Embryo development in vitro of cat oocytes cryopreserved at different maturation stages

The purpose of this study was to evaluate the ability of cat oocytes, at different stages of maturation, to survive after cryopreservation and to assess their subsequent development following IVM and IVF. In the initial toxicity trial, immature oocytes were exposed to different concentrations of DMSO and ethylene glycol (EG). Resumption of meiosis and metaphase II were evaluated after removal of the cryoprotectant and IVM. The highest rates of resumption of meiosis (51.4%) were achieved after exposure to 1.5 mol l(-1) of cryoprotectants, and no difference was observed with control oocytes. Metaphase II was obtained in 25.7% (P<0.01) and 22.9% (P<0.005) of oocytes exposed to 1.5 mol l(-1) of DMSO and ethylene glycol, although at lower rates than in control oocytes (54.4%). On the basis of this finding, 1.5 mol l(-1) of cryoprotectant was chosen for freezing cat oocytes at the germinal vesicle stage (immature) or at metaphase II stage (mature). Post-thaw viability was assessed by the evaluation of the embryo development in vitro. After fertilization, mature oocytes frozen in ethylene glycol cleaved in better proportions (38.7%) than immature oocytes (6.8%, P<0.001), and no differences were observed in the cleavage rate of oocytes frozen at different maturation stages with DMSO (immature 12.8%; mature 14.1%). Embryonic development beyond the 8-cell stage was obtained only when mature oocytes were frozen with ethylene glycol (11.3%). This study suggests that cryopreserved cat oocytes can be fertilized successfully and that their development in vitro is enhanced when mature oocytes are frozen with ethylene glycol. The stage of maturation may be a key element in improving cat oocyte cryopreservation.     

小鼠卵母细胞体外成熟、体外受精的效果观察

目的　研究不同培养条件对小鼠卵母细胞体外成熟及体外受精率的影响。方法　小鼠卵母细胞分别在含有FSH、BSA和胰岛素的培养液中体外成熟,在Whitten 氏液中体外受精,比较体外成熟率、体外受精率。结果　1- 裸卵(DO) 的体外成熟率、体外受精率(81-4% ,31-0 % ) 均高于卵丘卵母细胞复合体(COC)(48-6 % ,27-1% ) 。2- 在培养液中添加FSH、胰岛素和BSA,卵母细胞的体外成熟率为77-9 % ,82-3% 、60-7% ;体外受精率为77-2 % 、72-6 % 、26-7% ;2 - 细胞率为49-2 % 、34-2 % 、10-0% 。胰岛素组的卵母细胞IVM 率最高,但IVF率、2 - 细胞率低于FSH 组。3- 添加BSA的两组的体外受精率只有26-7 % 、25-8 % ,显著低于其他组,其体外成熟率也较添加FSH 和胰岛素的组成。4- 排出第一极体(PbI) 的卵母细胞的体外受精率和2 - 细胞率(85-9 % ,22-4% ) 均高于GV期卵母细胞(71-1 % ,12-9 % ) 。结论　1- 卵丘卵母细胞(COC) 较裸卵(DO) 的体外成熟率、体外受精率都低,差异显著(P成熟< 0-01;P受精< 0-05) 。2-FSH 和胰岛素均能提高小鼠卵母细胞的体外成熟率、体外受精率。3-BSA可以降低小鼠卵母细胞体外受精率,差异极显著。4-GV 期卵母细胞的体外受精率显著低于体外培养的排出第一极体的卵母细胞(P2 - cell < 0-05,P受精<0-05)     

In vitro viability and ultrastructural changes in bovine oocytes treated with a vitrification solution

