Production of dromedary (Camelus dromedarius) embryos by IVM and IVF and co-culture with oviductal or granulosa cells

The general objective of this work was to produce dromedary embryos from cumulus-oocyte complexes (COCs) that were matured, fertilized and co-cultured in vitro. A total of 1598 COCs were recovered from 457 ovaries; 1308 were deemed suitable for IVM and were cultured at 38.5 degrees C, 5% CO2, and >95% humidity for 36 h in TCM-199 supplemented with 10% heat-treated fetal calf serum (FCS), 10 ng/ml epidermal growth factor (EGF), 1 microg/ml FSH, and 500 microM cysteamine. Matured COCs (n = 88) were denuded, fixed, and stained to determine nuclear status; 63% (56/88) had reached metaphase II (MII) at 36 h. Overall, 1135 COCs were inseminated with ejaculated fresh semen (0.5 x 10(6)spermatozoa/ml in modified TALP-solution). Inseminated oocytes (n = 155) were examined for evidence of fertilization; 68% (106/155) were penetrated by spermatozoa, including 52% (55/106) with two pronuclei and 34% (36/106) with polyspermy. Inseminated, denuded oocytes (n = 819) were co-cultured with dromedary oviductal epithelial or granulosa cells in TCM-199 supplemented with 10% heat-treated FCS. Although the rate of first cleavage (two to eight cells) was similar for the two co-culture systems (32 versus 33%, respectively), more embryos (two-cell to blastocyst stage) were obtained from oocytes co-cultured with oviductal versus granulosa cells (61 versus 45%; P < 0.05). The proportions of fertilized oocytes developing to the early morula stage were 19% (80/417) and 12% (48/402) for oocytes co-cultured for 7 days with oviductal or granulosa cells, respectively (P > 0.05). However, development to the blastocyst stage (10% of fertilized oocytes) occurred only in oocytes co-cultured with oviductal cells. In conclusion, dromedary embryos were produced in vitro using abattoir-derived oocytes, fresh (ejaculated) semen, and oviductal cell co-culture.     

Low temperature preservation of mouse fetal germ cells at 4 degrees C

Mouse fetal germ cells(FGCs) isolated or within genital ridges from male fetuses at 15.5 d post coitum were preserved at 4 degrees C for 3-15 d with TCM-199 medium supplemented with 10, 50 or 100% fetal bovine serum (FCS) and 0.1, 0.6 or 1.0 M sucrose. The viability of FGCs was assessed by the dye exclusion test with trypan blue and nuclear transfer into enucleated oocytes and then into enucleated 2-cell embryos. Forty-one to 45 % of FGCs survived for 5 d after preservation in the medium containing 50% FCS and 0.1 M sucrose according to the results of the dye exclusion test. But significant good effect of sucrose was not observed regardless of its concentration. When genital ridges were preserved, 15-22% of FGCs survived even 10 d after preservation. The efficiency of the in vitro development of FGCs to blastocysts was similar when they were fused with enucleated oocytes and 2-cells blastomeres after 2 to 4 d storage(27-33%) compared with that of control FGCs(56%).     

In vitro maturation and fertilization of buffalo oocytes (Bubalus bubalis ): Effects of media, hormones and sera

Media (TCM-199 and Ham's F-10); sera (fetal calf serum, FCS, and buffalo estrous serum, BES); and hormones (FSH, 0.5 ug/ml, LH, 5 ug/ml and estradiol 1 ug/ml) were tested to determine the efficiency of in vitro maturation and fertilization of buffalo follicular oocytes. Immature good quality cumulus-oocyte complexes (COCs) were randomly assigned to 1 of 4 experiments. Each experiment consisted of 6 treatment groups. Oocytes cultured for 24 hours in medium (TCM-199 or Ham's F-10) containing 10% FCS or BES had a significantly higher maturation rate than those in medium alone (P < 0.05). However, the maturation rate was higher in medium supplemented with 10% FCS than with 10% BES. Addition of hormones alone or in combination with sera further improved the maturation rate, but no significant difference was observed in the maturation rate among the 3 hormone-treated groups. Immature oocytes matured in the various cultures were fertilized with frozen-thawed buffalo spermatozoa. Our findings show that hormone and/or serum supplementation of TCM-199 did not improve the fertilization rate. Supplementation of Ham's F-10 with LH alone or in combination with LH + FSH + E(2) and with FCS significantly improved the fertilization rate of oocytes while medium with FSH, E(2) or no hormones did not (P < 0.05); same media supplemented with BES resulted in lower fertilization rates both in the presence or absence of hormones. The results indicate that the culture medium has a marked effect on the fertilization rate of buffalo oocytes. Ham's F-10 + LH + FSH + E(2) supplemented with FCS was the most efficacious culture system of those studied for the in vitro maturation of buffalo oocytes.     

