Maintenance of meiotic arrest and developmental potential of porcine oocytes after parthenogenetic activation and somatic cell nuclear transfer

Several studies report that meiotic maturation of porcine oocytes can be reversibly preserved. The present study examined how long meiotic maturation can be suppressed. The first experiment determined the preservation medium suitable for reversibly suppressing meiotic maturation of porcine oocytes. The second experiment examined the in vitro developmental potential of oocytes maintained in meiotic arrest after parthenogenetic activation and nuclear transfer of somatic cells. Preservation of cumulus-oocyte complexes with NCSU-37 medium containing 10% follicular fluid, 1 mM dibutyryl cyclic AMP, and follicular shell pieces for 24-96 h at 39 degrees C did not affect oocyte maturation compared with controls (94-98% vs. 98%). The potential of parthenogenetically activated and nuclear-transferred oocytes maintained in meiotic arrest for 24-48 h to develop into blastocysts was not significantly different from that of controls (20-25% vs. 18% and 8-11% vs. 9%, respectively). The present study demonstrated that meiotic maturation of porcine oocytes can be suppressed after preservation for 48 h at 39 degrees C without decreasing oocyte maturation competence or the ability of oocytes to develop to at least the blastocyst stage.     

Transport of equine ovaries for assisted reproduction

Use of assisted reproduction to obtain foals from valuable mares post-mortem typically necessitates holding of ovaries during shipment to a laboratory. The present study evaluated whether holding ovaries briefly at a warm ( approximately 30 degrees C) temperature improves meiotic and developmental competence of oocytes, as determined after maturation in vitro and intracytoplasmic sperm injection. Ovaries were packaged in pairs in insulated containers, and held either at 24 or 25-35 degrees C for 4h, followed by cooling. Ovaries in both treatments were held for either a short (mean, 7-7.4h) or long (mean, 20.6-20.7h) duration before oocyte recovery. Control ovaries were collected en masse at the abattoir. The ovary temperature in this treatment slowly decreased to approximately 27 degrees C; oocyte recovery was performed after 3.5-7h total holding. There was no effect of temperature on oocyte meiotic or developmental competence within either treatment time period. Oocytes in the short duration holding group had similar meiotic competence to controls, but had a significantly decreased rate (P<0.05) of blastocyst development. Oocytes in the long duration holding group had decreased (P<0.05) meiotic competence and blastocyst development compared to controls. These findings indicate that storage of equine ovaries for only 7h may decrease blastocyst development, and that longer storage reduces both rate of oocyte maturation and blastocyst development. Further work is needed to determine if there is a critical time before 7h post-mortem by which equine oocytes should be recovered to maximize developmental competence.     

Changes in ovarian, follicular, and oocyte morphology immediately after the onset of puberty are not accompanied by an increase in oocyte developmental competence in the pig

This study was conducted to determine whether ovarian morphology and developmental competence of in vitro-matured (IVM) oocytes is immediately affected by the onset of puberty in the pig. Ovaries of peri-pubertal pigs were sorted into two groups according to the presence or absence of corpora lutea presence (CL and NCL, respectively. Ovary dimensions, follicle diameter and number, and oocyte diameter (with and without zona pellucidae) were determined. The developmental competence of in vitro-matured oocytes from these two groups was evaluated following parthenogenetic activation and culture in vitro. CL ovaries were significantly (P<0.01) larger than NCL ovaries (width: 22.3+/-0.9 mm versus 15.9+/-0.4 mm, length: 33.2+/-1 mm versus 24.1+/-0.4 mm). Although CL ovaries had fewer antral follicles in total compared with NCL ovaries (21.1+/-1.8 mm versus 46.8+/-2.2 mm), they had a similar number of follicles 3-8mm in diameter. The mean diameter of follicles that were aspirated was greater for CL ovaries than for NCL ovaries (4.5+/-0.1 mm versus 3.3+/-0.02 mm). Oocytes from CL ovaries were greater in diameter compared with those from NCL ovaries (zona retained: 159+/-1.3 microm versus 146.1+/-1.5 microm, zona free: 124.7+/-1.8 microm versus 113.1+/-1.6 microm). No differences were found between oocytes from CL and NCL ovaries for rates of meiotic maturation (91.6+/-3.2% versus 92.4+/-3.2%), cleavage (88.4+/-11% versus 90.7+/-2.6%) and blastocyst formation (21.0+/-3.7% versus 23.7+/-5.7%). Therefore, the onset of puberty coincides with immediate changes in ovarian morphology, increased ovary size, follicle and oocyte diameter, but not with improved oocyte developmental competence. This suggests that the higher developmental competence usually observed in adult oocytes is acquired gradually and requires exposure to multiple estrus cycles.     

