV light using a fluorescence microscope. The percentage of oocytes at the metaphase II stage was not different among the four groups 6.8, 1.8, 5.4, and 2.1% in the control, 10, 100, and 1000 ng/mL insulin groups, respectively. The incidence of oocytes with pronuclear-like structures or cleaving beyond the two-cell stage was not significant higher in the 10 and 100 ng/mL insulin treatment groups than in the control and 1000 ng/mL insulin groups 20.0 and 19.6% vs. 6.8 and 6.4%, respectively. These results indicate that the addition of insulin to the in vitro maturation medium of dog oocytes had no effect on the incidence of meiotic maturation to metaphase II, nor did it affect the frequency of occurrence of spontaneous oocyte activation.

224 LOCALIZATION OF NITRIC OXIDE SYNTHASE ACTIVITY IN BUFFALO (BUBALUS BUBALIS) OOCYTES AND EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 211
Author(s):  
K. R. Babu ◽  
R. Sharma ◽  
K. P. Singh ◽  
A. George ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Epifluorescence Microscopy ◽  
Rt Pcr ◽  
Cell Stage ◽  
Vitro Fertilization ◽  
Nos Isoforms

Ovarian nitric oxide (NO) and that produced within the oocytes and embryos have been reported to play important roles in oocyte meiotic maturation and embryo development. Production of NO is catalyzed by NO synthase (NOS), which exists in 3 isoforms, the constitutive endothelial (eNOS) and neuronal (nNOS) isoforms and the inducible (iNOS) isoform. We have previously shown that low concentrations of NO stimulate and high concentrations inhibit embryo development, and that endogenous NO produced by iNOS is necessary for optimal embryo development in the buffalo. The present study was aimed at localizing different isoforms of NOS and examining their relative mRNA abundance in buffalo oocytes and embryos. Oocytes from slaughterhouse ovaries were subjected to in vitro maturation in 100-μL droplets (10 to 15 oocytes/droplet) of in vitro maturation medium (TCM-199 + 10% FBS + 5 μg mL–1 of pFSH + 1 μg mL–1 of oestradiol-17β + 0.81 mM sodium pyruvate + 10% buffalo follicular fluid + 50 μg mL–1 of gentamicin) for 24 h in a CO2 incubator (5% CO2 in air) at 38.5°C. In vitro fertilization was carried out by incubating in vitro-matured oocytes with 2 to 4 million spermatozoa mL–1 for 18 h. The presumed zygotes were cultured on original beds of cumulus cells in in vitro culture medium (mCR2aa + 0.6% BSA + 10% FBS) for up to 8 days post-insemination. Immature and in vitro-matured oocytes and embryos at the 2-cell, 4-cell, 8- to 16-cell, morula, and blastocyst stages were examined for the presence of NOS isoforms by indirect immunofluorescence staining using epifluorescence microscopy and RT-PCR. Each experiment was repeated in triplicate, and data were analysed using one-way ANOVA, after arcsine transformation of percentage values. Expression of all 3 NOS isoforms was detected inside the cytoplasm, in all the stages of oocytes and embryos examined, by both immunofluorescence and RT-PCR. Abundance of the iNOS transcript was significantly higher (P ≤ 0.01) in the morula and blastocyst stages compared with that in immature and in vitro-matured oocytes and in embryos at the 2-cell, 4-cell, and 8- to 16-cell stages, indicating that its expression was up-regulated at the 8- to 16-cell stage. The expression of eNOS was significantly higher (P ≤ 0.05) in the immature and mature oocytes and in 8- to 16-cell stage embryos, morulae, and blastocysts than in the early-cleavage embryos at the 2- and 4-cell stages, indicating that it was down-regulated after fertilization and was up-regulated again at the 8- to 16-cell stage. Abundance of the nNOS transcript was not significantly different among all the stages of oocytes and embryos examined. These results demonstrate that different NOS isoforms are expressed in a dynamic manner during embryonic development in the buffalo. The role of an increase in expression of iNOS and eNOS at the 8- to 16-cell stage, at which a developmental block occurs in this species, needs to be examined.

scholarly journals 271 ASSESSMENT OF NUCLEAR STATUS OF ACTIVATED BOVINE OOCYTES MATURED IN DIFFERENT MATURATION CONDITIONS IN VITRO

