Excessive concentration of glucose during in vitro maturation impairs the developmental competence of bovine oocytes after in vitro fertilization: relevance to intracellular reactive oxygen species and glutathione contents

The effect of glucose (0, 1.5, 5.6 or 20.0 mM) in synthetic oviduct fluid supplemented with 20 amino acids (SOFaa) on the developmental competence of bovine oocytes after in vitro fertilization was investigated. Intracellular reactive oxygen species (ROS) and the glutathione content of bovine oocytes matured in SOFaa containing 0-20.0 mM glucose were also examined. When oocytes were matured in SOFaa without glucose, the nuclear maturation rate was lower than that in oocytes matured in glucose-containing medium. The developmental competence to the blastocyst stage of oocytes matured in 1.5 mM glucose was higher than that of oocytes matured in 20.0 mM glucose. In addition, the intracellular ROS content of oocytes matured in 0, 1.5 or 5.6 mM glucose was lower than that of oocytes matured in 20.0 mM glucose. Furthermore, the intracellular glutathione content of oocytes matured in 0, 1.5 or 5.6 mM glucose was higher than that of oocytes matured in 20.0 mM glucose. These results show that excessive glucose in the medium for oocyte maturation impairs the development of bovine oocytes to the blastocyst stage, possibly due to the increase of ROS and the decrease in the intracellular glutathione content of bovine oocytes.     

Low oxygen tension during in vitro maturation is beneficial for supporting the subsequent development of bovine cumulus-oocyte complexes

The effects of carbohydrates on meiotic maturation and ATP content of bovine oocytes under low oxygen tension (5%) were investigated. Furthermore, the developmental competence or intracellular H(2)O(2) contents of the oocytes matured under 5% or 20% O(2) was assessed. In vitro maturation of bovine cumulus-oocyte complexes was performed in synthetic oviduct fluid (SOF) containing 20 amino acids and hormones (SOFaa). The proportion of the oocytes that matured to the metaphase II stage in SOFaa containing 1.5 mM glucose, 0.33 mM pyruvate, and 3.3 mM lactate under 5% O(2) was dramatically lower than that of oocytes matured under 20% O(2) (P < 0.01). Similarly, the ATP content of the oocytes that matured under 5% O(2) was much lower than that of oocytes matured under 20% O(2) (P < 0.05). Under 5% O(2) the proportion of metaphase II oocytes increased with increasing glucose concentration (0-20 mM) in SOFaa without pyruvate or lactate. In addition, the ATP content of oocytes cultured in 20 mM glucose was higher (P < 0.05) than that of oocytes cultured in 1. 5 mM glucose. Two glucose metabolites (pyruvate and lactate) and a nonmetabolizable glucose analog (2-deoxy-glucose), however, had no noticeable effects on meiotic maturation under 5% O(2). These results suggest that ATP production under 5% O(2) is not dependent on the TCA cycle. Addition of iodoacetate, a glycolytic inhibitor, to SOFaa containing 20 mM glucose significantly reduced (P < 0.01) the proportion of metaphase II and ATP content. Moreover, the proportion of the development to the blastocyst stage of oocytes matured under 5% O(2) was higher (P < 0.05) than that of oocytes matured under 20% O(2). H(2)O(2) contents of oocytes matured under 5% O(2) was lower (P < 0.05) than that of oocytes matured under 20% O(2). The results of the present study demonstrate that glucose plays important roles in supporting the completion of meiotic maturation in bovine cumulus-oocyte complexes under low oxygen tension and that low oxygen tension during in vitro maturation is beneficial for supporting the subsequent development of bovine oocytes.     

