Effect of the cumulus on in vitro fertilization of bovine matured oocytes

The effect of the cumulus on in vitro fertilization in bovines was examined. Follicular oocytes were cultured in medium 199 plus OCS and extra granulosa cells. Frozen-thawed bovine spermatozoa was separated by the swim-up technique, suspended in Talp medium and capacitated with heparin. Fresh sheep and goat semen was incubated for 4 h at room temperature, washed and spermatozoa were then suspended in Talp medium and capacitated by incubation at 38.5 °C and 5% CO₂ in air and heparin.

In experiment 1, cumulus-enclosed oocytes, denuded oocytes and denuded oocytes plus additional cumulus cells were incubated with a reduced concentration of bovine spermatozoa for 8 or 18 h. In Experiment 2, cumulus enclosed and denuded oocytes were incubated with bovine spermatozoa for 4, 6, 8 and 18 h using a sperm concentration adjusted to secure high fertilization rates. In Experiment 3, cumulus-enclosed and denuded bovine oocytes were incubated with either sheep or goat spermatozoa for 18 h. Fertilization rates were then calculated and compared statistically. The results showed that 1) the cumulus improved the fertilization rate only when cumulus cells were associated with the oocytes 2) the timing of sperm penetration was not modified by the cumulus and started at 4 h after sperm incubation and 3) the presence of the cumulus improved the heterologous fertilization rate only when sheep spermatozoa were used. The results suggest that the cumulus improves fertilization rate by providing a capacitation-inducing mechanism and by facilitating the interaction between capacitated spermatozoa and the zona pellucida surface.     

In vitro fertilization and cleavage capability of bovine follicular oocytes classified by cumulus cells and matured in vitro

Bovine oocytes were collected from ovaries obtained from an abattoir. They were classified according to the character of the cumulus cells using a stereomicroscope, and cultured in 25 mM Hepes buffered Tissue Culture Medium 199 supplemented with 10% fetal calf serum at 39 degrees C and inseminated by capacitated sperm. Maturation rates of Class A oocytes, with compact, dense cumulus cells; Class B, partially naked oocytes with thin cumulus layers or small remnants of cumulus cells and Class C, naked oocytes were 97.4% (38/39), 89.8% (106/118) and 52.9% (45/85), respectively. The fertilization rates for the three classes were 86.8%, 85.8% and 53.3%, respectively. The naked oocytes had a significantly lower fertilization rate than oocytes of the other two classes. Significantly more Class A oocytes cleaved (63.7%, 232/364) than those of Class B (29.5%, 36/122) and Class C (17.7%, 28/158).     

In vitro fertilization and cleavage of in vivo matured bovine oocytes

The effect of the interval between onset of estrus and oocyte collection on in vitro fertilization (IVF) rates has been investigated. The oocytes were surgically collected 6-18 h (Group I), 19-24 h (Group II), 25-29 h (Group III) and 30-36 h (Group IV) after the beginning of estrus. Recognizable stages of nuclear maturation were identified in 54.9% of the oocytes used for IVF (5.9% at germinal vesicle, 31.4% at metaphase I, 17.6% at metaphase II); the other 45.1% were degenerate. Considerable between- and within-cow variation in oocyte morphology, oocyte maturation and IVF results was observed. The cverall fertilization and cleavage rates (to four-cell stages) were 26.5 and 6.0%, respectively. The fertilization rate increased as the interval between onset of estrus and collection increased and was optimal 30-36 h after onset. Thus, onset of estrus proved an effective means of timing oocyte collection for IVF.     