2005 ◽  
Vol 17 (2) ◽  
pp. 285
Author(s):  
J.I. Park ◽  
Y. Jang
Keyword(s):  
Oocyte Maturation ◽  
Polar Body ◽  
Calf Serum ◽  
Chi Square ◽  
Cell Stage ◽  
Maturation Medium ◽  
Second Polar Body ◽  
Humidified Air ◽  
Bovine Oocytes

This study was carried out to assess the nuclear status after parthenogenetic activation in in vitro matured oocytes under different conditions. Bovine ovaries were collected from slaughtered cows at a local abattoir. Oocytes were aspirated from follicles of 3–8 mm in diameter and transferred to maturation medium: tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal calf serum, 100 mg/mL l-cysteine, 20 mg/mL sodium pyruvate, gonadotropins (each 250 IU of eCG and hCG/mL), and 10 mg/mL epidermal growth factor, with or without 5 mM hypotaurine and taurine. Oocytes were cultured at 38.9°C in 5% CO2 in humidified air. After 24 h of culture, oocytes with polar body were selected and submitted to activation treatments. Oocytes were exposed to calcium ionomycin (5 μM for 5 min) followed by incubation with 6-DMAP (2 mM), roscovitine (50 μM), or 6-DMAP + roscovitine for 3.5 h. After activation, oocytes were cultured in mSOF medium containing 0.8% BSA at 38.9°C in 5% CO2, 5% O2 in humidified air for 16 h and stained with Hoechst 33342 or aceto-orcein for assessment of nuclear status. Nuclear status was recorded as follows: 1PB (polar body) + 1PN (pronucleus), 2PB + 1PN and others. Data were analyzed using chi-square test. The maturation rate of bovine oocytes cultured in maturation medium containing hypotaurine/taurine (89.3%, n = 84) was higher (P < 0.05) than those cultured without hypotaurine/taurine (72%, n = 93). In the oocytes matured with hypotaurine/taurine, the rates of diploid activation (1PB + 1PN) were 84% (n = 50) in oocytes treated with 6-DMAP + roscovitine, 78.6% (n = 56) with 6-DMAP, and 52% (n = 50) with roscovitine. In the oocytes matured without hypotaurine/taurine, the rates of diploid activation were 80% (n = 60) in oocytes treated with 6-DMAP + roscovitine, 72% (n = 50) with 6-DMAP, and 54% (n = 50) with roscovitine. The rates of diploid activation were not different in oocytes matured with or without hypotaurine/taurine and among activation treatments. The oocytes treated with roscovitine showed a lower rate (P < 0.05) of diploid activation and higher rate (39.3–40%) of second polar body extrusion (1PN + 2PB) than the other activation groups in both maturation conditions. Cleavage rates to 2-cell stage were 40–45% in all groups. Development rate of blastocysts were 7–10% in all the groups treated with 6-DMAP and 6-DMAP + roscovitine and no blastocysts were obtained from the groups treated with roscovitine alone. Hypotaurine/taurine are known to be stable and potent antioxidants, and have shown the properties of supporting oocyte maturation and further embryonic development (Guerin and Menezo 1995 Zygote 3, 333–43; Mizushima and Fukui 2001 Theriogenology 55, 1432–45). In this study, although the effectiveness of hypotaurine/taurine on promoting oocyte maturation was observed, there were no significant improvements in the rate of diploid activation in oocytes matured with hypotaurine/taurine. These results suggest that the nuclear status of activated oocytes may not have a direct relationship with the enhanced maturation condition. This work was supported by BioGreen 21 Program(#1000520030100000-1), Republic of Korea.

scholarly journals In vitro maturation of domestic cat oocytes subjected to different incubation times

2021 ◽  
Vol 10 (3) ◽  
pp. e15710313074
Author(s):  
Denilsa Pires Fernandes ◽  
Fernanda Araujo dos Santos ◽  
Luã Barbalho de Macêdo ◽  
Roberta Gonçalves Izzo ◽  
Brenna de Sousa Barbosa ◽  
...  
Keyword(s):  
Incubation Period ◽  
Cell Expansion ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Domestic Cat ◽  
First Polar Body ◽  
The Ideal