Involvement of apoptosis in disruption of developmental competence of bovine oocytes by heat shock during maturation

Various pathological stimuli such as radiation, environmental toxicants, oxidative stress, and heat shock can initiate apoptosis in mammalian oocytes. Experiments were performed to examine whether apoptosis mediated by group II caspases is the cause for disruption of oocyte function by heat shock applied during maturation in cattle. Bovine cumulus-oocyte complexes (COCs) were cultured at 38.5, 40, or 41 degrees C for the first 12 h of maturation. Incubation during the last 10 h of maturation, fertilization, and embryonic development were at 38.5 degrees C and 5% (v/v) CO2 for all treatments. In the first experiment, exposure of COCs to thermal stress during the first 12 h of maturation reduced cleavage rate and the number of oocytes developing to the blastocyst stage. In the second experiment, a higher percentage of TUNEL-positive oocytes was noted at the end of maturation for oocytes matured at 40 and 41 degrees C than for those at 38.5 degrees C. In addition, the distribution of oocytes classified as having high (>25 intensity units), medium (15-25 intensity units), and low (<15 intensity units) caspase activity was affected by treatment, with a greater proportion of heat-shocked oocytes having medium or high activity. In the third experiment, COCs were placed in maturation medium with vehicle (0.5% [v/v] DMSO) or 200 nM z-DEVD-fmk, an inhibitor of group II caspases. The COCs were matured at 38.5 or 41 degrees C, fertilized and cultured for 8 days. The inhibitor blocked the effect of heat shock on cleavage rate and the percentage of oocytes and cleaved embryos developing to the blastocyst stage. In conclusion, heat shock during oocyte maturation can promote an apoptotic response mediated by group II caspases, which, in turn, leads to disruption of the oocyte's capacity to support early embryonic development following fertilization.     

Effect of maturation media on male pronucleus formation in pig oocytes matured in vitro.

The present study was carried out to examine the effect of maturation media on male pronucleus formation of pig oocyte matured and fertilized in vitro. Follicular oocytes collected from prepubertal gilts at a local slaughter house were cultured (36 h) in three different media (mTCM-199, Waymouth MB 752/l, and mTLP-PVA), fertilized in vitro, and assessed for nuclear maturation and male pronucleus formation. The addition of 10% (v/v) pig follicular fluid (pFF) to maturation media significantly increased the rate of nuclear maturation of pig oocytes (P less than 0.01), whereas the rate of nuclear maturation of pig oocytes among three different media did not differ. However, the rate of male pronucleus formation of pig oocytes was significantly higher in pig oocytes matured in Waymouth MB 752/l with or without pFF than in oocytes matured in the other two media (P less than 0.01). In experiment 2, the addition of cysteine (the same concentration as in Waymouth medium, 0.57 mM), to mTLP-PVA significantly increased the rate of male pronucleus formation of pig oocytes compared with the control (P less than 0.01). The results indicate that the composition of maturation medium affects the ability of pig oocytes to form male pronuclei following sperm penetration; media containing a high concentration of cysteine (possibly as a substrate of glutathione), such as Waymouth MB 752/l, can remarkably promote this ability.     

Expression pattern of glucose metabolism genes in relation to development rate of buffalo (Bubalus bubalis) oocytes and in vitro–produced embryos

The expression pattern of glucose metabolism genes (hexokinase, phosphofructokinase, glucose-6-phosphate dehydrogenase [G6PDH], lactate dehydrogenase [LDH], and pyruvate dehydrogenase [PDH]) were studied in buffalo in vitro–matured oocytes and in vitro–produced embryos cultured under different glucose concentrations (0 mM, 1.5 mM, 5.6 mM, and 10 mM) during in vitro maturation of oocytes and culture of IVF produced embryos. The expression of the genes varied significantly over the cleavage stages under different glucose concentrations. Developmental rate of embryos was highest under a constant glucose level (5.6 mM) throughout during maturation of oocytes and embryo culture. Expression pattern of glucose metabolism genes under optimum glucose level (5.6 mM) indicated that glycolysis is the major pathway of glucose metabolism during oocyte maturation and early embryonic stages (pre-maternal to zygotic transition [MZT]) and shifts to oxidative phosphorylation during post-MZT stages in buffalo embryos. Higher glucose level (10 mM) caused abrupt changes in gene expression and resulted in shifting toward anaerobic metabolism of glucose during post-MZT stages. This resulted in decreased development rate of embryos during post-MZT stages. High expression of LDH and PDH in the control groups (0 mM glucose) indicated that in absence of glucose, embryos try to use available pyruvate and lactate sources, but succumb to handle the post-MZT energy requirement, resulting to poor development rate. Expression pattern of G6PDH during oocyte maturation as well early embryonic development was found predictive of quality and development competence of oocytes/ embryos.     

