Diploid Oocytes Research Articles

380 FULL-TERM DEVELOPMENT OF MOUSE EMBRYOS PRODUCED FROM RECONSTRUCTED GIANT OOCYTES BY INTRACYTOPLASMIC SPERM HEAD INJECTION

In mammals, unusually large diploid giant oocytes are ovulated occasionally, but as any fertilized embryos would be triploid, the potential of the cytoplasm to support normal development is unknown. However, if such giant oocytes possess normal cytoplasm, the oocytes or their excessive cytoplasm would have potential applications in the treatment of human infertility and would enhance the study of basic biology, such as the influence of cytoplasmic factors on genomic reprogramming. We reconstructed oocytes 2 to 9 times normal volume by electrofusion or mechanical fusion between intact and enucleated oocytes. First, we examined the in vitro developmental potential of 2- to 9-times giant oocytes after parthenogenetic activation. Second, 2-times giant oocytes were reconstructed by fusing with intact and enucleated oocytes; then we examined the in vitro and in vivo developmental potential of these giant oocytes after intracytoplasmic sperm injection (ICSI). Third, somatic cell nuclear transfer (SCNT) was carried out using enucleated giant oocytes. When the giant oocytes were activated parthenogenetically, most developed into morulae or blastocysts, irrespective of the original size. When sperm heads were injected into 2-times giant oocytes, these were fertilized and developed normally in vitro; after embryo transfer, we obtained 12 healthy offspring (3 female, 3 male, and 6 cannibalized the next day) by Caesarian section. All survivors grew to adulthood and demonstrated normal fertility. However, we failed to generate cloned mice by SCNT. In conclusion, reconstructed giant oocytes have normal potential for development after activation and fertilization. The reconstructed giant oocytes will provide us with interesting tools for basic biology, such as providing new material for the study of nuclear reprogramming and genomic imprinting, and may be important in widening the treatment options in human assisted reproductive technology.

IVM media, oocyte diameter and donor genotype at RYR1 locus in relation to the incidence of porcine diploid oocytes after maturation in vitro

In the present study three factors were investigated that may affect the process of the first polar body extrusion in pig oocytes matured in vitro: IVM medium, oocyte diameter and donor genotype at the ryanodine receptor (RYR1) locus. In the first experiment, COCs were collected by the aspiration of slaughterhouse ovaries. Oocytes were matured in vitro at 39 °C, in humidified 5% CO 2 atmosphere for 44 h using the following media: (1) TCM199 + hCG + eCG + follicular fluid (FF), (2) TCM199 + hCG + 17β-estradiol and (3) NCSU23 + hCG + eCG + FF. According to cytogenetic analysis, 98.1% of cells reached the second metaphase stage (MII). No significant differences were observed among IVM groups in terms of diploidy level. In the second experiment, oocytes collected by the aspiration or slicing of individual ovaries were matured in vitro in groups reflecting their origin. One ovary was considered a donor. IVM was carried out under conditions described in experiment I, with the use of TCM199 + hCG + 17β-estradiol. A total of 68 ovaries/donors were included in this study. Granulosa cells collected from each ovary were used as DNA source in molecular (RFLP) analysis. Genotype frequencies at the RYR1 locus were as follows: CC, 0.46; CT, 0.48 and TT, 0.06. After maturation the diameter of each denuded oocyte was determined with the use of a computer aided system. Five size categories were distinguished: <90, 90–100, 100.1–110, 110.1–120 and >120 μm. The average diameter of haploid oocytes at MII stage was 111.7 μm, whereas that of diploid cells was 110.4 μm. According to statistical analysis, diploidy was not related to the oocyte diameter. That trait, however, was influenced by the donor genotype at the RYR1 locus. The TT genotype was associated with a higher rate of diploidy.