Abattoir-derived oocytes were exposed to a concentrated cryoprotectant solution (DAP213: 2 M DMSO, 1 M acetamide, 3 M propanediol, and 10% FCS in TCM199) for 1.5 or 5 min at the germinal vesicle (GV) stage or after maturation in vitro (IVM). Their viability was assessed by in vitro fertilization (IVF) and culture (IVC) to blastocysts. To investigate the effect of DAP213 on the ultrastructure, GV and IVM oocytes were processed for transmission electron microscopy (TEM) before (control) or after exposure to the cryoprotectant. DAP213 induced profound ultrastructural modifications to the microvilli and mitochondria, resulted in large vesicle formation, and, most significantly, caused the premature release of the cortical granules (CG). In IVM oocytes exposed to the cryoproteclant for 5 min, exocytosis of CG into the perivitelline space was common and the IVF rate was reduced (P <.05). After exposure for 5 min, GV oocytes displayed clusters of CG comparable to controls, but after IVM-IVF, polyspermy rate was increased (P <.05). Furthermore, treated GV oocytes showed a reduced rate of cleavage and blastocyst formation and an increased percentage of oocytes exhibiting alterations in organelles, whereas the viability and ultrastructure of IVM oocytes treated for 1.5 min was not different from controls. These observations demonstrate that (1) cortical granule kinetics is one of the key elements controlling fertilizability of bovine oocytes treated with cryoprotectant, and (2) GV oocytes are more sensitive to the cryoprotectant than those that have already been matured in vitro.     

In vitro fertilization and development of frozen-thawed bovine oocytes.

Bovine oocytes were vitrified (V-oocytes) or frozen slowly (S-oocytes) at the germinal vesicle (GV) stage or after maturation in vitro (IVM) and their survival assessed morphologically and also by in vitro fertilization (IVF) and culture. The morphological survival of S-oocytes was 30.7% after freezing at the GV stage and 53.3% after IVM. The corresponding survival rates of V-oocytes were significantly lower, viz. 14.6 and 14.0%, respectively. The fertilization rate of S-oocytes frozen after IVM (51.0%) was lower than that of unfrozen controls (75.8%), but higher than after other treatments. Development continued in 16.0% of the fertilized S-oocytes, compared to 39.4% of control IVF zygotes and 1.6% developed into morulae or blastocysts (4.5% in controls). Only 0.8% of frozen-thawed GV stage oocytes and 4.6% of post-IVM V-oocytes cleaved after IVF and none formed morulae or blastocysts. Transfer of four embryos (two morulae and two blastocysts) derived from post-IVM S-oocytes into a recipient heifer resulted in pregnancy and the birth of twin calves.     

Effects of EGTA and slow freezing of bovine oocytes on post‐thaw development in vitro

Experiments were conducted to assess the morphological viability and in vitro developmental potential of bovine oocytes after exposure to Ethylene Glycol‐bis(‐aminoethyl Ether) N,N,N,N‐Tetra‐acetic Acid (EGTA) prior to slow freezing. Different concentrations of EGTA (0, 1, 5 and 10 mM) and exposure intervals (5, 10 and 15 min) were tested on immature (GV) and in vitro matured (IVM) oocytes equilibrated in 1.5 mM propylene glycol (PG) without (experiment 1) or with slow freezing (experiment 2). In addition, PG and ethylene glycol (EG) were compared for cryoprotective efficacy. In vitro maturation (IVM), in vitro fertilization (IVF) and embryo culture (IVC) were performed in defined conditions. Pretreatment of both types of oocytes with 1 mM EGTA for 5 min without freezing yielded morphological and functional results comparable to those obtained for controls while results from higher concentrations of EGTA were lower (P < 0.05). Higher rates of freeze‐thaw survival and embryonic development were obtained after pretreating GV oocytes with 1 or 5 mM EGTA for 5 min. Similarly, better results were obtained when IVM oocytes were pretreated with 1 mM EGTA for either 5 or 10 min. When pretreated with 1 mM EGTA for 5 min and frozen with PG IVM oocytes exhibited higher survival rates (P < 0.05) than those frozen with EG. However, no significant differences were observed in the in vitro development of surviving GV or IVM oocytes frozen with either PG or EG. Results suggest that a prefreeze treatment with 1 mM EGTA for 5 min can enhance oocyte viability. Conditions described enabled blastocyst development of 2.9% of GV oocytes and 8.0% of IVM oocytes after cryopreservation and IVF. Mol. Reprod. Dev. 52:86–98, 1999. © 1999 Wiley‐Liss, Inc.     

