Developmental competence of prepubertal goat oocytes selected with brilliant cresyl blue and matured with cysteamine supplementation

The aim of this study was to assess the effect of oocyte selection using the brilliant cresyl blue (BCB) test plus the addition of cysteamine to the in vitro maturation (IVM) medium to improve the in vitro embryo development of prepubertal goat oocytes. The oocytes were exposed to 26 microM BCB and classified according to their cytoplasm coloration: BCB+ (oocytes with blue cytoplasm) and BCB- (unstained oocytes). The oocytes were matured in a conventional IVM medium supplemented with cysteamine 100 microM. The control group consisted of oocytes not exposed to BCB and matured without cysteamine. The IVM-oocytes were inseminated and cultured in synthetic oviductal fluid (SOF) for 7 days. The normal fertilisation rate (oocytes showing 2 pronuclei and 1 sperm tail) of BCB+ oocytes (40%) was higher than those of BCB- (21%) and control oocytes (22%). The percentage of morulae plus blastocysts was higher (P < 0.05) in the BCB+ group than in the BCB- group (23.8 vs. 5.1%, respectively). In conclusion, the integration of the BCB test and the addition of cysteamine in the protocol of in vitro embryo production from prepubertal goat oocytes has improved the developmental rates of embryo development.     

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.     

Selection of prepubertal goat oocytes using the brilliant cresyl blue test

Brilliant cresyl blue stain allows us to determine the activity of glucose-6-phosphate dehydrogenase (G6PD), an enzyme synthesized in growing oocytes but with decreased activity in oocytes that have finished their growth phase. The objective of this study was to evaluate the utility of the brilliant cresyl blue (BCB) test as an indirect measure of oocyte growth, in order to select competent prepubertal goat oocytes for in vitro embryo production. Oocytes were exposed to BCB diluted in PBS and were classified according to their cytoplasm coloration: oocytes with a blue cytoplasm or grown oocytes (BCB+) and oocytes without a blue cytoplasm or growing oocytes (BCB-). After exposure to different BCB concentrations, we evaluated in vitro maturation (IVM), in vitro fertilization (IVF) and embryo development parameters. We defined matured oocytes as those oocytes that reached the metaphase II (MII) stage after being cultured for 27 h. Oocytes showing two pronuclei at 20 h post-insemination were classified as normally fertilized oocytes. We assessed embryo development 8 days post-insemination and recorded the percentage of total embryos, morale and blastocysts. The mean percentage of BCB+ oocytes was 29.4%. Mean diameter of BCB+ oocytes (136.6+/-6.3 microm) was higher (P < 0.001) than that of BCB- oocytes (125.5+/-10.2 microm). The percentage of BCB+ oocytes reaching the MII stage (81.4%) was higher (P < 0.05) than that of BCB- (52.5%) and control oocytes (72.4%). Normal fertilization rate of BCB+ oocytes was also higher (23.5%) than that of BCB- (8.2%; P < 0.0001) and control oocytes (11.9%; P < 0.05). The percentages of total embryos undergoing development to >8-cell and the morula plus blastocyst stages were higher (P < 0.05) in the group of BCB+ (41.3 and 12.0%, respectively) than in BCB- oocytes (21.3 and 3.6%, respectively). In conclusion, the BCB test is a useful way to select more competent prepubertal goat oocytes for in vitro embryo production.     

Effects on in vitro embryo development and intracellular glutathione content of the presence of thiol compounds during maturation of prepubertal goat oocytes