Meiotic stages and incidence of diploid oocytes in egyptian cattle and buffaloes

Meiotic stages and incidence of diploid oocytes in egyptian cattle and buffaloes

Administration of increasing amounts of gonadotrophin compromises preimplantation development of parthenogenetic mouse embryos

The aim of the present study was to examine the effect of ovarian stimulation with increasing amounts of pregnant mare's serum gonadotrophin (PMSG) on preimplantation development of diploid parthenogenetic embryos in vitro. Administration of 5, 10 and 20 IU PMSG significantly increased the number of oocytes obtained per mouse in a dose-dependent manner. The amount of PMSG administered did not alter the proportion of degenerate oocytes. However, there was a significant decrease in the proportion of 8-cell/compacted embryos after 53 h of culture with the administration of increasing amounts of PMSG. Proportion of embryos reaching at the blastocyst stage after 79 h of culture was reduced significantly in both the 10 and 20 IU PMSG groups. Reduced blastocyst development after 96 h of culture, however, was significant only in the 20 IU PMSG group. Total blastocyst, trophectoderm and inner cell mass numbers were also reduced significantly with the administration of 20 IU PMSG. It is concluded on the basis of these observations that preimplantation development of diploid parthenogenetic oocytes, which depends virtually entirely on maternal molecules accumulated during oogenesis along with gene products derived from the maternal genome, is compromised with the administration of increasing amounts of PMSG.

Case report: evidences of parthenogenetic origin of ovarian teratoma

Case report: evidences of parthenogenetic origin of ovarian teratoma

Growth and reproductive traits of diploid and triploid forms of the Squalius alburnoides cyprinid complex in a tributary of the Guadiana River, Portugal

The Squalius alburnoides hybrid complex has diploid, triploid and some rare tetraploid male and female forms, with triploid females predominating in most populations. The reproductive traits of diploids and triploids from a population in a tri- butary of the Guadiana River, Portugal, were compared, during a two-years period. Few differences were found in the growth and reproductive traits of diploid and trip- loid females. Marginal differences were found in the longevity, with a few triploid females living up to six years compared with a maximum of five years for diploid females and a maximum of four years for diploid males. Both diploid and triploid females exhibited rapid growth in the first two years of life followed by rapid drop off linked to attaining sexual maturity. Reproductive effort increased with age but was mainly linked to number of eggs produced and not to egg size, since oocyte size did not vary with age or length of fish in either ploidy forms. Both forms exhibited a pro- tracted spawning period between March and June, suggesting multiple-spawning be- haviour. Triploid females had a slightly smaller oocyte diameter that may be due to the production of reduced (haploid) oocytes while diploid females produced diploid (unre- duced) oocytes The potentially higher production of diploid oocytes (which become triploid eggs after fertilisation) by diploid females could at least partially account for the higher percentage of triploids in natural populations. In addition, the production of bigger (diploid) oocytes, with possibly higher energetic content could lead to bigger fish larvae, which tend to have a higher survival rate. The time of spawning of both diploids and triploids appears to be synchronised, although differences were found in

Mechanisms of non-disjunction in human female meiosis: the co-existence of two modes of malsegregation evidenced by the karyotyping of 1397 in-vitro unfertilized oocytes

Although numerous studies have been published on the chromosomal constitution of in-vitro unfertilized human oocytes, data remain highly variable and controversial because of the size of oocyte samples, technical reservations and potential misinterpretation. A cytogenetic study was undertaken on 3042 unfertilized human oocytes recovered from 792 women participating in an IVF programme for various infertility problems. Both a gradual fixation technique and an R-banding procedure were used. The analysis was successful in 1397 oocytes (45.9%) for which interpretable metaphases were obtained. Of the 1397 oocyte karyotypes, 1088 (77.9%) were normal (23,X). The overall frequency of chromosomal abnormality was 22.1%. No correlation was found between the rate of abnormalities and the type of infertility. Aneuploidy was observed in 151 cells (10.8%), consisting of 5.4% hypohaploidies, 4.1% hyperhaploidies, 0.8% complex aneuploidies and 0.05% extreme aneuploidies with less than 18 chromosomes. Both whole chromosome non-disjunction and chromatid predivision contributed to the formation of aneuploid oocytes, but the numerical abnormalities due to single chromatids significantly exceeded conventional non-disjunctions. Abnormalities also included 5.4% diploid oocytes, 3.8% sets of chromatids alone and 2.1% structural aberrations. Aneuploidy was found in all chromosome groups. However, groups E and G exhibited significantly higher frequencies of non-disjunction than expected, whereas groups A and B showed a significantly low incidence of aneuploidy. The implication of both chromosome and chromatid abnormalities in the occurrence of non-disjunction are discussed in relation to the recent data on chromatid cohesion throughout cell division. The results were consistent with the hypothesis of an unequal occurrence of non-disjunction among the chromosome groups in female meiosis.