Developmental competence and post-thaw survivability of buffalo embryos produced in vitro: effect of growth factors in oocyte maturation medium and of embryo culture system

The present study was conducted to examine the effects of supplementation to IVM medium of epidermal growth factor (EGF), fibroblast growth factor (FGF) and vasoactive intestinal peptide (VIP) along with pregnant mare serum gonadotrophin (PMSG) on oocyte maturation and cleavage of buffalo embryos (experiment 1). The developmental competence of cleaved embryos cultured in either a complex co-culture system (TCM-199+10% serum+oviduct cell monolayer) or defined media (a) modified form of synthetic oviductal fluid (mSOF) was evaluated (experiment 2). The post-thaw morphology and survivability of frozen blastocysts developed from embryos cultured either in complex or defined medium was compared (experiment 3). Aspirated oocytes were cultured in maturation medium (TCM-199+PMSG (40 IU/ml—control)) supplemented with EGF (20 ng/ml), FGF (20 ng/ml) and VIP (20 ng/ml), either alone or in combination, in a CO₂ incubator at 38.5 °C for 24 h. Maturation rate was assessed and oocytes were inseminated in vitro with frozen–thawed sperm processed in Brackett and Oliphant (BO) medium. The cleaved embryos were cultured either in complex co-culture system or mSOF. Results suggested that EGF had more beneficial effect on buffalo oocyte maturation, and embryo cleavage than FGF. Addition of VIP to the oocyte maturation medium did not improve the results. Blastocyst yields from buffalo oocytes were significantly higher in a complex co-culture system than in defined media (mSOF) when oocytes were matured in presence of EGF either alone or in combination with FGF and VIP. The mean percent of morphologically normal blastocysts after thawing and their survivability were significantly higher in blastocysts obtained from embryos cultured in mSOF than those cultured in complex co-culture system.     

Effects of hexoses on in vitro oocyte maturation and embryo development in pigs

The objective was to determine the effects of supplementing hexoses in oocyte maturation and embryo culture medium on in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes and in vitro development of in vitro produced (IVP) porcine embryos. In the first experiment, oocytes were matured in vitro in modified North Carolina State University (NCSU)-37 medium, supplemented with hexoses (glucose, fructose or galactose) at various concentrations: 0 (control), 2.5, 5.5 and 10 mM. Supplementing the maturation medium with either glucose or fructose (5.5 mM) increased the percentages of oocytes that matured to metaphase II (79.4 and 70.2%, respectively), as compared with the control group (P < 0.05). However, supplementing galactose had no effects on meiotic maturation and fertilization. In the second experiment, cleaved embryos were collected 3 days after IVF of oocytes matured in the maturation medium supplemented with 5.5 mM of glucose; they were cultured for an additional 4 days in modified NCSU-37 medium, supplemented with 5.5mM of glucose, fructose or galactose. The incidence of blastocyst formation was higher (P < 0.05) in the glucose and fructose groups (18.6 and 18.2%, respectively) than in the galactose group and non-supplemented control group (12.9 and 9.2%). Moreover, fructose supplementation increased the total cell number/blastocyst (48.0 versus 37.6) and reduced the index of DNA-fragmented nucleus in the blastocysts (7.6% versus 11.8%), as compared with glucose supplementation (P < 0.05). In conclusion, fructose was a practical alternative to glucose for supporting IVM of porcine oocytes and fructose was superior to glucose for producing high-quality porcine embryos in vitro.     