The effects of ultrarapid freezing on meiotic and mitotic spindles of mouse oocytes and embryos

Preovulatory mouse oocytes and 2-cell embryos were frozen with dimethyl sulfoxide and propanediol by an ultrarapid method. The survival of frozen oocytes was low (33–34%) compared to that of 2-cell embryos (78–79%) with either cryoprotectant. Development to blastocysts after postthaw culture was about 7–15% for oocytes and 79–80% for the embryos. Ultrarapid freezing preserves cell structure quite well as revealed by electron microscopy, but meiotic oocytes and late 2-cell embryos undergoing mitosis showed evidence of spindle disorganization involving loss or clumping of microtubules resulting in some scattering of chromosomes. Embryos developed from frozen eggs showed clear evidence of micronuclear formation and incomplete incorporation of chromosomal material into main nuclei. These experiments confirm our observations on freezing of human oocytes and show that spindle microtubules are sensitive to freeze-thawing and that cryopreservation could cause chromosomal aberrations during early development. A cautious approach to the introduction of oocyte freezing in human in vitro fertilization (IVF) programs is advocated.     

Effect of cryoprotectant concentration, equilibration time and thawing procedure on survival and development of rapid frozen-thawed mature mouse oocytes

Experiments were conducted to develop a simple rapid-freezing protocol for mature mouse oocytes that would yield a high proportion of oocytes with developmental potential. The effects of concentration (3.5, 4.5 and 6.0 M dimethyl sulfoxide (DMSO) all with 0.5 M sucrose) and the duration of exposure (2.5 min vs 45 sec) of oocytes to the cryoprotectant and its extraction after thawing in 2, 3 or 4 steps of descending sucrose concentration were studied. The most effective of the rapid-freezing and thawing protocols (4.5 M DMSO; 45 sec exposure and 3-step thawing) was compared to slow freezing protocols using 1.5 M DMSO and 1.0 M 1,2 propanediol as cryoprotectants. The DMSO concentrations had an effect on survival, fertilization and embryo development using short (45 sec) but not long (2.5 min) exposure. The rate of morphological oocyte survival was significantly higher using 4.5 M DMSO than 3.5 or 6.0 M (92% vs 82 and 73%, respectively). The development of fertilized embryos to blastocysts was also significantly higher at 4.5 M than at 3.5 or 6.0 M (68% vs 42 and 53%, respectively). The extraction of cryoprotectant in 3 or 4 steps of descending sucrose concentration resulted in higher survival (P < 0.01) and fertilization than in 2 steps. The best survival, fertilization and development was achieved with the 3-step procedure. Optimal combinations of conditions were 4.5 M DMSO at 45 sec prefreeze exposure and 3-step extraction of the cryoprotectant. Oocytes frozen by conventional methods had a survival, fertilization and development to blastocyst rate significantly lower than those frozen under the optimal rapid conditions. Thus rapid freezing of mature mouse oocytes with 4.5 M DMSO + 0.5 M sucrose and short prefreeze exposure is effective and has the additional advantage of being less time-consuming than slow freezing methods.     

Solvent effects on cytoskeletal organization and in-vivo survival after freezing of rabbit oocytes

NBD-phallacidin revealed a polymerized actin distribution in the cortical region of the rabbit egg and along junctional feet. Staining with anti-alpha-tubulin antibody showed that the microtubule distribution was restricted to the barrel-shaped spindle. After cryoprotective treatment in the presence of propanediol, cortical polymerized actin was no longer visible within the egg and along junctional feet but filamentous actin was still present after treatment with dimethylsulphoxide. However, exposure to dimethylsulphoxide or propanediol led to the appearance of microtubules in the cytoplasm and to a disassembly of the spindle often associated with anomalies in chromosome position. Cytoplasmic microtubules formed by the action of propanediol were still present after freezing, thawing, and removal of the cryoprotectant, but after recovery of eggs in culture, they disappeared and barrel-shaped spindles were able to reform. When the effect of propanediol addition on in-vivo fertilization and development of frozen oocytes was examined, 39% (79/200) of frozen oocytes were fertilized and 9% (9/105) developed to normal fetuses, compared to 81% (38/47) and 32% (12/38) respectively for unfrozen control oocytes.     

