Morphological and biochemical analysis of immature ovine oocytes vitrified with or without cumulus cells

The cryopreservation of oocytes is an open problem as a result of their structural sensitivity to the freezing process. This study examined (i) the survival and meiotic competence of ovine oocytes vitrified at the GV stage with or without cumulus cells; (ii) the viability and functional status of cumulus cells after cryopreservation; (iii) the effect of cytochalasin B treatment before vitrification; (iv) chromatin and spindle organization; (v) the maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK) activity of vitrified oocytes after in vitro maturation. Sheep oocytes were vitrified at different times during in vitro maturation (0, 2, and 6 h) with (COCs) or without cumulus cells (DOs). After warming and in vitro maturation, oocytes denuded at 0 h culture showed a significantly higher survival and meiotic maturation rate compared to the other groups. Hoechst 33342/propidium iodide double staining of COCs and microinjection of Lucifer Yellow revealed extensive cumulus cell membrane damage and reduced oocyte-cumulus cell communications after vitrification. Cytochalasin B treatment of COCs before vitrification exerted a negative effect on oocyte survival. After in vitro maturation, the number of vitrified oocytes with abnormal spindle and chromatin configuration was significantly higher compared to control oocytes, independently of the presence or absence of cumulus cells. The removal of cumulus cells combined with vitrification significantly decreased the MPF and MAPK levels. This study provides evidence that the removal of cumulus cells before vitrification enhances oocyte survival and meiotic competence, while impairing the activity of important proteins that could affect the developmental competence of oocytes.     

Detection of DNA damage in bovine metaphase II oocytes resulting from cryopreservation

Developmental competence of mammalian oocytes is compromised by currently available oocyte cryopreservation protocols. Experiments were designed to examine the effect of three cryopreservation protocols on the integrity of bovine oocyte DNA. In vitro matured bovine oocytes were cryopreserved either by slow cooling, vitrification in 0.25 ml straws, or in open pulled straws. After thawing/warming, recovered oocytes were immediately subjected to morphological evaluation. Morphologically intact oocytes underwent comet assay to detect cryoinjury at DNA level. All cryopreservation protocols resulted in significant morphological damage as well as DNA damage compared to unfrozen control. Among the morphologically intact oocytes, there was no difference among protocols in the number of oocytes displaying DNA damage. However, oocytes that had been cryopreserved by slow cooling or by vitrification in open pulled straws exhibited more damage than those vitrified in 0.25 ml straws in the extent of DNA damage. If we combine the number of oocytes with morphological damage and oocytes with DNA damage, oocytes cooled by slow cooling resulted in the most damage. This experiment demonstrated that oocyte DNA is a target of cryoinjury and different protocols result in different degrees of damage.     

Cryopreservation of Human Oocytes and Ovarian Tissue

Oocyte cryopreservation has the potential to be an important adjunct to assisted reproductive technologies and bypasses some ethical, moral, and religious dilemmas posed by human embryo cryopreservation. The success of human oocyte cryopreservation depends on morphological and biophysical factors that could influence oocyte survival after thawing. Among the morphological factors, the maturity, quality, size of the oocyte, the presence or the absence of the cumulus oophorus seems to play an important role in oocyte survival after thawing. The main biophysical factor of cellular disruption during cryopreservation process in the intracellular ice formation that can be avoided by an adequate cell dehydration; thus reducing the intracellular water by increasing the dehydration process we can limit the damages of the cryopreservation procedure. The dehydration process can be affected by the presence and concentration of the cryoprotectants in the freezing solutions (equilibration and loading solutions), and by the freezing and thawing rate. Two additional properties of cryoprotectants help to protect cells during slow cooling, when the cells are very dehydrated and are surrounded by concentrated salts. The cryoprotectants appear to reduce damage caused by high levels of salt, a property known as salt buffering. Some events occurring to the oocyte during cryopreservation procedure has been found to be a premature exocitosis of cortical granules, leading to an intempestive zona hardening and consequently to a reduction of fertilization rate, and the cryoinjury to the zona pellucida leading to a polispermic fertilization. ICSI is an efficient method to by pass these two events and to achieve a satisfactory outcome in terms of normal fertilization of cryopreserved oocytes. The application of the ICSI to cryopreserved oocytes did not seem to increase the degeneration rate after insemination with respect to fresh oocytes. The increased oocyte survival rate and the use of ICSI have facilitated the recent increase in the number of pregnancies and live birth.     

