Evaluation of different culture systems on the in vitro production of bovine embryos

The objective of this study was to determine the development potential and quality of in vitro produced bovine embryos cultured individually or in groups. After IVM and IVF, presumptive zygotes were cultured in groups or individually, either in drops or in the modified "well of the well" (mWOW) system. In Experiment 1, four culture systems were utilized: T1: drop in group (control); T2: mWOW in groups; T3: mWOW individually; and T4: drop individually. Cleavage and blastocyst rates at Days 6, 7 and 8 and total cell number of Day 6 blastocysts were similar (P > 0.05) for all treatments. However, in Day 7 blastocysts, total cell number was lower (P < 0.05) in embryos cultured individually in a small drop than those cultured in the mWOW. In Experiment 2, blastocysts of T1, T2 and T3 were allocated into two groups, control and vitrified. After warming, the vitrified embryos were cultured for 72 h. At 48 h, the development of the Days 6 and 7 embryos was similar (P > 0.05) for all treatments in the control group. For the vitrified embryos, lower hatching rates (P < 0.05) were observed in the T3 group. In conclusion, embryos cultured in groups in the mWOW system had the same blastocyst rates but better quality (measured by their survival after vitrification) than those cultured individually in the mWOW system.     

Effects of ammonia during different stages of culture on development of in vitro produced bovine embryos

The effects of various concentrations of ammonia in the media during in vitro fertilization (IVF), culture (IVC), and throughout maturation (IVM), IVF, and IVC were evaluated using a randomized complete block design. Ammonia was added to the media at various concentrations during IVF (experiment 1), during IVC (experiment 2), and throughout IVM, IVF, and IVC (experiment 3). In the first experiment, there was a significant (P<0.05) increase in embryos developed to blastocyst, and to expanding and hatching blastocyst, in IVF media containing moderate concentrations of ammonia compared with that in the IVF control media. In the second experiment, ammonia in the IVC media increased (P<0.05) the proportion of degenerate ova and decreased (P<0.05) the proportion of ova that developed to blastocysts. In experiment 3, cleavage rates tended (P=0.06) to be greater for control groups than for treatment groups. The proportion of ova developing to morula was greater (P<0.05) in media containing moderate concentrations of ammonia than that in the control groups. These results indicate that the effect of ammonia on development of preimplantation bovine embryos depends on the concentration of ammonia and the stage of development when exposure to ammonia occurs.     

Development and de novo protein synthetic activity of bovine embryos produced in vitro in different culture systems

In vitro matured (IVM) and fertilized (IVF) putative Day 1 zygotes (Day 0 = IVF) were allocated randomly to culture in formulations based on Synthetic Oviduct Fluid (SOF) medium and identified on the basis of their contrasting principal supplements, which were 10% v/v steer serum (SS; n = 558) or 4 mg/mL crystalline BSA (SBSA; n = 531) or 3 mg/mL polyvinyl alcohol (SPVA; n = 607) in 9 replicates. SBSA and SPVA also contained 10 microg/mL non-essential amino acids, while the former was further supplemented with 20 microL/mL essential amino acids and the latter with 0.5 mmol/L sodium citrate and 5 ng/mL epidermal growth factor. Zygotes were cultured in 20 microL drops (4 zygotes per drop) until Day 8 in an atmosphere of 5% CO2, 5% O2 and 90% N2 at 39 degrees C and droplets were renewed every 48 hours. The incidence of zygote cleavage was lower (P < 0.05) in SS (mean +/- SEM = 61 +/- 3%) than in SBSA (76 +/- 3%) but not in SPVA (72 +/- 4%) up to Day 3. The SPVA generated a lower yield of blastocysts on Day 7 (12 +/- 2%; P < 0.001) and by Day 8 (21 +/- 4%; P < 0.01) than did SS (33 +/- 3%; 40 +/- 3%) and SBSA (30 +/- 3%; 37 +/- 4%). Cell numbers (n) and diameters (d) of blastocysts on Day 8 were greater (P < 0.001; Replicates 1 to 5) in embryos from SBSA (n, 156 +/- 9; d, 203 +/- 4 microm) than in those from SS (n, 81 +/- 4; d, 177 +/- 3 microm) and SPVA (n, 76 +/- 5; d, 167 +/- 3 microm). Embryos produced in SS incorporated less 3H-phenylalanine into PCA-precipitable protein (replicates 6 to 9; log10 dpm = 3.03 +/- 0.04) than did embryos cultured in SBSA (3.21 +/- 0.03; P < 0.001) or in SPVA (3.14 +/- 0.03; NS). In conclusion, blastocyst yield was poor in SPVA, but the embryos had metabolic activities similar to those of embryos produced in SBSA. Blastocyst yields from SS were not compromised but their capacity for de novo protein synthesis was reduced significantly.     

