Meiotic competence of bovine fetal oocytes following in vitro maturation

The in vitro ability between fetal and cow oocytes to resume meiosis and progression to metaphase-II (M-II) was compared. Cumulus oocyte complexes (COCs) were harvested from 2 to 6 mm follicles from ovaries of 7.5 month to term fetuses and adult cows. Cumulus cells were removed using 3 mg/ml hyaluronidase and repeated pipetting. Denuded oocytes were fixed in 3% glutaraldehyde, stained with DAPI and evaluated under fluorescent microscopy for nuclear status before in vitro maturation (IVM). COCs from fetal and adult ovaries were also matured in 200 microl droplets of medium 199 supplemented with 10 microg/ml FSH, 10/ml LH, 1.5 microg/ml estradiol, 75 microg/ml streptomycin, 100 IU/ml penicillin, 10 mM hepes and 10% FBS for 24 h at 39 degrees C and 5% CO(2). Matured oocytes were fixed, stained and evaluated as explained above for nuclear status namely stage of germinal vesicle (GV) development and subsequent meiotic competence. Data were analyzed using chi-square analysis. The majority of fetal oocytes (P<0.05) before IVM were at GV stages GV-I (27.7%), GV-II (37.6%) and GV-V (22.8%) compared to cow oocytes, which were at GV stages IV (28.3%) and V (46.7%). After IVM, fewer fetal oocytes were at earlier stages of GV development and majority (P<0.05) were at GV-V (24.0%), premetaphase (17.4%) and metaphase-I (M-I: 7.2%) stages. However, after IVM, more cow oocytes matured to M-II than did fetal oocytes (93.7% versus 26.9%; P<0.05). In conclusion, fetal oocytes do not mature in vitro as well as cow oocytes. Our findings suggest that the low meiotic competence of fetal oocytes can be attributed to their being at earlier stages of GV development before IVM.     

The effect of hyaluronan concentrations in hST-supplemented TCM 199 on in vitro nuclear maturation of bitch cumulus-oocyte complexes

In this study, the effect of hyaluronan (HA) on in vitro nuclear maturation of bitch oocytes was evaluated. Cumulus-oocyte complexes (COCs) were cultured for 48 h at 37 degrees C in a humidified atmosphere of 5% CO2 in air. Oocytes with one or more layers of intact cumulus cells and dark cytoplasm were allocated to the following treatments: (A) TCM 199 supplemented with 25 mM Hepes/L (v/v), with 10% heat inactivated estrous cow serum (ECS), 50 microg/mL gentamycin, 2.2 mg/mL sodium bicarbonate, 22 microg/mL pyruvic acid, 20 microg/mL estradiol, 0.5 microg/mL FSH, 0.03 IU/mL hCG and 1 microg/mL human somatotropin (hST) (control medium), (B) Treatment A + 0.5 mg/mL HA and (C) Treatment A + 1.0 mg/mL HA. Supplementation with HA did not increase the number of oocytes that resumed meiosis. Additionally, there were no differences among treatments in the cumulus cell expansion of oocytes. In conclusion, the addition of HA to hST-supplemented TCM 199 did not improve the in vitro nuclear maturation of bitch oocytes, and therefore appeared to be unsatisfactory as supplement for in vitro maturation (IVM) of canine oocytes.     

Effect of co-culturing with embryonic fibroblasts on IVM, IVF and IVC of canine oocytes

