The objective of this study was to determine if insulin–transferrin–selenium (ITS) promoted a nuclear and cytoplasmic maturation of porcine oocytes that better supports subsequent embryonic development. The rate of oocyte in vitro maturation (IVM) in an experimental group treated with hormones for 42 h was significantly increased compared with that in a control group without hormone treatment (47.8% vs. 11.7%, respectively, p < 0.05). Following reduction of the hormone treatment period from 42 h to 21 h, which included both the first 21 h period of hormones treatment (45.4%) and the second 21 h period of hormone treatment (44.8%), the rate of oocyte IVM was still higher than that of the control group (p < 0.05). To improve porcine oocyte nuclear maturation, 1% ITS was added to medium supplemented with hormones. The rate of nuclear maturation in the ITS-treated group was significantly higher than in the ITS-untreated group (78.6% vs. 54.4%, respectively, p < 0.05). ITS treatment also significantly reduced the per cent of oocytes with type I and type III cortical granule (CG) distribution, respectively, and significantly increased the per cent of oocytes with type II CG distribution (85.3%). These observations indicated that the synchronization rates of nuclear and ooplasmic maturation reached 67.04% (78.56 × 85.33%). In conclusion, the combination of modified Tissue Culture Medium-199 (mM199) + 10 ng/ml epidermal growth factor (EGF) + 10 IU/ml pregnant mare serum gonadotrophin (PMSG) + 10 IU/ml human chorion gonadotrophin (hCG) + 2.5 IU/ml follicle stimulating hormone (FSH) + 1% ITS is suitable for culturing porcine oocytes in vitro, and effectively enhances porcine oocyte nuclear and cytoplasmic maturation.

Shortly after the implementation of comprehensive chromosome screening (CCS) techniques for preimplantation genetic testing for aneuploidies (PGT-A), the discussion about the transition from day 3 to blastocyst stage biopsy was initiated. Trophectoderm biopsy with CCS is meant to overcome the limitations of cleavage-stage biopsy and single-cell analysis. The aim of this study was to assess the results obtained in our PGT-A programme after the implementation of this new strategy. Comparisons between the results obtained in 179 PGT-A cycles with day 3 biopsy (D+3) and fresh embryo transfer, and 204 cycles with trophectoderm biopsy and deferred (frozen–thawed) embryo transfer were established. Fewer embryos were biopsied and a higher euploidy rate was observed in the trophectoderm biopsy group. No differences in implantation (50.3% vs. 61.4%) and clinical pregnancy rate per transfer (56.1% vs. 65.3%) were found. Although the mean number of euploid embryos per cycle did not differ between groups (1.5 ± 1.7 vs. 1.7 ± 1.8), the final number of euploid blastocysts available for transfer per cycle was significantly higher in the trophectoderm biopsy group (1.1 ± 1.3 vs. 1.7 ± 1.8). This factor led to an increased cumulative live birth rate in this last group (34.1% vs. 44.6%). Although both strategies can offer good results, trophectoderm biopsy offers a more robust diagnosis and the intervention is less harmful for the embryos so more euploid blastocysts are finally available for transfer and/or vitrification.

After in vitro maturation, the unfertilized pig oocytes underwent the process called ageing. This process involves typical events such as fragmentation, spontaneous parthenogenetic activation or lysis. Inhibition of histone deacetylase, using its specific inhibitor trichostatin A (TSA), significantly delayed the maturation of pig oocytes cultured in vitro. The ageing of oocytes matured under the effect of TSA is the same as the ageing in oocytes matured without TSA. The inhibition of histone deacetylase during oocyte ageing significantly reduced the percentage of fragmented oocytes (from 30% in untreated oocytes to 9% in oocytes aged under the effect of 100 nM of TSA). Oocytes matured in vitro and subsequently aged for 1 day under the effects of TSA retained their developmental capacity. After parthenogenetic activation, a significantly higher portion (27% vs. 15%) of oocytes developed to the blastocyst stage after 24 h ageing under 100 nM TSA when compared with oocytes activated after 24 h ageing in a TSA-free medium. The parthenogenetic development in oocytes aged under TSA treatment is similar to the development of fresh oocytes (29% of blastocyst) artificially activated immediately after in vitro maturation.