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 heparin dosage and sperm capacitation time on in vitro fertilization and cleavage of bovine oocytes matured in vitro

The present study was conducted to clarify the effect of heparin dosage and sperm capacitation time on in vitro fertilization (Experiment 1) and cleavage (Experiment 2) rates of bovine oocytes matured in vitro. For in vitro fertilization, seven dosages of heparin (0, 5, 10, 25, 50, 100 and 200 mug/ml) and nine incubation periods (0, 5, 15, 30, 45, 60, 120, 180 and 240 min) in a capacitation medium were examined, using 6,634 oocytes. The mean proportions of fertilized oocytes in 25, 50 and 100 mug/ml of heparin were significantly (P<0.05) higher (53 to 59%) than in the other dosages (3 to 44%). Incubation with heparin for longer than 60 min lowered the frequencies of fertilization (20 to 36%) compared with the shorter incubation periods (38 to 49%). Higher proportions of fertilized oocytes were obtained by 5, 15, 30 or 45 min of incubation (42 to 49%) than by the other time periods (20 to 38%). Cleavage rates were found by using 2,098 oocytes in a factorial study (4 x 4 x 15: dosages -25, 50, 100 and 200 mug/ml; incubation periods -0, 15, 30 and 60 min; and replicates). The incubation periods and replicates resulted in highly significant differences (P<0.001) in development rates to eight-cell stage, but the four dosages of heparin showed no significant differences. The present results indicate that heparin dosage and sperm capacitation time are important factors influencing in vitro fertilization and cleavage rates. Optimal heparin dosages for the capacitation of bull spermatozoa ranged from 25 to 100 mug/ml; optimal incubation periods ranged from 5 to 60 min.     

Ram-specific effects on in-vitro fertilization and cleavage of sheep oocytes matured in vitro

The present experiments were designed to identify possible male-specific effects on early embryonic development in vitro: Sheep oocytes were matured in vitro for 24-26 h and then fertilized in vitro using equal numbers of viable spermatozoa from 1 of 6 Clun Forest rams. At 15-18 h after insemination, oocytes were either fixed and examined for fertilization and polyspermy or further cultured in modified M 199 medium for 3 days in an oviduct epithelial co-culture system. There were significant differences in 5 separate trials between the rams with respect to the rate of fertilization, degree of polyspermy and cleavage rate after monospermic fertilization. The mean rate of fertilization varied from 89% in Ram B to 43% in Ram C while the percentage of polyspermic eggs varied from 5 to 34%. Both the absolute number of embryos cleaving to the 16-cell stage and the calculated percentage of monospermic eggs reaching the 16-cell stage differed markedly between groups of eggs fertilized by different rams. The results indicate that the development of sheep eggs in vitro is differentially affected by the ram from which the spermatozoa are collected.     

Influence of cumulus cells and sperm concentration on cleavage rate and subsequent embryonic development of buffalo (Bubalus bubalis) oocytes matured and fertilized in vitro

The objective of the present study was to investigate the effects of sperm concentration and presence or absence of cumulus cells on fertilization, cleavage rate and subsequent embryonic development upto the blastocyst stage in buffalo. Cumulus-oocyte-complexes (COCs) obtained from slaughterhouse ovaries were matured in vitro in TCM-199 + 10% FBS + 5 micrograms/mL FSH-P for 24 h. After maturation the COCs were either used as such (cumulus-intact) or freed from attached cumulus cells by repeated pipetting (cumulus-free). Frozen-thawed buffalo spermatozoa were treated with 10 micrograms/mL heparin and 2.5 mM caffeine for sperm capacitation. Oocytes were fertilized in vitro with 1 to 2, 4 to 5 or 9 to 10 million sperm/mL and the cleavage rate was recorded 42 to 44 h post insemination. The cleaved embryos were co-cultured with buffalo oviductal epithelial cells for 10 d post insemination, and the uncleaved oocytes were fixed and stained with aceto-orcein for determination of the penetration rate. The cleavage rate and the proportion of cleaved embryos that developed to morula and blastocyst stages were significantly higher (P < 0.05) whereas the proportion of degenerated oocytes and those that became arrested at the 2 to 16-cell stage were significantly lower (P < 0.05) with cumulus-intact than with cumulus-free oocytes at the 3 sperm concentrations. Increasing the sperm concentration increased the cleavage rate significantly (P < 0.05) from 1 to 2 million through 9 to 10 million sperm/mL but had no effect on the proportion of cleaved embryos that developed to morula and blastocyst stages. In conclusion, the results of the present study suggest that cumulus cells have a positive influence on fertilization, cleavage and subsequent embryonic development. Increase in sperm concentration increases cleavage rate without affecting subsequent embryonic development.     