Bovine oocyte vitrification: effect of ethylene glycol concentrations and meiotic stages

Success in oocyte cryopreservation is limited and several factors as cryoprotectant type or concentration and stage of oocyte meiotic maturation are involved. The aim of the present study was to evaluate the effect of maturation stage and ethylene glycol (EG) concentration on survival of bovine oocytes after vitrification. In experiment 1, kinetics of oocyte in vitro maturation (IVM) was evaluated. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII) oocytes were found predominantly at 0, 0–10, 10–14, and 18–24 h of IVM, respectively. In experiment 2, in vitro embryo development after in vitro fertilization (IVF) of oocytes exposed to equilibrium (ES) and vitrification solution VS-1 (EG 30%), or VS-2 (EG 40%) at 0, 12 or 18 h of IVM was evaluated. Only blastocyst rate from oocytes vitrified in SV-2 after 18 h of IVM was different from control oocytes. Hatched blastocyst rates from oocytes vitrified in VS-1 after 12 and 18 h, and SV-2 after 18 h of IVM were different from unvitrified oocytes. In experiment 3, embryo development was examined after IVF of oocytes vitrified using VS-1 or VS-2 at 0, 12 or 18 h of IVM. Rates of blastocyst development after vitrification of oocytes in VS-1 at each time interval were similar. However, after vitrification in VS-2, blastocyst rates were less at 18 h than 0 h. Both cleavage rates and blastocyst rates were significantly less in all vitrification groups when compared to control group and only control oocytes hatched. In conclusion, both EG concentration and stage of meiotic maturation affect the developmental potential of oocytes after vitrification.     

Effects of oocyte activation and sperm preparation on the development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection

The objective was to determine the effects of various methods of oocyte activation and sperm pretreatment on development of porcine embryos derived from in vitro-matured oocytes and intracytoplasmic sperm injection (ICSI). The second polar body was extruded in the majority (>78.4%) of in vitro-matured (IVM) oocytes 4h after electrical pulse activation. In embryos generated by ICSI and sham-ICSI, a combination of an electrical pulse, with various chemical activators 4 h later, improved (P < 0.05) blastocyst formation rate compared to activation only with a pulse. Treatment with 6-dimethylaminopurine (DMAP) after electrical activation significantly increased the oocyte activation rate. The effects of exposure of sperm to repeated freeze-thaw cycles (without cryoprotectant) on oocyte activation and the effects of sperm pre-incubated with dithiothreitol (DTT) or Triton X-100 on early embryo development were also examined. Blastocyst formation rates after ICSI did not differ between motile sperm and those rendered immotile by one-time freezing and thawing without cryoprotectant. However, sperm rendered immotile by three cycles of freezing/thawing without cryoprotectant had a significantly lower blastocyst formation rate. Although oocytes injected with sperm pre-incubated with Triton X-100 had a higher normal fertilization rate than those pre-incubated with DTT or one-time frozen/thawed sperm, rates of blastocyst formation and cell numbers were similar among the three groups. In conclusion, various methods of oocyte activation and sperm preparation significantly affected the developmental capacity of early porcine embryos derived from IVM and ICSI.     

