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.     

The effects of follicular fluid on in vitro maturation, oocyte fertilization and the development of bovine embryos

We determined the effects of follicular fluid in the maturation medium on bovine oocyte maturation, fertilization and subsequent development, as well as on the number of cells in blastocysts following culture. Fluid and oocytes from bovine follicles less than 5 mm in diameter were collected from the ovaries of slaughtered cows. For the maturation medium, follicular fluid at concentrations of 10, 30 or 60% (v/v) was added to Medium 199 with Earle's salts supplemented with 0.1 microg/ml estradiol-17 beta (E(2), Experiment 1) or 0.1 microg/ml E2 and 100 IU/ml hCG (Experiment 2). The control medium contained polyvinylpyrrolidone (PVP; 3 mg/ml) instead of follicular fluid. After maturation for 24 h, oocytes were fertilized in vitro with bull frozen-thawed spermatozoa and cultured on a monolayer of granulosa cells for 9 d. There were no differences in maturation or fertilization rates of oocytes. In Experiment 1, maturation medium containing 10% follicular fluid did not affect the developmental rate of the oocytes to > 2-cell, 8 to 16-cell, blastocyst and hatched blastocyst stage embryos, respectively; whereas 60% decreased embryonic development (P < 0.05) compared with the control. Blastocysts and hatched blastocysts developed from fertilized oocytes which had been matured in medium containing 10 and 30% follicular fluid/E(2) had more cells than the controls (P < 0.01). In Experiment 2, maturation medium containing 10 or 30% follicular fluid did not affect the development fertilized oocytes to the blastocyst stage compared with the control, but decreased at 60% (P < 0.01). There were no differences in the number of cells from Day 9 blastocysts and hatched blastocysts from fertilized oocytes matured in maturation medium containing follicular fluid and E(2) + hCG. The results of these experiments suggest that the addition of bovine follicular fluid to the maturation medium enhances the cell numbers in blastocysts from bovine follicular oocytes matured in vitro.     

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.     

Comparison of the effects of using standard and simple portable CO2 incubators on the in vitro developmental competence of bovine embryos reconstituted by somatic cell nuclear transfer

In an attempt to determine the cultural factors that would improve cloning efficiency, we compared the effects of two incubation systems-a simple portable system and a standard CO2 incubator-on the production of bovine embryos by electrofusion of quiescent fetal fibroblast nuclei to enucleated oocytes matured in vitro. While the temperature (38.5 degrees C) and CO2 concentration (5%) were similar in both systems, the portable incubator operated in a vacuum of 300 mmHg and at an O2 level of 8-10%, which is lower than the standard. Although there were no significant differences between the two systems in terms of in vitro oocyte maturation (MII stage), fusion rates, and the number of cells in Day 7 blastocysts, significantly higher proportions of nuclear-transferred oocytes cleaved (P < 0.05) and developed to the blastocyst stage (P < 0.01) in the portable incubator (70.5 +/- 0.6 and 36.1 +/- 1.4%, respectively) than in the standard incubator (64.1 +/- 3.2 and 23.5 +/- 1.4%, respectively). Following the transfer of six blastocysts from the portable incubator group to three recipients, survival rates on Days 60, 90, and 120 were 100, 66.7 and 33.3%, respectively. This relatively high early embryonic loss may be associated with multiple pregnancy complications or other abnormalities of placentation frequently observed in cloned embryos. Further studies using this portable incubator system are needed to determine the optimum levels of O2, CO2, and air pressure.     