The influence of different types of media supplement on the meiotic maturation of bovine oocytes in vitro

This work was designed to evaluate the influence of different types of media supplements in the maturation of preovulatory oocytes in cattle. We studied 4 different supplements: serum from estrous cattle (ECS), serum from fetal calves (FCS), amniotic fluid from cattle (bAF) and follicular fluid from cattle (bFF). Preovulatory bovine oocytes recovered from ovaries of mature cows after slaughter were cultured for 24 h in TCM-199 medium containing 20% of one of these protein supplements. The percentages of oocyte maturation were significantly higher (P < 0.001) in ECS (78%), FCS (79%) and bAF (77%) than in bFF (52%). These data indicate that supplementation with ECS, FCS or bFF is adequate for maturation of bovine oocytes without addition of other compounds.     

Cryo-survival and development of bovine blastocysts are enhanced by culture with recombinant albumin and hyaluronan

Recombinant albumin can be used to supplement culture medium for the maturation and fertilization of bovine oocytes and subsequent embryo development to the blastocyst stage. Recombinant albumin was able to support blastocyst development at rates equivalent to that of bovine serum albumin (BSA) supplemented media. Supplementation of media containing recombinant albumin and citrate stimulated blastocyst expansion. Culture with recombinant albumin and citrate significantly increased the ability of the resultant blastocysts to re-expand and hatch following cryopreservation. The further addition of the glycosaminoglycan hyaluronan to the culture medium containing either BSA or recombinant albumin also increased the ability of blastocysts to survive cryopreservation. Inclusion of recombinant albumin and hyaluronan in culture media facilitates the development of physiological defined culture conditions. For bovine embryos this has implications for both research and commercial applications where defined reproducible conditions are desirable.     

Cryopreservation of oocyte and ovarian tissue

Cryopreservation of reproductive cells (i.e., oocytes, spermatozoa) and tissues (i.e., ovarian and testicular tissue) is a developing technology that has tremendous implications for rapid advancement of biomedical research in general. Since the early 1980s, advances have been made in establishing optimal conditions for in vitro oocyte maturation, fertilization, and culture of resulting embryos. These in vitro systems have contributed significantly to the utilization of these cells and tissues after thawing and have made it possible to evaluate protocols designed to cryopreserve such biomaterials more effectively. Although cryopreservation of preimplantation embryos from various species including mouse, human, and farm animals has been successful, cryopreservation of oocytes from most mammalian species has been more challenging due to their extreme sensitivity to suboptimal conditions during the cryopreservation process. Cryopreservation on mouse oocytes have been well documented and have resulted in greater success than studies with other mammalian species. Ovarian tissue cryopreservation and transplantation techniques have recently received much scientific and public attention due to their great potential use in human infertility treatment, in safeguarding the reproductive potential of the endangered species, and in genome banking of genetically important lab animal strains. A review of past and current research in the field of oocyte and ovarian tissue cryopreservation and transplantation and discussion of possible strategies for oocyte and ovarian tissue banking are provided.     

Effects of different protein supplements in fertilization medium on in vitro penetration of cumulus-intact and cumulus-free bovine oocytes matured in culture

Bovine oocytes matured in culture were inseminated with frozen-thawed spermatozoa in BO medium containing 5 mM-caffeine, 10 mug/ml of heparin and different protein supplements at various concentrations. When cumulus-enclosed oocytes were inseminated, no significant differences were observed in the penetration rates (89 to 100%) between media with and without protein supplements and among the different concentrations of each protein supplement, except for 20% calf serum (CS), in which the penetration rate decreased drastically (43%). Notably higher incidences of polyspermy were obtained in medium with FCS (75 to 86%) than with either no supplement (25%) or with BSA (20 to 24%) and CS (13 to 49%). On the other hand, there was almost no penetration of cumulus-free oocytes in the nonsupplemented control medium. Concentration-dependent increases in penetration and polyspermy occurred with BSA, FCS and CS supplementation. A high concentration (5%) of FCS yielded a high incidence (97%) of polyspermy. A decrease in the penetration of cumulus-enclosed oocytes was observed when spermatozoa were capacitated with a high concentration (20%) of CS; difficulty of sperm penetration of cumulus-free oocytes occurred when the capacitation medium lacked protein supplementation; and an increased rate of polyspermy was observed following supplementation with FCS in both cumulus-enclosed and cumulus-free oocytes after insemination with spermatozoa from 5 different bulls.     