Effects of oxygen tension on the development and quality of porcine in vitro fertilized embryos

The present study was conducted to examine the effect of oxygen tension during in vitro culture (IVC) of porcine oocytes/embryos on their development and quality using two different culture systems. Porcine cumulus oocyte complexes (COCs) were matured (IVM) and fertilized (IVF) in vitro, and subsequently cultured for 6 days in a simple and economical portable incubator or a standard CO(2) incubator. While the same temperature (38.5 degrees C) and CO(2) concentration (5%) were used in the both systems, the portable incubator was operated in a negative air pressure (- 300 mmHg) to create an O(2) level at 8-10% (low O(2) concentration), or in a positive air pressure (high O(2) concentration). To compare the two culture systems, IVM and IVF of COCs and subsequent IVC of in vitro produced (IVP) embryos were carried out in the portable incubator with a low O(2) concentration (Group I) or in the standard incubator with a high O(2) concentration (Group II). To assess the effect of O(2) concentration on IVC of IVP embryos, some oocytes that had been cultured in the standard incubator for IVM and IVF were subsequently cultured in the portable incubator with a low O(2) concentration (Group III) or a high O(2) concentration (Group IV). The occurrence of DNA fragmentation in the blastocysts produced under different culture conditions was examined by TUNEL staining to assess embryo quality. The rates of oocytes that reached MII and were penetrated by spermatozoa following IVF did not differ between the two incubation systems. In contrast, the proportions of development to blastocysts and the mean cell number of blastocysts in Group I were higher than those in Group II and Group IV. The index of DNA-fragmented nucleus in the blastocysts of Group I was significantly lower than that in the blastocysts of Group II. Therefore, low oxygen tension during IVM, IVF and IVC enhanced the subsequent development of IVP embryos to the blastocyst stage and improved their quality.     

Effects of different cryopreservation methods on post-thaw culture conditions of in vitro produced bovine embryos

The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7-9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen-thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows?. Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.     

Influence of oxygen tension on apoptosis and hatching in bovine embryos cultured in vitro

Various oxygen tensions are employed for in vitro embryo production. Since it is known that oxygen tension can influence the efficiency of embryo production and embryo quality, the aim of our study was to define an optimal oxygen concentration for bovine embryo production in vitro in synthetic oviduct fluid (SOF). Embryo quality criteria were hatching ability and the degree of apoptosis as assessed by TUNEL staining and Bax gene expression. In Experiment 1, the effects of 2, 5 and 20% O(2) tensions on embryo development were compared. The highest rate of eight-cell embryos (47%) at 72 hpi was obtained under 20% O(2). However, it seemed that 2 and 5% O(2) were also suitable as assessed by embryo survival rates at 144 hpi (29 and 30% at morula stage), 168 hpi (21 and 19% at blastocyst stage) and 216 hpi (14 and 17% at hatched blastocyst stage). In Experiment 2, comparisons were made between effects of 5, 20% and alternating O(2) (20% O(2) to 72 hpi and then changed to 5% O(2) up to 216 hpi) on embryo development. Alternating the O(2) tension significantly reduced the number of hatching blastocysts to 7%. Staining with TUNEL revealed that apoptosis occurred in all tested hatched blastocysts, but a significantly lower apoptotic cell ratio was found in embryos cultured under 5% O(2) (P<0.05). Total cell number of embryos cultured under 5% and alternating oxygen was significantly higher than that of other groups (P<0.05). Bax gene expression was detected by means of RT-PCR in only 2 of 66 hatched blastocysts. It can be concluded that 5% oxygen is optimal for bovine embryo culture in cell free media. Moreover, it is very likely that the apoptosis detected by TUNEL staining in this study is Bax-independent.     