The aim of this study was to evaluate the effect of three different incubation times on in vitro maturation of domestic cat oocytes. Thus, ovaries (n = 42) were submitted to slicing procedure and the oocytes recovered were classified; only good quality oocytes (Grade I and II) underwent in vitro maturation for three different periods (24 vs. 30 vs. 36 h) in supplemented TCM-99 medium. After, oocytes were evaluated for cumulus cell expansion and presence of the first polar body. After six replicates (7 ± 1,7 ovaries per replicate), a total of 334 viable oocytes were recovered. Differences (p <0.05) were observed regarding the percentage of oocytes presenting expansion of the cumulus cells, where higher values were observed in the group of oocytes incubated for 36 h (84.3%), when compared to 30 (73.4%) and 24 h (71.0%). Moreover, differences were also observed regarding the presence of the first polar body (24 h: 29.7%; 30 h: 58.2%; 36 h: 69.8%). We conclude that the incubation period influenced the maturation rates, indicating 36 h as the ideal period for the in vitro maturation of domestic cat oocytes in supplemented TCM-199 medium.

scholarly journals The Number of LH Receptor could Predict the Success of Oocyte Maturity in the Process of In Vitro Maturation

2016 ◽  
Author(s):  
Adek Amansyah
Keyword(s):  
In Vitro Maturation ◽  
Clinical Laboratory ◽  
Polar Body ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Maturity ◽  
Lh Receptor ◽  
First Polar Body ◽  
Significant Difference

Objective: To evaluate the relationship between the number of LH receptor and the success of oocyte maturity in the process of in vitro maturation (IVM). Method: This experimental study was conducted in the Permata Hati Infertility Clinical Laboratory, Dr. Sardjito General Hospital, Yogyakarta, with the samples of 300 oocytes obtained through collecting immature cow’s oocytes from the abattoir and grouped the oocytes into 3 (three) groups based on the pattern of oocyte cumulus cells on the vesicle germinal stage 2 - 8 mm with three layers of cumulus cell. The sample of the cumulus cells from these three groups were taken and the LH receptor examination was done with immunohistochemistry. After that, the IVM process was performed to the three groups and its development for 24 hours was evaluated. Its maturation quality was evaluated with the emergence of the first polar body (1PB) and compared to the other groups and related to the number of LH receptor in the three groups. Result: The result of this study indicated that the oocyte cumulus cells showed a difference of function during IVM process. The maturity rate in this study showed that the number of LH receptor was related to the morphological pattern of oocyte cumulus cells with oocyte maturity. The maturity of the cumulus cells which 100% covered the oocyte was higher than that of the cumulus cells which > 50% and < 30% covered the oocytes, namely, 74% compared to 60% and 12%. The result of this study also showed that the average number of LH receptors in the three groups (A, B, and C) was 183.4, 78.8, and 24.0 respectively. A significant difference was found in the three groups (p < 0.0001). When related to IVM maturity, this difference showed that the bigger number of oocyte cumulus cells influenced the oocyte maturity. Conclusion: The number of LH receptor can be used as a prediction to determine the success of oocyte maturation in the process of in vitro maturation. [Indones J Obstet Gynecol 2013; 1-4:183-7] Keywords: IVM, LH receptor, oocyte cumulus cell

39 CLONAL OFFSPRING DERIVED FROM SEPARATED BLASTOMERES IN CYNOMOLGUS MONKEYS (MACACA FASCICULARIS)

2008 ◽  
Vol 20 (1) ◽  
pp. 100
Author(s):  
C. Iwatani ◽  
J. Okahara-Narita ◽  
J. Yamasaki ◽  
H. Tsuchiya ◽  
R. Torii
Keyword(s):  
Cell Lines ◽  
Zona Pellucida ◽  
Cynomolgus Monkey ◽  
Behavioral Science ◽  
Polar Body ◽  
Calf Serum ◽  
Es Cell ◽  
Cell Stage ◽  
Splitting Technique