Vitrification of immature and mature equine and bovine oocytes in an ethylene glycol, ficoll and sucrose solution using open-pulled straws

Studies were conducted to compare viability of immature and mature equine and bovine oocytes vitrified in ethylene glycol. Ficoll using open-pulled straws. Oocytes from slaughterhouse ovaries (N=50/group) with >2 layers of compact cumulus cells were vitrified immediately after collection (immature groups) or vitrified after 36 to 40 (equine) or 22 to 24 (bovine) h of maturation (mature groups). Immature oocytes were matured after thawing. Before vitrification, oocytes were exposed to TCM-199 + 10 FCS + 2.5 M ethylene glycol + 18% Ficoll + 0.5 M sucrose (EFS) for 30 sec and then to 5 M ethylene glycol in EFS for 25 to 30 sec at 37 degrees C. Oocytes were loaded into straws in approximately 2 microL of cryoprotectant and plunged directly into LN2. Warming straws and dilution of cryoprotectant was at 37 degrees C in TCM-199 + 10% FCS + 0.25 M sucrose for 1 min and then TCM-199 + 10% FCS + 0.15 M sucrose for 5 min. Non-vitrified oocytes undergoing the same maturation protocol for both species were used as controls. Oocytes were stained with orcein for nuclear maturation and live/dead status was determined using Hoechst 33342. Maturation of oocytes to MII after thawing was similar (P>0.05) among groups within species. All equine treatment groups had lower (P<0.01) maturation rates than bovine groups. Live/dead status did not differ among vitrification treatments within species. The percentage of oocytes that survived and reached MII did not differ (P>0.05) within treatment groups of each species. Rates of mature cortical granule distribution did not differ (P>0.05) within species; however, more bovine oocytes (P<0.05) had mature cortical granule distribution and nuclear maturation than equine oocytes. When concurrent cortical granule distribution and nuclear maturation were examined, there was no difference within species; however, only 30% of equine oocytes had nuclear and cytoplasmic maturation compared with 70% of bovine oocytes (P<0.05). In summary, both immature and mature equine and bovine oocytes survived cryopreservation using vitrification in open-pulled straws. However, survival rates were lower for equine than for bovine oocytes.     

Nuclear maturation kinetics and in vitro embryo development of cattle oocytes prematured with butyrolactone I combined or not combined with roscovitine