Effect of ovary storage and oocyte transport method on maturation rate of horse oocytes

Two experiments were conducted to determine the effects of storage on equine ovaries or isolated oocytes. Ovaries were collected at an abattoir and were maintained at room temperature during collection and transport (3-9h total). After arrival at the laboratory, ovaries were divided into three groups: immediate oocyte collection (control), storage at room temperature overnight (15-18 h) before oocyte collection, or storage at 4 degrees C overnight before oocyte collection. Collected oocytes were cultured in maturation medium for 24h. There was a significant increase in the proportion of oocytes classified as having compact cumuli in the two storage groups when compared with the controls. For oocytes originally having expanded cumuli, the rate of maturation to MII was significantly higher in the control group (72%) than in either storage group, and the maturation rate for oocytes from ovaries stored at room temperature (27%) was significantly higher than that for ovaries stored at 4 degrees C (10%). A similar trend was seen for oocytes originally having compact cumuli (24, 11, and 3% in MI-II for control, room temperature, and cold groups, respectively). In Experiment 2, we evaluated the effect of different packaging systems on the maturation of horse oocytes within a portable incubator. Use of 1 ml of equilibrated maturation medium in a 1 ml glass vial was associated with maturation equivalent to that for standard incubation.     

Storage of bovine isolated follicles: a new alternative way to improve the recovery rate of viable embryos from ovarian follicles of slaughtered cows

The vitality of bovine oocytes stored in isolated follicles was examined. The aim of this work was to prolong the time of in vitro manipulation of oocytes before their maturation and develop a new alternative of oocyte "capacitation" to improve the quality of in vitro produced embryos. Follicles were dissected from the ovaries of slaughtered cows; subsequently, follicles were divided according to their diameter into three categories (2-3, 3-4 and 4-6 mm), and stored at 17-18 degrees C for 24 or 48 h in a modified tissue culture medium-199 (TCM-199) with reduced pH. After that time, the cumulus-oocyte complexes (COCs) were isolated, matured, fertilized, and embryos cultured in vitro for a total of 9 days. The percentage of total blastocysts, and hatched blastocysts developed from oocytes, initially kept ("capacitated") for 24h at 17-18 degrees C, within follicles of 3-6mm size categories, were significantly higher than that oocytes of the control [of control oocytes] (44.9 and 30.3% versus 36.2 and 20.4%, respectively). The oocytes of follicles stored for 48 h at 17-18 degrees C already had decreased developmental capacity. Interesting data were obtained when COCs of the 3-4 and 4-6 categories were additionally divided into two subgroups according to their presumed developmental history (originating from the supposed growing "fit" in contrast to the supposed regressing "unfit" follicles). The higher improvement in the rate of hatched blastocysts from 24h stored oocytes was observed only in the subgroup originated from "fit" COCs (15.3 versus 25.0%, and 20.0 versus 34.4%, in the 3-4 and 4-6mm categories, respectively). The transfer of 26 blastocysts (developed of follicles kept for 24h at 17-18 degrees C) to 26 recipient heifers resulted in 18 pregnancies. Storage of follicles at 17-18 degrees C in vitro resulted not only in recovery of higher numbers of blastocysts of better quality but also facilitated the safe transport of follicles for a long distance. The extended, time of follicle storage before the proper oocyte maturation allowed also the synchronization of an appropriate number of recipient animals according to the number of isolated follicles.     