We studied the effects of mouse embryonic fibroblasts (MEF) and canine embryonic fibroblasts (CEF) on IVM, IVF and IVC of canine oocytes. Cumulus-oocyte complexes were harvested from ovaries by slicing, and in vitro maturation was evaluated in three different conditions: culture media only (control), co-culture with MEF, or co-culture with CEF. The oocytes were cultured for 48 or 72 h. Only oocytes larger than 100 microm in diameter with a homogeneous dark cytoplasm and two or more layers of cumulus cells were used. The culture medium was TCM 199+10% fetal bovine serum (FBS) with 100 IU/mL penicillin and 100 microg/mL streptomycin. After 48 h of IVM, the oocytes were fertilized in vitro with fresh canine spermatozoa that had been selected by a swim-up method, and the oocytes and spermatozoa were co-cultured in modified Krebs-Ringer bicarbonate solution (TYH) for up to 20 h in 5% CO2 in air at 38.5 degrees C. After insemination, oocytes were transferred to three different conditions (the same as for IVM) and were cultured. After 48 or 72 h of maturation in vitro, the maturation rate of MII oocytes cultured in co-culture of MEF and CEF was higher than for oocytes cultured in control (P<0.05). Although the rate that reached the MII stage was not different in the 48 and 72 h cultures, the percentage of degenerated oocytes was greater at 72 h in all three treatment groups. The proportion of monospermic and polyspermic oocytes was not different among the three treatment groups. Cleavage rates were higher in the MEF and CEF treatment groups than in the control group (P<0.05). Co-culture with CEF developed the embryo up to the 16-cell stage, and with MEF up to morula stage. In conclusion, co-culture of embryonic fibroblast cells enhanced nuclear and cytoplasmic maturation of canine oocytes.     

Effect of incubation temperature on in vitro maturation of porcine oocytes: nuclear maturation, fertilisation and developmental competence.

The present study examined the effect of low culture temperature during in vitro maturation (IVM) of pig oocytes on their nuclear maturation, fertilisation and subsequent embryo development. In experiment 1, oocytes were cultured at 35 or 39 degrees C for 44 h in modified tissue culture medium 199 supplemented with 10 ng/ml epidermal growth factor, 0.57 mM cysteine, 75 microg/ml potassium penicillin G, 50 microg/ml streptomycin sulphate, 0.5 microg/ml LH and 0.5 microg/ml FSH to examine the nuclear maturation status. In experiment 2, oocytes were cultured at 35 degrees C for 44 or 68 h and nuclear maturation was examined. In experiment 3, oocytes matured for 44 or 68 h at 39 degrees C and for 68 h at 35 degrees C were co-incubated with frozen-thawed spermatozoa for 5-6 h. Putative embryos were transferred into North Carolina State University (NCSU) 23 medium containing 0.4% bovine serum albumin. At 12 h after insemination, some oocytes were fixed to examine the fertilisation rate and the remaining embryos were examined at 48 and 144 h for cleavage and blastocyst formation rate, respectively. Compared with 39 degrees C, culture of oocytes at 35 degrees C for 44 h significantly (p < 0.05) reduced the metaphase II (M II) rate (79% vs 12%). However, extension of culture time to 68 h at 35 degrees C significantly increased (p < 0.05) the M II rate (7% vs 58%). In experiment 3, compared with other groups, fewer (p < 0.05) oocytes reached M II when cultured at 35 degrees C for 68 h (69-81% vs 49%). Extension of culture duration to 68 h at 39 degrees C stimulated spontaneous activation (28%) of oocytes. No difference in cleavage rates was observed among different groups. Compared with oocytes matured for 44 h at 39 degrees C (31%), the proportion of blastocysts obtained was low (p < 0.05) for oocytes matured at 35 degrees C (13%) or 39 degrees C (3%) for 68 h. The results indicate that lower culture temperature can delay nuclear maturation of pig oocytes. However, extension of culture time can stimulate nuclear maturation and these oocytes are capable of fertilisation and development to the blastocyst stage at moderate rates.     