Effect of sperm exposure time on in vitro fertilization and embryo development of bovine oocytes matured in vitro

This study was designed to investigate the effect of sperm exposure time on the fertilization rate and subsequent developmental capacity of bovine oocytes matured in vitro. Cumulus oocyte complexes (COCs) obtained from 2 to 6 mm follicles were matured for 24 h in TCM-199 supplemented with fetal bovine serum (FBS) and hormones (FSH, LH and estradiol 17-beta). In vitro fertilization (IVF) was performed by incubating 15 to 20 matured oocytes with 1 x 10(6) percoll separated frozen-thawed spermatozoa in 1 ml of IVF-TL medium for either 4, 8, 12, 16, 20, 24 or 28 h. Following sperm exposure for different periods of times, the presumptive zygotes were co-cultured with Buffalo Rat Liver cells (BRLC) monolayers in CZB medium without glucose, a simple semi-defined medium developed for mouse embryo culture, for 3 d post-insemination and then in M199/FBS (TCM-199-HEPES supplemented with 20% heat-treated FBS and 1 mM sodium pyruvate) for 5 d. The fertilization rates differed significantly among the 7 treatment groups, with higher frequencies obtained by co-incubation of gametes for 20, 24 or 28 h (67 to 76%) than for 4, 8 and 12 h (26 to 54.5%), with 16 h (57%) being intermediate. However, the length of sperm exposure time did not significantly affect subsequent embryo development, although an increasing trend was noted from 4 h to 20 h. The number of fertilized oocytes at 3 d post-insemination cleaving to 2- to 4-cell vs 8-cell stage was not different among treatment groups. Development of 8-cell embryos to morulae and blastocysts did not differ among the treatment groups. These data suggest that the optimum duration of sperm-oocyte incubation is 24 h, and periods shorter than 16 h may result in a reduced fertilization rate.     

In vitro fertilization and development of bovine oocytes matured in serum-free medium

This study was undertaken to investigate the effects of supplementation of serum (fetal calf serum), gonadotropins (LH, FSH, prolactin) and estradiol-17 beta (E2) to culture medium during in vitro maturation of bovine cumulus oocyte complexes on subsequent fertilization and development to the blastocyst stage in vitro. Serum supplementation during bovine oocyte maturation was not required but hormonal supplementation, gonadotropins (LH + FSH) and E2, enhanced the fertilizability and developmental ability of bovine oocytes matured in vitro. The addition of prolactin to maturation medium containing LH, FSH, and E2 did not further enhance frequencies of fertilization and development.     

In vitro fertilization of pig oocytes matured in vitro

The development of in vitro fertilization (IVF) techniques in pigs as well as in other species is of great importance because of the possible applications of this technology in different research fields. Methods of IVF vary in different incubation periods and temperatures, in the hormone concentrations used, and in the treatment of the sperm samples. It has been particularly difficult to succeed in the achievement of fertilization in the pig. In the present study we used FSH and LH concentrations of 2 IU/ml for oocyte maturation, an incubation temperature of 37°C, and dilution of spermatozoa for capacitation, and we achieved a high fertilization rate (50 to 75%) with no cases of polyspermy.     

Effect on fertilization and development by re-culture after freezing and thawing of bovine oocytes matured in vitro