Our previous study indicated that thimerosal is one of the most effective artificial activators to mimic sperm-induced increases in the intracellular free calcium concentration ([Ca2+]i) and other activation events in pig oocytes (Macháty et al., 1997). The present study was conducted to examine the temporal relationship between intracellular calcium transients, cortical granule (CG) exocytosis and the zona reaction induced by thimerosal. When pig oocytes matured in vitro were exposed to 200 μM thimerosal the first intracellular calcium transient, with a mean peak ratio of 4.97 ± 1.14, was observed 509.64 ± 122.03 s after addition of thimerosal. The density of CGs fell significantly from 63.3 ± 11.7 CGs/100 μm2 of cortex in control oocytes to 25.7 ± 19.2 CGs/100 μm2 of cortex (59.4% release) at 2 min after the first intracellular calcium transient. At 5 min after the calcium transient the residual CG density had been reduced to 10.7 ± 10.4 CGs/100 μm2 of cortex (83.1% release). This degree of CG exocytosis was the same as that in oocytes penetrated by sperm (9.5 ± 5.1 CGs/100 μm2 of cortex). No further decrease in residual CG density was observed at 10 min (10.3 ± 14.8 CGs/100 μm2 of cortex). Whereas 77.4% (120/155) of control oocytes were penetrated by spermatozoa only 1.4% (2/144) of thimerosal-treated oocytes were penetrated. Further experimental results obtained by in vitro fertilisation of oocytes with preincubated (capacitated) spermatozoa suggested that the zona block to sperm penetration in thimerosal-treated oocytes occurred within 35 min after CG exocytosis and 40 min after the first calcium transient. These results indicate that polyspermic penetration of pig oocytes inseminated in vitro is not due to delayed or incomplete CG exocytosis but more likely to a delayed zona reaction and/or simultaneous sperm penetration.

Offspring gender preselection has applications of considerable economic, health and ecological interest. In this study we analysed modifications of the percentages of spermatozoa bearing Y and X chromosomes when semen samples are submitted to a double swim-up technique as a possible method for producing embryos of known sex with in vitro fertilisation protocols. As an initial experiment to provide accurate evaluation of the method we determined the possible incidence of natural deviations in the primary sex ratio between bulls or ejaculates, analysing the percentage of Y-chromosome DNA bearing spermatozoa (%Y-CDBS) with a polymerase chain reaction (PCR) amplification of X- and Y-specific fragments. Ejaculates were tested by direct semiquantitative PCR sexing and by sexing blastocysts produced in vitro with these spermatozoa. Bulls and ejaculates did not have any effect on the %Y-CDBS or on the sex ratio of embryos produced in vitro using these ejaculates. However, our double swim-up sperm preparation method produced differences in %Y-CDBS in some of the sperm fractions, suggesting that there are intrinsic differences in capacitation of X- and Y-bearing spermatozoa that might be used to produce embryos of the desired sex with in vitro fertilisation.

Heat stress (HS) is especially harmful for bovine ovarian follicle development and oocyte competence. Furthermore, HS causes premature aging in oocytes due to high levels of reactive oxygen species (ROS), involved in the harmful effects over the oocyte maturation and the steroidogenic activity of follicular cells. In this study, the presumptive protective effects of antioxidant agents on heat-stressed oocytes were evaluated. Heifer oocytes were matured for 22 h under control (38°C) and HS conditions (41.5°C at 18–21 h of maturation). For each oocyte, nuclear stage and cortical granule (CG) distribution were evaluated. Steroidogenic activity of cumulus cells was also recorded. The antioxidant agents used in the study were: retinol (1.43 μg/ml), retinyl (0.28 μg/ml) and oleic acid (0.05 mg/ml). Based on a chi-squared test (P < 0.05), HS affected negatively the metaphase II (MII) progression and produced a premature CG exocytosis. Retinol improved the oocyte MII progression. However, retinyl and oleic acid, at the concentrations used in this study, could not counteract adverse effects of HS. A decrease in progesterone and increase in estradiol availability were observed when retinyl and oleic acid were supplemented to the maturation medium, respectively. In conclusion, retinol proved to be valuable in heat-stressed oocytes protecting nuclear maturation.