Effect of 17beta-estradiol on the in vitro maturation of bovine oocytes

Although 1 microg/ml of 17beta-estradiol (E2) is often used in routine in vitro maturation (IVM) and in vitro fertilization (IVF), its effect remains controversial. The objective of our study was to investigate the effects of E2 on bovine oocyte IVM and subsequent embryo development, using a defined medium. Bovine cumulus oocyte complexes (COCs), aspirated from 2 to 8 mm follicles of slaughterhouse ovaries, were matured in TCM199 in the presence of 1 microg/ml E2 with or without 0.05 IU/ml recombinant hFSH. Cultures without E2, FSH or both served as controls. COCs were matured for 22 h at 39 degrees C in a humidified atmosphere of 5% CO2 in air. To investigate the effect of E2 with and without FSH on nuclear maturation, COCs were fixed after maturation and the nuclear stage was assessed following DAPI staining. Similarly, denuded oocytes (DO) were matured in the presence of E2 and the nuclear stage assessed after 22 h. To investigate the effect of E2 with and without FSH during IVM on subsequent embryo development, in vitro matured COCs were fertilized in vitro and after removal of the cumulus cells, the presumed zygotes were cocultured on BRL monolayer for 11 days. At Day 4, the number of cleaved embryos, and at Days 9 and 11, the number of blastocysts, were assessed. Addition of 1 microg/ml E2 to TCM199 significantly decreased the percentage of Metaphase II (MII) compared to control (56.3 and 74.0%, respectively), and increased the percentage of nuclear aberrations compared to control (13.3 and 2.1%, respectively). The negative effect of E2 on nuclear maturation was stronger when DO were matured; 25.1 and 60.0% of the oocytes reached MII stage for the E2 and control groups, respectively. When COCs were matured in TCM199 supplemented with FSH, the addition of 1 microg/ml E2 did not influence the proportion of MII oocytes, although a higher percentage of nuclear aberrations as compared to control was observed. Presence of E2 during IVM also decreased the blastocyst rate (14.4 and 10.0% for control and E2 groups, respectively). However, when FSH was present, the addition of E2 had no effect on the cleavage rate and blastocyst formation (20.3 and 21.7% for control and E2 groups, respectively). In conclusion, supplementation of 1 microg/ml E2 to a serum free maturation medium negatively affects bovine oocyte nuclear maturation and subsequent embryo development. Although these effects are attenuated in the presence of FSH, we strongly suggest omission of E2 in routine maturation protocols of bovine oocytes.     

Cytogenetic evaluation of in vitro matured bovine oocytes collected from ovaries of individual donors

Oocytes were collected after slaughter by aspiration from pairs of ovaries of individual donors. A total of 656 oocytes was selected for IVM from 74 pairs of ovaries (8.9 oocytes per pair, ranging between 1 and 25). The oocytes were matured in droplets of maturation medium (TCM-199 medium supplemented with 20% estrous cow serum (ECS), 50 microg/ml gentamycin, 10 microg/ml FSH, 1 microg/ml estradiol-17beta). Cytogenetic analysis of 348 oocytes showed 79 at the first metaphase (MI; 22.7%, 79 348 ), 11 at the first telophase (TI; 3.2%, 11/348 ), and 258 at the second metaphase (MII; 74.1% 258/348 ). Significant differences (P < 0.01) were shown among the donors regarding the number of oocytes selected for IVM and the number of oocytes matured for IVF.     

In vitro maturation, in vitro fertilization and embryonic development of canine oocytes

In this study we have investigated the efficiency of in vitro maturation (IVM) as a basic way to study the development of canine oocytes after in vitro fertilization (IVF). We decided, therefore, to perform two-part experiments. Firstly, experiment I compared the effects of TCM199 without fetal bovine serum (FBS) with TCM199 supplemented with 5% FBS on the in vitro nuclear maturation rate of canine oocytes. For the efficiency of meiotic development to the metaphase II (MII) stage, we found that 4.7% (4/64) of all oocytes grown in TCM199 without FBS developed to the MII stage compared with only 1.7% (1/59) of those grown in TCM199 with 5% FBS for 48 h. Therefore, FBS did not increase in vitro nuclear maturation. In experiment II, the cleavage rate of canine oocytes used for IVF was investigated following heparin treatment. Canine oocytes were fertilized in four groups: Fert-TALP medium without heparin (Fert I) or Fert-TALP medium supplemented with 10, 20 or 30 microg/ml heparin (Fert II, Fert III, Fert IV, respectively). Oocytes that were grown for 24 h in Fert I following fertilization showed the highest rate of all of the groups, 6.5% (5/77) and developed to the early morula stage. Markedly, the oocytes cultured in Fert I for 24 h following insemination had a higher rate of embryonic development than other groups. We can assert that, unlike findings in other mammals, heparin treatment in canine IVF does not increase the efficiency of the fertilization rate and is therefore not an important factor.     