Effects of prolactin on intracellular stored calcium in the course of bovine oocyte maturation in vitro

At present there are divergent opinions as to the role of prolactin (PRL) in the mechanisms of meiotic regulation in mammals. We investigated the effects of bovine PRL (bPRL) on bovine oocyte maturation in different culture systems and varying levels of intracellular stored calcium ([Ca2+]is) in the oocytes. Cumulus-oocyte complexes (COC) were incubated in TCM 199 containing either 10% fetal calf serum (FCS) in the absence (System 1) or presence (System 2) of FSH and estradiol, or 6 mg/mL bovine serum albumin (BSA) in the presence of FSH and estradiol (System 3). Levels of [Ca2+]is in oocytes were determined by using the fluorophore chlortetracycline. The addition of 50 ng/mL bPRL to different culture media increased the percentage of oocytes at telophase I and metaphase II stages (Systems 1 and 2) and/or decreased the percentage of oocytes with degenerated chromosomes (Systems 1 and 3). Compared with the control, lower levels of [Ca2+]is were observed in oocytes cultured for 2.5 h in those systems in which bPRL decreased the rate of oocytes with degenerated chromosomes (1.27+/-0.11 vs. 1.67+/-0.09 arbitrary units (AU) in System 1, P<0.001 and 1.27+/-0.12 vs. 1.52+/-0.04 AU in System 3, P<0.001). These findings show that the effects of bPRL on bovine oocyte maturation depend on the composition of the culture system and that the decline in the rate of oocytes with degenerated chromosomes in response to bPRL may be the result of the decrease in [Ca2+ ]is levels at early stages of oocyte maturation.     

Changes in the reciprocal position of the first polar body and oocyte chromosome set in golden hamsters

The golden hamster is an attractive model organism for studying reproductive physiology, oncology, genetics and virology. In an effort to establish experimental protocols necessary for cloning golden hamsters, we examined changes in the reciprocal position of the FPB (first polar body) and chromosome set of MII (the second meiotic metaphase) oocytes of golden hamsters. Oocytes were collected under three different conditions: (i) oocyte direct recovery from the oviduct of hormonally treated donor; (ii) oocyte recovery from the oviduct of hormonally treated donor followed by 5 h/10 h in vitro culture; and (iii) oocyte recovery from ovaries of hormonally treated donors and in vitro maturation. Then oocyte recovery was performed from the oviduct of hormonally treated donors, followed by 5 h in vitro culture with colchicine and/or CB (cytochalasin B). Denuded oocytes were stained with Hoechst 33342 and propidium iodide and evaluated under a microscope. Our results demonstrate that the change in FPB position in relation to the MII oocyte chromosome set increases with age of in vivo-matured oocytes. Cumulus cells can protect the FPB of in vitro-cultured oocytes from degeneration but do not significantly affect its repositioning, and in vitro-matured oocytes age slower. The colchicine has a stronger effect on cytoplasmic protrusions of golden hamster oocytes when compared with CB. These results define conditions for changes in FPB position relative to the MII oocyte chromosome set. Early ovulated oocytes, in vitro-matured oocytes and oocytes treated with colchicine should improve the effectiveness of the cloning procedure in golden hamsters as an animal model for human diseases.     

Cryopreservation of Unfertilized Mouse Oocytes: The Effect of Replacing Sodium with Choline in the Freezing Medium

Although embryo cryopreservation has become commonplace in many species, effective methods are not available for routine freezing of unfertilized eggs. Cryopreservation-induced damage may be caused by the high concentration of sodium ions in conventional freezing media. This study investigates the effect of a newly developed low-sodium choline-based medium (CJ2) on the ability of unfertilized, metaphase II mouse eggs to survive cryopreservation and develop to the blastocyst stagein vitro.Specifically, the effects of cooling to subzero temperatures, thawing rate, LN₂plunge temperature, and equilibration with a low-sodium medium prior to freezing are examined. In contrast to cooling to 23, 0, or −7.0°C in a sodium-based freezing medium (ETFM), cooling in CJ2 had no significant negative effect on oocyte survival or development. Oocytes frozen in CJ2 survived plunging into LN₂from −10, −20, or −33°C at significantly higher rates than oocytes frozen in ETFM. With the protocol used (1.5 M PrOH, 0.1 M sucrose, −0.3 C/min, plunging at −33°C) rapid thawing by direct submersion in 30°C water was more detrimental to oocyte survival than holding in air for 30 or 120 s prior to transfer to water. Equilibration of unfertilized oocytes with a low-sodium medium prior to cryopreservation in CJ2 significantly increased survival and blastocyst development. These results demonstrate that the high concentration of sodium in conventional freezing media is detrimental to oocyte cryopreservation and show that choline is a promising replacement. Reducing the sodium content of the freezing medium to a very low level or eliminating sodium altogether may allow oocytes and other cells to be frozen more effectively.     