Sexing in vitro produced bovine embryos, at different stages of development, using rat H-Y antiserum

The male-specific H-Y antigen is present on mammalian cell membranes and has been identified by various methods, including antiserum cytotoxicity. The objective of the present study was to determine the sex of in vitro produced (IVP) bovine embryos, at varying stages of development, by culturing in the presence of rat monoclonal H-Y antibodies. Embryos derived from IVM/IVF were classified according to the interval after IVF (48, 96 or 120 h) as Category 1, 2 or 3 if they had 4 to 8, <32, and >32 cells, respectively. Embryos of each category were cultured for 24h in TCM-199 supplemented with bovine oviductal epithelial cells, fetal calf serum (FCS), and antibiotics (Control group), to which the following had been added: guinea pig serum (GPS; C' group); H-Y antiserum (HY group); or GPS and H-Y antiserum (C' + HY group). After culture, embryos were designated as "affected" when development was arrested or one or more blastomeres was degenerate; embryos lacking these changes were designated "unaffected." The sex of each embryo was subsequently determined by chromosome analysis. After 48h of IVF (Category 1), within each of the four treatments, the proportion of unaffected embryos was higher than the proportion of unaffected embryos (81% versus 19%, P < 0.05). Similarly, the Control, C' and HY groups of Categories 2 and 3 embryos had different proportions of unaffected versus affected embryos (75% versus 25%, P < 0.05). In all these groups, the male:female ratio did not significantly differ from 1:1. In contrast, in the C' + HY group of Categories 2 and 3 embryos, the ratio of unaffected versus affected embryos was 41% versus 59% (P < 0.05) and the male:female ratio differed (P < 0.05) from the expected 1:1 ratio (approximately 0.3:1 and 4.5:1 for unaffected versus affected, respectively). In conclusion, when bovine embryos were cultured in the presence of rat monoclonal H-Y antibodies and compliment, alterations occurred in embryos that were beyond the 8-cell stage; we inferred that the antibodies cross-reacted with H-Y antigens.     

Effects of oxygen tension and supplements to the culture medium on activation and development of bovine follicles in vitro

Our laboratory developed a method for culturing small pieces of bovine and baboon ovarian cortex, rich in primordial follicles, that supports the initiation of follicle growth and development to the primary stage. However, only a few follicles progressed to the secondary stage. The purpose of the current experiments was to determine if changes in culture conditions, specifically oxygen concentration and supplements to the culture medium, would facilitate the primary to secondary follicle transition. In Experiment 1, ovarian cortical pieces from late-gestation bovine fetuses were cultured with 2, 5, 20, or 60% oxygen in Waymouth's medium plus ITS+ (insulin, transferrin, selenium plus linoleic acid and BSA). Although the three lower concentrations of oxygen were generally equivalent in promoting follicle activation and growth, the highest concentration (60%) had deleterious effects on follicle survival after 7 days in culture, reducing the number of healthy follicles to about 35% of the number observed with 20% oxygen (P<0.05). In Experiment 2, bovine ovarian cortical pieces were cultured in the standard gas mixture (5% CO(2) in air) with graded doses of fetal bovine serum (FBS, 2.5, 5, or 10%) in the presence or absence of 0.5 or 1x ITS+. All concentrations of FBS alone were much less effective at maintaining follicular health and supporting the initiation and progression of follicular growth than was ITS+. However, 5 and 10% FBS alone increased the percentage of healthy primordial and primary follicles by about twofold (P<0.05) in the absence of ITS+ and in the presence of 0.5x ITS+, they enhanced the primary to secondary follicle transition by 10- and 9-fold, respectively. Thus, of the culture conditions evaluated, 20% oxygen and medium containing 0.5x ITS+ plus 5% or 10% FBS were the most effective for promoting follicular health and development.     