This study evaluated the effect of 6-h repeat culture before insemination of frozen-thawed, in vitro-matured oocytes on their fertilizability and developmental capacity. Immature oocytes were cultured for 18 h or 24 h and were frozen incrementally. Post-thaw oocytes were repeat cultured for 0 h (control) or 6 h before insemination. With fertilizability, the proportion of enlarged sperm head was significantly (P<0.05) higher in oocytes cultured for 24 h without repeat culture (24:0 h; 51.8%) than those cultured for 24 h with repeat culture (24:6 h; 26.1%) and nonfrozen oocytes (25.9%). However, the proportion of male pronucleus (MPN) in the group of 24:0 h (32.4%) was significantly (P<0.05) lower than that of nonfrozen oocytes (64.3%); the formation of the female pronucleus and MPN were also significantly (P<0.01) lower (17.2%) than that of nonfrozen oocytes (56.1%). Polyspermic oocytes cultured for 18 h with repeat culture for 6 h (18:6 h) were significantly (P<0.05) higher (47.5%) than for nonfrozen oocytes (25.6%). Development to 8-cell stage in the group of 18:6 h was significantly (P<0.05) lower (1.6%) than that of nonfrozen oocytes (18.5%). The cleavage rates in the groups of 24:0 h (16.3%) and 24:6 h (23.4%) were also significantly (P<0.05) lower than for nonfrozen oocytes (63.3%). Development to blastocysts was low (3.9%), but hatched blastocysts were observed in frozen-thawed oocytes cultured for 18:0 h. These results indicate the post-thaw 6-h repeat culture did not greatly improve the fertilizability and embryonic development of oocytes cultured for 18 h or 24 h before freezing. Frozen-thawed oocytes after 24 h in vitro maturation without repeat culture especially had impaired capacities for fertilizability and development.     

Pentoxifylline improves in vitro fertilization and subsequent development of bovine oocytes

The purpose of this study was to examine whether pentoxifylline improves in vitro fertilization and developmental rates of bovine oocytes. In the first experiment, we examined the effects on the fertilization rate of various concentrations of pentoxifylline (0-7.5 mM) combined with heparin (10 IU/mL). In the second experiment, we examined fertilization cleavage and blastocyst rates after frozen-thawed spermatozoa, obtained from four different bulls, were incubated with heparin (10 IU/mL) with or without caffeine (5 mM) or pentoxifylline (5 mM). In the first experiment, a significantly higher fertilization rate was obtained in heparin containing 5 mM pentoxifylline compared to that in heparin alone or in heparin containing 7.5 mM pentoxifylline (86% vs 60% vs 64%, respectively). The percentage of monospermy in 5 mM pentoxifylline (81%) was significantly higher than in heparin alone (57%). In the second experiment, the interactions among Bulls A, B, C, and D; between treatments (pentoxifylline-with-heparin, caffeine-with-heparin and heparin alone), and between bulls and treatments were analyzed for the number of oocytes penetrated, monospermic oocytes, cleaved oocytes and blastocysts. Among bulls, there was a significant difference in the number of oocytes penetrated (P < 0.01), monospermic oocytes (P < 0.05), cleaved oocytes (P < 0.001), and blastocysts (P < 0.001). Between treatments, there was a significant difference in the number of oocytes penetrated (P < 0.001), monospermic oocytes (P < 0.01) and cleaved oocytes (P < 0.001). Interaction between bulls and treatments was observed for the number of oocytes penetrated (P < 0.05). Individually, for Bulls A, C and D, the numbers of oocytes penetrated and monospermic oocytes in pentoxifylline-with-heparin were significantly higher than in heparin alone. For Bull D, significantly higher results were obtained for the number of oocytes penetrated, monospermic oocytes, cleaved oocytes and blastocysts in pentoxifylline-with-heparin compared to caffeine-with-heparin and heparin alone (P < 0.05). These results suggest that treating sperm with 5 mM pentoxifylline in combination with heparin is effective for bovine in vitro fertilization and it that this treatment is effective even for bulls that produce low fertilization and blastocysts after sperm treatment with caffeine-with-heparin or heparin alone.     

Coculturing cumulus oocyte complexes with denuded oocytes alters zona pellucida ultrastructure in in vitro matured bovine oocytes