Cyclin dependent kinase inhibitors (CDKIs) may be used for pre-maturation culture, but can accelerate nuclear maturation. The aim of the present research was to compare the effect of butyrolactone I (BLI) alone or combined with roscovitine (ROS) at lesser than typically used concentrations on nuclear maturation kinetics and embryo development. To assess maturation kinetics (Experiment 1), oocytes were cultured in 100 microM BLI (B) or 6.25 microM BLI+12.5 microM ROS (BR) in TCM-199 for 24 h. Oocytes were subsequently submitted to in vitro maturation (IVM) in TCM-199+0.5 microg/ml FSH, 50 microg/ml LH and 10% FCS for another 24 h, during which oocytes were fixed every 3 h. In Experiment 2, oocytes were submitted to 24h pre-maturation treatments, with the inhibitors being diluted in TCM-199 or DMEM. IVM lasted 21 h in the culture media DMEM+0.5 microg/ml FSH, 50 microg/ml LH, 5% FCS and 50 ng/ml EGF. After IVM, oocytes from all groups were fertilized in vitro. Oocytes and sperm (2x10(6) sperm cells/ml) were co-cultured for 18 h. Embryos were co-cultured with granulosa cells in CR2aa for 8 days. All cultures were in droplets under oil, at 38.5 degrees C and 5% CO2 in air. In both experiments, control oocytes (C) were submitted only to IVM. In Experiment 1, at 0 h, C and B oocytes were all (100%) at the germinal vesicle stage (GV) of development. BR had fewer GV oocytes (89%, P<0.05). After 3 h IVM, B and BR had fewer oocytes in GV (84.7 and 79.6%, P>0.05) than C (100%, P<0.05). At 12 h, most oocytes were at intermediate stages (metaphase to telophase I) in all groups (approximately 80%, P>0.05). After 21 (77-89%) and 24 h (85-95%), all groups had similar metaphase II (MII) rates of development (P>0.05). In Experiment 2, cleavage (79-84%, P>0.05) and Day 7 blastocyst rates (26-36%, P>0.05) were similar. After 8 days, the group pre-matured with BR in DMEM had lesser blastocyst rates of development (32.3%) lower than C (40.1%, P<0.05). The other groups were similar to C (35-38%, P>0.05). Hatching rates were similar (10-15%, P>0.05) as were total cell numbers (141-170). In conclusion, BR is less effective in maintaining meiosis block; B and BR accelerate meiosis resumption; and use of pre-maturation medium may affect developmental rates.     

Influence of oviductal cells and conditioned medium on porcine gametes

The aim of this study was to optimise porcine in vitro fertilisation (IVF) with cryopreserved semen with the exploitation of the oviduct secretion. The oocytes were cultured in NCSU37 supplemented with db-cAMP (1 mM), porcine follicular fluid (pFF; 10%), cysteine (0.1 mg/ml) and beta-mercaptoethanol (25 microM) for 44 h (the first 20 h with 10 IU/ml hCG and PMSG). The oviductal epithelial cells (OEC) were cultured in TCM-199 medium (with 10% FCS, 0.2 mM pyruvate and 50 microg/ml gentamicin) for 48 h. To determine the effects of OEC and conditioned medium, oocytes were separated into five groups for the last 3 h of maturation and placed in: fresh maturation medium (controls), OEC-cNCSU with OEC in the maturation medium for 24 h; OEC-fNUSU with fresh OEC in maturation medium; cTCM with TCM-199 conditioned with OEC for 48 h; or fTCM with fresh TCM-199. Results indicate that OEC-cNCSU and OEC-fNCSU increase the number of oocytes reaching the two pronucleus (2PN) stage (p < 0.01) and decrease the polyspermy rate (p < 0.01) compared with controls. The rates are significantly lower than controls when cTCM and fTCM were used (p < 0.01). As regards blastocyst rates, an increase was observed in the OEC-cNCSU and cTCM groups (p < 0.05). For the second experiment, spermatozoa were incubated with OEC in IVF medium (mTBM medium supplemented with 0.1% BSA) without caffeine for 4 h prior to IVF. Results indicate that sperm treatment with OEC increases the 2PN rate (p < 0.01) compared with controls and reduces the polyspermy rate (p < 0.01). In conclusion, our study shows that co-incubation of OEC with both oocytes and sperm before IVF reduces polyspermy rates and improves embryo development.     

Effects of preservation media on in vitro maturation of porcine oocytes

The effects of preservation media for ovaries on in vitro maturation of porcine oocytes was studied. The cumulus-oocyte complexes (COCs) obtained from ovaries that had been preserved in three different media at various temperatures for different time intervals were cultured in the M199 maturation medium. The preservation media used were 0.9% saline solution, BCS (Braun-Collins solution) and Dulbecco's phosphate buffered saline solution (PBS). Mature oocytes obtained from the ovaries preserved in three preservation media for 8 h were electrically activated. The activated oocytes were then cultured in the NCSU23 embryo culture medium for 16 h to observe activation, or for 144 h to observe embryo development. It was found that the preservation temperature significantly affected maturation of the porcine oocytes. A preservation temperature of about 25 degrees C showed an optimal maturation rate for a preservation time of 8 h for the three preservation media. Although the preservation temperature was a major factor influencing the maturation rate, different preservation media at 25 degrees C for 8 h also significantly affected the maturation rate, activation rate and embryo development. Among these three preservation media, PBS exhibited the highest cleavage rate indicating that PBS should be a better preservation medium for porcine ovaries at 25 degrees C for 8 h or longer periods.     