Retinol improves bovine embryonic development in vitro

Retinoids are recognized as important regulators of vertebrate development, cell differentiation, and tissue function. Previous studies, performed both in vivo and in vitro, indicate that retinoids influence several reproductive events, including follicular development, oocyte maturation and early embryonic development. The present study evaluated in vitro effects of retinol addition to media containing maturing bovine oocytes and developing embryos in both a low oxygen atmosphere (7%) and under atmospheric oxygen conditions (20%). In the first experiment, abbatoir collected bovine oocytes were matured in the presence or absence of varying concentrations of retinol. After a 22–24 hour maturation period the oocytes were fertilized, denuded 18 hours later and cultured in a modified synthetic oviductal fluid (mSOF) in a humidified atmosphere at 38.5 degrees C, 5% CO2, 7% O2 and 88% N2. Cleavage rates did not differ among control and retinol-treated oocytes in all three experiments. Addition of 5 micromolar retinol to the maturation medium (IVM) tended (p < 0.07) to increase blastocyst formation (blastocyst/putative zygote; 26.1% +/- 2.2%) compared to the controls (21.9% +/- 1.9%). Further analysis revealed when blastocyst development rates fell below 20% in the control groups, 5 micromolar retinol treatment dramatically improved embryonic development, measured by blastocyst/putative zygote rate (14.4 +/- 2.1 vs 23.7 +/- 2.5; p < 0.02). The 5 micomolar retinol treatment also enhanced the blastocyst/cleaved rate by nearly 10% (23.7% vs 34.6%; p < 0.02). In the second and third experiments addition of 5 micromolar retinol to the embryo culture medium (IVC) under low oxygen conditions did not significantly improve cleavage or blastocyst rates, but 5 micromolar retinol significantly increased blastocyst development under 20% O2 conditions (p < 0.001). These studies demonstrate that supplementation of 5 micromolar retinol to the maturation medium may improve embryonic development of bovine oocytes indicated by their increased blastocyst rate. A significant improvement in the blastocyst development with the 5 micromolar retinol treatment under atmospheric conditions suggests a beneficial antioxidant effect during embryo culture.     

Sphingosine 1-phosphate protects bovine oocytes from heat shock during maturation

Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite that can block apoptosis by counteracting the proapoptotic effects of ceramide. Experiments were performed to evaluate whether S1P blocks the disruption in oocyte developmental competence caused by heat shock. Cumulus-oocyte complexes (COCs) were placed in maturation medium and cultured at 38.5 or 41 degrees C for the first 12 h of maturation. Incubation during the last 10 h of maturation, fertilization, and embryonic development were performed at 38.5 degrees C. Heat shock during the first 12 h of maturation reduced cleavage rate, the number of oocytes developing to the blastocyst stage, and the percentage of cleaved embryo that subsequently developed to blastocysts. Addition of 50 nM S1P to maturation medium had no effect on oocytes matured at 38.5 degrees C but blocked effects of thermal stress on cleavage and subsequent development. The blastocysts formed at Day 8 did not differ between S1P and control groups in caspase activity, total cell number, or percentage of cells that were apoptotic. Blocking endogenous generation of S1P by addition of 50 nM N1N-dimethylsphingosine, a sphingosine kinase inhibitor, reduced or tended to reduce cleavage rate and blastocyst development regardless of whether maturation of COCs was at 38.5 or 41 degrees C. Results demonstrate that S1P protects oocytes from a physiologically relevant heat shock and affects oocyte maturation even in the absence of heat shock. The S1P-treated oocytes that survived heat shock and became blastocysts had a normal developmental potential as determined by caspase activity, total cell number, and percentage of apoptotic cells. Thus, modulation of developmental competence of oocytes using S1P may be a useful approach for enhancing fertility in situations where developmental competence of oocytes is compromised.     

Effects of the addition of glutathione during maturation on in vitro fertilisation of prepubertal goat oocytes.

Glutathione (gamma-glutamyl-cysteinyl-glycine; GSH) is a ubiquitous intracellular free thiol that improves development of the male pronucleus at fertilisation and has also been implicated in promoting the development of preimplantation embryos. The objective of this study was to evaluate the effects of adding GSH or cysteine to the in vitro maturation medium on intracellular GSH amounts after in vitro maturation and fertilisation of prepubertal goat oocytes. Oocytes were matured in TCM199 medium supplemented with 10% bovine fetal serum, 1 mg/ml 17beta-estradiol, 10 microg/ml o-FSH, 10 microg/ml LH and 50 mg/ml gentamicin. In vitro maturation medium was completed with two independent treatments: GSH at different concentrations (0, 0.25, 0.50 and 1.00 mM) and L-cysteine at different concentrations (0, 150, 300, 600 and 900 microM). After 27 h of culture at 38.5 degrees C in 5% CO2 in air, the nuclear stage was evaluated. Simultaneously, another sample of oocytes was frozen and the intracellular GSH level was evaluated with spectrophotometric methodology. Oocytes were inseminated with fresh semen (2-3 x 10(6) sperm/ml) in TALP medium supplemented with 1 mg/ml hypotaurine. Oocytes were fixed at 20 h post-insemination to evaluate the in vitro fertilisation. Oocytes matured in 1.00 mM GSH-supplemented medium exhibited higher amounts of intracellular GSH (3.23 pmol per oocyte). The percentage of normal fertilisation (17-27%) was similar for the treatment groups. In conclusion, the addition of 1.00 mM GSH to the maturation medium could be a useful method for increasing the intracellular GSH levels of prepubertal goat oocytes. However, this increase was not associated with a higher normal fertilisation rate of prepubertal goat oocytes.     