Timing of sequential changes in cumulus cells and first polar body extrusion during in vitro maturation of buffalo oocytes

Studies were conducted to investigate the degree of the cumulus cell expansion and expulsion of the first polar body in relation to time of incubation in three different culture media during in vitro maturation of buffalo oocytes and to suggest a suitable practical method for assessment of in vitro maturation rate of buffalo oocytes. Buffalo oocytes were aspirated from ovaries collected from a local slaughterhouse. Only oocytes with more than two layers of cumulus cells and homogenous ooplasm were cultured into 50 microl droplets of three different culture systems: (1) TCM-199 + steer serum (10%): (2) TCM-199 + steer serum (10%) + PMSG (40 IU/ml); and (3) TCM-199 + steer serum (10%) + PMSG (40 IU/ml) + estradiol 17beta (1 microg/ml) in a 35 mm Petri dish. The droplets were covered with warm (39 degrees C) mineral oil and incubated in a CO2 incubator (39 degrees C, 5% CO2 in air, 90-95% relative humidity) for 16-18, 20, 22, and 24 h. The maturation rate was assessed by evaluation of degree of cumulus cells expansion and identifying first polar body extrusion into the perivitelline space under stereo zoom microscope. Matured oocytes were inseminated in vitro with 9-10 million sperm/ml of Brackett and Oliphant (BO) medium. Cleaved embryos were cultured in TCM-199 supplemented with steer serum (10%) for 8 days. Cumulus expansion and extrusion of first polar body commenced at 16 and 17 h, respectively, of buffalo oocyte culture. These events mainly exhibited during 22-24 h of culture. Oocytes with Degrees 1 and 2 cumulus cells expansion and extruded first polar body in degree 0 oocytes may be considered as matured and can be used in IVF studies.     

Optimum gas atmosphere for in vitro maturation and in vitro fertilization of bovine oocytes

The objective of this study was to determine optimal gas atmosphere conditions for in vitro maturation (IVM) and in vitro fertilization (IVF) of bovine oocytes. In Experiment 1, groups of 10 to 12 cumulus-oocyte complexes (COCs) were matured (24 h) and fertilized (18 h) under 1) 5% CO(2), 5% O(2;) 2) 5% CO(2), 10% O(2) or 3) 5% CO(2), 20% 0(2.) The COCs were cultured in 50 microl drops of maturation medium (TCM-199 + 10% bovine calf serum + oLH, oFSH and estrogen) or fertilization medium (TALP + swim-up separated spermatozoa +1 microg/ml heparin sulfate) under a layer of 10 ml paraffin oil at 39 degrees C with saturated humidity. Half of the oocytes in each drop were assigned randomly for maturation scoring and the remainder were inseminated. Reduced atmospheric O(2) drastically decreased proportions of oocytes reaching MII (71.4, 26.9 and 9.3% with 20, 10 and 5% O(2), respectively; P < 0.05). The percentages of total fertilization in 10 and 20% O(2) were similar and considerably higher than in 5% O(2) (80.3, 87.0 and 53.1%, respectively; P < 0.05). In addition, the percentage of polyspermy markedly increased when IVF was conducted in reduced O(2) (26.6 and 28.8% in 5 and 10% O(2) vs 15.4% in 20% O(2;) P < 0.05). Experiment 2 was similar to Experiment 1 except that CO(2) was the variable: 1) 2.5% CO(2) in air, 2) 5% CO(2) in air and 3) 10% CO(2) in air. The proportion of MII oocytes did not differ across treatments (64.9, 68.9 and 61.9%, respectively; P > 0.05). Although the percentages of total fertilization among treatments were not different (75.4, 80.9 and 76.1%, respectively), the proportion of normal fertilization was significantly reduced in 10% C0(2) (55.1%) when compared with that of either 2.5% CO(2) (62.7%) or 5% CO(2) (68.7%; P < .05). This study indicates that low O(2) is detrimental for IVM/IVF of bovine oocytes and that optimal atmospheric conditions are either 2.5 or 5% CO(2) and 20% O(2).     