A statistical approach using sequentially principal component analysis (PCA) clustering and discriminant analysis was developed to disclose morphometric sperm subpopulations. In addition, we used a similar approach to disclose subpopulations of spermatozoa with different degrees of DNA fragmentation. It is widely accepted that sperm morphology is a strong indicator of semen quality and since the sperm head mainly comprises the sperm DNA, it has been proposed that subtle changes in sperm head morphology may be related to abnormal DNA content. Semen from four mongrel dogs (five replicates per dog) were used to investigate DNA quality by means of the sperm chromatin structure assay (SCSA), and for computerized sperm morphometry (ASMA). Each sperm head was measured for nine primary parameters: head area (A), head perimeter (P), head length (L), head width (W), acrosome area (%), midpiece width (w), midpiece area (a), distance (d) between the major axes of the head and midpiece, angle (θ) of divergence of the midpiece from the head axis; and four parameters of head shape: FUN1 (L/W), FUN2 (4π A/P2), FUN3 ((L – W)/(L + W)) and FUN 4 (π LW/4A). The data matrix consisted of 2361 observations, (morphometric analysis on individual spermatozoa) and 63 815 observations for the DNA integrity. The PCA analysis revealed five variables with Eigen values over 1, representing more than 79% of the cumulative variance. The morphometric data revealed five sperm subpopulations, while the DNA data gave six subpopulations of spermatozoa with different DNA integrity. Significant differences were found in the percentage of spermatozoa falling in each cluster among dogs (p < 0.05). Linear regression models including sperm head shape factors 2, 3 and 4 predicted the amount of denatured DNA within each individual spermatozoon (p < 0.001). We conclude that the ASMA analysis can be considered a powerful tool to improve the spermiogram.

The duration of M-phase is largely determined by the time necessary for the formation of a functional metaphase spindle and the correct alignment of all chromosomes on the metaphase plate. The spindle assembly checkpoint prevents the exit from M-phase before the proper alignment of all chromosomes on a metaphase plate in many cell types. In the present paper we show that the first mitotic M-phase of the mouse embryo lasts about 119 min, while the second embryonic M-phase lasts only about 70 min. Histone H1 kinase is activated rapidly during nuclear envelope breakdown in both mitoses. Its maximum, however, is followed by a plateau only during the first mitosis. In the second mitosis, the inactivation of histone H1 kinase activity follows its maximum directly. Histone H1 kinase is more stable in the cytoplasts obtained from mouse embryos during the first embryonic M-phase than during the second one. The stability of histone H1 kinase is greatly increased by the presence of the mitotic apparatus in both M-phases. The mitotic spindle assembly during the first and the second mitoses differs and the first metaphase spindle is stabilised during the period of maximum histone H1 kinase activity. These data show that an unknown developmentally regulated mechanism controls the duration of the two first mitoses in the mouse embryo.

The spermatoozoon is an excitable cell that responds to specific effectors by rapidly changing its behaviour. In species with external fertilisation, spermatozoa are stored in a quiescent state in the testis, but within seconds after spawning, dilution into water triggers several activation events such as increases in motility and respiration. In some species, these are followed by the acrosome reaction, an exocytotic process that allows the spermatozoon to penetrate the egg investments and activate the egg (Dale, 1983). The majority of information on sperm activation has come from the sea urchin; secondarily teleosts and starfish have proved to be useful models.

In a Graafian follicle, granulosa cells are classified into two principal cell subpopulations: cumulus cells, which are closely associated with the oocyte to form the cumulus cell-oocyte complex (COC), and mural granulosa cells, which are organised as a stratified epithelium at the periphery of the follicle. Following the preovulatory gonadotropin surge, cumulus cells lose contact with mural granulosa cells and start to synthesise and secrete a large amount of hyaluronan (HA), a glycosaminoglycan with high molecular weight and large hydrodynamic domains (Salustri et al., 1992). Proteins derived from serum (Chen et al., 1992, 1994) and synthesised by cumulus cells (Camaioni et al., 1993, 1996) organise the strands of HA into an intercellular elastic network that traps the cumulus cells and the oocyte in a unit which can not be mechanically dissociated – a process also referred to as cumulus expansion. At ovulation, the expanded COC is released through the ruptured follicle wall and transferred to the oviduct. The matrix in the expanded COC facilitates its extrusion from the follicle and its capture by oviductal fimbria, and provides, together with the cumulus cells, a suitable microenvironment for sperm penetration and fertilisation (for references see Salustri et al., 1993).