Follicle size-dependent effects of sow follicular fluid on in vitro cumulus expansion, nuclear maturation and blastocyst formation of sow cumulus oocytes complexes

Follicular fluid from 2 to 4 and 5 to 8 mm diameter non-atretic follicles (SFF and LFF, respectively) of sows was added during IVM of cumulus oocytes complexes (COCs) to study its effects on cumulus expansion, nuclear maturation, and subsequent fertilization and embryo development in presence or absence of recombinant human FSH. COCs aspirated from 2 to 5 mm follicles of sow ovaries, were cultured for the first 22 h in TCM-199 and 100 microM cysteamine, with or without 10% pFF and/or 0.05 IU/ml recombinant hFSH. For the next 22 h, the COCs were cultured in the same medium, but without pFF and FSH. After culture, cumulus cells were removed and the oocytes were either fixed and stained to evaluate nuclear stages or co-incubated with fresh sperm. Twenty-four hours after fertilization, presumptive zygotes were fixed to examine fertilization or cultured for 6 days to allow blastocyst formation. Subsequently, embryos were evaluated and the blastocysts were fixed and stained to determine cell numbers. When LFF was added to maturation medium, cumulus expansion and percentage of nuclear maturation (277 +/- 61 microm and 72%, respectively) of COCs were significantly higher (P < 0.05) than those in SFF (238 +/- 33 microm and 55%, respectively). However, in the presence of FSH both FF stimulated cumulus expansion and nuclear maturation to a similar degree. No differences were observed with regards to sperm penetration, male pronucleus formation, and to polyspermia between fertilized oocytes matured either in SFF or LFF. Fertilized oocytes matured in the presence of LFF without or with FSH showed a higher cleavage (45 +/- 7% and 51 +/- 7%, respectively) and blastocyst (14 +/- 4% and 22 +/- 6%, respectively) formation rate compared to SFF (cleavage, 35 +/- 8% and 41 +/- 4%, blastocyst: 8 +/- 3 and 13 +/-3, respectively; P < 0.05). The mean number of cells per blastocyst did not differ significantly between treatments. These findings indicate that factor(s) within follicles at later stages of development play an important role during oocyte maturation and thereby enhance developmental competence to occur.     

In vitro maturation of porcine oocytes in follicular fluid with subsequent effects on fertilization and embyo yield in vitro

The objective of the present study was to test the ability of porcine follicular fluid (pFF) to improve maturation of porcine cumulus-oocyte-complexes (COC) in vitro and to observe subsequent effects on fertilization and development to late morula/blastocyst stages under in vitro conditions. The COC were incubated in Tissue Culture Medium (TCM) 199, supplemented with 1% fetal calf serum (FCS), 10% pFF collected from immature follicles (2 to 5 mm), with or without addition of 1microg/ml FSH. Control groups were matured in TCM 199 with or without FSH. Follicular aspirates were centrifuged (1700 x g, 5min.) and the supematants were stored at -20 degrees in 1.5-ml Eppendorff cups until used. On 7 experimental days a total of 3849 immature COC was aspirated from follicles ranging from 2 to 5 mm in diameter. A total of 1117 COC was selected for the experiments, and 239 COC were fixed and stained with 1.5% aceto-orcein after 48 h of in vitro maturation at 39 degrees C with 5% CO(2) in humidified air. Germinal vesicle breakdown (GVBD; 91.7%) and development to metaphase II (60.4%) were superior (P 相似文献   

Glutathione and adenosine triphosphate content of in vivo and in vitro matured porcine oocytes