Influence of cysteamine supplementation and culture in portable dry-incubator on the in vitro maturation, fertilization and subsequent development of mouse oocytes

The need to transport oocytes and embryos between two laboratories have prompted us to evaluate the effects of in vitro maturation of immature mouse oocytes in a CO2-deficient dry heat portable incubator and subsequent in vitro development of these fertilized mouse oocytes in a standard CO2 incubator. In addition, the effects of cysteamine supplementation on maturation rate and embryonic development during in vitro maturation (IVM) and culture of embryos in the portable incubator were also investigated. Germinal vesicle stage mouse oocytes, recovered at 40-h post-FSH from 6- to 8-week-old C57BL/6xCBA F1 healthy female mice, were matured in vitro in a modified TCM-199 supplemented with or without 100 microM cysteamine in a standard incubator (5% CO2; 37 degrees C) or cultured in a CO2-deficient dry heat portable incubator for 5 h at 37 degrees C and thereafter transferred to a standard incubator for further culture. The addition of cysteamine in the IVM medium significantly improved maturation rates of the GV mouse oocytes to metaphase II stage. However, cysteamine supplementation in the culture medium did not significantly improve fertilization and blastocyst formation rates of IVM and ovulated oocytes, and in vivo-derived zygotes. Culture conditions in a CO2-deficient dry heat portable incubator did not adversely affect the developmental competence of in vivo-derived zygotes and in vitro matured mouse oocytes after IVF or parthenogenetic activation. Cysteamine supplement in the IVM medium could enhance nuclear maturation of these immature oocytes during shipment.     

Effects of thiol compounds on in vitro maturation of canine oocytes collected from different reproductive stages

Various thiol compounds are known to improve cytoplasmic and/or nuclear maturation of oocytes in vitro. The present study examined the effects of two thiol compounds, cysteine (0.1, 0.5, and 1.0 mM) and cysteamine (50, 100, and 200 microM), on cytoplasmic and nuclear maturation of canine oocytes. Oocytes collected from different reproductive stages were cultured in TCM-199 supplemented with 10% fetal bovine serum, 2.2 mg/ml sodium carbonate, 2.0 microg/ml estrogen, 0.5 microg/ml FSH, 0.03 IU/ml hCG, and 1% penicillin-streptomycin solution for 72 h. Data were analyzed by two-way ANOVA after arcscine transformation and protected by Bonferroni post hoc test. The effects of cysteine and cysteamine on canine IVM were varied depending on the reproductive stage of oocyte donor bitches. In the follicular stage, significantly more oocytes reached the metaphase II (M II) stage when cultured with 0.5 or 1.0 mM cysteine (16.7% and 16.9%, respectively) compared to the control (6.2%). In the follicular stage, cysteamine increased oocyte maturation rate upto the M II stage (15.1% to 17.0%) compared to the control (4.4%). Both the 0.5 mM cysteine and 100 microM cysteamine, alone or together, increased the intracellular GSH level of canine oocytes compared to the control. Irrespective of reproductive stage, no further beneficial effects on nuclear or cytoplasmic maturation were observed when 0.5 mM cysteine and 100 microM cysteamine were supplemented together. In conclusion, addition of 0.5 mM cysteine and 100 microM cysteamine to the maturation medium improved IVM of canine oocytes.     