Many biotechnologies are currently used in livestock breeding with the aim of improving reproductive efficiency and increasing the rate of genetic progress in production animals. Semen cryopreservation is the most widely used cryobiotechnology, although vitrification techniques now allow embryos and oocytes to be banked in ever-increasing numbers. Cryopreservation of other types of germplasm (reproductive tissue in general) is also possible, although the techniques are still in the early stages of development for use in livestock species. Although still in their infancy, these techniques are increasingly being used in aquaculture. Germplasm conservation enables reproductive tissues from both animals and fish to be preserved to generate offspring in the future without having to maintain large numbers of living populations of these species. However, such measures need careful planning and coordination. This review explains why the preservation of genetic diversity is needed for livestock and fish, and describes some of the issues involved in germplasm banking. Furthermore, some recent developments in semen handling leading to improved semen cryopreservation and biosecurity measures are also discussed.

Pseudoplatystoma coruscans is a very popular species for tropical fish culture as it has boneless meat of delicate taste and firm texture. Few studies on fish reproductive biology refer to the morphological features of eggs. The goal, therefore, of this present work was to perform a structural and ultrastructural analysis of fertilization and embryonic development in P. coruscans. The incubation period, from fertilization to hatching, lasts 13 h at 28/29 °C and 18 h at 27 °C. The oocytes had a mean diameter of 0.95 mm and hatched larvae were 2.55 mm in diameter. Analysing their development, we observed round, yellow oocytes that bore a double chorion membrane and a single micropyle. At 10 s after fertilization, several spermatozoa were detected attached to the oocyte surface. After 1 min of development, a fertilization cone that obstructed the micropyle could be observed. Segmentation started between 20 and 30 min after fertilization, when the egg cell was then formed. The first cleavage occurred between 30 and 45 min after fertilization, prior to reaching the morula stage (75 and 90 min after fertilization). The epiboly movement started at 120 and 180 min after fertilization and ended at 360 and 480 min after fertilization. Differentiation between cephalic and caudal region was detected after 420 and 600 min after fertilization and larvae hatched between 780 and 1080 min after fertilization. Seven main embryonic development stages were identified: egg cell, cleavage, morula, blastula, gastrula, segmentation with differentiation between cephalic and caudal regions, and hatching.

Bisphenol AF (BPAF), a homolog of bisphenol A (BPA), is a widely used environmental chemical that has adverse effects on reproduction. The aim of this study was to analyse the effects of BPA and BPAF exposure on oocyte maturation in vitro. Oocytes were cultured in the presence of BPA or BPAF (2, 20, 50 or 100 μg/ml) for 18 h. At concentrations of 50 and 100 μg/ml, BPA and BPAF inhibited oocyte maturation, with BPAF treatment causing a sharp decrease in the number of oocytes reaching maturity. Oocytes were exposed to BPA or BPAF at 2 μg/ml and cultured for different durations (6, 9, 12, 15 or 18 h). Both BPAF and BPA caused a cell cycle delay under these conditions. Oocytes cultured in the presence of BPA or BPAF (50 μg/ml) for 21 h were tested for the localization of α-tubulin and MAD2 using immunofluorescence. High concentrations of BPAF induced cell cycle arrest through the activation of the spindle assembly checkpoint. After 12 h of culture in BPAF (50 μg/ml), oocytes were transferred to control medium for 9 h. Only 63.3% oocytes treated in this manner progressed to metaphase II (MII). Oocytes exposed to high doses of BPA experienced a cell cycle delay, but managed to progress to MII when the culture period was prolonged. In addition, MAD2 was localized in the cytoplasm of these oocytes. In conclusion, both BPAF and BPA exposure affected oocyte maturation, however BPAF and BPA have differential effects on SAC activity.

This study aimed to compare the neonatal outcomes of transplanted fifth day (D5) or sixth day (D6) frozen–thawed blastocysts, aiming to provide guidance when choosing blastocyst recovery methods. The clinical data of 1109 patients that were performed for recovered blastocyst transplantation, as well as the data of 515 patients who had already delivered were analyzed retrospectively. The patients were divided into single- or twins-birth groups according to blastocyst formation time (D5 or D6) and the number of fetus(es). The implantation rate, clinical pregnancy rate and live birth rate of the D5 group were all significantly higher than those in the D6 group (P < 0.05), and the biochemical pregnancy rate in the D5 group was significantly lower than that in the D6 group (P = 0.049). Other factors, including birth weight, birth length, incidence of low birth weight and sex ratio, differed between the D5 and D6 groups, but the difference was not statistically significant (P > 0.05). In conclusion, the implantation rate, clinical pregnancy rate and live birth rate in the D5 group were all significantly higher than that in the D6 group. The birth weight at D6 was prone to be heavier no matter the birth group, and the sex ratio in the D5 group was higher than that at D6, the difference in neonatal outcomes between the two groups was not statistically significant.