Effect of sperm capacitation and fertilization media on IVF and early embryo development of prepubertal goat oocytes

Experiments were carried out to develop an improved IVF system for prepubertal goat oocytes matured in vitro. Cumulus oocyte complexes (COC) were obtained by slicing ovaries from slaughtered prepubertal goats. Oocytes were matured in TCM199 supplemented with 20% estrous goat serum (EGS) + 10 micrograms/mL FSH + 10 micrograms/mL LH + 1 microgram/mL estradiol 17 beta for 27 h at 38.5 degrees C in 5% CO2 in air. In Experiments 1 and 2, freshly ejaculated spermatozoa were capacitated in 1 of 3 media: TALP/H, modified Defined Medium (mDM) and mH-M199 with 50 micrograms/mL heparin for 45 min. Matured oocytes were fertilized in TALP, mDM or mH-M199 in Experiment 1 and in TALP in Experiment 2. In Experiment 3, three media were used for sperm capacitation and fertilization: Treatment A (control group): spermatozoa were capacitated in mDM with 50 micrograms/mL heparin for 45 min and fertilized in TALP medium with 1 microgram/mL hypotaurine; Treatment B: spermatozoa were capacitated in mDM with 50 micrograms/mL heparin + 388 micrograms/mL caffeine for 30 min and fertilized in TALP medium without hypotaurine; Treatment C: spermatozoa were capacitated in mDM with 50 micrograms/mL heparin for 45 min and fertilized in TALP medium with PHE (20 microM penicillamine, 10 microM hypotaurine and 2 microM epinephrine). At 24 h post insemination, the ova were transferred to a granulosa cell monolayer, and early embryo development was evaluated until Day 8. In experiment 2, the results show, that mDM plus heparin for sperm capacitation and TALP medium with hypotaurine for oocyte fertilization provided the highest proportion of penetrated oocytes, both total number (79.6%) and normal fertilization (55.1%), whereas the use of caffeine (44.6 and 31.2%, total and normal fertilization rate, respectively) and PHE (31.8 and 20.6%, total and normal fertilization rate, respectively) as motility enhancers did not improve the results obtained in the control group (48.7% and 37.2%, total and normal fertilization rate, respectively). These were no differences for the results of morulae and blastocysts.     

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.     

Maturation and fertilization of porcine oocytes in vitro

Four experiments were conducted to study 1) factors affecting porcine oocyte maturation in culture medium and 2) a new method for oocyte maturation outside the porcine body. In Experiment 1, five groups of oocytes were cultured in m-TCM199 or m-KRB medium for 24 to 28, 32 to 36 or 40 to 42 hours and then were fertilized in vitro. The cleavage rate (two to four-cell stage) of oocytes cultured for 32 to 36 hours was significantly higher than those of the other oocytes. The results indicate that a suitable culture period for the in vitro maturation of porcine oocytes is 32 to 36 hours. In Experiment 2, four groups of oocytes were cultured in m-KRB or m-KRB supplemented with PFF, PMSG or FSH for in vitro maturation, and the cleavage rates of oocytes were 7.94, 22.56, 30.23 and 23.26%, respectively, after in vitro fertilization. The results show that porcine follicular fluid (PFF) and gonadotrophins added to the culture medium promote porcine oocyte maturation in vitro. In Experiment 3, oocytes were cultured in m-KRB or m-TCM199, supplemented with both gonadotrophin and pocine folliclar fluid for maturation in vitro. After fertilization in vitro, the cleavage rates of oocytes were 26.32 and 27.93% for the two media. The results indicate that the difference between m-KRB and m-TCM199 was insignificant when the media were used to culture porcine oocytes. But there was a significant difference when PFF and gonadotrophins were added to the basic media. In Experiment 4, porcine oocytes were transferred into the reproductive tracts of other animals for maturation. After 34 to 36 hours, the oocytes were collected and fertilized in vitro. The cleavage rates of oocytes were 10.42, 28.45, 3.33 and 36.36%, respectively, for the oocytes matured in mouse uterine horns, rat uterine horns, rat oviducts or rabbit oviducts. The results show that porcine oocytes can be matured in the reproductive tracts of other animals.     

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.     