In vitro maturation, fertilization and early cleavage rates of bovine fetal oocytes

The in vitro developmental competence of oocytes harvested from 3 to 6 mm follicles from ovaries of 7.5 months to term fetuses and adult cows was compared. Cumulus oocyte complexes (COCs) were washed and placed in 200 microl droplets of maturation medium 199, supplemented with 10 microg/ml FSH, 10 microg/ml LH, 1.5 microg/ml estradiol, 75 microg/ml streptomycin, 100 IU/ml penicillin, 10 mM Hepes, and 10% fetal bovine serum (FBS) under oil and incubated for 24 h at 39 degrees C and 5% CO2. Matured oocytes were exposed to frozen-thawed TALP swim-up, heparin-capacitated sperm (20 h, 39 degrees C, 5% CO2). Presumptive zygotes were cultured in medium 199 containing 8 mg/ml BSA-V, 100 IU/ml penicillin G, 75 microg/ml streptomycin, and 10 mM Hepes (48 h, 39 degrees C, 5% CO2). Oocytes/embryos were fixed, stained with DAPI, and evaluated under fluorescent microscopy to assess maturation, fertilization, and subsequent embryonic development. There was a difference (P<0.05) between fetal and adult cow oocytes for in vitro maturation (IVM; 80.1% versus 92.0%), fertilization (69.3% versus 79.9%), and cleavage rates (36.7% versus 49.9%), respectively. Poor IVM, fertilization and embryonic development of fetal oocytes may be due to a higher incidence of blockage at germinal vesicle (GV) and metaphase-I (M-I) stage after IVM (12.0% versus 2.3% for fetal versus adult oocytes, respectively, P<0.05). Although the IVF results with fetal oocytes are poorer than with adult cow oocytes, they were still high enough to be considered for use in research and when death of the dam and/or fetus is pre-mature or sudden.     

Timing of nuclear maturation of nonstored and stored domestic cat oocytes

In this study we compared the effects of preculture storage of ovaries, IVM medium, a reduced O(2) atmosphere and duration of culture on in vitro maturation (IVM) of domestic cat oocytes. One randomly selected ovary of each pair (69 pairs) was stored in PBS at 10 degrees C for 16-24h before oocyte recovery. The second ovary from each pair was used as a nonstored control. In Experiment I, we investigated the effect of culture media (TCM 199 versus SOF) and a reduced O(2) atmosphere (a humidified gas atmosphere of either 5% CO(2) in air or 5% CO(2):5% O(2):90% N(2)) on IVM of both stored and nonstored oocytes. In the second experiment, we compared timing of nuclear maturation of both stored and nonstored oocytes cultured for 17-18, 20-21, 24-26, 28-30, 33-34 or 42-45 h before being evaluated for meiotic status. In both, Experiments I and II, the recovery rate, quality and competence for maturation of oocytes originating from stored ovaries did not differ (P>0.05) compared with nonstored. In Experiment I, neither culture medium (37.5 versus 43.2% of Metaphase II, respectively in TCM 199 versus SOF) or gas atmosphere (40.0 versus 32.5% of Metaphase II, respectively in 5% CO(2) in air versus 5% CO(2):5% O(2):90% N(2)) affected oocyte maturation. In Experiment II, the mean proportion of oocytes achieving Metaphase II within 17-18 h of culture was 36.1% and did not significantly increase (P>0.05) over time up to 28 h. The highest proportion of oocytes (67.3%) reached Metaphase II stage after 42-45 h of culture. Therefore, we conclude that two "waves" of nuclear maturation of cat oocytes can be distinguished. The first wave takes place within 26 h and it is likely that most oocytes of this wave mature by 17-18 h; the second wave occurs after 28-30 h of IVM. It can be assumed that this double wave may reflect the presence of two oocyte populations with two different degrees of "prematuration" which require different lengths of IVM.     