Chromosomal analysis of mammalian oocytes matured in vitro with various culture systems

The objective of this study was to evaluate oocyte maturation in vitro. Ten virgin CD-1 mice were used with 3 replications for in vitro with 4 different culture media. Media were minimal essential medium (MEM) with Earl's salt, Waymouth MB 752/1 (MB 752/1), BGjb medium (BGjb), and tissue culture medium-199 (TCM-199). The oocyte chromosomes were C-banded to enable an objective analysis of the chromosome abnormality and number. There was a percentage of blockage at metaphase I (M I), in matured oocytes in all culture media. Metaphase II (M II) was reached by 70.9 to 87.3% of oocytes in 4 different culture media. The frequencies of hyperploid M II oocytes were 0.0, 1.1, 2.8 and 2.6% for TCM-199, MEM, MB 752/1 and BGjb, respectively. A small proportion of oocytes was also found to be polyploid in 4 different culture media. There was an occurrence of premature centromere separation among oocytes. It was concluded that the chromosomes of the oocytes matured in vitro were not all in the normal condition (being at M II). The media used in this study for oocyte maturation caused maturation delay (being blocked at M I), premature centromere separation, polyploidy, and aneuploidy (such as, hyperploid, hypoploid).     

Ultrastructure of human mature oocytes after vitrification

Since the introduction of human assisted reproduction, oocyte cryopreservation has been regarded as an attractive option to capitalize the reproductive potential of surplus oocytes and preserve female fertility. However, for two decades the endeavor to store oocytes has been limited by the not yet optimized methodologies, with the consequence of poor clinical outcome or of uncertain reproducibility. Vitrification has been developed as the promising technology of cryopreservation even if slow freezing remains a suitable choice. Nevertheless, the insufficiency of clinical and correlated multidisciplinary data is still stirring controversy on the impact of this technique on oocyte integrity. Morphological studies may actually provide a great insight in this debate. Phase contrast microscopy and other light microscopy techniques, including cytochemistry, provided substantial morpho-functional data on cryopreserved oocyte, but are unable to unraveling fine structural changes. The ultrastructural damage is one of the most adverse events associated with cryopreservation, as an effect of cryo-protectant toxicity, ice crystal formation and osmotic stress. Surprisingly, transmission electron microsco py has attracted only limited attention in the field of cryopreservation. In this review, the subcellular structure of human mature oocytes following vitrification is discussed at the light of most relevant ultrastructural studies.Key words: oocyte, MII, vitrification, ultrastructure, TEM, human.     

Cryopreservation of zebrafish (Danio rerio) oocytes using improved controlled slow cooling protocols

Cryopreservation of gametes provides a promising method to preserve fish genetic material. Previously we reported some preliminary results on cryopreservation of zebrafish (Danio rerio) oocytes using controlled slow cooling and determined the optimum cryoprotective medium and cooling rate for stage III zebrafish oocytes. In the present study, the effects of two different cryopreservation media, cryoprotectant removal method, final sample freezing temperature before LN₂ plunge, warming rate, and the post-thaw incubation time on oocyte viability were investigated. Commonly used cryoprotectant methanol and glucose were used in this study. Stage III zebrafish oocytes were frozen in standard culture medium 50% L-15 or in a sodium-free KCl buffer medium. Oocyte viability was assessed using trypan blue staining and ATP assay. The viability of oocytes frozen in KCl buffer was significantly higher than oocytes frozen in L-15 medium. The results also showed that fast thawing and stepwise removal of cryoprotectant improved oocyte survival significantly, with highest viability of 88.0 ± 1.7% being obtained immediately after rapid thawing when assessed by trypan blue staining. However, after 2 h incubation at 22 °C the viability of freeze-thawed oocytes decreased to 29.5 ± 5.1%. Results also showed that the ATP level in oocytes decreased significantly immediately after thawing. All oocytes became translucent after freezing which complicated the use of GVBD test (in vitro maturation of oocytes followed by observation of germinal vesicle breakdown which results in oocytes becoming translucent). New oocyte viability assessment methods are urgently needed.     