The influence of reduced glutathione in fertilization medium on the fertility of in vitro–matured C57BL/6 mouse oocytes

It is well known that IVM oocytes show a decreased potential for fertility and development compared with in vivo–matured oocytes. In this study, we added reduced glutathione (GSH) to the fertilization medium during IVF to investigate its effect on the fertility and early embryo development of IVM oocytes. The fertilization rate for IVM oocytes and fresh sperm increased with the addition of GSH (0, 1.0, and 2.0 mM: 51%, 76%, and 70%). Moreover, the addition of GSH to the fertilization medium also improved the developmental potential compared with the control sample (0 mM). In addition, we performed IVF using IVM oocytes and frozen/thawed sperm that had been cryopreserved in a mouse bank. Results indicated a marked increase in the fertilization rate when 1.0 mM GSH was added to the fertilization medium compared with when no GSM was used (0.0 mM GSH: 2% (3/195); 1.0 mM GSH: 33% (156/468)). Furthermore, the fertilization rate improved dramatically via zona drilling using laser equipment (52%: 267/516), whereas normal offspring were obtainsed after transferring embryos created via IVF using IVM oocytes and frozen/thawed sperm. This is the first report in which offspring have been obtained via IVF using IVM oocytes and frozen/thawed sperm.     

In vitro maturation and fertilization of prepubertal goat oocytes

The aim of this work was to study the IVM-IVF of prepubertal goat oocytes collected from a slaughterhouse as an alternative source of oocytes to those of FSH-primed adult goats. In Experiment 1, IVM of prepubertal goat oocytes in co-culture with granulosa cells were compared with IVM in 50 microl microdrops of medium. There was no significant difference in the percentage of maturation (72.0 vs 76.9%) between the 2 groups. In Experiment 2, a low percentage of normal fertilization (24.4%) was observed for prepubertal goat oocytes matured with granulosa cells from prepubertal goats. This result was significantly lower than that obtained for ovulated (62.2%) or in vitro-matured (48.7%) oocytes from adult goats. There were no significant differences with respect to the oocytes from adult goats matured in vitro when prepubertal goat oocytes were cultured with adult goat granulosa cells (33.3%) or in microdrops (29.7%). No differences were observed among the treatments in the percentage of oocytes showing evidence of fertilization (normal fertilization + abnormal fertilization + polyspermy). In Experiment 3, it was shown that there were no differences in the percentage of normally fertilized oocytes after in vitro maturation in microdrops containing oocytes with 1 to 2 and 3 or more complete layers of cumulus cells (32.1 and 33.3% respectively). In conclusion, the ovaries of prepubertal slaughterhouse goats were found to be an economical alternative for an abundant source of oocytes for IVM-IVF research. In vitro maturation of oocytes in microdrops yielded maturation and fertilization rates comparable to those obtained with oocytes from FSH-primed adult goats. Moreover, similar maturation and fertilization rates were obtained using oocytes with 1 to 2 layers or 3 or more layers of cumulus cells.     

Supplementation with cysteamine during maturation and embryo culture on embryo development of prepubertal goat oocytes selected by the brilliant cresyl blue test

Our previous studies have shown that the addition of 100 mircroM cysteamine to the in vitro maturation (IVM) medium increased the embryo development of prepubertal goat oocytes. The aim of the present study was to evaluate the effect of adding different concentrations of cysteamine to the IVM medium and to the in vitro embryo culture medium (IVC) on the embryo development of prepubertal goat oocytes selected by the brilliant cresyl blue (BCB) test. Oocytes were exposed to BCB and classified as: oocytes with a blue cytoplasm or grown oocytes (BCB+) or oocytes without blue cytoplasm or growing oocytes (BCB-). In Experiment 1, oocytes were matured in a conventional IVM medium supplemented with 100 microM, 200 microM or 400 microM cysteamine. In Experiment 2, oocytes were matured with 400 microM cysteamine and following in vitro fertilization (IVF) were cultured in SOF medium supplemented with 50 microM and 100 microM cysteamine. In Experiment 1, BCB+ oocytes matured with 100 microM and 200 microM cysteamine showed higher normal fertilization and embryo development rates than BCB- oocytes. Oocytes matured with 400 microM cysteamine did not present these differences between BCB+ and BCB- oocytes. In Experiment 2, the addition of 50 microM and 100 microM cysteamine to culture medium did not affect the proportion of total embryos obtained from BCB+ oocytes (35.89% and 38.29%, respectively) but was significantly different in BCB- oocytes (34.23% and 29.04%, respectively, P < 0.05). In conclusion, the addition of 400 microM cysteamine to the IVM improved normal fertilization and embryo development of BCB- oocytes at the same rates as those obtained from BCB+ oocytes. The proportions of morulae plus blastocyst development were not affected by the treatments.     

