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.     

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.     

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.     

Kinetics of nuclear maturation and effect of holding ovaries at room temperature on in vitro maturation of camel (Camelus dromedarius) oocytes

Experiments were conducted to investigate kinetics of in vitro nuclear maturation and the effect of storing ovaries at room temperature on initial chromatin configuration and in vitro maturation of dromedary camel oocytes. Cumulus oocyte complexes (COCs) were collected from slaughterhouse ovaries and matured in vitro for 4-48h. At every 4h interval (starting from 0 to 48 h), groups of oocytes were fixed, stained and evaluated for the status of nuclear chromatin. Oocytes were categorized as germinal vesicle (GV), diakinesis (DK), metaphase-I (M-I), anaphase-I (A-I), metaphase-II (M-II) stage and those with degenerated, fragmented, activated or without a visible chromatin as others. At the start of culture, 74% (66/89) oocytes were at GV stage, 13% (12/89) at DK and 12% (11/89) were classified as others. Germinal vesicle breakdown started spontaneously in culture and at 20 h of culture 97% oocytes had already completed this process. After 8 and 16 h of maturation the highest proportion of oocytes (42%, 48/114 and 41%, 51/123) were at DK and M-I stage, respectively. The proportions of oocytes reaching M-II stage at 32 (42%, 50/118), 36 (45%, 47/104), 40 (49%, 57/117), 44 (52%, 103/198) and 48 h (46%, 55/120) of culture were not different from each other (P>0.05). The proportion of oocytes categorized as others, however, increased after 40 h of culture and was higher (P<0.05) at 48 h compared with other maturation periods. There was no difference (P>0.05) in the proportion of oocytes reaching M-II stage from the ovaries collected and stored in normal saline solution (NSS) at room temperature for 12h (43%, 64/148) and those collected in warm NSS (37 degrees C) and processed immediately after arrival in laboratory (49%, 57/117). However, low number of oocytes reached M-II stage from ovaries collected in warm NSS but stored at room temperature (29%, 37/128) compared with other two groups (P<0.05). It may be concluded that dromedary oocytes require 32-44h of in vitro culture to have an optimum number of oocytes in M-II stage. However, further studies are required to find out the most appropriate maturation period, which will result in the further development of these oocytes after IVF, ICSI, parthenogenetic activation or nuclear transfer. Ovaries can be collected and stored in normal saline solution at room temperature for 12h without any appreciable effect on the nuclear maturation of the oocytes.     

Possible role of 5'AMP-activated protein kinase in the metformin-mediated arrest of bovine oocytes at the germinal vesicle stage during in vitro maturation

The 5'AMP-activated protein kinase (AMPK) activation is involved in the meiotic maturation of oocytes in the ovaries of mice and pigs. However, its effects on the oocyte appear to be species-specific. We investigated the patterns of AMPK and mitogen-activated protein kinases (MAPK3/1) phosphorylation during bovine in vitro maturation (IVM) and the effects of metformin, an AMPK activator, on oocyte maturation in cumulus-oocyte complexes (COCs) and denuded bovine oocytes (DOs). In bovine COCs, PRKAA Thr172 phosphorylation decreased, whereas MAPK3/1 phosphorylation increased in both oocytes and cumulus cells during IVM. Metformin (5 and 10 mM) arrested oocytes at the GV stage in COCs but not in DOs. In COCs, this arrest was associated with the inhibition of cumulus cell expansion, an increase in PRKAA Thr172 phosphorylation, and a decrease in MAPK3/1 phosphorylation in both oocytes and cumulus cells. However, the addition of compound C (10 muM), an inhibitor of AMPK, accelerated the initiation of the GV breakdown (GVBD) process without any alteration of MAPK3/1 phosphorylation in oocytes from bovine COCs. Metformin decreased AURKA and CCNB1 protein levels in oocytes. Moreover, after 1 h of IVM, metformin decreased RPS6 phosphorylation and increased EEF2 phosphorylation, suggesting that protein synthesis rates were lower in oocytes from metformin-treated COCs. Most oocytes were arrested after the GVBD stage following the treatment of COCs with the MEK inhibitor, U0126 (100 micromoles). Thus, in bovine COCs, metformin blocks meiotic progression at the GV stage, activates PRKAA, and inhibits MAPK3/1 phosphorylation in both the oocytes and cumulus cells during IVM. Moreover, cumulus cells were essential for the effects of metformin on bovine oocyte maturation, whereas MAPK3/1 phosphorylation was not.     