Lowering storage temperature during ovary transport is beneficial to the developmental competence of bovine oocytes used for somatic cell nuclear transfer

The objective of this study was to determine the effect of storage temperature during ovary transport on the developmental competence of bovine oocytes for use in somatic cell nuclear transfer (SCNT). Ovaries obtained from a slaughterhouse were stored in physiological saline for 3-4h at one of the three temperatures: 15 °C, 25 °C, or 35 °C. The developmental competence of oocytes used for SCNT was ascertained by cleavage and blastocyst formation rate, total cell number, apoptosis index, and the relative abundance of Bax and Hsp70.1 in day 7 blastocysts. Ovaries stored at 35 °C for 3-4h reduced the recovery rate of grade I and II oocytes compared with those stored at 25 °C or 15 °C (45.1±0.7% vs. 76.7±1.2% or 74.8±2.0%, P<0.05). The proportion of oocytes matured to the MII stage (maturation rate) for oocytes stored at 35 °C was significantly lower than those stored at 25 °C or 15 °C (51.3±0.9% vs. 75.1±1.4% or 71.7±1.3%, P<0.05). Cleavage rate (77.7±2.1%, 77.9±1.1% and 72.1±0.7% for 15 °C, 25 °C and 35 °C groups, respectively) and blastocyst formation rate (39.1±0.5%, 36.8±1.4% and 32.2±0.9% for 15 °C, 25 °C and 35 °C groups, respectively) following SCNT were not significantly different between treatments. Oocytes from ovaries stored at 15 °C, however, produced blastocysts with higher cell numbers (97.3±8.6 vs. 80.2±10.8 or 77.4±11.7; P<0.05) and lower apoptotic index (5.1±1.3 vs. 13.5±1.6 or 18.6±1.1, P<0.05) than those stored at 25 °C or 35 °C. The relative abundance of Bax and Hsp70.1 in day 7 blastocysts produced from oocytes derived from ovaries stored at 15 °C was lower than those stored at 25 °C or 35 °C (P<0.05). It was concluded that a storage temperature of 15 °C for a 3-4h period had a significant beneficial effect on the quality and developmental competence of oocytes used for SCNT due to the alleviation of stresses on the oocytes compared with those subjected to storage temperatures of 25 °C or 35 °C.     

Effect of meiotic stages and maturation protocols on bovine oocyte's resistance to cryopreservation

We investigated the effect of meiotic stages and two maturation protocols on bovine oocyte's resistance to cryopreservation. Oocytes at germinal vesicle breakdown (GVBD) and metaphase II (MII) stage as well as oocytes matured for 22 h in media supplemented with FSH or LH were vitrified by the open pulled straw method. After warming, oocytes underwent additional 16 h (GVBD group) or 2 h (MII group) maturation. Then they were subjected to in vitro fertilization and culture. Some oocytes that matured in the medium supplemented with LH were subjected to parthenogenetic activation after vitrification to determine their developmental potential in absence of fertilization. Survival of oocytes after vitrifying/warming was determined after 22 h in fertilization medium. Cleavage and blastocyst formation rates were used to assess their developmental competence. In both experiments, a portion of unvitrified MII oocytes were subjected to in vitro fertilization and culture as control groups. In Experiment 1, similar cleavage rates were obtained for both GVBD and MII oocytes (53.56 versus 58.01%, P > 0.05). However, significantly higher proportion of cleaved embryos from vitrified MII oocytes developed into blastocysts than those from vitrified GVBD oocytes (1.06 versus 8.37%, respectively, P < 0.01). In Experiment 2, vitrified MII oocytes matured in medium supplemented with LH were superior to vitrified MII oocytes matured in FSH supplementation not only in cleavage rates (61.13 versus 50.33%), but in blastocyst formation rates (11.79 versus 5.19%, P < 0.01) as well. Cleavage and blastocyst formation rates of parthenogenetically activated oocytes were similar to those that were fertilized. Nevertheless, the vitrifying/ warming process significantly compromised the oocytes' developmental capacity since the vitrified oocytes showed significant reduction in both cleavage and blastocyst rates compared to those of not vitrified controls in both experiments (P < 0.01). We showed that oocytes at different maturation stages respond to cryopreservation differently and MII stage oocytes have better resistance to cryopreservation than GVBD stage oocytes. The maturation protocols also influence oocyte's ability to survive cryopreservation. Poor developmental potential after vitrification seem to have resulted from the cryodamage to the oocyte itself. These results suggested the importance of maturation on the developmental competence of cryopreserved oocytes.     