The low number of embryos produced from in vitro matured, fertilized, and cultured (IVM-IVF-IVC) oocytes of prepubertal goat is mainly due to a low incidence of sperm head decondensation at fertilization (Martino et al., 1995: Theriogenology 43:473-485; Mogas et al., 1997: Theriogenology 48:815-829). Thiol compounds stimulate glutathione (GSH) synthesis and improve the rates of male pronucleus (MPN) formation and embryo development. The present study was carried out to determine whether supplementation of the IVM medium with 100 microM of cysteamine, 100 microM of beta-mercaptoethanol, 0.57 mM of cysteine, and 0.57 mM cystine might improve the embryo development and intracellular GSH level of prepubertal goat oocytes. After 27 hr post IVM, a sample of oocytes was frozen and the intracytoplasmic GSH content was evaluated by spectrophotometry. IVM-oocytes were inseminated with fresh semen and cultured in SOF medium. Only the addition of cysteamine to IVM media significantly improved the percentage of the morula plus blastocyst yield compared to the control group (oocytes matured in absence of thiol compounds) (22.2 vs. 6.4%, respectively; P < 0.05). The percentage of expanded blastocysts in cysteamine and control groups was 13.0 and 2.6%, respectively, and the mean cell number per blastocyst was 86.8 and 60.5, respectively. None of the other thiol compounds studied significantly improved the percentage of embryos obtained. It has been demonstrated that prepubertal goat oocytes synthesize GSH during IVM and that thiol compounds increase this GSH synthesis. In conclusion, only the addition of 100 microM of cysteamine to the maturation medium improves embryo development from prepubertal goat oocytes although all the thiol compounds used in this study increased intracellular GSH content.     

Selection of developmentally competent buffalo oocytes by brilliant cresyl blue staining before IVM

The brilliant cresyl blue (BCB) test determines the activity of glucose-6-phosphate dehydrogenase (G6PDH); the activity of this enzyme is greatest in growing oocytes, but it declines as oocytes mature. The objective was to develop and evaluate this test for assessing development of buffalo oocytes (to select developmentally competent oocytes for increased in vitro embryo production). Oocytes were exposed to BCB stain diluted in mDPBS (DPBS with 0.4% BSA) for 90 min at 38.5 degrees C in a humidified air atmosphere; those with or without blue coloration of the cytoplasm were designated as BCB+ and BCB-, respectively. In Experiment 1, oocytes were exposed to 13, 26, or 39 microM BCB. There were fewer BCB+ oocytes after exposure to 13 microM BCB (10%) than after exposure to 26 or 39 microM BCB (57.2 and 61.8%; P<0.05), but there was no significant difference among treatments for blastocyst production rate. In Experiment 2, the diameter of BCB+ oocytes (144.4+/-4.2 microm; mean+/-S.E.M.) was higher (P<0.05) than that of BCB- oocytes (136.8+/-4.6 microm). In Experiment 3, oocytes were allocated into three groups: control (immediately cultured); holding-control (kept in mDPBS for 90 min before cultured); and treatment-incubation (incubated with 26 microM BCB). After IVM, oocytes were fertilized in vitro and cultured on an oviductal monolayer. The nuclear maturation rate was higher (P<0.05) in BCB+ (86.2%), control (83.4%) and holding-control (82.6%) oocytes than BCB- (59.2%) oocytes. The BCB+ oocytes yielded more blastocysts than control or holding-control oocytes (33.4, 20.2, and 21.0%, P<0.05); blastocyst development was lowest in BCB- oocytes (5.2%). In conclusion, staining of buffalo oocytes with BCB before IVM may be used to select developmentally competent oocytes for increased in vitro embryo production.     

Effect of glutathione synthesis stimulation during in vitro maturation of ovine oocytes on embryo development and intracellular peroxide content

Cysteamine and beta-mercaptoethanol supplementation of in vitro maturation (IVM) medium has been found to increase intracellular glutathione (GSH) content in oocytes and to improve embryo development and quality in several species. The objective of this experiment was to study the effect of cysteamine and beta-mercaptoethanol added during IVM of sheep oocytes on GSH synthesis and embryo development. Furthermore, we examined if cysteamine addition (hence GSH production) had an effect on the reduction of the intracellular peroxide content. We matured oocytes obtained from ovaries collected at a slaughterhouse in vitro in the presence of 0, 50, 100, and 200 microM cysteamine (Experiment 1) or with 0, 50, 100, and 200 microM beta-mercaptoethanol (Experiment 2). Following fertilization and embryo development, there was a increasing level of morula and blastocyst development in the presence of cysteamine, reaching significance in the presence of 200 microM (P < 0.05). However, beta-mercaptoethanol did not influence on the rate of embryo development. GSH levels were measured in oocytes matured in the presence or absence of 200 microM cysteamine (Experiment 3) or 50 microM beta-mercaptoethanol (Experiment 4), with or without buthionine sulfoximide (BSO), an inhibitor of GSH synthesis. Results demonstrated that for both cysteamine and beta-mercaptoethanol, intracellular GSH levels increased against control values (P < 0.01), which was abolished in the presence of BSO. Finally, we reduced intracellular peroxide levels, as measured by the relative fluorescence of the intracellular peroxide probe, carboxy-H2DCFDA, in the presence of either 200 microM cysteamine or 50 microM beta-mercaptoethanol (Experiment 5). These results demonstrate that cysteamine, but not beta-mercaptoethanol, when present during IVM, stimulates sheep embryo development; both cysteamine and beta-mercaptoethanol stimulate GSH synthesis; the increase in intracellular GSH is associated with a decrease in peroxide levels within oocytes.     