Glutathione (GSH) content in mature porcine oocytes is correlated with subsequent fertilization and developmental success. Adenosine triphosphate (ATP) is an important energy source for maintaining cellular activities and protein synthesis. The objective of this study was to compare GSH and ATP concentrations of in vivo and in vitro matured porcine oocytes. Ovulated, in vivo matured oocytes were frozen at -80 degrees C in groups of 10-20 (GSH) or 5-10 (ATP). In vitro oocytes were matured in either tissue culture medium-199 (TCM199) supplemented with polyvinyl alcohol (PVA) or hyaluronic acid (MAP5), or North Carolina State University-23 (NCSU23) supplemented with porcine follicular fluid (pFF) and frozen as described, or fertilized and cultured. GSH content was determined by the dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. ATP content was determined by using the Bioluminescent Somatic Cell Assay Kit. Oocytes matured in vitro in defined TCM199 with PVA or hyaluronic acid, or NCSU23 with pFF had significantly lower concentrations (P < 0.05) of GSH (n = 207, 9.82 +/- 0.71 pmol/oocyte; n = 104, 9.73 +/- 0.81 pmol/oocyte; n = 108, 7.89 +/- 0.66 pmol/oocyte, respectively) compared to in vivo matured oocytes (n = 217, 36.26 +/- 11.00 pmol/oocyte). Concentrations of ATP were not different between treatments (in vivo, n = 70, 0.97 +/- 0.07 pmol/oocyte; TCM-PVA, n = 117, 0.81 +/- 0.13 pmol/oocyte; TCM-MAP, n = 107, 1.02 +/- 0.18 pmol/oocyte; NCSU-pFF, n = 134, 0.71 +/- 0.08 pmol/oocyte). Intracellular ATP content does not appear to be related to developmental potential in porcine oocytes. Low intracellular GSH may be responsible, in part, for lower developmental competence observed in in vitro matured porcine oocytes.     

Effects of porcine follicular fluid on male pronucleus formation in porcine oocytes matured in vitro

Porcine follicular oocytes, collected from antral follicles (2–5 mm in diameter) of gilt ovaries, were matured in vitro with or without porcine follicular fluid (pFF), gonadotrophins (GTH) or fetal calf serum (FCS) for 48 hours at 37°C under 5% CO₂ in air, and their ability of male pronucleus (mPN) formation was examined after in vitro fertilization. Formation of mPN was observed in 38.6% of penetrated oocytes matured in modified Krebs-Ringer bicarbonate solution (TYH) 18 hours after insemination. The addition of GTH into the maturation medium did not improve the proportion of mPN-formed oocytes (20–30%). In contrast, the mPN formation rate elevated significantly (59.5%) when the oocytes were cultured with pFF, and the addition of follicle-stimulating hormone (FSH) enhanced this pFF action (the rate became 81.0%). In the presence of FSH, significant pFF effect was observable at the concentration of 5%, and its efficiency was elevated with the increase of pFF concentration. When the oocytes were matured with FCS, the mPN formation rate was unchanged or decreased rather than improved (0–25%). These results suggest that pFF, but not FCS, have substance(s) stimulating the ability of mPN formation in porcine oocytes.     

The effect of macromolecular supplementation on the surface tension of TCM-199 and the utilization of growth factors by bovine oocytes and embryos in culture

The objectives of this study were to determine the surface tension of bovine follicular fluid (BFF) and TCM-199, and the effects of the synthetic surfactant, Twin-80, or FCS, in TCM-199 on surface tension measurements and subsequent effects on bovine oocyte and embryo development. The surface tension of BFF was determined to be approximately 45.5 mN m(-1) at 25 degrees C and approximately 42.7 mN m(-1) at 39 degrees C, which was comparable to the surface tension of TCM-199 containing Twin-80 (45.7 and 43.2 mN m(-1), respectively). There was no difference in surface tension measurements of BFF from follicles 2-7 mm or 8-15 mm in diameter. Both Millipore water and phosphate buffered saline (PBS) had a surface tension measurement of 69.5 mN m(-1) at 39 degrees C. Although the presence of Twin-80 in TCM-199 resulted in a reduction in surface tension measurement as compared with unsupplemented TCM-199, there was no effect on the number of oocytes reaching metaphase II. However, the addition of epidermal growth factor (EGF) to TCM-199 containing Twin-80 did result in increased maturation rates of oocytes in vitro. There was no effect of insulin, transferrin, and selenium (ITS), or EGF on surface tension measurement of TCM-199, but significantly more zygotes cleaved and developed to morulae/blastocysts in TCM-199 containing both Twin-80 and growth factors. The addition of 20 microg EGF ml(-1) to TCM-199 containing Twin-80 was as efficacious in supporting bovine embryos in culture as was the addition of 5% or 10% fetal calf serum. This study demonstrates that surface-active components in culture media positively affect bovine oocyte maturation and embryo development in culture. Data also suggest that non-ionic surfactants, such as Twin-80 in TCM-199, may successfully replace the surface-active properties but not the embryotrophic properties of serum in embryo maturation/culture media. Although commercial TCM-199 (containing Twin-80) did not require the addition of other surface-active compounds to lower surface tension, it did benefit from the addition of growth promoting factors, which were also provided by serum.     