In vitro fertilization of goat oocytes

Experiments were carried out to achieve fertilization (IVF) and initial embryonic development of goat oocytes in vitro. Oocyte/cumulus complexes were recovered from large follicles (greater than 7 mm) of hormonally treated doses and from 1-6-mm follicles of ovaries from hormonally superstimulated and nontreated goats. Three different sperm treatment/IVF media were used: defined medium (Brackett and Oliphant, Biol Reprod 1975; 12:260-274) with modifications (mDM); TALP (Bavister and Yanagimachi, Biol Reprod 1977; 16:228-237), as modified by Parrish et al. (Theriogenology 1986; 25:591-600), i.e. modified TALP (mTALP); and HEPES-buffered M199 with modifications (mH-M199). Immature oocytes (from 1-6 mm, small antral follicles) were cultured for in vitro maturation (IVM) in M199 buffered with bicarbonate and with modifications including supplementation with 20% (v/v) goat serum (mB-M199) with either (a) 100 micrograms LH/ml, (b) 5 micrograms FSH/ml, or (c) no added gonadotropin control. Insemination of (in vivo or in vitro) matured oocytes was performed with swim-up separated and heparin-treated freshly ejaculated sperm; additionally, caffeine was included in the mDM treatment. Use of mDM yielded better results than mTALP or mH-M199 (p less than .05). Results with oocytes after IVM were significantly better than those obtained with oocytes matured in vivo (68.4% vs. 45.5%, p less than 0.05). Presence of LH or FSH during oocyte maturation improved both the IVM and IVF results over those of the control (p less than 0.05). The highest proportion of fertilized oocytes (fertilization rate) was achieved by combining the use of mDM for sperm and IVF with IVM in the presence of LH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Is it best to cryopreserve human cumulus-free immature oocytes before or after in vitro maturation?

Freezing unfertilized oocytes is an option for females without a partner, either to preserve their fertility prior to sterilizing cancer treatment or for social reasons. Our study considered whether it is best to freeze immature human oocytes at the germinal vesicle (GV) stage, prior to in vitro maturation (IVM) or at metaphase-II (M-II), after IVM. Sibling GV-stage oocytes from stimulated ICSI cycles were allocated to freezing either prior to (n = 109) or after (n = 107) IVM. Cumulus-free oocytes were cryopreserved using a choline-substituted slow-freezing protocol and matured in a defined medium, with analysis of chromatin, microtubules, and microfilaments by three-dimensional imaging. Cryopreserved oocytes were compared with oocytes matured in vitro but never frozen (n = 114). Survival was similar between oocytes frozen before or after IVM (69.7% vs. 70.5%). Polar body extrusion after IVM was lower in oocytes frozen at the GV stage versus those matured and then frozen (51.3% vs. 75.7%) or not frozen (75.4%). Stratification by patient age (<36 and ?36 year) showed no difference in oocyte survival or maturation. Oocytes frozen as GVs showed elevated proportions of spontaneous activation (with or without polar body), an effect augmented by patient age. Spindle and chromosome configurations were disrupted to similar extents in both groups of frozen oocytes, with no further detrimental effect of patient age. The length, width, and volume of bipolar M-II spindles were comparable in all three groups. When frozen as GVs, oocytes exhibited decreased maturation and increased spontaneous activation, suggesting that it is best to freeze oocytes at M-II.     