Development of in vitro embryo production systems for red deer (Cervus elaphus). Part 2. The timing of in vitro nuclear oocyte maturation

The time course of in vitro red deer nuclear oocyte maturation was determined. Ovaries were obtained at slaughter and oocytes were aspirated from follicles greater than 2mm in diameter. Oocytes with compact cumulus cells were matured in 50 microl microdrops (10 per drop) under mineral oil containing TCM 199 supplemented with 0.33 mM pyruvate, 10 microg LH and FSH, 1 microg oestradiol and 10% foetal bovine serum. Oocytes were matured at 39 degrees C and 5% CO(2) in air. At 3h intervals (0-27 h) oocytes were removed from incubation, cumulus expansion scored and removed, and fixed oocytes in ethanol:acetic acid (3:1) for 48 h. Oocytes were stained with lacmoid (1%) and nuclear maturation assessed. Oocytes were arrested in the germinal vesicle (GV) stage at aspiration and up to 6h of incubation. The nuclear membrane began to disperse after 6h and by 10.6+/-0.6h of incubation 75% of the oocytes exhibited germinal vesicle breakdown (GVBD). The mean time for 50% of the oocytes to reach metaphase one (MI) and metaphase two (MII) was 11.7+/-0.4 and 24.8+/-0.9h, respectively. Cumulus oophorus were tightly compacted at aspiration and did not begin expansion until 12h of culture. Full expansion was complete by 18 h of culture. Corona radiata cells did not begin expansion until 15 h and were fully expanded by 24h. Results indicate that in vitro red deer oocyte maturation follows a similar time course of nuclear maturation as reported for bovine and ovine oocytes.     

Effect of holding technique and culture drop size in individual or group culture on blastocyst development after ICSI of equine oocytes with low meiotic competence

The effect of medium-to-embryo ratio on blastocyst development of equine embryos from oocytes with compact cumuli was evaluated in the present experiment. In addition, two methods for holding oocytes before in vitro maturation were compared. In Experiment 1, oocytes cultured with roscovitine for 16-18h before maturation were fertilized by intracytoplasmic sperm injection and cultured individually in 2.5, 5, 10 or 50microl droplets. In Experiment 2, oocytes were either cultured with roscovitine or held in a modified M199 with 20% serum at room temperature (EH treatment) for 16-18h, then matured, fertilized and cultured in groups at 5microl medium per embryo. In Experiment 3, oocytes were held in the EH treatment, then were matured and fertilized. In Study 3.1, injected oocytes were cultured individually in drop sizes as for Experiment 1; in Study 3.2, groups of 2-7 oocytes were cultured in fixed drop sizes of 5 or 50microl. Blastocyst development rates of individually-cultured embryos were not significantly different among drop sizes in either Experiment 1 or 3 (15-29%). In Experiment 2, blastocyst rates were not significantly different between holding treatments (17-23%). In Experiment 3, for group-cultured oocytes, blastocyst development was not significantly different between 5 and 50microl drops (39 and 27%, respectively). In conclusion, compact-cumulus oocytes may be effectively held in the EH treatment before maturation, and single culture of equine embryos yields acceptable blastocyst development. The greatest blastocyst rate (39%) was obtained with group culture in a 5microl droplet.     

Holding bovine oocytes in the absence of maturation inhibitors: kinetics of in vitro maturation and effect on blastocyst development after in vitro fertilization

Holding immature oocytes before maturation simplifies the transport of oocytes and aids in scheduling later manipulations. We examined the effect of holding bovine oocytes in the absence of meiotic inhibitors on their subsequent meiotic and developmental competence. Oocytes were matured immediately after recovery (control) or were held in a mixture of 40% TCM 199 with Earle's salts, 40% TCM 199 with Hanks' salts, and 20% FBS, at room temperature for 16 to 18h (EH-held) and then matured. Chromatin status was determined at 0, 10, 14, 18, and 22h of maturation culture. Oocytes were fertilized in vitro after either 18 or 22-24h maturation. The EH treatment maintained oocytes at the germinal vesicle stage (79.3%, vs. 87.7% for control oocytes at 0h; P>0.05). Upon culture, held oocytes matured more quickly than did control oocytes. The proportions of mature oocytes were not significantly different between groups at 18h (EH-held, 80.6% and control, 79.3%); however, after 22h significantly more EH-held than control oocytes had degenerated (24.1% vs. 4.5%, P<0.0001). Blastocyst development was similar between groups for oocytes fertilized after 18h maturation (EH-held, 29.6% and control, 27.8%). When oocytes were fertilized after 22-24h maturation, EH-held oocytes yielded lower blastocyst development than did control oocytes (16.5% vs. 29.3%, P<0.05). In conclusion, bovine oocytes may be effectively held in the EH treatment before maturation without adversely affecting meiotic or developmental competence. However, holding affects the kinetics of maturation and this must be taken into account when subsequent manipulations are performed.     