Effect of hyaluronic acid on development of in vitro produced bovine embryos

The present study was carried out to evaluate the effect of hyaluronic acid (HA) added to the culture medium on bovine embryo development to the blastocyst stage as well as embryo quality and viability after freezing and thawing. In vitro matured and fertilized (IVM/IVF) bovine oocytes from slaughterhouse ovaries were cultured for 8 d in SOFm supplemented with 4 mg/mL fatty acid-free BSA, either in the absence or presence of 1 or 0.5 mg/mL HA. There was a significant increase in blastocyst yield in the presence of 1 mg/mL HA (P < 0.01), whereas 0.5 mg/mL HA was ineffective. Cleavage rate and mean number of days to blastocyst formation were unaffected by HA at any concentration. At 1 mg/mL, HA did not affect either post-freeze survival of Grade 1 and 2 blastocysts or the number of nuclei per blastocyst. Supplementation with HA at 1 mg/mL also significantly enhanced embryo development up to the blastocyst stage (P < 0.05) in a chemically-defined culture medium without a protein source. It is concluded that supplementation of both semi-defined and defined culture media with 1 mg/mL HA improves the development of IVM/IVF bovine embryos to the blastocyst stage, without affecting embryo quality and post-freeze survival. These results open the possibility of including HA in culture media in order to increase the efficiency of in vitro blastocyst production from in vitro-matured bovine oocytes.     

Interaction between embryos and culture conditions during in vitro development of bovine early embryos

Various factors such as embryo density and substances in the medium can influence embryo development in vitro. These factors and the embryos probably interact with each other, however the interactions are not fully understood. To investigate the interactions, we examined the effects of the number of embryos, drop size, oxygen concentration and glucose and inorganic phosphate in the medium during protein-free culture of bovine IVM/IVF embryos. In Experiment 1, different numbers of embryos were cultured in a 50 microl drop of medium. The frequencies of blastocyst development in the groups with 25, 50 and 100 embryos per drop were higher than in the other groups. One, five and 25 embryos were cultured in different drop sizes (Experiment 2), a 50 microl drop of medium at different O2 concentrations (Experiment 3) and a 50 microl drop of medium excluding glucose and/or inorganic phosphate (Experiment 4). In Experiment 2, the size of the medium drops did not improve blastocyst development. In Experiment 3, the highest frequency of blastocyst development for one, five and 25 embryos per drop was obtained at 1, 2.5 and 5% O2, respectively. In Experiment 4, blastocyst development for one and five embryos per drop were improved in the medium excluded inorganic phosphate. These results indicate that there is a cooperative interaction among embryos during culture and that this interaction may be mediated by reduction of toxic factors in the medium. At low embryo density, reduced oxygen concentration or the exclusion of inorganic phosphate enhanced blastocyst development.     

Effect of culture system on survival rate of vitrified bovine embryos produced in vitro

This study was designed to evaluate the effect of in vitro culture system on bovine blastocyst yield and quality after vitrification. In Experiment 1, IVM/IVF zygotes were allocated to three culture conditions: (I) Oviductal cells-SOF (OCM-SOF); (II) Oviductal cells-TCM (OCM-TCM); and (III) SOF for 8 days. There was no significant difference between blastocyst rates among groups.In Experiment 2, the IVP-blastocysts in three above culture conditions were vitrified within groups segregated according to age (Day 7 and 8) and blastocoelic cavity size (early and expanded blastocysts). A trend of higher survival rate was obtained in vitrified/warmed early blastocysts compared with expanded ones, so that the difference in OCM-TCM group was significant (P < 0.001). Higher survival and hatching rates (P < 0.001) were obtained in OCM-SOF and OCM-TCM groups (co-culture) compared with SOF group and the age of blastocyst had no effect on post-thaw survival and hatching rates. In Experiment 3, after staining of blastocysts, in fresh blastocysts the highest number of trophectoderm cells was observed in OCM-TCM group and the number of inner cell mass (ICM) was higher in co-culture groups than SOF group (P < 0.001). In vitrified/warmed blastocysts the number of ICM and trophectoderm cells in co-culture groups was higher than SOF group (P < 0.001) except for the ICM of expanded blastocysts. In conclusion, in our culture conditions, the blastocyst yield is not influenced by culture system, while the cryotolerance of IVP-blastocysts is positively influenced by the presence of somatic cells. Moreover, the expanded blastocysts are more susceptible to cryoinjury than early blastocysts.     