Cysteamine supplementation during in vitro maturation and embryo culture: a useful tool for increasing the efficiency of bovine in vitro embryo production

Cysteamine when added during in vitro maturation (IVM) or in vitro embryo culture (IVC) stimulates glutathione (GSH) synthesis and improves embryo developmental rates. This suggests that GSH synthesis is decreased in the in vitro produced embryo. The present study was carried out to evaluate if addition of cysteamine to culture medium at the same time, during IVM and IVC of bovine oocytes, may promote an overall improvement on the developmental rate and embryo quality. Oocytes were matured in TCM 199 supplemented with 10% (v/v) fetal calf serum, hormones, and 0 or 100 microM of cysteamine for 24 hr. After IVM, the oocytes were fertilized (day 0). Day 2 embryos (2-8 cell) were washed and transferred to fresh IVC medium supplemented with 0, 25, 50, or 100 microM of cysteamine and cultured for 48 hr. After this, embryos were cultured in IVC medium without cysteamine until day 8 of IVC. In the present study, we confirmed our previous results by demonstrating that the percentage of embryos that developed to the blastocyst stage was significantly higher (P < 0.05) when 100 microM of cysteamine was added during IVM, and this was further improved when 100 and 50 microM of cysteamine where present during IVM and IVC, respectively (P < 0.05). After cryopreservation, no differences were observed on embryo development, but a significant increase on embryo hatching was found between unsupplemented and supplemented oocytes with 100 and 50 microM of cysteamine during IVM and IVC, respectively (P < 0.05). We can conclude that GSH synthesis stimulation during bovine IVM with cysteamine, concomitant with GSH stimulation during IVC, will be a useful and simple tool for increasing the efficiency of in vitro bovine embryo production.     

Effects of oocyte quality, semen donor and embryo co-culture system on the efficiency of blastocyst production in goats

The aim of the study was to determine whether the selection of immature oocytes by a combination of cumulus-oocyte-complexes (COCs) morphology and staining with brilliant cresyl blue (BCB) would be helpful in selecting developmentally competent oocytes, and thereby increase the efficiency of blastocyst production from ovarian oocytes of FSH-primed, adult goats. In a second experiment the interaction between oocyte quality and semen donor was assessed. In a third experiment the usefulness of Vero cells for co-culture with goat embryos was investigated. In the pool of morphologically normal COCs recovered from ovaries following slicing (21.9+/-11.0), the mean rate of COCs classified as BCB+ was 85.6%, and the BCB- was approximately 11%. Oocytes classified as grade 1 and BCB+ exhibited the highest developmental competence (P<0.001) after in vitro maturation and fertilization compared with oocytes of grade 1 BCB- and grade 2 BCB+ or BCB-. There were no significant differences in developmental competence in grade 2 oocytes, regardless of BCB coloration. No significant differences in embryo cleavage and blastocyst formation rates among three bucks were observed when morphologically normal, BCB+ oocytes were used. For all tested bucks, differences in embryo production efficiency were related only to the oocyte quality. Similar blastocyst rates were developed from embryos co-cultured with goat oviduct epithelial cells (34.3%) and with Vero cells (33.3%). These results show that the most important criterion for selection of COCs before maturation is the visual assessment of morphological features. Staining with BCB of COCs recovered from adult goats does not enhance efficiency of selection of developmentally competent oocytes for IVF.     