The Development of Porcine Parthenogenetic Diploid Oocytes with Homogeneous Genomic Components In Vitro.

The aim of this study was to determine the influence of the homogeneity of genomic components and ploidy on the in vitro development of porcine parthenogenetic oocytes to the blastocyst stage. In vitro matured oocytes were subjected to a single pulse of electro-stimulation (El-St; 100 μsec, 1,500 V/cm) for activation. First, the activated oocytes were cultured for 6, 16, 18, 20 and 22 h after El-St, and examined for the timing of the first cleavage of parthenogenetic haploids. Next, the effects of the timing and duration of cytochalasin B (CB) treatment on inhibition of the first cleavage of haploids were examined in order to produce homogenous parthenogenetic diploids. Then the developmental ability to the blastocyst stage was compared among activated oocytes with genomic components of haploid (without CB treatment), homogeneous diploid (nn-diploid, CB treatment for 6 h from 20 h after El-St), and heterogeneous diploid (nn'-diploid, CB treatment for 4 h immediately after El-St). Most haploids were at the prophase to telophase of the first cleavage between 16 and 22 h after El-St. When the haploids were treated with 5.0 μg/ml CB for 6 h from 20 h after El-St, their first cleavage was efficiently suppressed, and most of them (84%) became diploids. The frequency of parthenogenetic development to the blastocyst stage was significantly lower in haploids (5%) than in nn-diploids (48%, P<0.01) or nn'-diploids (57%, P<0.01). These results shows that ploidy of activated oocytes, but not the homology of genomic components, affects the development of porcine parthenogenetic oocytes to the blastocyst stage.

Influence of stage of maturation of bovine oocytes at time of vitrification on the incidence of diploid metaphase II at completion of maturation

The present study was to verify the incidence of bovine diploid oocytes when vitrified at various maturation stages. Bovine cumulus-oocyte complexes were recovered from ovaries at a slaughterhouse and then divided into five groups: control group (unvitrified oocytes), 0 h group (composed of oocytes vitrified before the onset of maturation) and 8, 12, and 22 h groups (vitrified respectively at 8, 12 and 22 h after the onset of maturation). The oocytes remained vitrified for 24 h, and then were thawed. In all groups, the oocytes completed 24 h of maturation. Subsequently, the cumulus cells were removed, and the denuded oocytes fixed on slides and stained with aceto-orcein. No differences ( P>0.05) in the incidence of diploid metaphase II oocytes were observed between the control, non-vitrified group (2.4%, 1/41) and oocytes vitrified at 12 h (6.9%, 3/43) or 22 h (2%, 1/50). However, significantly ( P<0.05) more diploid oocytes were detected after vitrification at 0 h (28.5%, 10/35) or 8 h (35.4%, 11/31) of maturation. These results suggest that the nuclear stage at which bovine oocytes are vitrified may affect the incidence of diploid oocytes, especially in oocytes vitrified before maturation or 8 h after the onset of maturation.

Reduced ovulation rate and cytogenetic analysis following tyrosine phosphatase inhibition in mouse oocytes.