Effects of leptin supplementation in in vitro maturation medium on meiotic maturation of oocytes and preimplantation development of parthenogenetic and cloned embryos in pigs

The objective of the present study was to investigate the effects of leptin addition in in vitro maturation (IVM) medium on meiotic maturation of oocytes and preimplantation development of parthenogenetic and cloned embryos in pigs. In experiment 1, oocytes were matured in North Carolina State University 23 (NCSU-23) medium supplemented with various concentrations of leptin: 0, 1, 10 and 100 ng/ml. IVM medium added with 10 or 100 ng/ml leptin significantly increased the rate of oocytes reaching metaphase II compared to the control (76.8% and 73.8% versus 61.7%). In experiment 2, the influence of the timing of leptin addition in IVM medium on meiotic maturation of porcine oocytes was assessed, and maximum maturation rate of oocytes developing to metaphase II was achieved when supplemented during the first half (0-22 h), the latter half (22-44 h) or the entire maturation period (0-44 h) compared to the control (80.5%, 84.7% and 78.1% versus 70.4%). In experiment 3, leptin strikingly increased the blastocyst rate of parthenogenetic embryos at the concentration of 10 ng/ml (37.5% versus 21.7%) and this increase was independent of the addition timing (0-44, 0-22, 22-44 h) compared to the control (32.5%, 34.6% and 31.5% versus 16.2%). Moreover, total cell number per blastocyst of parthenogenetic embryos was obviously increased in the 10 and 100 ng/ml leptin treatments as compared with the control (36, 38 versus 28). In experiment 4, 10 ng/ml leptin treatment significantly increased the rate of cleavage (72% versus 56%) of cloned embryos. Meanwhile, the rate of blastocyst formation was also improved although no significant difference was found (12.8% versus 7.1%). Collectively, our results indicate that leptin supplementation in IVM medium may be beneficial not only for developmental potential of oocytes but for subsequent developmental competence of embryos produced by parthenogenetic activation and the cleavage of embryos derived by somatic cell nuclear transfer (SCNT).     

Effects of cumulus cell density during in vitro maturation of the developmental competence of bovine oocytes

To determine the role of cumulus cells in oocyte maturation, we carried out an investigation on the effects of addition of cumulus cells to the maturation medium on the developmental competence of corona-enclosed oocytes and oocytes denuded from their somatic cells. The addition of cumulus cell (1.6 x 10(6) cells/mL) improved the development of bovine corona-enclosed oocytes, however, addition of a similar number of cumulus cells as cumulus-oocyte-complexes (COCs, cumulus cell density: 4.2 x 10(6) cells/mL) had no effect on the development of oocytes denuded from their somatic cells. To determine if corona-enclosed oocytes can obtain developmental competence without the addition of extra cumulus cells, the effects of cell density during in vitro maturation on the developmental competence were studied. A density of 1.6 to 3.2 x 10(6) cumulus cells/mL was the most effective for in vitro maturation of oocytes with intact gap junctions. The effects of the medium conditioned by COCs on the developmental competence of oocytes was also examined. It was demonstrated that COC-conditioned medium improved the development of bovine oocytes to the blastocyst stage. These data suggest that the developmental competence of bovine oocytes surrounded with corona cells is supported in a cell density-dependent manner in the maturation medium. In addition, the data indicate that cumulus cells benefit bovine oocyte development either by secreting soluble factors which induce developmental competence or by removing an embryo development-suppressive component from the medium.     