Germinal-vesicle-stage oocytes enclosed with compact cumulus cell layers (COCs) were recovered from adult or prepubertal minke whale ovaries, and were vitrified in a solution containing 15% ethylene glycol, 15% DMSO and 0.5 M sucrose using either a Cryotop or an open-pulled straw (OPS) as the cryodevice. The post-warm COCs with normal morphology were cultured for 40 h in a 390 mosmol in vitro maturation medium, and oocytes extruding the first polar body were considered to be matured. The proportion of morphologically normal COCs after vitrification and warming was higher when the COCs were cryopreserved by Cryotop (adult origin, 88.4%; prepubertal origin, 80.8%) compared with the OPS (adult origin, 67.7%; prepubertal origin, 64.2%). The oocyte maturation rate was higher in the adult/Cryotop group (29.1%) compared with those of the prepubertal/Cryotop group (14.4%), the adult/OPS group (14.3%) and the prepubertal/OPS group (10.6%). These results indicate that the Cryotop is a better device than the OPS for vitrification of immature oocytes from adult minke whales.

The aim of the present study was to investigate the stability of six housekeeping genes, and the relative expression of growth factors (EGF, GDF-9, BMP-15, VEGF, FGF-2, BMP-6, IGF-1 and KL) and hormone receptors (FSH, LH and GH) in goat preantral follicles. To evaluate to stability of housekeeping genes micro-dissected fresh follicles (150–200 μm) as well as follicles that have been in vitro cultured for 12 days were used. In addition, isolated fresh follicles were used to compare expression of various growth factors and hormone receptors before culture. Both fresh and cultured follicles were subjected to total RNA extraction and synthesis of cDNA. After amplification of cDNA by real-time PCR, the geNorm software program was used to evaluate the stability of glyceraldehyde-2-phosphate dehydrogenase (GAPDH), β-tubulin, β-actin, phosphoglycerokinase (PGK), 18S rRNA, ubiquitin (UBQ) and ribosomal protein 19 (RPL-19). In addition, follicular steady-state levels of mRNA from the various growth factors under study were compared. Results demonstrated that, in goat preantral follicles, UBQ and β-actin were the most suitable reference genes and thus could be used as parameters to normalize data from future in vitro studies. In contrast, 18S RNA appeared the least stable gene among the tested housekeeping genes. Analysis of mRNA for several hypophyseal hormone receptors in fresh preantral follicles showed significantly higher FSH-R mRNA levels than those of LH-R and GH-R, and no difference between GH-R and LH-R mRNA levels. In regard growth factor mRNA expression in goat preantral follicles, EGF mRNA levels appeared significantly lower than those of the other studied growth factors. Increasingly higher relative mRNA levels were observed for GDF-9, BMP-15, BMP-6, FGF-2, VEGF, Kl and IGF-1, successively. In conclusion, UBQ and β-actin are the most stable housekeeping genes in fresh and 12-days cultured caprine preantral follicles. Furthermore, in fresh follicles, high levels of FSH-R mRNA are detected while among eight growth factors, IGF-1 is the most highly expressed and EGF the weakest expressed compound.

The objective of the present study was to elucidate whether resveratrol could facilitate the survival of boar sperm during liquid preservation and fast cooling processes. Boar semen were diluted with Modena extender containing different concentrations of resveratrol. Sperm motility was evaluated by visual estimation. Membrane integrity, acrosome integrity and mitochondrial membrane potentials were measured by SYBR-14/PI, FITC-PNA and JC-1 staining, respectively. Moreover, the levels of reactive oxygen species (ROS), malonaldehyde (MDA) and total antioxidant capacity (T-AOC) were measured using commercial assay kits. B-cell lymphoma protein-2 (BCL2) content was determined by western blotting. During liquid preservation at 17oC, the addition of 50 μM resveratrol to the Modena extender significantly improved sperm motility, membrane integrity, acrosome integrity, and sperm mitochondrial membrane potentials. Similar results were also observed in the 150 μM resveratrol group during the fast cooling process. Furthermore, addition of resveratrol led to a decrease of ROS and MDA, and an increase in the content of T-AOC and BCL2. These observations suggest that addition of resveratrol to Modena extender protects boar sperm against oxidative stress. The optimal concentrations of resveratrol are 50 μM and 150 μM during liquid preservation and fast cooling process, respectively.