Effect of incubation temperature on in vitro maturation of porcine oocytes: nuclear maturation, fertilisation and developmental competence.

The present study examined the effect of low culture temperature during in vitro maturation (IVM) of pig oocytes on their nuclear maturation, fertilisation and subsequent embryo development. In experiment 1, oocytes were cultured at 35 or 39 degrees C for 44 h in modified tissue culture medium 199 supplemented with 10 ng/ml epidermal growth factor, 0.57 mM cysteine, 75 microg/ml potassium penicillin G, 50 microg/ml streptomycin sulphate, 0.5 microg/ml LH and 0.5 microg/ml FSH to examine the nuclear maturation status. In experiment 2, oocytes were cultured at 35 degrees C for 44 or 68 h and nuclear maturation was examined. In experiment 3, oocytes matured for 44 or 68 h at 39 degrees C and for 68 h at 35 degrees C were co-incubated with frozen-thawed spermatozoa for 5-6 h. Putative embryos were transferred into North Carolina State University (NCSU) 23 medium containing 0.4% bovine serum albumin. At 12 h after insemination, some oocytes were fixed to examine the fertilisation rate and the remaining embryos were examined at 48 and 144 h for cleavage and blastocyst formation rate, respectively. Compared with 39 degrees C, culture of oocytes at 35 degrees C for 44 h significantly (p < 0.05) reduced the metaphase II (M II) rate (79% vs 12%). However, extension of culture time to 68 h at 35 degrees C significantly increased (p < 0.05) the M II rate (7% vs 58%). In experiment 3, compared with other groups, fewer (p < 0.05) oocytes reached M II when cultured at 35 degrees C for 68 h (69-81% vs 49%). Extension of culture duration to 68 h at 39 degrees C stimulated spontaneous activation (28%) of oocytes. No difference in cleavage rates was observed among different groups. Compared with oocytes matured for 44 h at 39 degrees C (31%), the proportion of blastocysts obtained was low (p < 0.05) for oocytes matured at 35 degrees C (13%) or 39 degrees C (3%) for 68 h. The results indicate that lower culture temperature can delay nuclear maturation of pig oocytes. However, extension of culture time can stimulate nuclear maturation and these oocytes are capable of fertilisation and development to the blastocyst stage at moderate rates.     

Effect of high molecular weight factors present in bovine oviduct-conditioned medium on in vitro bovine embryo development

To investigate the presence of embryotrophic factors in bovine oviduct-conditioned medium (BOCM), the high molecular weight fraction (> 10 KDa) from BOCM was added to 3 chemically defined embryo culture media (TCM199, DMEM/F12 and modified synthetic oviduct fluid [mSOF]). Zygotes were obtained by in vitro maturation and fertilization of oocytes. Conditioning of TCM199 with oviduct cells increased both cleavage to the 5- to 8-cell stage (59 vs 37%) and further development to the blastocyst stage (19 vs 4%). The low molecular weight fraction (< 10 KDa) of BOCM maintained development to the 5- to 8-cell stage but did not allow development to the blastocyst stage. Adding the high molecular weight fraction to the inactive low molecular weight fraction restored bovine embryo development up to the blastocyst stage. This embryotrophic effect of the high molecular weight fraction was not observed when this fraction was added to TCM199 or DMEM/F12 medium. Whereas adding this fraction to mSOF medium significantly (P<0.05) increased embryo development up to the blastocyst stage (36%) in comparison with that of mSOF (15%) or BOCM (14%). These results show that BOCM contains high molecular weight factors promoting embryo development up to the blastocyst stage. Some chemically defined media mask the effect of these embryotrophic factors.     