There are no reports of cloning by embryo splitting in the cynomolgus monkey, but production of genetically identical monkeys would have tremendous implications for biomedical research, especially for immunological studies, production of disease models, and behavioral science. Cloning would also reduce the number of animals required for the above research by increasing experimental reproducibility. In this study, we tried to produce cynomolgus monkey offspring by embryo splitting and embryo transfer. Controlled ovarian stimulation and oocyte recovery have been previously described by Torii et al. (2000 Primates 41, 39–47). Cumulus-free mature oocytes were fertilized by intracytoplasmic sperm injection. Single spermatozoa were individually immobilized by scoring their tails and picking them up with the injection pipette. The denuded oocyte was held by the holding pipette with the polar body in the 12 o'clock position. The injection pipette was then inserted at the 3 o'clock position and was introduced into the cytoplasm, breaking the ooplasmic membrane by pulling gently. One spermatozoon was injected into the cytoplasm. The injected oocytes were cultured at 38�C in 5% CO2, 5% O2 and 90% N2 in CMRL-1066 medium (Invitrogen, Grand Island, NY, USA) containing 20% calf serum (CS, Invitrogen) for 2–3 days. Splitting was performed using 4- to 7-cell-stage embryos. The zona pellucida was disrupted with acidic Tyrode's solution, and individual blastomeres were separated from the zona-free embryos by 0.25% trypsin-EDTA with added CaCl2 (<1 min). After transferring the zona-free embryos into TALP-HEPES medium, blastomeres were dissociated by pipetting with a 40–50 µm micropipette 4–5 times. These blastomeres were then transferred into empty zonae that had been produced from immature oocytes by the aspiration of ooplasm with a micromanipulator. Sixteen embryos underwent blastomere separation and a total of 33 split embryos were produced. After being cultured for 2–3 h in CMRL-1066 medium containing 20% CS, 30 of these split embryos, comprising 1–4 blastomeres each, were transferred into the oviducts of 23 fertile surrogate mothers at 0 to 5 days after ovulation. Pregnancy was confirmed in two animals (8.7%; 2/23) by ultrasound approximately 30 days after transfer. The pregnancies were uneventful and two normal healthy babies were born without any assistance 159 days after transfer. The low pregnancy rate could be due to the presence of fewer cells in the smaller split embryos, the ruptured zona pellucida, or the in vitro micromanipulation of embryos during blastomere separation and reconstruction. Here we report the first production of viable cloned offspring produced by blastomere separation in the cynomolgus monkey. Since we have previously succeeded in establishing ES cell lines from isolated blastomeres, in the future we will be able to produce genetically identical monkeys from a single 4- to 8-cell-stage embryo using those ES cell lines and the embryo splitting technique.

211 IN VITRO PRODUCTION OF SWAMP BUFFALO EMBRYOS BY INTRACYTOPLASMIC SPERM INJECTION: EFFECT OF CHEMICAL ACTIVATION TREATMENTS

2009 ◽  
Vol 21 (1) ◽  
pp. 203
Author(s):  
Y. Y. Liang ◽  
D. N. Ye ◽  
C. Laowtammathron ◽  
T. Phermthai ◽  
R. Parnpai
Keyword(s):  
Polar Body ◽  
Chemical Activation ◽  
Cumulus Cells ◽  
Success Rates ◽  
In Vitro Production ◽  
Cell Stage ◽  
Sham Injection ◽  
Swamp Buffalo ◽  
Second Polar Body

Intracytoplasmic spern injection (ICSI) in the buffalo has not yet been well examined. Several factors involved affect the success rates of this technique, particularly the postinjection activation procedure. The objective of this study was to evaluate the effects of chemical activation treatments on in vitro development of oocytes after ICSI. A single spermatozoa was injected into the cytoplasm of an in vitro-matured oocyte using a micromanipulator under an inverted microscope. The ICSI oocytes were assigned to the following chemical activation treatments: (1) exposed to 5 μm ionomycin (Io) in Emcare medium for 5 min and placed in Emcare medium for 3 h, or (2) exposed to 7% ethanol (EtOH) in Emcare medium for 5 min and placed in Emcare medium for 3 h. The treated oocytes that extruded a second polar body were then selected and cultured either in (A) 1.9 mm 6-dimethylaminopurine (6-DMAP) in mSOF medium for 3 h, or (B) 10 μg mL–1 of cychloheximide (CHX) for 5 h. The treated oocytes were further cultured in mSOF medium supplemented with 3 mg mL–1 of fatty acid-free BSA at 38.5°C under a humidified atmosphere of 5% O2, 5% CO2, and 90% N2 for 2 d. Thereafter, 8-cell-stage embryos were selected and co-cultured with buffalo cumulus cells in mSOF medium at 38.5°C under a humidified atmosphere of 5% CO2 in air for another 5 d. The medium was changed daily and the development of embryos was recorded at the same time the medium was changed. The sham-injected oocytes were treated and cultured along with ICSI oocytes. With 8 replications for each activation treatment, 336 oocytes were used for ICSI. With 6 replications for each activation treatment, 211 oocytes were used for sham injection. The cleavage of ICSI oocytes treated with Io + 6-DMAP, EtOH + 6-DMAP, and EtOH + CHX was 76.2, 69.4, and 78.3%, respectively, which was significant higher (P < 0.01) than ICSI oocytes treated with Io + CHX (52.4%) and also significant higher (P < 0.01) than sham-injected oocytes in all treatments. The highest blastocyst rate was observed in ICSI oocytes treated with Io + 6-DMAP (28.6%), which was not significantly different from ICSI oocytes treated with EtOH + CHX (24.4%). The blastocyst rates of ICSI oocytes treated with Io + 6-DMAP and EtOH + CHX were significantly higher than ICSI oocytes treated with Io + CHX (5.9%) and EtOH + 6-DMAP (16.5%) and also were significantly higher than sham-injected oocytes in all treatments. In conclusion, our study demonstrated that activated ICSI of swamp buffalo oocytes with Io + 6-DMAP or EtOH + CHX gave the highest cleavage and blastocyst rates. This work was supported by the Thailand Research Fund and Suranaree University of Technology.