A chronologic review of mature oocyte vitrification research in cattle, pigs, and sheep

Vitrification as a means of cryopreservation has become a standard approach for oocytes from livestock. This paradigm shift occurred primarily as a result of the demonstration in 1996 that bovine oocytes are extremely susceptible to chilling injury. Since that early work, numerous devices have been used as supports for oocytes during so-called “ultra-rapid cooling”, and occasionally, trials involving the deposition of small volumes of media containing oocytes directly into liquid nitrogen to facilitate cooling have been reported. Results reporting blastocyst development exceeding 10% are common, but variability remains high, and a standard method for bovine oocytes remains to be established. Oocytes from pigs are particularly difficult to cryopreserve, even with the use of ultrarapid cooling approaches. Few reports have demonstrated blastocyst development exceeding 5%. The application of hydrostatic pressure before vitrification appears to impart stress tolerance to porcine oocytes, as the results of some treatments have shown development to blastocysts at proportions >10%. Work on sheep oocyte vitrification is relatively new, and a few articles have reported blastocyst development at 10% or more. Messenger RNA levels are reportedly altered in sheep oocytes as a result of vitrification, and damage to the cytoskeleton is common across species.     

Evaluation of cell viability and apoptosis in human amniotic fluid-derived stem cells with natural cryoprotectants

A previous study demonstrated that disaccharides, antioxidants, and caspase inhibitors can be used in freezing solutions to reduce the concentration of Me₂SO from the current standard of 10% (v/v) to 5% (v/v) or 2.5% and to eliminate fetal bovine serum (FBS) for the cryopreservation of human amniotic fluid-derived stem cells (AFSCs). Hence, this study investigated whether an irreversible inhibitor of caspase enzymes, benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone (zVAD-fmk), could be used in post-thaw culture media to increase the survival rate of AFSCs. Our results showed that AFSCs cryopreserved in freezing solution containing trehalose, catalase, and 5% (v/v) Me₂SO and then supplemented with zVAD-fmk in the post-thaw culture media showed similar post-thawing viability, proliferation, and apoptosis than cells cryopreserved in the control solution (10% (v/v) Me₂SO and 20% FBS). The caspase-3 activity in all the cryopreservation solutions tested was similar to that of the control. Caspase-3, caspase-8, caspase-9, and PARP expression was not found in the cryopreserved cells. In addition, no difference was found in the survival rate and apoptosis between short-term (3 weeks) and long-term (1 year) storage of AFSCs cryopreserved in the solutions used in this study. The results of the present study demonstrate that recovery of cryopreserved cells was enhanced by using a caspase inhibitor in the post-thaw culture media.     

Human oocyte morphometry before and after cryopreservation: A prospective cohort study

The cryopreservation of human oocytes is an important strategy to spare fertility in women submitted to gonadotoxic therapy, ovarian surgery, or even to allow gestation by assisted reproduction technology after natural ovarian senescence. Methods to predict oocyte resistance to cryopreservation are still based on qualitative morphological assessment. In this study we evaluated whether morphometric characteristics of mature oocytes before vitrification and after warming are related to successful fertilization by intracytoplasmic sperm injection (ICSI). This was a prospective cohort study including 28 infertile women and 71 oocytes. Morphometric assessments included oocyte diameter, perivitelline space (PS), zona pellucida (ZP) and first polar body (PB). Out of 49 warmed oocytes, 27 (55%) survived cryopreservation and their pre-vitrification measures were similar to those of the 22 oocytes that perished. However, the oocytes that eventually failed to be fertilized had undergone more enlargement of the total diameter (p = 0.029) and shrinking of the PS (p = 0.033) after cryopreservation, compared to oocytes that were successfully fertilized. These findings suggest that the morphometric characteristics of fresh oocytes do not predict their survival to vitrification, while fertilization failure is associated with oocyte enlargement and PS shrinking after cryopreservation.     