Effects of beta-mercaptoethanol on formation of pronuclei and developmental competence of swamp buffalo oocytes

This study was conducted to determine the effect of supplementing maturation medium with beta-mercaptoethanol (betaME) on pronuclei formation and developmental competence of swamp buffalo oocytes. Buffalo oocytes were matured in TCM199 medium either with 10mM betaME or without betaME supplementation for 24h. In Experiment 1, oocytes were fixed and stained for cytological evaluation after in vitro fertilization (IVF). In Experiment 2, presumptive zygotes were cultured and their developmental competency was assessed. It was found that betaME significantly improved the proportion of oocytes that exhibited synchronous pronuclei formation (31.8+/-5.1% versus 17.9+/-3.3%, P<0.05). There were no significant differences between oocytes matured with or without betaME in their capability of developing into blastocyst-stage embryos (3.0+/-1.3% versus 1.8+/-0.9%). However, blastocysts produced from oocytes matured in the presence of betaME appeared to develop faster than those from oocytes matured in the absence of betaME (P<0.05). Cavitation of embryos from oocytes matured in the presence of betaME occurred at 156 hpi, whereas those matured in the absence of betaME occurred at 180 hpi. Although in vitro production of blastocysts did not increase by addition of betaME to maturation medium, quality of blastocysts produced from oocytes matured in the presence of betaME was improved. This study provides information for further investigations on optimizing a system for in vitro production of swamp buffalo embryos.     

Effect of glucose levels during the in vitro culture in synthetic oviduct fluid medium on in vitro development of bovine oocytes matured and fertilized in vitro

The present study was conducted to determine the optimal glucose levels during the in vitro culture of bovine oocytes matured and fertilized in vitro for blastocyst development. Oocytes matured in TCM-199 + 10% FCS + hormones and granulosa cells were fertilized in vitro in a TALP medium with frozen-thawed, swim-up separated, and heparin-treated spermatozoa. After insemination, 1199 oocytes were cultured for 3 days in synthetic oviduct fluid medium (SOFM) supplemented with 10% human serum (HS) and with 10 different glucose levels (0 to 5 mM), and further cultured for 5 days in SOFM + 10% HS containing 1.5 mM glucose (Experiment 1). In Experiment 2, 739 oocytes were cultured for 3 days following insemination in either SOFM + human serum albumin or SOFM + 10% HS containing 0.188 mM glucose. From Days 4 to 8, the oocytes were cultured in SOFM containing 4 different glucose levels. A high level of glucose (3.0 and 5.0 mM) at Days 0 to 3 significantly reduced the rate of blastocyst development (3.0 to 4.2%), and a yet higher (5.0 mM) glucose level at Days 4 to 8 also significantly lowered the rate of blastocyst development as compared with 1.5 mM glucose (19.5% vs 29.3%). The present results indicate that a lower level (0.188 mM: 28.8% in blastocyst development) of glucose is preferable in SOFM for the in vitro development to blastocysts at Days 0 to 3 after insemination. At Days 4 to 8, the original level (1.5 mM) of glucose contained in SOFM appears to be the most effective treatment.     

Heat shock at the germinal vesicle breakdown stage induces apoptosis in surrounding cumulus cells and reduces maturation rates of porcine oocytes in vitro