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.     

Epidermal growth factor enhances meiotic resumption of canine oocytes in the presence of BSA

Despite many attempts to improve the in vitro maturation (IVM) of canine oocytes using various culture conditions, the efficiency of canine IVM remains very low compared with that of other domestic animals. In the present study we examined the effect of ovarian estrus stage on oocyte quality, and the effect of epidermal growth factor (EGF) in the presence and absence of macromolecules on the IVM of canine oocytes. More oocytes >or=100 microm in diameter were obtained from follicular ovaries than from ovaries at other estrus stages. After 72 h of culture, significantly more oocytes recovered from follicular ovaries than from anestrous and luteal ovaries were in germinal vesicle break down (GVBD). Bovine serum albumin (BSA) or fetal bovine serum (FBS) supplementation improved meiotic resumption as compared to polyvinyl alcohol (PVA) supplementation; however, there was no difference between the BSA and FBS supplements. The oocytes matured in North Carolina State University (NCSU) 37 medium containing 0.4% BSA and 100 ng/ml EGF showed the highest rates of development to the metaphase II (MII) stage when compared with the control treatment (P < 0.05). These results suggest that the estrous cycle of bitches influences the meiotic resumption of oocytes cultured in vitro, and EGF increases the meiotic resumption of canine oocytes in the presence of BSA in vitro.     

In vitro maturation and fertilization of porcine oocytes after a 48 h culture in roscovitine, an inhibitor of p34cdc2/cyclin B kinase

Maintaining oocytes at the germinal vesicle (GV) stage in vitro may permit enhanced acquisition of the developmental competence. The objective of the current study was to evaluate the nuclear and cytoplasmic maturation in vitro of porcine oocytes after pretreatment with S-roscovitine (ROS). Cumulus oocyte complexes (COC) were treated with 50 microM ROS for 48 h and then matured for various lengths of time in a conventional step-wise in vitro maturation (IVM) system by using dibutyryl cyclic AMP. The COC that were matured in the same system for 44 h without pretreatment with ROS were used as the control group. At various periods after the start of IVM, oocytes were assessed for the meiotic stages and subjected to in vitro fertilization (IVF) with fresh spermatozoa. The ROS treatment inhibited GV breakdown of 94.4% oocytes, with the majority arrested at the GV-I stage (67.4%). Maximum maturation rate to the metaphase-II stage after ROS treatment was achieved by 44 h of IVM (92.1%) and no differences were observed with control oocytes (95.0%). Penetration rate was correlated to the maturation rate. The duration of IVM had no effects on polyspermy and male pronuclear (MPN) formation rates at 8 h post insemination (hpi), whereas both rates increased at 22 hpi. Direct comparison with controls assessed at 22 hpi confirmed a lesser MPN formation in ROS-treated oocytes (73.7% compared with 53.6%). Glutathione (GSH) concentrations were less in oocytes treated with ROS than in control oocytes (5 compared with 7.7 pmol/oocyte) as well as blastocyst rate (22.0% compared with 38.1%, respectively). These results demonstrate that cytoplasmic maturation in porcine oocytes pretreated with ROS for 48 h did not equal that of control oocytes in the current IVM system.     

Changes in cumulus-oocyte complexes of pregnant and non-pregnant camels (Camelus dromedarius) during maturation in vitro