Kinetics of oocyte maturation and subsequent development of IVF,parthenogenetic, and NT bovine embryos after meiotic inhibition with roscovitine

The developmental competence of bovine oocytes meiotically arrested with specific cdk2 inhibitor roscovitine was studied. After removal of the 32-h block with roscovitine, 82.7 +/- 5.4% reached the metaphase II stage at the end of maturation, which was lower than in controls (96.3 +/- 1.3%, p < 0.001). The process of polar body formation started at 11 h of maturation in the roscovitine group, that is 4 h earlier than in controls and its kinetics was quite similar to controls up to 16 h of maturation, when nearly 70% of oocytes extruded their polar bodies. The rate of blastocyst formation of roscovitine oocytes and their cell number after IVF, parthenogenetic activation, and nuclear transfer (NT) were equal to controls, which demonstrates the possibility of artificially maintaining bovine oocytes in the GV stage for 32 h without altering their preimplantation developmental competence. This approach can be very useful for the management of an NT program where enucleated oocytes are required at specific times or locations.     

Improvement of an electrical activation protocol for porcine oocytes

Factors influencing pig oocyte activation by electrical stimulation were evaluated by their effect on the development of parthenogenetic embryos to the blastocyst stage to establish an effective activation protocol for pig nuclear transfer. This evaluation included 1) a comparison of the effect of epidermal growth factor and amino acids in maturation medium, 2) an investigation of interactions among oocyte age, applied voltage field strength, electrical pulse number, and pulse duration, and 3) a karyotype analysis of the parthenogenetic blastocysts yielded by an optimized protocol based on an in vitro system of oocyte maturation and embryo culture. In the first study, addition of amino acids in maturation medium was beneficial for the developmental competence of activated oocytes. In the second study, the developmental response of activated oocytes was dependent on interactions between oocyte age at activation and applied voltage field strength, voltage field strength and pulse number, and pulse number and duration. The formation of parthenogenetic blastocysts was optimal when activation was at 44 h of maturation using three 80-microsec consecutive pulses of 1.0 kV/cm DC. Approximately 84% of parthenogenetic blastocysts yielded by this protocol were diploid, implying a potential for further in vivo development.     

Sphingosine 1-phosphate protects bovine oocytes from heat shock during maturation

Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite that can block apoptosis by counteracting the proapoptotic effects of ceramide. Experiments were performed to evaluate whether S1P blocks the disruption in oocyte developmental competence caused by heat shock. Cumulus-oocyte complexes (COCs) were placed in maturation medium and cultured at 38.5 or 41 degrees C for the first 12 h of maturation. Incubation during the last 10 h of maturation, fertilization, and embryonic development were performed at 38.5 degrees C. Heat shock during the first 12 h of maturation reduced cleavage rate, the number of oocytes developing to the blastocyst stage, and the percentage of cleaved embryo that subsequently developed to blastocysts. Addition of 50 nM S1P to maturation medium had no effect on oocytes matured at 38.5 degrees C but blocked effects of thermal stress on cleavage and subsequent development. The blastocysts formed at Day 8 did not differ between S1P and control groups in caspase activity, total cell number, or percentage of cells that were apoptotic. Blocking endogenous generation of S1P by addition of 50 nM N1N-dimethylsphingosine, a sphingosine kinase inhibitor, reduced or tended to reduce cleavage rate and blastocyst development regardless of whether maturation of COCs was at 38.5 or 41 degrees C. Results demonstrate that S1P protects oocytes from a physiologically relevant heat shock and affects oocyte maturation even in the absence of heat shock. The S1P-treated oocytes that survived heat shock and became blastocysts had a normal developmental potential as determined by caspase activity, total cell number, and percentage of apoptotic cells. Thus, modulation of developmental competence of oocytes using S1P may be a useful approach for enhancing fertility in situations where developmental competence of oocytes is compromised.     

Modification of ovary stock solution with magnesium and raffinose improves the developmental competence of oocytes after long preservation