Maturation of porcine oocytes after cooling at the germinal vesicle stage

Maturation of porcine oocytes was examined after oocytes were cooled at the germinal vesicle stage. Cumulus-oocyte complexes (COCs) collected from medium-sized follicles were cooled at 24 degrees C or 4 degrees C for 5, 30 or 120 min in a solution with or without 1.5 M dimethylsulfoxide (DMSO). After rewarming, COCs were cultured in maturation medium at 39 degrees C, 5% CO2 in air for 44 h. Meiotic spindle organisation (by immunostaining and confocal microscopy), nuclear maturation (by orcein staining) and cytoplasmic maturation (by intracellular glutathione assay) of oocytes were examined after maturation. When COCs were cooled at 24 degrees C for various times in the medium without DMSO, a tendency to decreased spindle formation, nuclear maturation and cytoplasmic maturation was observed, but there was no statistical difference compared with controls. Addition of DMSO during cooling inhibited subsequent nuclear maturation and spindle formation. When COCs were cooled at 4 degrees C, both nuclear and cytoplasmic maturation as well as spindle formation were inhibited in most oocytes in a time-dependent manner. DMSO during cooling did not have any beneficial effect on subsequent oocyte maturation and spindle formation. These results suggest that porcine oocytes are very sensitive to a drop in the temperature before exposure to culture. Cooling oocytes before maturation inhibits their subsequent spindle organisation, nuclear and cytoplasmic maturation. Addition of DMSO to the cooling solution did not protect porcine oocytes from cooling-induced damage.     

Effect of growth hormone releasing hormone (GHRH) and vasoactive intestinal peptide (VIP) on in vitro bovine oocyte maturation

The aim of this study was to investigate the effects of growth hormone releasing hormone (GHRH) and the structural-related peptide vasoactive intestinal peptide (VIP) on nuclear maturation, cortical granule distribution and cumulus expansion of bovine oocytes. Bovine cumulus oocyte complexes (COCs) were cultured in M199 without FCS and gonadotropins and in the presence of either 100 ng/mL bovine GHRH or 100 ng/mL porcine VIP. The COCs were incubated at 39 degrees C in a humidified atmosphere with 5% CO2 in air, and the nuclear stage was assessed after 16 or 24 h of incubation using DAPI staining. Cortical granule distribution was assessed after 24 h of incubation using FITC-PNA staining. To assess the effects of GHRH and VIP on cumulus expansion, COCs were incubated for 24 h under the conditions described above. In addition, 0.05 IU/mL recombinant human FSH was added to GHRH and VIP groups. Cultures without GHRH/VIP/FSH or with only FSH served as negative and positive controls, respectively. At 16 h neither GHRH (42.9%) nor VIP (38.5%) influenced the percentage of MII stage oocytes compared with their respective controls (44.2 and 40.8%). At 24 h there also was no difference in the percentage of MII oocytes between GHRH (77.0%), VIP (75.3%) and their respective controls (76.0 and 72%). There was no significant cumulus expansion in the GHRH or VIP group, while FSH induced significant cumulus expansion compared with the control groups, which were not inhibited by GHRH or VIP. Distribution of cortical granules was negatively affected by GHRH and VIP. The percentage of oocytes showing more or less evenly dispersed cortical granules in the cortical cytoplasm aligning the oolemma (Type 3) was lower in the GHRH (2.7%) and VIP (7.8%) groups than in the control group (15.9%). In conclusion, GHRH and VIP have no effect on nuclear maturation or cumulus expansion of bovine COCs but retard cytoplasmic maturation, as reflected by delayed cortical granule migration.     