Insulin-transferrin-selenium (ITS) improves maturation of porcine oocytes in vitro

The objective of this study was to determine if insulin-transferrin-selenium (ITS) promoted a nuclear and cytoplasmic maturation of porcine oocytes that better supports subsequent embryonic development. The rate of oocyte in vitro maturation (IVM) in an experimental group treated with hormones for 42 h was significantly increased compared with that in a control group without hormone treatment (47.8% vs. 11.7%, respectively, p < 0.05). Following reduction of the hormone treatment period from 42 h to 21 h, which included both the first 21 h period of hormones treatment (45.4%) and the second 21 h period of hormone treatment (44.8%), the rate of oocyte IVM was still higher than that of the control group (p < 0.05). To improve porcine oocyte nuclear maturation, 1% ITS was added to medium supplemented with hormones. The rate of nuclear maturation in the ITS-treated group was significantly higher than in the ITS-untreated group (78.6% vs. 54.4%, respectively, p < 0.05). ITS treatment also significantly reduced the per cent of oocytes with type I and type III cortical granule (CG) distribution, respectively, and significantly increased the per cent of oocytes with type II CG distribution (85.3%). These observations indicated that the synchronization rates of nuclear and ooplasmic maturation reached 67.04% (78.56 × 85.33%). In conclusion, the combination of modified Tissue Culture Medium-199 (mM199) + 10 ng/ml epidermal growth factor (EGF) + 10 IU/ml pregnant mare serum gonadotrophin (PMSG) + 10 IU/ml human chorion gonadotrophin (hCG) + 2.5 IU/ml follicle stimulating hormone (FSH) + 1% ITS is suitable for culturing porcine oocytes in vitro, and effectively enhances porcine oocyte nuclear and cytoplasmic maturation.     

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.     

Influence of reproductive status on in vitro oocyte maturation in dogs

In the bitch, oocytes need 48-72 h to complete post-ovulatory maturation to the metaphase II stage in the isthmus of the oviduct, an interval similar to that found in in vitro studies. The effect of estrous cycle stage on in vitro meiotic competence of dog oocytes has been described in several studies. However, there are no reports evaluating the possible effects of pyometra or pregnancy on subsequent potential of oocytes recovered from such females to undergo in vitro maturation.In this study, immature cumulus-oocyte complexes (COCs) were recovered from fresh excised domestic dog ovaries in various reproductive states. The donor females were classified into groups based on stage of the estrous cycle: follicular (proestrus or estrus), luteal (diestrus) or anestrus or at the clinical conditions of pregnancy and pyometra. Grades 1 and 2 oocytes were cultured in vitro at 37 degrees C in TCM-199, supplemented with 25 mM Hepes/l (v/v), and with 10% heat inactived estrous cow serum (ECS), 50 microg/ml gentamicin, 2.2 mg/ml sodium carbonate, 22 microg/ml pyruvic acid, 1.0 microg/ml estradiol, 0.5 microg/ml FSH and 0.03 IU/ml hCG. The nuclear maturation rate was evaluated at 72 h of incubation under Hoechst 33342 (10 microg/ml) staining for fluorescence microscopy. There was no statistical difference in nuclear progression to the MII stage among the various reproductive states (follicular phase, 5.4%; diestrus, 4.2%; anestrus, 4.4%; pyometra, 8.1% and pregnancy, 4.7%). Resumption of meiosis was 24.6% at the follicular phase, 19.6% for diestrus, 16.4% for anestrus, 37.1% for pyometra and 29.2% for pregnancy. Positive and higher numbers of residue above the expected value were observed for the pyometra and pregnancy conditions at the metaphase/anaphase I (MI/AI) stages.Our results indicate that in vitro nuclear maturation of dogs oocytes is not influenced by the in vivo reproductive status of the female. The quality of the oocyte is a more reliable indicator of its potential for meiotic maturation in vitro than the hormonal environment of the donor female at the time of oocyte retrieval.     

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.     