Nuclear maturation and development of IVM/IVF canine embryos in synthetic oviductal fluid or in co-culture with buffalo rat liver cells

In vitro embryo production in the domestic bitch can provide valuable insights for conservation of endangered canids. In the present study, canine oocytes underwent in vitro maturation (IVM) in simple or complex media, with production of in vitro matured and fertilized (IVM/IVF) canine embryos. Cumulus–oocyte complexes (COCs) were harvested from ovaries by slicing and subjected to IVM in four media (SOF, TCM 199, Ham-F10, and DMEM/F12). After culture for 48 h, oocytes were stained and examined for nuclear maturation. There were no significant differences in the mean (±S.D.) percentage of nuclear maturation (metaphase II) of oocytes cultured in SOF (18.6 ± 7.6%), TCM 199 (18.3 ± 4.5%), Ham-F10 (13.9 ± 8.2%), or DMEM/F12 (11.9 ± 4.2%). For assessment of embryo development, oocytes were matured for 48 h in synthetic oviductal fluid (SOF), fertilized with frozen-thawed sperm, and presumptive zygotes were cultured for 7 d, either in SOF or as co-cultures with BRL cells in TCM 199. Percentages of IVM/IVF oocytes that developed to the 2-cell, 3–4-cell, and 5–7-cell stages were higher (P < 0.05) following culture in SOF versus BRL cell co-cultures (33.6 ± 1.2% vs 13.7 ± 1.2%, 24.7 ± 0.5% vs 8.7 ± 1.1%, and 15.1 ± 2.2% vs 4.3 ± 1.3%, respectively). However, none of the embryos developed beyond the 8–16-cell stage. In conclusion, simple or complex media successfully induced resumption of meiosis and nuclear maturation of canine oocytes. Furthermore, SOF supported in vitro development of IVM/IVF canine embryos to the 8–16-cell stage.     

In vitro fertilization of ovine oocytes vitrified by solid surface vitrification at germinal vesicle stage

Cryopreservation of immature oocytes at germinal vesicle (GV) stage would provide a readily available source of oocytes for use in research and allow experiments to be performed irrespective of seasonality or other constraints. This study was designed to evaluate the recovery, viability, maturation status, fertilization events and subsequent development of ovine oocytes vitrified at GV stage using solid surface vitrification (SSV). Cumulus oocyte complexes (COCs) obtained from mature ewes were randomly divided into three groups (1) SSV (oocytes were vitrified using SSV), (2) EXP (oocytes were exposed to vitrification and warming solutions without vitrification) or (3) Untreated (control). Following vitrification and warming, viable oocytes were matured in vitro for 24h. After that, nuclear maturation was evaluated using orcein staining. Matured oocytes were fertilized and cultured in vitro for 7days. Following SSV, 75.7% 143/189 oocytes were recovered. Of those oocytes recovered 74.8%, 107/143 were morphologically normal (viable). Frequencies of in vitro maturation were significantly (P<0.01) decreased in SSV and EXP groups as compared to control. In vitro fertilization rates were significantly (P<0.01) decreased in SSV (39.3%) group as compared to EXP (56.4%) and control (64.7%) groups. Cleavage at 48h post insemination (pi) and development to the blastocyst stage on day 7 pi were significantly (P<0.001) decreased in SSV oocytes as compared to EXP and control groups. In conclusion, immature ovine oocytes vitrified using SSV as a simple and rapid procedure can survive and subsequently be matured, fertilized and cultured in vitro up to the blastocyst stage, although the frequency of development is low.     