Objective: To investigate the effects of sodium orthovanadate (NaV)-induced tyrosine phosphatase inhibition on ovulation rate and aneuploidy during mouse oocyte maturation (OM). Design: A controlled NaV dose response cytogenetic study involving ovulated mouse oocytes. Materials/Methods: ICR female mice were superovulated with intraperitoneal injections of 10.0 IU PMSG and 5.0 IU hCG 48 hrs later. NaV was given immediately after hCG. Ovulated oocytes were collected and counted 16 hrs after hCG and processed for cytogenetic analysis. Based on preliminary cell and animal toxicity data, NaV doses of 5, 15, and 25 mg/kg were selected. Controls received the solvent distilled water. Chromosomes were C-banded to distinguish between complete dyads (chromosomes) and those separated into two chromatids. Data were analyzed by Chi-square and Fisher’s Exact tests. Results: NaV-induced tyrosine phosphatase inhibition led to a significant reduction in the number of oocytes ovulated but did not increase the frequencies of cytogenetic aberrations. The number of oocytes ovulated per number of females for the control group (535/20, 26.8%) was significantly (p < 0.01) greater than that in the 5 mg/kg NaV group (372/20, 18.6%), the 15 mg/kg NaV group (365/20, 18.3%), and the 25 mg/kg NaV group (537/30, 17.9%). The frequencies of hypoploidy (n = 10–19), hyperploidy (n = 20–29), premature centromere separation, premature anaphase, and single chromatids were neither significantly (p 0.05) different between the controls and each of the NaV groups nor among the NaV groups. Ovulated MI oocytes, diploid oocytes, and structural chromosome aberrations were not found. Conclusions: These in vivo oocyte data appear unique in the literature. Although phosphatase and kinase activities are essential for oocyte maturation, the tyrosine phosphatase inhibitor, NaV, did not alter the temporal sequence of OM and increase the frequency of numerical or structural chromosome aberrations. However, NaV-inhibition of tyrosine phosphatase activity resulted in a significant decrease in the number of mouse ovulated oocytes. Supported by: Department of Obstetrics and Gynecology, Louisiana State University Health Sciences Center, Shreveport, LA.

Successful implantation of in vitro-matured, electro-activated oocytes in the pig

In the present study, we derived parthenogenetic porcine fetuses from in vitro-matured oocytes following a simple activation process in order to evaluate their developmental limitations in-vivo. Follicular oocytes collected from gilts at local slaughterhouses were matured for 48 h. They were subjected to a single square pulse of direct current for 100 μsec at 1,500 V/cm and then treated with 5 μg/mL cytochalasin B for 4 h to obtain activated diploid oocytes. The diploids were cultured in modified Whitten's medium until transfer. Diploids which had cleaved to the 2 and 3 to 4-cell stages were transferred to oviducts of recipients. Live and/or dead parthenogenetic fetuses were recovered in 6 of 8 trials at 17, 18, 19, 24 and 29 d post activation. The total proportion of fetuses to transferred diploids was 31.3% (62/198). When fetuses were recovered at 19 d post activation, the proportion of development into fetuses was 71% (15/21). Our results, however, suggest that periods of gestation longer than 19 d resulted in a decrease of these proportions to 45% (18/40) at 24 d and to 18% (7/40) at 29 d. The hearts were beating in nearly all of the fetuses recovered at 19, 24 and 29 d post activation. Thus, parthenogenetic porcine diploids developed to at least the stage of limb-bud formation beyond the early heart-beating stage. Abnormalities were also externally visible on some fetuses. Formation of cyst-like structures in the heart and liver, and insufficient development of the head region and acephali were observed in some cases.