Capacity of adult and prepubertal mouse oocytes to undergo embryo development in the presence of cysteamine

The present study was carried out to study de novo glutathione (GSH) synthesis and to evaluate the effect of stimulating GSH synthesis during in vitro maturation (IVM) of adult and prepubertal mouse oocytes on the embryo developmental rate. Adult (8 weeks old) and prepubertal mice (24-26 days old) were primed with 5 IU of PMSG and oocytes were retrieved from the ovary 48 hr later for IVM. After IVM (18 hr) Cumulus oocyte complexes (COC) were in vitro fertilized (IVF) and in vitro culture (IVC) in order to observe embryo development. The IVM medium was supplemented with: 0, 25, 50, 100, or 200 microM of cysteamine. To study the novo GSH synthesis, 5 mM BSO was added during IVM of adult or prepubertal oocyte. Developmental rates up to blastocyst were recorded for each group. Experiments also included a group of ovulated oocytes (in vivo matured) after priming with PMSG and HCG. After IVM of adult mice oocytes, an improvement was observed on embryo development in all the supplemented groups when compared with the untreated group (P < 0.05). No differences were observed in blastocyst rate among IVM oocytes with cysteamine and ovulated oocytes. Prepubertal IVM mouse oocytes had a lower cleavage rate compared with ovulated oocytes (P < 0.05). Cysteamine failed to improve prepubertal oocytes developmental rates (P > 0,05). 2-cell embryos, coming from IVM prepubertal oocytes and ovulated oocytes had the same preimplantation developmental rate up to the blastocyst stage. In prepubertal, and adult oocytes an inhibition of embryo development was observed when buthionine sulfoximide (BSO), a specific inhibitor of the gamma-glutamylcysteine synthetase, was added during oocyte maturation (P < 0.01). In conclusion, an improvement in mouse embryo development was observed when cysteamine was added to the IVM medium of adult mice oocytes. In prepubertal oocytes cysteamine addition during oocyte maturation failed to improve embryo developmental rates. The presence of BSO lowered or completely blocked blastocyst development. This proves that, de novo GSH synthesis during oocyte maturation of adult and prepubertal oocytes undoubtedly plays an important role in embryo development. The improvement on oocyte competence observed in adult mice oocytes is probably related to intracellular GSH synthesis stimulated by cysteamine. Nevertheless the reason why cysteamine failed to improve prepubertal oocytes competence remains as an open question.     

Enrichment of in vitro maturation medium for buffalo (Bubalus bubalis) oocytes with thiol compounds: effects of cystine on glutathione synthesis and embryo development

The purpose of this study was to evaluate whether enriching the oocyte in vitro maturation medium with cystine, in the presence of cysteamine, would improve the in vitro embryo production efficiency in buffalo by further increasing the GSH reservoir created by the oocyte during maturation. Cumulus-oocytes complexes were matured in vitro in TCM 199 + 10% FCS, 0.5 microg/ml FSH, 5 microg/ml LH and 1 microg/ml 17beta-estradiol in the absence or presence of cysteamine (50 microM), with or without 0.3mM cystine. In Experiment 1, glutathione content was measured by high-performance liquid chromatography and fluorimetric analysis in representative samples of oocytes matured in the four different experimental conditions. In Experiment 2, oocytes were fixed and stained to assess nuclear maturation and normal pronuclear development following IVM and IVF respectively. In Experiment 3, mature oocytes were in vitro fertilized and cultured to assess development to blastocysts. In all supplemented groups the intracytoplasmic GSH concentration was significantly higher than the control, with the highest GSH levels in oocytes matured in the presence of both thiol compounds (3.6, 4.7, 5.4 and 6.9 picomol/oocyte in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Cystine supplementation of IVM medium, both in the presence or absence of cysteamine, significantly increased the proportion of oocytes showing two normal synchronous pronuclei following fertilization. In all supplemented groups, cleavage rate was significantly improved compared to the control (55, 66.1, 73.5 and 78.4% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Similarly, blastocyst yield was also increased in the three enriched groups compared to the control (17.1, 23.8, 29.3, 30.9% in the control, cysteamine, cystine and cystine+cysteamine groups, respectively; P < 0.05). Overall, the addition of cystine to a cysteamine-enriched medium resulted in a significant increase of cleavage rate and transferable embryo yield compared to the medium supplemented with only cysteamine.     