The aim of the present study was to examine the cumulus morphology and the oocyte chromatin quality of camel cumulus-oocyte complexes (COCs) at the time of recovery, and to monitor changes in oocyte chromatin configuration and apoptosis in cumulus cells from camel COCs during in vitro maturation (IVM) (0, 12, 24, 32, 36, 42, and 48 p.IVM) depending on pregnancy of donors. A total of 1023 COCs were isolated from sliced ovaries after slaughtering of 47 pregnant and 43 non-pregnant camels in an abattoir. The mean number of COCs per donor was 10.3 in pregnant and 12.5 in non-pregnant donors. The cumulus morphology of COCs was independent of the type of donor and was divided in COCs with compact (26.9 and 28%), dispersed (39.3 and 46%), corona radiata cumulus investment (27.9 and 21.7%) and without cumulus (6 and 4.2%), respectively for pregnant and non-pregnant donors. The highest proportion of COCs exhibited dispersed cumulus (P<0.05). Oocytes with meiotic stages of diplotene >50% were found only in compact (55 and 56.5%) and in dispersed COCs (58.4 and 60%), respectively for pregnant and non-pregnant donors. During IVM (0-48h) the first significant onset of specific meiotic stages were different in oocytes from pregnant donors: metaphase 1 (24-32h), metaphase 2 (36-42h), versus oocytes from non-pregnant donors: metaphase 1 (24h), metaphase 2 (32-48h) (P<0.05). The level of apoptotic cells in cumuli of matured COCs increased during IVM and was higher in matured COCs from non-pregnant donors for each time point during IVM (P<0.01). Camel oocytes meiosis during IVM is accompanied by a drastic increase of apoptosis in the surrounding cumulus cells 0-32 and 0-24h during IVM, respectively for pregnant and non-pregnant donors. The oocytes of pregnant camels require 36h of maturation to reach levels of >50% metaphase 2 stage in comparison to oocytes from non-pregnant donors where 32h are sufficient. The earlier onset of apoptosis in the COCs derived from non-pregnant donors possibly determines the faster progression of the oocytes through the final stages of meiosis.     

Kinetics of meiotic progression, M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase) activities during in vitro maturation of porcine and bovine oocytes: species specific differences in the length of the meiotic stages

The kinetics of nuclear maturation, M-phase promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase) activities during in vitro maturation of porcine and bovine oocytes were examined. A further objective was to determine the duration of the meiotic stages during the maturation process. Porcine and bovine cumulus-oocyte complexes (COCs) were incubated in TCM 199 supplemented with 20% (v/v) heat inactivated fetal calf serum (FCS), 0.05microg/ml gentamycin, 0.02mg/ml insulin, 2.5microg/ml FSH and 5microg/ml LH. COCs were removed from the culture media in hourly intervals starting immediately after recovery from the follicle until 24 (bovine) or 48h (porcine) of culture. Oocytes were either fixed to evaluate the maturation status or the activity of MPF, assessed by its histone H1 kinase activity, and MAP kinase were determined by a radioactive assay simultaneously. In oocytes of both species, the MPF activity oscillated during the culture period with two maxima corresponding with the two metaphases: between 27-32 and after 46h (porcine) and between 6-9 and after 22h (bovine). There was a temporary decline in activity after 33-38 (porcine) and after 19h (bovine), which corresponded with anaphase I and telophase I. MAP kinase activity increased during the whole culture period and reached maximum levels after 47 (porcine) and after 22h (bovine). In porcine oocytes, the MAP kinase was activated before GVBD and MPF activation. In bovine oocytes, MPF and MAP kinase were activated at approximately the same time as the GVBD (8-9h of incubation). In average porcine, oocytes remain 23.4h in the germinal vesicle (GV) stage (13h in GV I, 5.7h in GV II, 3.2h in GV III and 1.5h in GV IV), 0.9h in diakinese, 9.6h in the metaphase I, 2.8h in anaphase I and 1.9h in telophase I of the first meiotic division. In bovine oocytes, the temporal distribution of the meiotic stages were 8.5h for the GV stage, 1.2h for diakinese, 8.3h for metaphase I, 1.6h for anaphase I and 1.9h for telophase I. These results indicate that the duration of the meiotic stages differs between the species and that MAP kinase is activated before MPF and GVBD in porcine oocytes.     

Effect of Hoechst 33342 staining on developmental competence of prepubertal goat oocytes

This study was undertaken to evaluate the effects of Hoechst staining on nuclear maturation and fertilisation when used at different stages of in vitro maturation (IVM) in prepubertal goat oocytes. Oocytes were matured in TCM1999 supplemented with 10% fetal bovine serum, 10 microg LH/ml, 10 microg FSH/ml and 1 microM 17beta-estradiol for 27 h. Frozen-thawed sperm cells were prepared by centrifugation in a discontinuous Percoll gradient and resuspended in DMH medium with 20% steer serum. Oocytes were fertilised in DMH medium with 7.75 mM calcium lactate. During IVM oocytes were exposed to 0.5 microg/ml of Hoechst 33342 staining and to ultraviolet light for a mean time of 3 s at 0 h, 8 h, 15 h, 20 h and 27 h. The percentage of metaphase II oocytes decreased significantly when oocytes were stained with Hoechst dye at 0 h, 8 h and 15 h of IVM. There was a decrease in total fertilisation rate and normal fertilisation rate of Hoechest-stained oocytes, independently of the time of Hoechst staining. Hoechst staining produces a significant reduction in oocyte viability when it is used in the early stages of in vitro maturation.     