In vitro fertilization and cleavage of bovine oocytes matured in medium supplemented with estrous cow serum

Bovine follicular oocytes were matured in TCM 199 supplemented with: (1) fetal calf serum (FCS, 20% v/v), Luteinizing Hormone (LH, 10 ug/ml), and Estradiol-17-beta (E(2), 1 ug/ml) in Experiment 1; (2) 20% cow serum recovered at standing estrus (Experiment 2); or (3) 20% FCS (Experiment 3). Maturation, fertilization, and initial cleavage development were evaluated at 16 and 48 h after in vitro insemination. The proportions of oocytes fertilized after maturation in the presence of added hormones (78.5%, Experiment 1) or estrous serum (71.3%, Experiment 2) were significantly higher (p < 0.01) than after use of FCS alone (39.3%, Experiment 3). Cleavage of zygotes within 48 h post-insemination differed significantly (p < 0.01) between maturation treatments, 27.3%, 75.5% and 6.6% for Experiments 1, 2, and 3, respectively. Results demonstrate a beneficial influence of estrous cow serum, characterized by an elevated concentration of LH, on bovine oocyte maturation in vitro.     

Effects of oxygen tension on the development and quality of porcine in vitro fertilized embryos

The present study was conducted to examine the effect of oxygen tension during in vitro culture (IVC) of porcine oocytes/embryos on their development and quality using two different culture systems. Porcine cumulus oocyte complexes (COCs) were matured (IVM) and fertilized (IVF) in vitro, and subsequently cultured for 6 days in a simple and economical portable incubator or a standard CO(2) incubator. While the same temperature (38.5 degrees C) and CO(2) concentration (5%) were used in the both systems, the portable incubator was operated in a negative air pressure (- 300 mmHg) to create an O(2) level at 8-10% (low O(2) concentration), or in a positive air pressure (high O(2) concentration). To compare the two culture systems, IVM and IVF of COCs and subsequent IVC of in vitro produced (IVP) embryos were carried out in the portable incubator with a low O(2) concentration (Group I) or in the standard incubator with a high O(2) concentration (Group II). To assess the effect of O(2) concentration on IVC of IVP embryos, some oocytes that had been cultured in the standard incubator for IVM and IVF were subsequently cultured in the portable incubator with a low O(2) concentration (Group III) or a high O(2) concentration (Group IV). The occurrence of DNA fragmentation in the blastocysts produced under different culture conditions was examined by TUNEL staining to assess embryo quality. The rates of oocytes that reached MII and were penetrated by spermatozoa following IVF did not differ between the two incubation systems. In contrast, the proportions of development to blastocysts and the mean cell number of blastocysts in Group I were higher than those in Group II and Group IV. The index of DNA-fragmented nucleus in the blastocysts of Group I was significantly lower than that in the blastocysts of Group II. Therefore, low oxygen tension during IVM, IVF and IVC enhanced the subsequent development of IVP embryos to the blastocyst stage and improved their quality.     

Effect of maturation media on male pronucleus formation in pig oocytes matured in vitro.

The present study was carried out to examine the effect of maturation media on male pronucleus formation of pig oocyte matured and fertilized in vitro. Follicular oocytes collected from prepubertal gilts at a local slaughter house were cultured (36 h) in three different media (mTCM-199, Waymouth MB 752/l, and mTLP-PVA), fertilized in vitro, and assessed for nuclear maturation and male pronucleus formation. The addition of 10% (v/v) pig follicular fluid (pFF) to maturation media significantly increased the rate of nuclear maturation of pig oocytes (P less than 0.01), whereas the rate of nuclear maturation of pig oocytes among three different media did not differ. However, the rate of male pronucleus formation of pig oocytes was significantly higher in pig oocytes matured in Waymouth MB 752/l with or without pFF than in oocytes matured in the other two media (P less than 0.01). In experiment 2, the addition of cysteine (the same concentration as in Waymouth medium, 0.57 mM), to mTLP-PVA significantly increased the rate of male pronucleus formation of pig oocytes compared with the control (P less than 0.01). The results indicate that the composition of maturation medium affects the ability of pig oocytes to form male pronuclei following sperm penetration; media containing a high concentration of cysteine (possibly as a substrate of glutathione), such as Waymouth MB 752/l, can remarkably promote this ability.     