During ovary storage oocytes lose some of their developmental competence. In the present study, we maintained storage solutions of phosphate-buffered saline (PBS) at various temperatures (20 or 35 degrees C) or supplemented them with magnesium (Mg), raffinose and sucrose. Subsequently, we examined the kinetics of electrolytes in the follicular fluid (FF) during the ovary storage period (9 h), the survival rate of granulosa cells in the follicles, and the developmental competence of oocytes after the storage. Lowering the temperature from 35 to 20 degrees C increased the total cell number of blastocysts that developed at 7 days after in vitro maturation and in vitro fertilization of oocytes. In stock solution with supplements of 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose or sucrose, a significantly higher number of oocytes developed into blastocysts with a large number of cells in each blastocyst, and a significantly higher number of living granulosa cells were obtained as compared with stock solutions without any supplements. During ovary storage, the concentrations of potassium and chloride in the FF were increased, and the addition of Mg to the stock solution increased the concentration of Mg in the FF. Germinal vesicle breakdown in oocytes that were collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM of raffinose occurred at a slower rate than that in oocytes collected from ovaries stored in PBS alone. On the other hand, the oocytes collected from ovaries stored in the solution supplemented with 15 mM Mg or a combination of 5 mM Mg and 10 mM raffinose reached the metaphase II (MII) stage more rapidly than the oocytes collected from ovaries stored in the PBS alone. In conclusion, the modification of stock solution by the addition of Mg and raffinose improved the developmental competence of oocytes obtained from ovaries preserved for a long period.     

Bovine parthenogenetic blastocysts following in vitro maturation and oocyte activation with ethanol

The appropriate in vitro bovine oocyte maturation and ethanol activation conditions for preimplantation bovine embryo parthenogenetic development to the blastocyst stage were investigated. A 7% ethanol concentration significantly enhanced (P<0.05) the proportion of activated, in vitro-matured bovine oocytes (7% ethanol, 83.4 +/- 3.2% versus 0% ethanol, 63.9 +/- 2.0%). The proportion of activated oocytes was significantly higher (P<0.05) by treatment with 7% ethanol for a minimum of 2 minutes (2 minutes, 89.8 +/- 4.0% versus 0.5 minutes 63.4 +/- 4.9%). Oocyte maturation for periods ranging from 30, 34, 38 and 44 hours resulted in a significant increase (P<0.05) in the proportion of activated oocytes, and in oocytes displaying 2 or 3 pronuclei versus oocytes matured for 26 hours. The proportion of cleaved, activated oocytes (2-cell stage), 4 -cell stage and parthenogenetic morula/blastocysts was significantly higher (P<0.05) within the 34-hour oocyte maturation treatment group. Although the 44-hour oocyte maturation treatment group displayed the highest proportion of activated oocytes with 2 pronuclei, it did not display the highest cleavage frequency, possibly due to the effects of postovulatory aging. Several morphologically normal parthenogenetic bovine blastocysts developed from oocytes that were in vitro matured for 34 hours. The ability to produce such parthenogenetic embryos will eventually facilitate investigation into the role(s) of the maternal and paternal genomes during bovine early development.     

In vitro maturation,fertilization and development of domestic cat oocytes recovered from ovaries collected at three stages of the reproductive cycle

This study was conducted to examine the effect of the donor cat's reproductive cycle stage on in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro development of oocytes recovered from ovaries that were collected and stored at 35 degrees C for a short period (1-6 h). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into inactive, follicular, or luteal stages. Nuclear status of 161 cumulus-oocyte complexes (COCs) were examined immediately after recovery; 91.3% of the oocytes were found to be at the immature germinal vesicle (GV) stage, and 3.7% of the oocytes were at metaphase II (MII) stage. The percentage of the oocytes at the GV stage was significantly lower in the follicular stage than in the inactive stage (P < 0.01). Of the oocytes from the follicular stage, 9.1% were at MII stage. After culture for 24 h, however, the proportions of oocytes that reached metaphase I and MII were not different among the reproductive cycle stages of the ovaries collected (P > 0.05). After co-incubation with sperm, 63.1% of oocytes were fertilized, but there were no significant differences among the reproductive cycle stages of the ovaries with respect to the proportions of normal and polyspermic fertilization. However, the number of oocytes reaching cleavage stage and development to the morula and blastocyst stages from follicular stage ovaries were significantly lower (P < 0.05) than those obtained from inactive and luteal stage ovaries. These results indicate that the reproductive cycle stage of donor cat ovaries, stored at 35 degrees C, has no apparent effects on the frequencies of maturation and fertilization of oocytes, but influences developmental competence of the oocytes following IVM or IVF.     