Effects of Postmortem Interval on Mouse Ovary Oocyte Survival and Maturation

To study the time- and temperature-dependent survival of ovarian oocytes collected from postmortem carcass, ICR mice were killed and placed for different periods (0, 1, 2, 4, 6, 8 and 10 h) at different temperatures (25°C, 4°C and 37°C). After preservation, oocyte morphology, germinal vesicle (GV) oocyte number, oocyte meiotic maturation percentage, mitochondrial distribution and intracellular glutathione (GSH) level were evaluated. The results showed no surviving oocytes could be collected by 2h, 6h, and 12 h after carcass preservation at 37°C, 25°C and 4°C, respectively. The number of collected GV oocytes in the ovary deceased as the preservation time lasted at the same temperature. Meanwhile at the same point in time, the ratio of germinal vesicle breakdown (GVBD) and the first polar body emission (PBE) gradually reduced as preservation temperature increased. In addition, the percentage of abnormal mitochondrial distribution in the preserved oocytes was obviously higher than that in the control oocytes, while GSH level was not altered in collected oocytes. Unexpectedly, neither chromosome arrangement nor spindle organization was affected as long as the oocytes from preserved carcasses could complete maturation. These data are helpful for proper use of ovary oocytes from postmortem carcass of valuable individuals.     

The effect of FF-MAS on porcine cumulus-oocyte complex maturation, fertilization and pronucleus formation in vitro

Follicular fluid meiosis-activating sterol (FF-MAS) has been isolated from the follicular fluid (FF) of several species including man. FF-MAS increases the quality of in vitro oocyte maturation, and thus the developmental potential of oocytes exposed to FF-MAS during in vitro maturation is improved. The aim of the present study was to investigate the effects of FF-MAS on porcine oocyte maturation and pronucleus formation in vitro. Porcine cumulus-oocyte complexes (COCs) were isolated from abattoir ovaries and in vitro matured for 48 h in NCSU 37 medium supplemented with 1 mg/l cysteine, 10 ng/ml epidermal growth factor and 50 microM 2-mercaptoethanol with or without 10% porcine follicular fluid (pFF). For the first 22 h, 1 mM db-cAMP and 10 I.E PMSG/hCG was added. The medium was supplemented with 1 microM, 3 microM, 10 microM, 30 microM or 100 microM FF-MAS dissolved in ethanol. After maturation the COCs were denuded mechanically using a fine glass pipette under constant pH and in vitro fertilized with fresh semen (5 x 10(5) spermatozoa/ml). The presumptive zygotes were evaluated 18 h after fertilization. The addition of pFF increased the monospermic as well as the polyspermic penetration of oocytes. In the absence of pFF, the addition of FF-MAS decreased the polyspermic penetration rate, whereas FF-MAS in combination with pFF decreased monospermic and increased polyspermic penetration. The degeneration rate of ova decreased in the presence of FF-MAS irrespective of the presence or absence of pFF. In the absence of pFF, FF-MAS at 3-10 microM increased the number of zygotes with advanced maternal pronuclear stages. In supraphysiological doses, i.e. 30-100 microM, FF-MAS dose-dependently and reversibly inhibited nuclear maturation in the absence of pFF.     

Regulation of oocyte mitochondrial DNA copy number by follicular fluid, EGF, and neuregulin 1 during in vitro maturation affects embryo development in pigs