Clomiphene citrate-induced perturbations during meiotic maturation and cytogenetic abnormalities in mouse oocytes in vivo and in vitro

Objective: To determine if clomiphene citrate induces temporal perturbations during meiotic maturation and aneuploidy in mouse oocytes. Design: A controlled dose study involving mouse oocytes in vivo and in vitro. Setting: Clinical and academic research setting in a university medical center. Intervention(s): Oocytes were obtained after superovulation and from mature follicles. Main Outcome Measure(s): Cytogenetic analysis of oocytes for aneuploidy, premature centromere separation, premature anaphase, and single chromatids, and the frequencies of metaphase I and diploid oocytes. Result(s): Clomiphene citrate resulted in a decrease in the number of ovulated oocytes and a significant ( P<.05) increase in hyperploidy at 100 mg/kg in vivo. In vitro, 5.0 μg/mL of clomiphene citrate significantly ( P<.05) increased hyperploidy and reduced the proportion of metaphase I oocytes. Conclusion(s): These findings suggest that clomiphene citrate has the potential for inducing aneuploidy in mouse oocytes both in vivo and in vitro and that the rate of oocyte maturation is altered after clomiphene exposure in vitro. Additional data are needed to support the results of this study.

Electroactivation of rabbit oocytes in an hypotonic pulsing medium and parthenogenetic in vitro development without cytochalasin B-diploidizing pretreatment

We investigated the electroactivation frequencies, type of activation and in vitro development of rabbit oocytes. In Experiment 1, activation (8 pulses, 12 min apart, 60 μsec, 0.6 kVcm −1) was performed by altering osmolarity (190 vs 320 mOsm kg −1) and Ca ++ concentration (10, 60 or 100 μM) in mannitol pulsing media. More oocytes were activated in hypotonic pulsing medium, regardless of Ca ++ concentration (96 to 100%). Both haploid and diploid parthenogenetic embryos developed to compacted morulae (57 to 92% and 63 to 100%, respectively) regardless of the activation treatment; however, the blastocyst rates were more variable (0 to 74% and 0 to 73%, respectively). In Experiment 2, the effects of pulse duration (30 or 60 μsec) and number of applied pulses (4, 8 or 12) under hypotonic conditions were studied. Activation frequencies were the lowest after four 30 μsec-pulses (58 vs 88 to 100%, respectively). A lower haploid frequency was obtained when more than four 30 or 60 μsec-pulses were applied (from 67 to 25% and 83 to 0%, respectively). Increasing the number of 60-μsec pulses improved the compacted morula rate of haploid and diploid oocytes (47 to 83% and 57 to 96%, respectively). Overall, haploid development to morulae and blastocysts was lower than diploid development to these stages (69 and 25 % vs 74 and 44 %, respectively).

Influence of follicle size, medium, temperature and time on the incidence of diploid bovine oocytes matured in vitro

The present work describes a cytogenetic study of in vitro-matured bovine oocytes designed to analyze the incidence of diploid oocytes induced by concentration of serum in the culture medium, follicle size, culture temperature and incubation time. In Experiment 1, immature follicular oocytes from follicles of the same size were cultured for 24 h in TCM-199 supplemented with increasing concentrations 0, 10, 20 and 50% of estrous cow serum (ECS). In Experiment 2, immature oocytes harvested from follicles of different sizes were cultured for 24 h in TCM-199 supplemented with 20% ECS at 39°C in 5% CO 2. In Experiment 3, immature follicular oocytes were matured in TCM-199 supplemented with 20% ECS at 2 different temperatures (37°C or 39°C) in 5% CO 2. In Experiment 4, immature oocytes were matured over 4 different incubation times (24, 36 and 48 h) in TCM-199 supplemented with 20% ECS in 5% CO 2. The highest concentration (50%) of ECS supplement in the culture medium induced the highest incidence of diploid oocytes. This incidence of diploid oocytes matured in vitro was higher in oocytes from follicles with a diameter between 11 and 15 mm. Finally, lower culture temperature (37°C) and prolonged incubation time (48 h) also significantly (P<0.01) increased the percentage of diploid oocytes.