Developmental capacity of in vitro matured and fertilized oocytes from prepubertal and adult goats

The developmental competence of oocytes from prepubertal and adult goats was studied through in vitro maturation, fertilization and embryo culture up to the blastocyst stage. Oocytes were recovered from antral follicles of prepubertal and adult goat ovaries, with or without ovarian stimulation with exogenous FSH. The effect of different sources of granulosa cells during IVM on the developmental competence of prepubertal goat oocytes was also noted. Oocytes were matured for 27 h at 38.5 degrees C in 5% CO(2) in air in 50-microl microdrops in TCM199 supplemented with 20% estrus goat serum, FSH, LH and estradiol-17beta or in 2 ml of the same medium supplemented with granulosa cells. Matured oocytes were inseminated with freshly ejaculated spermatozoa following capacitation At 24 h post-insemination, the oocytes were transferred to a granulosa cell monolayer, and early embryo development was evaluated until Day 10. Results show that the developmental ability of embryos from prepubertal goats after IVM and IVF is similar to those from adult goats. Treatment of the prepubertal and adult goats with FSH did not improve the developmental capacity of the resulting embryos. On studying the addition of different sources of granulosa cells to a maturation system of 2 ml of medium, a significantly positive effect of the cells from primed females was observed on the percentage of maturation, on embryo cleavage and on the percentage of embryos that overcame the in vitro developmental block from 8 to 16 cells.     

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.     

Effect of sheep and human follicular fluid on the maturation of sheep oocytes in vitro

This study examines the effect of sheep and human follicular fluid on the in vitro maturation (IVM) of sheep follicular oocytes. Oocyte cumulus complexes recovered post mortem were matured for 24 to 26 h at 38.6 degrees C, 5% CO(2) in air, in TCM-199 bicarbonate medium supplemented with 20% fetal calf serum (FCS) and, where stated, with maturation hormones, including FSH (5.0 ug/ml), LH (5.0 ug/ml) and estradiol (1 ug/ml), or with sheep follicular fluid recovered from large (>5mm) or small (2 to 5mm) ovarian follicles post mortem, or with human periovular follicular fluid obtained during routine IVF procedures. The matured oocytes were then denuded, and their maturation stage and developmental capacity were assessed by in vitro fertilization (IVF) and culture (IVC). It was found that inclusion of sheep or human follicular fluid or hormone supplements in the IVM media more than doubled the number of oocytes completing maturation (FCS alone 33%, compared with 76.2% for maturation hormones, 84.2% for fluid from large and 69.6% for fluid from small sheep follicles and 82.6% for human follicular fluid), and significantly increased fertilization rates (FCS alone 51.6%, compared with 71.9% for maturation hormones, 78.4% for fluid from the large and 75.7% for fluid from small sheep follicles and 73.1% for human follicular fluid) without discernible adverse effects on the development of the cleaving embryos to the morula or blastocyst stage in culture. Omission of FCS and supplements from the IVM medium resulted in a marked reduction (56%) in the number of oocytes maturing. This reduction could be offset to a large part, but not completely, by inclusion of human follicular fluid or human follicular fluid plus LH (5 ug/ml) in the medium. The results of this study show that addition of sheep or human follicular fluid to maturation medium can enhance rather than inhibit the maturation and fertilizability of sheep follicular oocytes in vitro.     