Little is known about mitochondrial DNA (mtDNA) replication during oocyte maturation and its regulation by extracellular factors. The present study determined the effects of supplementation of maturation medium with porcine follicular fluid (pFF; 0, 10%, 20%, and 30%) on mtDNA copy number and oocyte maturation in experiment 1; the effects on epidermal growth factor (EGF; 10 ng/mL), neuregulin 1 (NRG1; 20 ng/mL), and NRG1 + insulin-like growth factor 1 (IGF1; 100 ng/mL + NRG1 20 ng/mL), on mtDNA copy number, oocyte maturation, and embryo development after parthenogenic activation in experiment 2; and effects on embryo development after in vitro fertilization in experiment 3. Overall, mtDNA copy number increased from germinal vesicle (GV) to metaphase II (MII) stage oocytes after in vitro maturation (GV: 167 634.6 ± 20 740.4 vs. MII: 275 131.9 ± 9 758.4 in experiment 1; P < 0.05; GV: 185 004.7 ± 20 089.3 vs. MII: 239 392.8 ± 10 345.3 in experiment 2; P < 0.05; Least Squares Means ± SEM). Supplementation of IVM medium with pFF inhibited mtDNA replication (266 789.9 ± 11 790.4 vs. 318 510.1 ± 20 377.4; P < 0.05) and oocyte meiotic maturation (67.3 ± 0.7% vs. 73.2 ± 1.2%, for the pFF supplemented and zero pFF control, respectively; P < 0.01). Compared with the control, addition of growth factors enhanced oocyte maturation. Furthermore, supplementation of NRG1 stimulated mitochondrial replication, increased mtDNA copies in MII oocytes than in GV oocytes, and increased percentage of blastocysts in both parthenogenetic and in vitro fertilized embryos. In this study, mitochondrial biogenesis in oocytes was stimulated during in vitro maturation. Oocyte mtDNA copy number was associated with developmental competence. Supplementation of maturation medium with NRG1 increased mtDNA copy number, and thus provides a means to improve oocyte quality and developmental competence in pigs.     

Generation of Live Piglets from Cryopreserved Oocytes for the First Time Using a Defined System for In Vitro Embryo Production

We report the successful piglet production from cryopreserved oocytes for the first time by using a simple, high capacity vitrification protocol for preservation and a defined system for in vitro embryo production. Immature cumulus-oocyte complexes (COCs) from prepubertal gilts were vitrified in microdrops and stored in liquid nitrogen. After warming, COCs were subjected to in vitro maturation (IVM), fertilization (IVF), and subsequent culture (IVC). Adjusting warmplate temperature to 42°C during warming prevented temperature drops in a medium below 34.0°C and significantly increased the percentage of oocyte survival and thus blastocyst yields obtained from total vitrified oocytes compared with that of warming at 38°C (87.1% vs 66.9% and 4.4% vs 2.7%, respectively). Nuclear maturation and fertilization of oocytes were not affected by vitrification and warming temperature. Blastocyst development on day 7 (day 0 = IVF) of the surviving oocytes after warming at 38°C and 42°C was not different but lower (P<0.05) than those of non-vitrified control oocytes (4.6%, 5.2% and 17.9%, respectively). However, blastocyst cell numbers in the control and vitrified groups were similar irrespective of warming temperature. Omitting porcine follicular fluid (pFF) from IVM medium (POM) did not affect maturation, fertilization and embryo development of vitrified-warmed oocytes. Transfer of blastocysts obtained on day 5 from vitrified oocytes matured either with or without pFF into 4 recipients (2 for each group) resulted in 4 pregnancies and the delivery of a total of 18 piglets. In conclusion, optimization of warming temperature was a key factor for achieving high survival rates, and surviving oocytes could be utilized in vitro using defined media. Using these modifications, live piglets could be obtained from cryopreserved oocytes for the first time.     

Developmental competence of frozen-thawed yak (Bos grunniens) oocytes followed by in vitro maturation and fertilization

In the present study, we examined the ability of immature germinal vesicle (GV) and subjected to in vitro matured (MII) yak oocytes to survive after cryopreservation as well as their subsequent development following in vitro maturation and fertilization. Both GV and MII oocytes were cryopreserved by using two different vitrification solutions (VS); VS-I contained 10% ethylene glycol (EG) and 10% dimethylsulfoxide (DMSO) in TCM-199 + 20% (v/v) fetal calf serum (FCS) whereas VS-II contained 40% EG + 18% Ficoll + 0.5 M sucrose in TCM-199 + 20% FCS. The percentage of oocytes found to be morphologically normal was greater (P < 0.01) in VS-I group than in VS-II group. Rates of cleavage (30.6–42.2%) and blastocyst formation (2.9–8.9%) did not differ among groups, but were lower than in unfrozen control (55.7% and 25.4%, P < 0.01). These results show that a combination of EG and DMSO or EG, Ficoll and sucrose can be used to cryopreserve yak oocytes in French straws.