Comparison of cytoskeletal integrity,fertilization and developmental competence of oocytes vitrified before or after in vitro maturation in a porcine model

Aim of the study was to investigate the effect of vitrification on viability, cytoskeletal integrity and in vitro developmental competence after in vitro fertilization (IVF) of oocytes vitrified before or after in vitro maturation (IVM) using a pig model. Oocytes from abattoir-derived porcine ovaries were vitrified at either the germinal vesicle (GV) or metaphase II (MII) stage by modified solid surface vitrification (SSV). Oocyte viability was evaluated by stereomicroscopic observation whereas their nuclear stage and morphology of microtubules and F-actin were observed by confocal microscopy after immunostaining. Fertilization was assessed by orcein staining. The survival rate after vitrification was higher for MII-stage than for GV-stage oocytes. However, the ability of surviving oocytes to reach the MII stage after vitrification at the GV stage (GV-vitrified oocytes) was similar to that of control oocytes. Furthermore, after IVM, GV-vitrified oocytes had better spindle and F-actin integrity than oocytes vitrified at the MII stage (MII-vitrified oocytes). In accordance with this result, GV-vitrified oocytes had better ability to extrude the second polar body and support male pronucleus formation after in vitro fertilization (IVF), in comparison to MII-vitrified oocytes. Fertilization rates did not differ among groups. Finally, the ability of GV-vitrified oocytes to develop into embryos was superior to that of MII-vitrified oocytes. However, both vitrified groups showed reduced blastocyst development compared with the control group. In conclusion vitrification of porcine oocytes at the GV stage is advantageous in conferring better cytoskeletal organization and competence to develop to the blastocyst stage in comparison with vitrification at the MII stage.     

Fertilizability and developmental capacity of individually cultured bovine oocytes

Culture of single oocytes throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC) provides detailed information on maturity, fertilizability and developmental capacity of individual bovine oocytes and embryos. In the present study, effects of sperm concentration (Experiment 1), microdrop size (Experiment 2), and the addition of hypotaurine (HT) or glutathione (GSH; Experiment 3) during IVF were investigated. In Experiment 4, in vitro maturity and developmental capacity of bovine oocytes cultured for IVM in a medium supplemented with fetal calf serum (FCS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA) during IVM were investigated. In Experiments 1 to 3, the percentages of normal (2 pronuclei with a spermtail) and polyspermic fertilization in singly cultured oocytes were similar to those of group IVF culture (5 oocytes/drop). The addition of GSH during single oocyte IVF significantly increased the proportion of normal fertilization and decreased the polyspermic fertilization compared with addition of HT or of the control. The rates of mature oocytes (62.4 and 67.7%) and blastocyst development (12.9 and 15.2%) for single oocyte IVM cultures (Experiment 4) were also similar compared with the group culture; PVA supplementation significantly increased the matured oocyte rate, but decreased blastocyst development significantly (7.1%) as compared with FCS (19.5%) or BSA (15.6%). These results indicate that a single oocyte culture system throughout in vitro production of bovine embryos provides similar maturity, fertilizability and developmental capacity to oocytes cultured in groups.     

Effects of electrical stimulation before or after in vitro fertilization on sperm penetration and pronuclear formation of pig oocytes