Oocyte quality is a key factor affecting success of in vitro embryo production in cattle. Improving the microenvironment of oocytes during in vitro maturation (IVM) can increase developmental rate and embryo quality. Therefore, the objective was to determine whether denuded oocytes (DO) affect embryo development and ultrastructure of the zona pellucida (ZP) in in vitro matured bovine oocytes. Intact immature cumulus-oocytes complexes (COC) obtained from a local abattoir or by ovum pick-up (OPU) were cocultured with and without abattoir-obtained DO at a COC:DO ratio of 1:5. After IVM, DO were removed and intact DO were either fertilized or observed by scanning electron microscopy. Blastocyst quality was evaluated using a TUNEL assay. The ZP pore size decreased after IVM in COC + DO coculture, regardless of their origin (OPU, 310.5 ± 92.5 vs. 428.9 ± 148.5 nm; abattoir, 317.5 ± 68.5 vs. 358.9 ± 128.5 nm; P < 0.05; mean values ± standard deviation). Moreover, the number of ZP pores in OPU COC + DO and COC + DO was greater than those in OPU COC and COC (control) groups (56 ± 4 and 55 ± 7 vs. 50 ± 6 and 42 ± 4; P < 0.05). The rate of blastocyst development in COC + DO and OPU COC + DO groups was greater those in control and OPU COC groups (36.6% and 55.5% vs. 28.1% and 40.0%; P < 0.05). Moreover, the total cell numbers of blastocysts in COC + DO group exceeded that of control (132.91 ± 30.90 vs. 115.44 ± 24.95; P < 0.05), with no significant between OPU COC + DO and OPU COC groups (139.31 ± 42.51 vs. 137.00 ± 61.34). In conclusion, in vitro embryo development competence and quality improved when oocytes were cocultured with DO. Furthermore, there more, but smaller, ZP pores.     

Investigation of means to improve rates of fertilization in in vitro matured/in vitro fertilized bovine oocytes

Experiments were conducted with 5,979 oocytes to determine whether detaching some of the cumulus cells from oocytes either before or after maturation would improve the fertilization rate and proportion of oocytes that developed to expanded blastocysts. Oocytes were aspirated from ovaries of slaughtered cows and matured, fertilized and cultured in vitro. Pipetting immature oocytes before maturation to detach some of the cumulus, with all cumulus cells left in the maturation wells, significantly increased fertilization rates, especially of oocytes that initially had a full cumulus investment. In further experiments, pipetting oocytes either before or after maturation to detach most of the cumulus, or treating with hyaluronidase after maturation to disperse the cumulus, significantly increased fertilization rates and proportions of oocytes developing to expanded blastocysts.     

The effect of endotoxin-contaminated medium on in vitro fertilization and development of bovine oocytes matured in vitro

The purpose of this study was to determine whether the presence of bacterial endotoxin in the bovine serum albumin (BSA) used to supplement media utilized for sperm preparation and co-culture of bovine sperm and oocytes affects in vitro penetration and embryonic development of oocytes matured in vitro. The chromogenic limulus amoebocyte lysate (LAL) test was used for quantification of the content of endotoxin. The proportion of penetrated ova was significantly greater (P greater than 0.0005) for the endotoxin-contaminated group (89%) versus the non-contaminated group (61%), but this was probably not due to endotoxin contamination. The presence of endotoxin resulted in a high rate of polyspermy (27% versus 4%, respectively; P greater than 0.0005), while the occurrence of parthenogenetic activation was the same for each group (8%). The proportion of total embryos put into culture that developed to the blastocyst stage by day 8 was similar (30% and 26%) for the contaminated and non-contaminated group, respectively. Fifty-three and 69%, respectively, hatched on day 10. These results suggest that endotoxin induces polyspermy, but has no adverse effect on embryonic development.     

In vitro fertilization of horse follicular oocytes matured in vitro

In vitro fertilizing ability of stallion spermatozoa was assessed using horse follicular oocytes matured in vitro. After collection, stallion spermatozoa were either: 1) washed and incubated in TALP medium with 3 mg/ml bovine serum albumin (BSA) and 10 micrograms/ml heparin for 4h, 2) washed and incubated in TALP with 3 mg/ml BSA for 3 h and cultured for a further 1 h with 1 mM caffeine and 5 mM dbcAMP, 3) washed and incubated in TALP medium with 3 mg/ml BSA at pH 7.9-8.2 for 2-4 h, or 4) diluted and incubated in TALP medium with 10 mg/ml BSA and 7.14 microM calcium ionophore A 23187 for 5-10 min followed by washing. After a given pretreatment, suspensions were diluted into B2 medium to a concentration of 5 x 10(6) sperm/ml and co-incubated with oocytes for 12 h or 24-48 h. In the ionophore-treated group, 18 of 54 oocytes (33%) were fertilized by 12 h, and 11 of 45 (24%) cleaved by 24-48 h. Evidence of fertilization was not found in the oocytes incubated with spermatozoa from other treatment procedures.     