The objectives were to determine the effects of cumulus cells (CC) on porcine oocyte maturation in vitro (IVM) after heat shock (HS). Treated oocytes were cultured at 39 degrees C for 20h, followed by HS treatment (42 degrees C for 1h), and then matured in vitro for 23h. The CC were removed before maturation (H1), after HS (H2), or after maturation (H3). Control oocytes were continuously cultured under the same conditions and CC were similarly removed before maturation (C1), after 21h of IVM (C2), and after maturation (C3). Maturation rates were affected by HS (P<0.01) and by an interaction between HS and CC (P<0.01). A significant decrease in maturation rate only occurred when CC were not removed from cumulus oocyte complexes during IVM after HS (H3, 39.2+/-5.7% versus C3, 78.2+/-8.2%, P<0.01). Mature oocytes in all treatment groups were electrically activated and cultured for 8 d in NCSU23. Blastocyst rates in group H1 (7.2+/-3.5%) and C1 (6.3+/-3.1%) were lower than in other groups (H2, 21.4+/-4.4%, C2, 20.5+/-7.0%, H3, 23.1+/-2.0%, C3, 24.3+/-3.1%, P<0.05). Damaged DNA was detected in CC by a comet assay at 0h after HS (60.8+/-12.5% compared with 9.2+/-2.2% in control, P<0.05); in HS groups, both DNA damage (comet assay, 74.9+/-6.3% compared with 10.0+/-2.1% in control) and apoptosis (TUNEL assay, 21.6+/-1.6% compared with 5.6+/-0.6% in control) in CC were increased (P<0.05) at 44h of maturation. In conclusion, heat shock (42 degrees C for 1h) during IVM induced DNA damage and apoptosis of porcine CC; furthermore, apoptotic CC may contribute to maturation failure of oocytes in vitro.     

Effects of exogenous hexoses on bovine in vitro fertilized and cloned embryo development: Improved blastocyst formation after glucose replacement with fructose in a serum-free culture medium

To evaluate the embryotrophic role of three hexoses (glucose, fructose, and galactose), bovine embryos derived from somatic cell nuclear transfer (SCNT) or in vitro-fertilization (IVF) were cultured in a modified synthetic oviductal fluid (mSOF), which contained either glucose (1.5 or 5.6 mM), fructose (1.5 or 5.6 mM), or galactose (1.5 or 5.6 mM). Compared to 1.5 mM glucose, use of 1.5 mM fructose significantly enhanced blastocyst formation in both SCNT (23 vs. 33%) and IVF embryos (26 vs. 34%), while 5.6 mM fructose did not improve blastocyst formation. Using 1.5 mM galactose did not improve blastocyst formation in SCNT embryos (22 vs. 23%), whereas it significantly inhibited blastocyst formation in IVF embryos (26 vs. 0%). In both SCNT and IVF embryos, 5.6 mM glucose or galactose significantly inhibited embryo development. In a second experiment, in glucose-free mSOF, fructose at concentrations of 0.75, 1.5, 3.0, or 5.6 mM was able to support to morula (32-42 vs. 12%) and blastocyst formation (30-38 vs. 12%) compared to 0 mM fructose. In Experiment 3, addition of fructose (1.5, 3.0, or 5.6 mM) to mSOF containing 1.5 mM glucose did not further promote blastocyst formation in SCNT embryos compared with replacement with 1.5 mM fructose only. Replacement of glucose with 1.5 mM fructose significantly increased total blastomeres (143 vs. 123 cells) and trophectodermal (TE) cells (116 vs. 94 cells) and decreased inner cell mass (ICM) to TE cell ratio (0.24 vs. 0.31) in blastocysts, compared to 1.5 mM glucose. The combined addition of 1.5 mM fructose and glucose significantly increased ICM cell number (36.7 cells) and ICM/TE ratio (0.46). In conclusion, fructose might be a more efficient energy substrate than glucose for producing large number of transferable blastocysts derived from SCNT.     

Effects of antioxidants on the development of bovine IVM/IVF embryos in various concentrations of glucose

This study was undertaken to determine the effects of glucose, antioxidants and different oxygen tensions on the development of bovine embryos cultured in modified synthetic oviduct fluid (m-SOF) medium. In vitro matured (IVM) and fertilized (IVF) oocytes were incubated for 48 h. Embryos reaching at least the 4-cell stage were selected for further culture under various conditions for 6 d. Supplementing the m-SOF media with 4.5 mM glucose resulted in a significantly lower (P < 0.01) embryo developmental rate (21%; Day 8) than was obtained with 1.5 mM glucose (58%; Day 8) or no glucose (53%; Day 8). Antioxidants such as SOD, catalase and mannitol had no positive effect on embryo development in m-SOF medium supplemented with 1.5 mM glucose. However, in m-SOF medium supplemented with 4.5 mM glucose, SOD and mannitol significantly (P < 0.05) improved embryo development: SOD increased the developmental rate from 19 to 35% (Day 8), while mannitol increased it from 13 to 30% (Day 8). Low oxygen concentration improved embryo development significantly (P < 0.05) in m-SOF medium supplemented with 4.5 mM glucose (low O2: 31% vs high O2: 14%; Day 8) but not 0 mM glucose (low O2: 58% vs high O2: 55%; Day 8). Our data suggest that low concentration of glucose during culture of bovine embryos is beneficial, and that generation of free oxygen radicals is partly caused by a high concentration of glucose in the medium.     