Bovine blastocyst development rate in vitro is influenced by selection of oocytes by brillant cresyl blue staining before IVM as indicator for glucose-6-phosphate dehydrogenase activity

The aim of this present study was to increase the efficiency of blastocyst production from cows after in vitro maturation/fertilization (IVM/IVF) by oocyte selection before maturation. Oocytes were selected on the basis of brillant cresyl blue (BCB) staining, used to indicate glucose-6-phosphate dehydrogenase (G6PDH) activity. To re-valuate the hypothesis that growing oocytes are expected to have a high level of active G6PDH, while mature oocytes have low G6PDH activity, cumulus oocyte complexes (COCs) were recovered from slaughterhouse ovaries by slicing the surface of the ovary. Only oocytes with a compact cumulus investment were used. Oocytes were placed into three groups: (1) control--placed immediately into culture; (2) holding control--COCs kept in PBS containing 0.4% BSA for 90 min before placement into culture; and (3) treatment--incubation with BCB for 90 min before culture. Treated oocytes were then divided into BCB- (colorless cytoplasm, increased G6PDH) and BCB+ (colored cytoplasm, low G6PDH) on their ability to metabolize the stain. Activity of G6PDH was determined via measurement of NADP reduction induced by G6P as substrate oxidized by G6PDH in the cytosol of control, BCB- and BCB+ groups; G6PDH activity was significant higher in BCB- COCs than in control and BCB+ COCs. After IVM, oocytes were fertilized in vitro. Embryos were cultured to day 8. The rate of maturation to metaphase II was significantly higher for control and BCB+ oocytes than for BCB- oocytes. The BCB+ oocytes yielded a significantly higher proportion of blastocysts (34.1%) than did control or holding control oocytes (18.3 and 19.2%); and both controls and BCB+ oocytes had significantly higher blastocyst development than did BCB- oocytes (3.9%). These results show that the staining of bovine cumulus oocyte complexes with BCB before in vitro maturation may be used to select developmentally competent oocytes for IVF. In addition, G6PDH activity may be useful as a marker for oocyte quality in future studies on factors affecting developmental competence.     

Developmental capacity of in vitro matured and fertilized oocytes from prepubertal and adult goats

The developmental competence of oocytes from prepubertal and adult goats was studied through in vitro maturation, fertilization and embryo culture up to the blastocyst stage. Oocytes were recovered from antral follicles of prepubertal and adult goat ovaries, with or without ovarian stimulation with exogenous FSH. The effect of different sources of granulosa cells during IVM on the developmental competence of prepubertal goat oocytes was also noted. Oocytes were matured for 27 h at 38.5 degrees C in 5% CO(2) in air in 50-microl microdrops in TCM199 supplemented with 20% estrus goat serum, FSH, LH and estradiol-17beta or in 2 ml of the same medium supplemented with granulosa cells. Matured oocytes were inseminated with freshly ejaculated spermatozoa following capacitation At 24 h post-insemination, the oocytes were transferred to a granulosa cell monolayer, and early embryo development was evaluated until Day 10. Results show that the developmental ability of embryos from prepubertal goats after IVM and IVF is similar to those from adult goats. Treatment of the prepubertal and adult goats with FSH did not improve the developmental capacity of the resulting embryos. On studying the addition of different sources of granulosa cells to a maturation system of 2 ml of medium, a significantly positive effect of the cells from primed females was observed on the percentage of maturation, on embryo cleavage and on the percentage of embryos that overcame the in vitro developmental block from 8 to 16 cells.     

Cysteamine, glutathione and ionomycin treatments improve in vitro fertilization of prepubertal goat oocytes

The aim of this study was to improve in vitro embryo development of prepubertal goat oocytes by studying the effect of adding cysteamine to in vitro maturation medium, glutathione (GSH) to in vitro fertilization medium and ionomycin to the sperm capacitation medium. In experiment 1, we analysed the effect of 1 mM GSH added to fertilization medium of oocytes matured with 400 microM cysteamine. The control group were oocytes without cysteamine and GSH. In experiment 2, oocytes matured and fertilized in the presence of 400 microM cysteamine and 1 mM GSH, respectively, were inseminated with spermatozoa treated with ionomycin or heparin. In experiment 1, the percentages of total and normal fertilized oocytes were significantly higher for oocytes supplemented with cysteamine and GSH (40.26% and 30.20%, respectively) than for oocytes from the control group (16.66%, and 10.61%, respectively). The percentage of total embryos obtained after 7 days of culture was significantly higher in the group supplemented with cysteamine and GSH (30.62%) than in the control group (8.09%). In experiment 2, percentages of total and normal fertilized oocytes were significantly higher for the group of spermatozoa capacitated with ionomycin (52.21% and 37.17%, respectively) than with heparin (38.62% and 28.35%, respectively). After 7 days of culture, total embryo rate was significantly higher in the group of sperm capacitated with ionomycin (44.91%) than with heparin (38.69%). However, the percentage of embryos developed to the blastocyst stage was not affected by any of the treatments studied.     