Development of a simple, portable carbon dioxide incubator for in vitro production of bovine embryos

The objective of this study was to develop a simple and portable CO2 incubator using effervescent granules (EG) and to examine the effect of negative and positive air pressure for in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of bovine oocytes. In experiment 1, cumulus-oocyte complexes (COCs) were matured (22 h), fertilized (5 h) and cultured (7 days) using 0.25, 0.5 or 1.0 g of EG per 0.6 l added to maintain an optimum level of CO2 (approximately 3, 6 or 12%, respectively) for in vitro production of embryos. Control oocytes, zygotes and embryos were cultured in a standard CO2 incubator. The blastocyst production rates observed on Days 7 to 9 after insemination were 20.5+/-4.2%, 18.5+/-3.9% and 28.7+/-5.1% for the 0.25 g EG, 0.5 g EG treatments and control, respectively. These rates were significantly higher (P < 0.05) than that of the 1.0 g EG treatment (8.7+/-2.6%). The number of cells in the inner cell mass (ICM) and trophectoderm (TE) produced from blastocysts using the control procedure were 40.8+/-2.9 and 81.2+/-5.3, respectively, and were higher (P < 0.05) compared to the 0.50 g EG (34.6+/-2.9 and 66.8+/-5.7) and 1.0 g EG treatments (33.4+/-3.4 and 67.2+/-7.3). In experiment 2, COCs were placed in a small box with 0.25 g of EG so that the effects on IVM, IVF and IVC of positive or negative air pressure could be compared. The blastocyst production rate observed in the negative air pressure treatment (29.6+/-4.6%) was higher (P < 0.01) than that of the positive air pressure treatment (6.2+/-1.5%) or the normal treatment pressure (P < 0.05; 18.7+/-4.2%) but did not differ from that of the control (30.7+/-4.4%). These results indicate that this simple type of incubator with negative air pressure can be successfully used for in vitro production of bovine embryos and could be used at the field level.     

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).     

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.     

Holding immature equine oocytes in the absence of meiotic inhibitors: effect on germinal vesicle chromatin and blastocyst development after intracytoplasmic sperm injection

Holding immature oocytes before the onset of maturation simplifies oocyte transport and aids in scheduling later manipulations. We report here a method for holding equine oocytes in the absence of meiotic inhibitors. In Experiment 1, immature oocytes with expanded cumuli were cultured at 38.2 degrees C in medium containing cycloheximide, or were held at room-temperature in M199 with Hanks' salts, for 16-18 h before maturation. Control oocytes were matured immediately after recovery. Oocytes were fertilized by intracytoplasmic sperm injection and cultured for 4d. Embryo development was not different among treatments. In Experiment 2, oocytes were treated as in Experiment 1, but embryos were cultured for 7.5d. Blastocyst development was significantly lower in the cycloheximide-treated group than in controls (7% versus 30%) with the room-temperature group intermediate (16%). In Experiment 3, oocytes were cultured at 38.2 degrees C in medium containing roscovitine, or were held at room temperature in sealed glass vials in a mixture of 40% M199 with Earle's salts, 40% M199 with Hanks' salts, and 20% FBS (EH treatment) for 16-18 h, before maturation, sperm injection, and embryo culture for 7.5d. Blastocyst development of oocytes in the EH treatment was significantly higher than that for roscovitine-treated oocytes (34% versus 12%), but not significantly different from that for controls (25%). Oocytes in the EH treatment did not mature during holding (70% germinal vesicle stage after 18 h holding). Whereas culture with cycloheximide or roscovitine of equine oocytes with expanded cumuli reduced subsequent blastocyst formation, these oocytes could be held in a modified M199 at room temperature overnight without adverse affecting meiotic or developmental competence.