Effect of the addition of glutathione and glucose to the culture medium on embryo development of IVM-IVF prepubertal goat oocytes

Our previous studies have shown that larger and more competent oocytes can be selected using the brilliant cresyl blue (BCB) test. The objective of this study was to assess, in BCB-selected oocytes, the effect on the embryo development of prepubertal goat oocytes of the addition to in vitro culture (IVC) medium of either glutathione (GSH) alone or GSH in combination with glucose. Oocytes were exposed to 26 mM BCB and were classified as: oocytes with a blue cytoplasm or grown oocytes (BCB+) and oocyteswithout blue cytoplasm or growing oocytes (BCB-). Oocytes were matured in TCM-199 with 100 microM cysteamine. Presumptive zygotes were cultured in synthetic oviductal fluid (SOF) in the presence or absence of 1 mM glutathione (experiment 1) for 7 days (8 days post-insemination, p.i.). In experiment 2 we tested the addition to culture of 2.78 mM glucose at day 5 p.i. BCB+ oocytes showed higher percentages of nuclear maturation than the BCB- and control groups (82.6%, 55.7% and 74.7%, respectively). The percentage of polyspermic oocytes was higher in BCB- than BCB+ oocytes. Supplementation of SOF medium with 1 mM GSH did not affect embryo development but the percentage of total embryos developed after culture was higher in BCB+ oocytes than in BCB- oocytes independently of the GSH supplementation. Glucose, alone or with GSH, added at 5 days p.i. did not affect embryo development. In conclusion, prepubertal goat oocytes were unable to develop beyond the 8-cell stage embryo under the culture conditions in this study.     

Energy substrate requirement for in vitro maturation of oocytes from unstimulated adult rhesus monkeys

The energy substrates lactate, pyruvate, and glucose were evaluated for supporting in vitro cytoplasmic maturation of rhesus monkey oocytes. A total of 321 cumulus-oocyte complexes (COCs) aspirated from > or = 1000 microm diameter follicles of unstimulated adult monkeys were matured in one of six media with various individual or combinations of energy substrates: (1) mCMRL-1066 (control); (2) HECM-10 (containing 4.5 mM lactate); (3) HECM-10+0.2 mM pyruvate; (4) HECM-10 + 5.0 mM glucose; (5) HECM-10+ 0.2 mM pyruvate + 5.0 mM glucose; and (6) HECM-10 minus lactate + 5.0 mM glucose. All media contained gonadotropins, oestradiol, and progesterone. Following maturation, all mature oocytes were subjected to the same in vitro fertilization and embryo culture procedures. Oocytes matured in control medium or in treatment groups 4 and 6 had the best morulae+ blastocysts developmental responses (35, 36, and 32%, respectively, P < 0.05). HECM-10 + 0.2 mM pyruvate + 5.0 mM glucose for COC maturation supported intermediate embryonic development (16% morulae + blastocysts). The lowest (P < 0.05) morula + blastocyst developmental responses were obtained after maturation of COCs in HECM-t10 and HECM-10 + 0.2 mM pyruvate (4 and 6%, respectively). The COCs matured in glucose-containing medium showed greater levels of cumulus expansion than those in glucose-free medium. These results indicate that (a) glucose is both necessary and sufficient as the energy substrate for supporting optimal cytoplasmic maturation in vitro of oocytes from unstimulated rhesus monkeys; (b) pyruvate suppresses the stimulatory effect of glucose on oocyte maturation; (c) glucose is involved in cumulus expansion; (d) cumulus expansion is not a reliable indicator of primate oocyte competence.