In the absence of protein, estradiol suppresses meiosis of porcine oocytes in vitro

The effect of estradiol on the spontaneous maturation of porcine oocytes was investigated. Cumulus-enclosed (intact) and cumulus-free (denuded) oocytes were cultured in the presence of estradiol-17 beta (0 to 10 microgram/ml) in a chemically defined bicarbonate-buffered medium that contained either dextran or BSA, or in a complex Hepes-buffered medium that was supplemented with serum. After 24 hr, chromatin spreads were prepared and meiotic maturation was scored. The biochemical integrities of the cumulus cells were assessed by determination of the estradiol and progesterone content of spent media after culture of intact oocytes in the presence of 0.5 X 10(-6) M testosterone and 10 microgram/ml follicle-stimulating hormone. Estradiol did not significantly affect the onset of maturation of either intact or denuded oocytes that were cultured in medium containing either BSA or serum. In serum-supplemented medium, however, the progression of maturation beyond metaphase I was significantly affected by the steroid in a dose-dependent manner. The steroid significantly inhibited the release from meiotic arrest of both types of oocyte cultured in medium supplemented with dextran. Supplementation of all media with testosterone and FSH significantly stimulated the synthesis of estradiol by the cumulus cells, compared with that of control groups. The synthesis of progesterone, however, was significantly stimulated by testosterone and FSH only in the BSA and serum-supplemented media. It is concluded that exogenous estradiol has the capacity to arrest meiosis in vitro but that this capacity can only be expressed if no exogenous protein(s) is present. In the absence of exogenous protein, progesterone synthesis by the adherent cumulus cells is minimal.     

Culture media for mouse oocyte maturation affect subsequent embryonic development

These experiments were done to determine whether the culture medium used for the spontaneous maturation of mouse oocytes can affect the subsequent capacity of the ova to become fertilized and complete preimplantation development in vitro and development to live young. Oocytes obtained from antral follicles of gonadotropin-primed immature mice underwent spontaneous maturation in control medium, i.e. Eagle's Minimum Essential Medium (MEM) supplemented with 5% fetal bovine serum, or in one of eight different media which were also supplemented with serum. All of the ova were fertilized in Whitten's medium and were assessed for cleavage to the 2-cell stage and for further preimplantation development to blastocysts during culture in Whitten's medium. Three of the eight media used for oocyte maturation improved the capacity of the ova to develop to the blastocyst stage when compared with the control: Waymouth MB 752/1, MEM with non-essential amino acids, and MEM Alpha; Waymouth medium promoted the highest frequency of development of ova to the blastocyst stage. Moreover, the blastocysts derived from oocytes that matured in Waymouth medium contained more cells than blastocysts derived from oocytes that matured in control medium. Although BGJb medium promoted the cleavage of eggs to the 2-cell stage when present during oocyte maturation, it had a detrimental effect on their subsequent preimplantation developmental capacity. Following transfer to foster mothers, more 2-cell stage embryos developed to live young after oocyte maturation in Waymouth medium (21%) than in control medium (13%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Epidermal growth factor enhances meiotic resumption of canine oocytes in the presence of BSA

Despite many attempts to improve the in vitro maturation (IVM) of canine oocytes using various culture conditions, the efficiency of canine IVM remains very low compared with that of other domestic animals. In the present study we examined the effect of ovarian estrus stage on oocyte quality, and the effect of epidermal growth factor (EGF) in the presence and absence of macromolecules on the IVM of canine oocytes. More oocytes >or=100 microm in diameter were obtained from follicular ovaries than from ovaries at other estrus stages. After 72 h of culture, significantly more oocytes recovered from follicular ovaries than from anestrous and luteal ovaries were in germinal vesicle break down (GVBD). Bovine serum albumin (BSA) or fetal bovine serum (FBS) supplementation improved meiotic resumption as compared to polyvinyl alcohol (PVA) supplementation; however, there was no difference between the BSA and FBS supplements. The oocytes matured in North Carolina State University (NCSU) 37 medium containing 0.4% BSA and 100 ng/ml EGF showed the highest rates of development to the metaphase II (MII) stage when compared with the control treatment (P < 0.05). These results suggest that the estrous cycle of bitches influences the meiotic resumption of oocytes cultured in vitro, and EGF increases the meiotic resumption of canine oocytes in the presence of BSA in vitro.