The effects of exposure of pig oocytes to an electrical pulse on sperm penetration and pronuclear formation were determined before or after in vitro fertilization (IVF). After in vitro maturation (IVM) or after collection from oviducts of unmated gilts, pig oocytes either were not exposed or were exposed to an electrical pulse (a 10 sec pulse at 4.0 V mm^?1 AC followed by a 30 μsec pulse at 120 V mm^?1 DC), followed 30 min later by IVF. The incidence of male pronuclear formation of both IVM and in vivo-matured oocytes at 12 hr after insemination was decreased from 59% and 100%, respectively, to 2% and 36%, respectively, by the electrical pulse, but the penetration rates (88–100%) and polyspermic rates (79–100%) were not affected by exposure to an electrical pulse. Similarly, when pig IVM oocytes were exposed to an electrical pulse at 6 hr after insemination, electrical activation did not decrease penetration rates (93% vs. 90%), polyspermic rates (83% vs. 91%), or number of spermatozoa in penetrated oocytes (4.0 ± 0.5 vs. 4.6 ± 0.5) but did decrease the rate of male pronuclear formation from 58% to 18%. When oocytes were examined at 6 hr after insemination, 75% of them had been penetrated and resumed meiotic progression, but all sperm heads in penetrated oocytes were fully condensed or only partially decondensed. The percentage of penetrated eggs with multiple female pronuclei was increased when oocytes were exposed to an electrical pulse in all experimental series. In summary, electrical activation of pig oocytes before or just after IVF does not prevent sperm penetration but does inhibit male pronuclear formation and increases the formation of multiple female pronuclei. © 1993 Wiley-Liss, Inc.     

In vitro maturation and fertilization of oocytes from Norwegian semi-domestic reindeer (Rangifer tarandus )

Reindeer oocytes were submitted to in vitro maturation, fertilization and culture (IVM,IVF,IVC) using the established procedures for bovine in vitro embryo production. The study was conducted outside the main breeding season. Semen was collected from epididymides immediately after slaughter, and was diluted in Tris-fructose-citric acid extender containing 6% glycerol and 20% egg-yolk and then frozen in liquid nitrogen. Following 24 h of maturation, cumulus expansion was complete, and 71% of the oocytes reached Metaphase II (MII), with extrusion of the first polar body. Of the remaining oocytes, 22% were at the germinal vesicle stage (GV), 2% at diakinesis and 5% at Metaphase I (MI). The percentages of fertilization and cleavage were 36.0 and 31.8%, respectively. Two of the fertilized oocytes developed to the morula stage after 7 d of culture.     

Influence of oocyte quality, culture media and gonadotropins on cleavage rate and development of in vitro fertilized buffalo embryos

The present study was designed to examine the influence of oocyte quality, culture media and gonadotropins on cleavage rate and development of in vitro fertilized buffalo embryos. Three experiments were conducted. In experiment 1, oocytes were classified by number of cumulus cell layers and morphology of the ooplasm as good, fair or poor. Oocytes were cultured for IVM, IVF and IVC in CR1aa medium. In experiment 2, good quality oocytes were cultured for maturation in: (1) CR1aa; (2) CR2aa; (3) TCM-199; (4) MEM or (5) RPMI-1640, and then fertilized using frozen thawed buffalo spermatozoa in CR1aa. The oocytes were cultured in the same medium used for maturation after fertilization. In experiment 3, oocytes were classified into three groups: group (1) was without gonadotropin and serve as a control; group (2) in which IVM medium was supplemented with 10microg/ml FSH and group (3) in which IVM medium was supplemented with 10IUml(-1) eCG. In all experiments, oocytes were kept at 38.5 degrees C under 5% CO(2) for IVM, IVF, IVC and examined for cleavage and embryo development rates on days 3 and 8, respectively. Good and fair quality oocytes produced a higher cleavage rate (P<0.01) than poor quality oocytes. Morula production rate was also higher (P<0.01) for good as compared with fair quality oocytes. Embryo development with poor quality oocytes was arrested at the two to sixteen cell stage. In experiment 2, the cleavage rate was higher (P<0.05) in CR1aa than CR2aa, and higher (P<0.01) than TCM-199, MEM and RPMI-1640. The numbers of morulae and blastocysts were higher (P<0.01) for oocytes cultured in CR1aa and CR2aa media than TCM-199 or MEM. In experiment 3, the addition of FSH or eCG to the maturation medium increased (P<0.01) cleavage and developmental rates of buffalo embryo compared with control medium. In conclusion, the IVM of good quality buffalo oocytes in CR1aa or CR2aa medium and the addition of FSH or eCG in maturation medium produced higher cleavage and developmental rates of IVF buffalo embryos.