The present study was undertaken to establish an effective method for in vitro maturation (IVM) of denuded oocytes (DOs) by simulating the ovarian three-dimensional status in vivo using buffalo ovarian tissues or cumulus cells, so as to provide a model for investigating the mechanisms of oocyte maturation. Buffalo cumulus–oocyte complexes from ovaries taken at slaughter were denuded by pipetting, and then allocated randomly into four groups for IVM by direct culture in maturation medium (M1, control group), co-culture with a monolayer of cumulus cells (M2), embedded in cumulus cell clumps (M3) and ovarian tissue (M4) for 24 h. The nuclear maturation of DOs was assessed by the extrusion of the first polar body and the cytoplasmic maturation was evaluated by subsequently developmental capacity after parthenogenetic activation. More DOs matured to MII (56.89%) and developed to blastocysts (25.75%) when they were matured in vitro with M3 in comparison with DOs matured in vitro with M1 (45.14 and 15.97%) and M4 (40.48 and 13.49%). Further detection of gap junctions by injecting Lucifer yellow directly into cytoplasm of matured DOs with adherent cumulus cells and scanning with confocal microscope showed that Lucifer yellow were found in nine out of 11 the adherent cumulus cells in M3, indicating that the gap junctions between oocytes and cumulus cells was reconstructed in vitro. These results indicate that co-culture of DOs embedded in cumulus cell clumps can improve their nuclear and cytoplasmic maturation of DOs, possibly through the reconstruction of gap junctions in vitro.

Xenopus laevis eggs are surrounded by an extracellular matrix consisting of a vitelline envelope, and three jelly layers, J1, J2, and J3 (from egg surface outward). The jelly layers vary in thickness (about 150, 15 and 200 μm for J1, J2 and J3 respectively) but all are translucent allowing observation of sperm penetration. Video microscopy demonstrated that sperm are able to penetrate and traverse J3 at velocities approaching 30 μm/s. Sperm swim through jelly in a corkscrew-like manner with their rotational and forward velocities being tightly coupled at about 30°/μm forward travel. They are propelled by whip-like power strokes involving hairpin bends in the flagellum that are generated every 180° of rotation and which are propagated from base to tip. The overall trajectories of individual sperm are quite variable. Many sperm head directly for J2 but some do not, these swimming circumferentially, or even away from the egg surface. Most sperm (over 97%) that enter the jelly do not get to the egg surface but are stopped at a variety of positions within J3 or at the outer surface of J2. Efficient sperm penetration and passage through the jelly layers requires a low electrolyte concentration in the surrounding medium, and is inhibited by the lectin wheat germ agglutin (WGA) in a dose-dependent manner. WGA does not block sperm penetration of J3 but does block further progression towards the egg surface. This observation suggests that sperm motility within the jelly is dependent on the carbohydrate moieties of the large glycoconjugates present, and that their alteration by WGA binding accounts for the inability of sperm to reach the egg surface and fertilise the egg.

The cortical cytoplasm and cortical granules (CGs) of mouse oocytes were analysed by electron microscopy. Oocytes were collected soon and 20 h after ovulation from adult young females (3–4 months old). In addition, gametes collected soon after ovulation from 12- to 14-month-old females were used. Ultrastructural analyses were undertaken using the conventional procedures and the alcoholic PTA method. PTA selectively stains the CGs indicating the presence of lysine-rich proteins in these granules. Oocytes from young females showed CGs as dense granules 300–500 nm in diameter linearly arranged under the oolemma. In oocytes recovered 20 h after ovulation 24.31% of CGs appeared vacuolated and 38.40% internalized in the cytoplasm. In gametes collected from old females several changes were observed in the cortical cytoplasm: (a) CGs appeared concentrated in some areas while others regions were devoid of granules; (b) groups of CGs appeared internalized in the egg cytoplasm; (c) the CG contents had swollen and changed, showing dense and clear areas; (d) numerous dense structures and vesicles (lysosome-like vesicles) were present; (e) cytoplasmic fragmentation was frequently seen. Fragments contained CGs, dense structures and vacuoles. These changes are closely related to the low fertilization rates shown by these oocytes when they were used for in vitro fertilization procedures.