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 maturation medium on in vitro cleavage of canine oocytes fertilized with fresh and cooled homologous semen

This study evaluated the effect of three maturation media on the development of in vitro-matured and in vitro-fertilized dog oocytes. In Experiment 1 (non-comparative experiment) canine cumulus-oocyte complexes (COCs) were matured in vitro in TCM199 supplemented with estrous cow serum (10%) + gonadotropins + steroid (treatment A), TCM199 + estrous cow serum (10%) (treatment B), or TCM199 + polyvinylpyrrolidone (PVP) (4%) (treatment C). All maturation media contained a final concentration of 1 microg/ml of human somatotropin (hST). Oocytes were fertilized with fresh ejaculated sperm and development was assessed by cleavage. The objective of Experiment 2 (comparative experiment) was to compare the rates of cleavage and developmental capacity of COCs matured in vitro in same medium as in Experiment 1, and fertilized either with fresh ejaculated or with cooled extended homologous spermatozoa. In Experiments 1 and 2, oocytes fertilized with fresh semen were in vitro-matured for 48 h, while in Experiment 2 COCs fertilized with cooled semen were matured in vitro for 72 h. The results of Experiments 1 and 2 demonstrated that cleavage was not influenced by the oocyte's maturation environment. The results of Experiment 1 showed that pronucleus formation + cleavage (day 7 after IVF) was similar among treatments A, B and C (p = 0.277). Also, in Experiment 2, pronucleus formation + cleavage (day 7 after IVF) was not different for oocytes fertilized in vitro either with fresh or cooled semen and maturated in media A (p = 0.190), B (p = 0.393) or C (p = 0.687). In both experiments, the numbers of embryos that developed to the 6-8-cell stage were higher for oocytes matured in medium A and fertilized with fresh semen, when compared with numbers of oocytes matured in media B and C. Embryo development to the 6-8-cell stage of oocytes fertilized either with fresh or cooled sperm was observed in treatments A and C in Experiment 2. Cumulus cell expansion was similar among treatments in Experiment 1. In Experiment 2, cumulus cell expansion among treatments A, B and C was similar after 48 h or 72 h of IVM. In both experiments, the greatest expansion category seen was for category 2 (outer cumulus cells slightly expanded). No correlation between cumulus expansion and cleavage were observed. Polyspermy rates in oocytes matured in medium A, and fertilized with fresh sperm were not significantly different from polyspermy rates observed using media B and C, in both experiments. Our findings indicate that treatments A, B and C are similarly effective for the cleavage of dog oocytes. Furthermore, it was demonstrated that canine oocytes matured in vitro could be fertilized by homologous cooled spermatozoa and progress to cleavage.     

Replacement of serum and hormone additives with follicular fluid in the IVM medium: effects on maturation, fertilization and subsequent development of buffalo oocytes in vitro

Buffalo follicular fluid was used in the IVM medium in place of serum and hormone additives for stimulating nuclear and cytoplasmic maturation of buffalo oocytes in vitro. Follicular fluid (buFF) was aspirated from visible surface follicles from buffalo ovaries. Cumulus oocyte complexes (COCs) were matured for 24 to 26 h at 38.5 degrees C, 5% CO(2) in air in the maturation medium (TCM-199). When used, the concentration of fetal bovine serum (FBS) was 10% and that of FSH-P was 5 mug/ml. In Experiment 1 TCM-199 was supplemented with 1) FBS, 2) FBS + FSH-P, 3) 20% buFF and 4) 40% buFF. The matured oocytes were denuded and stained with Giemsa stain to study nuclear maturation. The proportion of oocytes which completed nuclear maturation was similar in medium containing FSH (74%) and 20 or 40% buFF (67%), which was higher (P < 0.05) than in medium with FBS but without FSH or buFF (47%). In Experiment 2, which was aimed at examining the effects of buFF on cumulus expansion and rates of fertilization and subsequent development to the blastocyst stage after IVF, the maturation medium was supplemented with 1) FBS + FSH-P, 2) 20% buFF and 3) 40% buFF. The COCs matured in medium containing 20 or 40% buFF had significantly higher (P < 0.01) cumulus expansion than those matured in medium with FBS + FSH-P. Of the COCs matured in medium with FBS + FSH-P and 20 or 40% buFF, the fertilization rates indicated by the incidence of cleavage (56, 51 and 52%, respectively) and the proportion of cleaved COCs developing to morula (58, 54 and 57%, respectively) and blastocyst stage (30, 31 and 35%, respectively) were not significantly different. In Experiment 3, supplementation of the maturation medium with 1) FBS + FSH-P and 2) FBS + FSH-P + 20% buFF resulted in similar rates of morulae (41 and 38%, respectively) and blastocysts (31 and 25%, respectively), indicating that simultaneous presence of FBS, FSH-P and buFF did not have an additive effect on embryo yield. The results show that the gonadotropin and serum source in the IVM medium can be replaced by buFF at the 20% level to achieve comparable morula and blastocyst yields.     