Cytogenetic study of bovine oocytes matured in vitro

Described in the present paper is a cytogenetic study of bovine oocytes matured in vitro. The cumulus-oocyte complexes (COC), punctured from ovaries recovered in a local slaughterhouse, were classified into 3 groups according to follicular diameter 1 to 4mm, 5 to 8mm and 9 to 13 mm. Metaphases available for observation were classified as metaphase I, haploid and diploid metaphase II. High levels of haploid metaphases II (90.6, 86.9 and 94.4 %) among the 3 groups of follicular sizes indicated successful meiotic resumption during in vitro maturation and suggested that cytoplasmic maturation may be responsible for low developmental rate after IVM, IVF and in vitro development (IVD).     

Effects of caffeine, cumulus cell removal and aging on polyspermy and embryo development on in vitro matured and fertilized ovine oocytes

The objectives of these studies were to determine the effects of cumulus cell removal and caffeine treatment on the development of in vitro matured ovine oocytes aged in vitro until until fertilization. Oocytes were denuded (DO) at 24 h post-onset of maturation (hpm), control cumulus oocyte complexes (COC's) and DO groups were fertilized at 24 hpm or returned to culture in the presence or absence of 10 mM caffeine and fertilized at 30 hpm. Removal of cumulus cells and aging both increased polyspermy, caffeine reduced this increase, however, with the exception of DO's (30 hpm) vs. COC's (24 hpm) the differences were not statistically significant. Aging significantly decreased cleavage between COC groups at 24 hpm and 30 hpm and caffeine did not affect this (68.4%, 73.4%, 74.0% respectively). In contrast, the frequency of cleavage was significantly reduced in the DO (24 hpm) group as compared to COC controls (45.6% vs. 68.4% (P < 0.05)), however, cleavage increased in the DO group on aging (73.4%) and this was not affected by caffeine (73.0%). The percentage of COC's and DO's developing to the blastocyst stage significantly decreased on aging, caffeine treatment of DO's prevented this (31.3%, 12.7% and 29.4% respectively (P < 0.05)) but had no effect on COC's (4.2% vs. 3.9%). Total cell numbers in blastocysts were not statistically different (92.4 ± 5.2, 84.7 ± 3.7 and 80.4 ± 5.8 (P > 0.05)). In summary caffeine treatment of aged COC's had no significant effect on the frequency of development, however, in aged DO's caffeine treatment statistically increased development to blastocyst and lowered the frequency of polyspermy.     

In vitro fertilization of sheep oocytes matured in vivo

Incubating washed ram spermatozoa in a modified Brackett's defined medium buffered with Hepes (DM-H) containing 20% of heat-inactivated sheep serum appears to be a reliable method of capacitating sperm for in vitro fertilization. Raising the Ca(++) concentration in the fertilization medium (DM-H-SS) to 10 mM stabilized the fertilization rate of various rams (2). This study was designed to determine if the developmental competence of the oocytes fertilized under such conditions was normal. Thirty-seven ewes, treated with progestagen sponges, were superovulated with porcine follicle stimulating hormone (pFSH: 16 mg). An intramuscular injection of gonadotropin releasing hormone (GnRH: 100 mug); given 24 to 26 h after sponge removal, induced the synchronization of ovulations 24 h later. Ovulated oocytes (n = 229) recovered with flushing of the oviducts were inseminated in vitro and 17 h later either fixed in acetic/alcohol (n = 115) to evaluate fertilization or transferred (n = 114) into 38 synchronized recipients (three oocytes/recipient) to evaluate their developmental competence. Of the fixed oocytes, 82.6% were fertilized and 61.7% were monospermic. Nineteen of the recipient ewes (50%) were pregnant at Day 18, and 16 ewes produced a total of 26 live young (mean: 1.63/ewe). The results showed a high efficiency of in vitro fertilization of ovulated oocytes in sheep following a pFSH-GnRH treatment and the in vivo developmental competence of oocytes fertilized in the presence of elevated Ca(++) concentration.