Germinal vesicle chromatin configuration and meiotic competence is related to the oocyte source in canine

This study investigated the effect of deriving oocytes from different stages of the estrous cycle on oocyte diameter, germinal vesicle (GV) chromatin configuration, and in vitro meiotic competence in canine oocytes. Cumulus oocyte complexes (COCs) were recovered from both ovaries during anestrous, follicular, and luteal phases and in vivo ovulated oocytes. The diameter of canine oocyte was compared with or without the zona pellucida (ZP) before in vitro maturation (IVM). Also, GV chromatin configuration was evaluated before (0 h) or 72 h after IVM by fixation with 3.7% formaldehyde supplemented with 10 microg/ml Hoechst 33342 for 30 min. COCs were matured in TCM199 supplemented with 10% fetal bovine serum (FBS), 0.6 mM cysteine, 0.2 mM pyruvic acid, 50 microg/ml gentamycin sulfate, and 20 microg/ml 17beta-estradiol (E(2)) at 39 degrees C and 5% CO(2) in air for 72 h. The diameter of in vivo ovulated oocytes with the ZP (167.5+/-12.7 microm) or without ZP (133.9+/-5.3 microm) was significantly greater (p<0.05) than those of anestrous, follicular, and luteal oocytes (with ZP, 151.2+/-7.4, 153.1+/-8.8 and 152.8+/-5.4 microm, respectively; without ZP, 115.3+/-7.6, 122.1+/-4.9 and 114.3+/-6.6 microm, respectively). At 0 h, the GV-II configuration was more prevalent in oocytes from anestrual ovaries than from follicular or luteal ovaries or in vivo ovulated oocytes (63.6% versus 14.8%, 33.0%, and 0.0%; p<0.05), whereas the proportion of oocytes with the GV-V configuration was higher in follicular phase and ovulated oocytes than in oocytes from anestrus and luteal phase (57.4% and 100% versus 2.0% and 22.7%; p<0.05). However, oocytes in luteal phase exhibited diverse GV configurations (10.3%, 33.0%, 16.5%, 13.4%, and 22.7% in GV-I, GV-II, GV-III, GV-IV, and GV-V, respectively). After 72 h post-IVM, a greater percentage of in vivo ovulated oocytes progressed to MII than those oocytes collected during anestrous, follicular, and luteal phases (50.0% versus 5.5%, 11.5%, and 9.1%; p<0.05). In conclusion, the oocyte diameter, GV chromatin configuration, and meiotic maturation of canine COCs are related to the oocyte source. These results indicated that the oocyte source could be critical to nuclear progression to MII stage in canines.     

Effects of cooling ovaries before oocyte aspiration on meiotic competence of porcine oocytes and of exposing in vitro matured oocytes to ambient temperature on in vitro fertilization and development of the oocytes

Experiments were conducted to evaluate the effects of cooling porcine ovaries to low temperature (4 degrees C, 15 degrees C, 20 degrees C, 25 degrees C or 30 degrees C) for 1 h on the meiotic competence of their oocytes. Moreover, it was determined whether or not the exposure of in vitro matured oocytes to ambient temperature (20 degrees C, 25 degrees C or 30 degrees C) for 1 h affects the fertilization and developmental competence of the oocytes. There was no difference between the proportions of oocytes that underwent maturation to metaphase II when isolated from control ovaries held at 35 degrees C and ovaries exposed to 30 degrees C. However, the percentages of oocytes from ovaries exposed to 25 degrees C or less were significantly lower than those of oocytes from ovaries exposed to 30 degrees C and control ovaries. The proportions of total and normal fertilization of oocytes that had been exposed to 20 degrees C before in vitro fertilization (IVF) were significantly lower than those of control oocytes maintained at 38.5 degrees C. However, cooling in vitro matured oocytes had no effects on their cleavage and development to blastocysts after IVF. These data suggest that exposing porcine ovaries to a low temperature of 25 degrees C or less before aspiration of oocytes may adversely affect their subsequent in vitro maturation. It may be necessary to maintain the oocytes at a temperature of more than 25 degrees C during manipulation of oocytes for retaining the fertilizability of the oocytes.