Taxol-induced meiotic maturation delay, spindle defects, and aneuploidy in mouse oocytes and zygotes

To increase our understanding about the potential risks of chemically-induced aneuploidy, more information about the various mechanisms of aneuploidy induction is needed, particularly in germ cells. Most chemicals that induce aneuploidy inhibit microtubule polymerization. However, taxol alters microtubule dynamics by enhancing polymerization and stabilizing the polymer fraction. We tested the hypothesis that taxol induces meiotic delay, spindle defects, and aneuploidy in mouse oocytes and zygotes. Super-ovulated ICR mice received 0 (control), 2.5, 5.0, and 7.5 mg/kg taxol intraperitoneally immediately after HCG. Females were paired (1:1) with males for 17 h after taxol treatment. Mated females were given colchicine 25 h after taxol and their one-cell zygotes were collected 16 h later. Ovulated oocytes from non-mated females were collected 17 h after taxol. Chromosomes were C-banded for cytogenetic analyses. Oocytes were also collected from another group of similarly treated females for in situ chromatin and microtubule analyses. Taxol significantly ( p<0.01) enhanced the proportion of oocytes exhibiting parthenogenetic activation, chromosomes displaced from the meiotic spindle, and sister-chromatid separation. Moreover, 7.5 mg/kg taxol significantly ( p<0.01) increased the proportions of metaphase I and diploid oocytes and polyploid zygotes. A significant ( p<0.01) dose response for taxol-induced hyperploidy in oocytes and zygotes was found. These results support the hypothesis that taxol-induced meiotic delay and spindle defects contribute to aneuploid mouse oocytes and zygotes.

Helium-Neon (He-Ne) Laser Irradiation Increases the Incidence of Unreduced Bovine Oocytes During the First Meiotic Division In Vitro

Low power He-Ne laser irradiation was applied to immature bovine oocytes to investigate the incidence of unreduced oocytes (diploid oocytes) during the first meiotic division in vitro. Immature bovine oocytes from cows killed at a slaughterhouse were irradiated with He-Ne laser irradiation at 0.05 and 0.25 J/cm(2) exposures. An oocyte group was left untreated serving as the control group. All oocytes were matured in TCM-199 medium supplemented with 10% fetal calf serum (FCS). Unreduced oocyte percentages obtained in the irradiated oocytes group were significantly higher (p<0.01 and p<0.001, respectively) than those of the control group. Furthermore, the laser-treated oocytes showed a degeneration rate significantly higher (p<0.01 and p<0.001, respectively) than those observed in the control group. It is concluded that the application of He-Ne laser irradiation at 0.05 and 0.25 J/cm(2) exposures increased the incidence of unreduced oocytes and the percentage of degenerated oocytes during the in vitro meiosis process of immature bovine oocytes.

Chromosome analysis in human oocytes remaining unfertilized after in-vitro insemination: effect of maternal age and fertilization rate.

The incidence of chromosomal abnormalities was studied in 719 unfertilized human oocytes obtained from our in-vitro fertilization (IVF) programme. To make chromosome preparations, a gradual fixation/air-drying method was utilized. Of 388 oocytes successfully karyotyped, 70 (18.0%) were abnormal. The abnormalities included 33 aneuploidies (8.5%) (14 hyperhaploidies and 19 hypohaploidies), 25 diploidies (6.4%) and 15 structural abnormalities (3.9%), three of them being accompanied by aneuploidy. Of the 33 aneuploidies, 16 (48.5%) showed the loss or gain of dyads (so-called non-disjunction), while 17 (51.5%) showed the loss or gain of monads (so-called predivision). There was no maternal age-dependent increase in the incidence of aneuploidy. Unfertilized oocytes from patients with a high fertilization rate (>25%) had a significantly higher (11.4%, P < 0.05) incidence of diploidy compared with the oocytes from the remaining patients (4.3 and 4.0%), suggesting that diploid oocytes might have a lower fertilizing ability.

Investigation of effects of pregnant mare serum gonadotropin (PMSG) on the chromosomal complement of CD-1 mouse embryos.