167 DEVELOPMENTAL COMPETENCE OF PORCINE OOCYTES DERIVED FROM SMALL- OR MEDIUM-SIZED FOLLICLES AND DENUDED OF CUMULUS CELLS BEFORE AND DURING IN VITRO MATURATION

2017 ◽  
Vol 29 (1) ◽  
pp. 192
Author(s):  
P. Ferré ◽  
K. X. Nguyen ◽  
T. Wakai ◽  
H. Funahashi
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Polar Body ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Cumulus Cells ◽  
Developmental Competence ◽  
Blastocyst Formation ◽  
First Polar Body

This experiment was undertaken to assess the meiotic and developmental competences of oocytes derived from different sized follicles and denuded of cumulus cells 0, 20, and 44 h after the start of culture for in vitro maturation (IVM). Groups of 60 oocyte-cumulus complexes from small- (SF; <3 mm) and medium-sized follicles (MF; 3–6 mm) were cultured for IVM in porcine oocyte medium with 50 μM β-mercaptoethanol supplemented with 1 mM dibutyryl-cyclic adenosine monophosphate, 10 IU mL−1 of eCG, and 10 IU mL−1 of hCG for 20 h at 39°C and 5% CO2 in air. Then, after washing, they continued culture in fresh β-mercaptoethanol without dibutyryl-cyclic adenosine monophosphate and gonadotropins under the same conditions for another 24 h. At 0, 20, and 44 h of IVM, cumulus cells were removed with 0.1% (wt/vol) hyaluronidase and the denuded oocytes continued IVM culture following the protocol. Mature oocytes with the first polar body were selected, parthenogenetically activated with a single electrical pulse (DC: 1.2 kV/cm, 30 µs), incubated with 4% (wt/vol) BSA and 5 μM cytochalasin B for 4 h, and cultured in porcine zygote medium for 5 days. Cleavage and blastocyst formation rates were observed on Day 2 and 5, respectively. Blastocysts were stained with 4’,6-diamidino-2-phenylindole for cell count assessment. The experiment was replicated 5 times and analysed with a 1- or 2-way ANOVA. If P < 0.05 in ANOVA, a Tukey multiple comparisons test was performed. Regardless of the time of cumulus cell removal, oocytes from MF had significantly higher in rates of maturation, cleavage, and blastocyst rates, as compared with those from SF, whereas there were no significant differences in the cell number of blastocysts between SF and MF (32 v. 34 cells, respectively). When oocytes were denuded before IVM culture, rates of oocyte maturation (37.6% in SF and 50.8% in MF), and blastocyst formation (2.7% in SF and 27.3% in MF) were significantly lower than controls (51.2% in SF and 76% in MF; 25.8% in SF and 48.5% in MF, respectively). When oocytes were denuded 20 h after the start of IVM, oocyte maturation rates were significantly increased (64.1% in SF and 82.5% in MF) as compared with controls, whereas no significant differences were observed in cleavage and blastocyst formation rates in comparison with controls. These results conclude that removing cumulus cells from oocyte-cumulus complexes 20 h after the start of IVM improves the meiotic competence of oocytes derived from both SF and MF, without any reduction of developmental competence of the oocytes following parthenogenetical activation.

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