Effect of gas atmosphere on the development of one-cell bovine embryos in two culture systems

The effect of two concentrations of oxygen on the development of bovine embryos was compared using two separate co-culture systems. In Experiment I, bovine oocytes were matured and fertilized in vitro and were then co-cultured for 7 days in 20 mul drops of M199 with 10% fetal calf serum containing oviduct cells. When cultures were performed in an atmosphere of 5% CO(2) in air (20% O(2)) or in a mixture of 5% CO(2), 5% O(2) and 90% N(2) (5% O(2)), 22 of 179 (12%) and 56 of 179 (31%) zygotes developed to or beyond the late morula stage (P<0.0001), respectively. After freezing, thawing and 48 hours of additional culture, 2 of 21 (10%) and 18 of 53 (34%) embryos were judged viable (P<0.001) within the respective treatment groups. In Experiment II, zygotes produced by the same means were co-cultured in 0.5 ml of M199 containing 10% fetal calf serum with monolayers of buffalo rat liver (BRL) cells. In 20% O(2), 51 of 177 (29%) zygotes developed into viable embryos, while in 5% O(2) only 9 of 177 (5%) were judged viable after 7 days of culture (P<0.0001). Post-freezing survival rates were 53% and 67% for embryos from the two respective oxygen concentration treatment groups. The transfer of 20 Grade 1 frozen/thawed embryos produced by co-culture with BRL cells produced six pregnancies (30%). These experiments show that the critical effect of oxygen concentration on embryo development in vitro and the ability of embryos produced by in vitro procedures to survive freezing can be influenced by the type of culture system employed.     

The differential requirement of albumin and sodium citrate on the development of in vitro produced bovine embryos

In vitro culture for bovine embryos is largely not optimal. Our study was to determine the components necessary for early embryo development. In experiment 1, IVF embryos were cultured for two days in CR1aa medium containing sodium citrate and BSA from two sources (Sigma vs. ICPbio), subsequently for additional five days with cumulus monolayer in 10% FBS CR1aa. We found that supplementation with both Sigma-BSA and sodium citrate significantly increased total blastocyst (BL) development compared with the ICPbio-BSA groups (37% vs. 19-21%), and enhanced the total number of high quality (C1 BL, IETS standard) blastocysts (26% vs. 11-17%) (P < 0.05). In experiment 2 with serum free and/or somatic free culture, we found that CR1aa culture can support a comparable embryo development with a supplement of Sigma BSA. The addition of sodium citrate did not increase blastocyst development in either the Sigma-BSA or the ICPbio-BSA groups. An inferior blastocyst development occurring in ICPbio-BSA culture (1-3%) could be rescued by culture in CRlaa supplemented with 10% FBS (29%), more importantly, by culture in CR1aa with a replacement of Sigma BSA (24%) (P <0.05). C1 blastocysts rescued by FBS and Sigma BSA in ICPbio-BSA culture possessed indistinguishable morphology to embryos developed in a Sigma-BSA, FBS and somatic co-culture system, showing similar cell number/blastocyst (129-180, P > 0.05). Our study found a beneficial effect of sodium citrate and BSA on the in vitro development of bovine IVF embryos during co-culture. We also determined that differential embryotrophic factor(s) contained in BSA and serum, probably not sodium citrate, is necessary for promoting competent morula and blastocyst development in cattle.     

Oxamflatin significantly improves nuclear reprogramming, blastocyst quality, and in vitro development of bovine SCNT embryos

Aberrant epigenetic nuclear reprogramming results in low somatic cloning efficiency. Altering epigenetic status by applying histone deacetylase inhibitors (HDACi) enhances developmental potential of somatic cell nuclear transfer (SCNT) embryos. The present study was carried out to examine the effects of Oxamflatin, a novel HDACi, on the nuclear reprogramming and development of bovine SCNT embryos in vitro. We found that Oxamflatin modified the acetylation status on H3K9 and H3K18, increased total and inner cell mass (ICM) cell numbers and the ratio of ICM∶trophectoderm (TE) cells, reduced the rate of apoptosis in SCNT blastocysts, and significantly enhanced the development of bovine SCNT embryos in vitro. Furthermore, Oxamflatin treatment suppressed expression of the pro-apoptotic gene Bax and stimulated expression of the anti-apoptotic gene Bcl-XL and the pluripotency-related genes OCT4 and SOX2 in SCNT blastocysts. Additionally, the treatment also reduced the DNA methylation level of satellite I in SCNT blastocysts. In conclusion, Oxamflatin modifies epigenetic status and gene expression, increases blastocyst quality, and subsequently enhances the nuclear reprogramming and developmental potential of SCNT embryos.     