Effects of gonadotropins on bovine oocytes matured in TCM-199

The effects of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) during in vitro maturation of bovine oocytes in TCM-199 without serum were evaluated. Bovine oocytes with compact cumulus cells were collected from slaughterhouse-derived ovaries and cultured in Hepes-buffered TCM-199 supplemented with 5 mg/mL BSA, 1 microg/mL estradiol-17beta, FSH (0, 0.015, 0.05, 0.15, 1.5 or 15 ng/mL; Experiment 1), LH (0, 0.14, 1, 7 or 49 microg/mL; Experiment 2) and combinations of 1 or 10 ng/mL FSH and 1 or 10 microg/mL LH (Experiment 3) at 39 degrees C in 5% CO2 in air. After 22 h of maturation, cumulus expansion was estimated by scoring from 0 (no expansion) to 4 (full expansion of cumulus mass). In vitro fertilization was done with Percoll (45/90%) separated bull sperm at 1 x 10(6) sperm/mL in fert-TALP with 5 U/mL heparin. At 18 to 20 h post-insemination, presumptive zygotes were transferred to a chemically defined medium (CDM-1) supplemented with 0.5% BSA and nonessential amino acids for 72 h and then moved to CDM-2, additionally supplemented with essential amino acids. Zygotes were cultured at 39 degrees C in 5% CO2, 5% O2 and 90% N2 for 8 days. During Experiments 1 and 2, cumulus expansion increased in proportion to concentrations of FSH and LH. Cleavage rates and development to blastocysts were not significantly different among FSH and LH treatments. In Experiment 3, cumulus expansion of bovine oocytes was maximal when 1 ng/mL FSH and 1 microg/mL LH were added to IVM medium, but cumulus expansion again was not related to developmental ability, although cleavage rates were improved slightly (P<0.05) by the combination of LH and FSH. Blastocyst quality, estimated by the size of inner cell mass, was not different between combinations of FSH and LH, and the numbers of nuclei were not different. Although expansion of cumulus cells surrounding bovine oocytes was altered in response to FSH and/or LH in semi-defined medium, cumulus expansion was not related to rates of cleavage or subsequent embryonic development in vitro. The effects of LH on cumulus expansion can be explained by as little as 1 part per 10, 000 contamination with FSH.     

Development of water buffalo (Bubalus bubalis) embryos from in vitro matured oocytes reconstructed with fetal skin fibroblast cells as donor nuclei

The present study was carried out to explore the feasibility of using buffalo fetal skin fibroblasts as donor nuclei and to find out the developmental competence of embryos following transfer of these nuclei to in vitro matured enucleated buffalo oocytes. Skin cells were isolated from 1 to 2-month-old fetuses obtained from slaughterhouse, by enzymatic digestion (0.5% w/v trypsin +0.05% w/v collagenase in Dulbecco's PBS) for 15-20 min. The cells were washed 4 times with Dulbecco's PBS and then once with RPMI-1640+10% FBS by centrifugation at 600 x g. The cells were then cultured in the same medium in a CO2 incubator (5% CO2 in air) at 38.5 degrees C for 2-3 days. Cumulus-oocyte complexes (COCs) collected from slaughterhouse buffalo ovaries were subjected to IVM in the IVM medium (TCM-199 + 5 microg/ml FSH-P + 10 microg/ml LH+10% FBS) for 20-22 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees C. Oocytes were denuded with 0.1% trypsin followed by repeated pipetting and then enucleated by aspirating the first polar body with 10-15% of nearby cytoplasm with a micromanipulator. Two different types of donor cells (growing cells and those arrested with cytochalasin-B) were used for reconstruction of oocytes. The reconstructs were electro fused and incubated in the activation medium (TCM-199 + 8 microg/ml cytochalasin-B+10% FBS) for 4 h. These were then cultured in IVC medium (TCM-199+10% FBS) in a CO2 incubator (5% CO2 in air) at 38.5 degrees C for 48 h. The cleaved embryos were then co-cultured with buffalo oviduct cells in embryo development media (EDM). Out of 119 denuded matured oocytes which were enucleated and reconstructed with growing cells, 78 (65.5%) were electro fused, activated and cultured, out of which 4 (5.1%) reconstructs cleaved and developed to 2-cell stage, 3 (3.8%) reached to 4-cell stage and 3 (3.8%) reached to 8-cell stage. In the synchronized group, out of 62 denuded matured oocytes which were reconstructed with cytochalasin-B blocked cells, 40 (65%) were electrofused, activated and cultured, out of which 4 (10%) developed to 2-cell stage, 3 (7.50%) to 4-cell stage, 2 (5.0%) to early morula stage and 1 (2.50%) to blastocysts stage. These results suggest that buffalo fetal skin fibroblasts could be used as donor nuclei for the production of buffalo embryos after nuclear transfer to enucleated in vitro matured buffalo oocytes.