The objective of this study was to examine the effect of superovulatory doses of gonadotropins on the frequency of chromosomal abnormalities of mouse embryos. Chromosome analysis of 8- to 16-cell stage mouse embryos and zygotes was performed by a cytogenetic method. There was no significant effect of the pregnant mare serum gonadotropin (PMSG) dose on the level of aneuploidy and structural abnormalities from 8- to 16-cell-stage embryos among superovulated groups. However, a simple dose-response relationship between the PMSG dose and the incidence of polyploidy was observed, with the level of polyploidy rising from 2.9% with 10 i.u. PMSG to 10.5% with 15 i.u. PMSG. In zygote stage, the proportion of polyploid embryos also increased as the dose increased, from 1.9% in 5 i.u. to 6.7% in 15 i.u. PMSG. It was observed that the extra chromosomal set in polyploidy embryos originated by both fertilization of a diploid oocyte and dispermy. These results indicate a dose-response relationship between the PMSG dose and the incidence of polyploidy in the CD-1 mouse. Both a disturbance at maturation division and an error at fertilization were the cause of polyploidy.

Development of porcine blastocysts from in vitro- matured and activated haploid and diploid oocytes

The purpose of this study was to determine the developmental capacity of electro-activated porcine oocytes. Follicular oocytes collected from gilts at local slaughterhouses were matured for 48 h and were then subjected to a single square pulse of direct current for 100 ρjusec at 1,500 V/cm for activation. To obtain activated diploid oocytes, some were treated with 5.0 μ/ml cytochalasin B for 4 h immediately after electro-activation. The frequency of activation ranged from 96 to 100%. While 91% of activated oocytes that had not been treated with cytochalasin B had 2 polar bodies and a nucleus (haploids), 92% of the oocytes treated with cytochalasin B had only the first polar body and 2 nuclei (diploids). Haploids and diploids were further cultured in TCM-199 medium that contained 10% (v/v) heat- treated fetal calf serum (FCS) and 0.1 mg/ml sodium pyruvate (mTCM) or in Whittenk medium plus 0.4% (w/v) bovine serum albumin (BSA). The frequency of abnormal oocytes was significantly higher in mTCM (83%) than in Whitten's medium (65%) 96 h after the electro-activation (P < 0.01), suggesting that Whitten's medium supported the development of activated oocytes beyond the morula stage. In all cases, several oocytes developed to the blastocyst stage 144 h after electro- activation (1 to 12%). The frequency was significantly higher in the case of diploids cultured in Whitten's medium (12%) (P < 0.01) than in the case of haploids cultured in Whitten's medium (4%), or in the case of haploids cultured in mTCM (1%). The mean number of nuclei per blastocyst was significantly lower in mTCM (haploids, 15; diploids, 16.1) than in Whitten's medium (haploids, 35.7; diploids, 40.1; P < 0.01), suggesting that the number of nuclei in blastocysts was affected by the culture medium.

The incidence of bovine diploid oocytes matured in vitro

The present work describes a cytogenetic study of in vitro matured bovine oocytes to determine the proportion of unreduced oocytes carrying the diploid number of chromosomes. Studied oocytes were derived either from a pool of oocytes collected from several different donors, or from oocytes collected separately from individual donors. In vitro maturation was performed by culturing immature oocytes for 24 h in TCM199-medium supplemented with estrous cow serum and hormones at 39 °C in 5% CO 2. Chromosomal complement of in vitro matured oocytes was studied by Giemsa-staining and produced analyzable results in approximately 60% of the cases. The results revealed that approximately 75% of oocytes had matured to the MII stage in both groups of oocytes studied. Of these MII oocytes, 11 and 12.4% (from the oocyte pool or from individual cows, respectively) contained the diploid set of chromosomes. The occurrence of diploid MII oocytes was not quite uniform among donors: 40.5% of all cows produced one, 18.9% produced two and 2.7% (one cow) produced three diploid MII oocytes. However, a positive relationship between the number of MII oocytes in general and diploid MII oocytes among individual donors was not found. The possible factors that may lead to the formation of diploid MII oocytes observed under IVM procedures are discussed. The results of this study showed a higher incidence of diploid oocytes in cattle than previously reported.