Improved in vitro development of OPU-derived bovine (Bos taurus) embryos by group culture with agarose-embedded helper embryos

The average number of oocytes collected by ovum pick up (OPU) from Bos taurus cattle is <8 per live donor. The objective was to determine whether development of small numbers of cattle embryos (produced by OPU and IVF), was enhanced by including “helper” embryos, produced from abbatoir-derived oocytes and embedded in agarose. Oocytes were from abbatoir-derived ovaries (Experiments 1 and 2) or OPU of elite donors (Experiment 3). In Experiment 1, cleaved embryos (2–8 cells), were randomly allocated. Controls were groups of 1, 3, 5, 10, and 20 cleaved embryos cultured in 50 μL serum-free SOF, whereas treatments were groups of 1, 3, and 5 embryos freely cultured along with helpers in groups of either 9, 7 or 5 embedded in agarose per droplet. Therefore, there were 10 cleaved embryos per droplet in combinations of 1 + 9, 3 + 7 or 5 + 5 (free + helper), respectively. There was an increase in the progression to blastocyst for 1–5 embryos per droplet, compared to 10 and 20 (6.6–24.2% vs. 39.2–43.3%, P < 0.05). For the tested free embryos, those cultured with helpers had increased blastocyst development over their control counterparts (39.3–49.5% vs. 6.6–24.2%, P < 0.05). When the number of embryos per droplet was 10 or 20, blastocyst percentage was similar (39.2–49.5%, P > 0.05). In Experiment 2, addition of an agarose chip to the culture medium did not significantly affect development to the blastocyst stage. In Experiment 3, after fertilizing OPU oocytes with sorted X-sperm, a group of three cleaved embryos were cultured in a droplet with either seven helpers (3 + 7) or alone (3 + 0). Blastocyst development of OPU oocytes in the 3 + 7 group was 37.1%, higher than that in the 3 + 0 group (11.8%, P < 0.05). In conclusion, limited numbers of OPU/IVF oocytes had competent development when cultured with helpers (embedded in agarose to provide physical separation).     

Promoting effect of beta-mercaptoethanol on in vitro development under oxidative stress and cystine uptake of bovine embryos

The effects of beta-mercaptoethanol (beta-ME) on in vitro development under oxidative stress and cystine uptake of bovine embryos were investigated. Bovine 1-cell embryos obtained by in vitro fertilization were cultured in TCM-199 or synthetic oviductal fluid (SOF) in 20% O(2) supplemented with beta-ME. Addition of beta-ME significantly (P < 0.01) promoted embryo development when cultured in both TCM-199 and SOF under high levels of O(2), to almost the same rates when they were cultured in 5% O(2). To investigate whether the growth-promoting effect of beta-ME was related to cystine uptake, which is an important amino acid for intracellular glutathione (GSH) synthesis, 1-cell, 8-cell, morula, and blastocyst stage embryos were incubated in cystine, cysteine-free TCM-199 containing radioisotope-labeled cystine supplemented with or without beta-ME. It was found that cystine uptake was consistently low in each embryo stage incubated without beta-ME. In contrast, addition of beta-ME significantly (P < 0.05 to 0.0001) promoted cystine uptake in each stage of embryo development. This increase of cystine uptake by beta-ME was significantly inhibited by supplementation of buthionine sulfoximine, a specific inhibitor of GSH biosynthesis (P < 0.0001). High-performance liquid chromatography (HPLC) analysis clearly revealed a decrease of cystine in culture medium after supplementation by beta-ME, thereby forming another peak. HPLC analysis also showed the incorporated cystine by supplementation of beta-ME was possibly metabolized for GSH synthesis in the embryos. These results indicate that beta-ME has a protective effect in embryo development against oxidative stress and that the effect of beta-ME is associated with the promotion of cystine uptake of low availability in embryos.