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.     

Supplements to in vitro maturation media affect the production of bovine blastocysts and their apoptotic index but not the proportions of matured and apoptotic oocytes

The objective of this study was to compare the effect of different supplements to the basic IVM medium (TCM199) on the efficiency of cattle oocyte maturation and blastocyst production, and the incidence of apoptosis in both oocytes and blastocysts. Two protein supplements (FBS and fafBSA) and a macromolecule (PVP40) were compared in a 3 treatmentsx9 replicates design. Cumulus-oocyte complexes (COCs) aspirated from slaughterhouse ovaries were matured for 24h in TCM199 medium supplemented with 10% FBS, 6% fafBSA or 4% PVP40 (50-70 COCs in each treatment/replicate), then inseminated and cultured in vitro for 8 days. Immature and mature oocytes as well as Day 8 blastocysts were subjected to TUNEL analysis. Cleavage rate was monitored on Day 2 post-insemination (pi), whereas blastocyst yield on Day 8 pi. The composition of maturation media did not affect zygotic cleavage rate on Day 2 (on average 71.0%), however the blastocyst rate on Day 8 pi was significantly lower (P<0.001) for embryos derived from oocytes matured with PVP40 (16.0%) than for those matured with FBS (22.4%) or fafBSA (22.1%). The rate of TUNEL positive oocytes differed significantly between immature (1.4%) and mature (11.2%) oocytes (P<0.01). Supplements to maturation medium were not related to the incidence of apoptosis in mature oocytes (11.2%) and the rate of oocytes at the second metaphase stage (71.5%). Cumulus cell expansion was reduced by maturation in medium supplemented with PVP40. This macromolecule was also correlated with higher apoptotic index in blastocysts (5.8%) when compared to FBS (3.2%) and fafBSA (3.1%; P<0.001). In conclusion, lower blastocyst rate and elevated apoptotic index in embryos derived from oocytes matured with PVP40 may suggest that synthetic macromolecule provides less balanced environment for oocyte maturation and therefore should be treated with caution.     

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.     

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.     

Glutathione synthesis during in vitro maturation of buffalo (Bubalus bubalis) oocytes: effects of cysteamine on embryo development

It was demonstrated that cysteamine supplementation during in vitro maturation (IVM) improves embryo development by increasing glutathione (GSH) synthesis in several species. An improved developmental competence of oocytes matured in the presence of cysteamine was also recorded in buffalo species. The purpose of this work was to investigate (1) if glutathione is de novo synthesized during in vitro maturation of buffalo oocytes, (2) if cysteamine improves buffalo embryo development via an increase in GSH synthesis, and (3) if the inhibition of glutathione synthesis by buthionine sulfoximide (BSO), in the presence or absence of cysteamine, affects subsequent embryo development and GSH synthesis.Cumulus-oocytes complexes (COCs), recovered from slaughtered animals, were matured in vitro in TCM199+10% fetal calf serum (FCS), 0.5 microg/ml FSH, 5 microg/ml LH and 1 microg/ml 17-beta-estradiol in the absence or presence of cysteamine (50 microM), with or without 5mM BSO. Glutathione content was measured by high-performance liquid chromatography (HPLC) and fluorimetric analysis in immature oocytes and in oocytes matured in the different experimental conditions.In a second experiment, the mature oocytes were in vitro fertilized and cultured for 7 days in order to assess development to blastocysts (BLs). It was demonstrated that buffalo oocytes synthesize glutathione during in vitro maturation and that cysteamine increases glutathione synthesis. Furthermore, the promoting effects of cysteamine on embryo development and GSH synthesis were neutralized by buthionine sulfoximide. These results indicate that glutathione plays a critical role on buffalo embryo development.