Selection of oocytes for in vitro maturation by brilliant cresyl blue staining： a study using the mouse model

Selecting oocytes that are most likely to develop is crucial for in vitro fertilization and animal cloning. Brilliant cresyl blue （BCB） staining has been used for oocyte selection in large animals, but its wider utility needs further evaluation. Mouse oocytes were divided into those stained （BCB＋） and those unstained （BCB-） according to their ooplasm BCB coloration. Chromatin configurations, cumulus cell apoptosis, cytoplasmic maturity and developmental competence were compared between the BCB＋ and BCB- oocytes. The effects of oocyte diameter, sexual maturity and gonadotropin stimulation on the competence of BCB＋ oocytes were also analyzed. In the large- and medium-size groups, BCB＋ oocytes were larger and showed more surrounded nucleoli （SN） chromatin configurations and higher frequencies of early atresia, and they also gained better cytoplasmic maturity （determined as the intracellular GSH level and pattern of mitochondrial distribution） and higher developmental potential after in vitro maturation （IVM） than the BCB-oocytes. Adult mice produced more BCB＋ oocytes with higher competence than the prepubertal mice when not primed with PMSG. PMSG priming increased both proportion and developmental potency of BCB＋ oocytes. The BCB＋ oocytes in the large-size group showed more SN chromatin configurations, better cytoplasmic maturity and higher developmental potential than their counterparts in the medium-size group. It is concluded that BCB staining can be used as an efficient method for oocyte selection, but that the competence of the BCB＋ oocytes may vary with oocyte diameter, animal sexual maturity and gonadotropin stimulation. Taken together, the series of criteria described here would allow for better choices in selecting oocytes for better development.     

Effect of sperm capacitation and fertilization media on IVF and early embryo development of prepubertal goat oocytes

Experiments were carried out to develop an improved IVF system for prepubertal goat oocytes matured in vitro. Cumulus oocyte complexes (COC) were obtained by slicing ovaries from slaughtered prepubertal goats. Oocytes were matured in TCM199 supplemented with 20% estrous goat serum (EGS) + 10 micrograms/mL FSH + 10 micrograms/mL LH + 1 microgram/mL estradiol 17 beta for 27 h at 38.5 degrees C in 5% CO2 in air. In Experiments 1 and 2, freshly ejaculated spermatozoa were capacitated in 1 of 3 media: TALP/H, modified Defined Medium (mDM) and mH-M199 with 50 micrograms/mL heparin for 45 min. Matured oocytes were fertilized in TALP, mDM or mH-M199 in Experiment 1 and in TALP in Experiment 2. In Experiment 3, three media were used for sperm capacitation and fertilization: Treatment A (control group): spermatozoa were capacitated in mDM with 50 micrograms/mL heparin for 45 min and fertilized in TALP medium with 1 microgram/mL hypotaurine; Treatment B: spermatozoa were capacitated in mDM with 50 micrograms/mL heparin + 388 micrograms/mL caffeine for 30 min and fertilized in TALP medium without hypotaurine; Treatment C: spermatozoa were capacitated in mDM with 50 micrograms/mL heparin for 45 min and fertilized in TALP medium with PHE (20 microM penicillamine, 10 microM hypotaurine and 2 microM epinephrine). At 24 h post insemination, the ova were transferred to a granulosa cell monolayer, and early embryo development was evaluated until Day 8. In experiment 2, the results show, that mDM plus heparin for sperm capacitation and TALP medium with hypotaurine for oocyte fertilization provided the highest proportion of penetrated oocytes, both total number (79.6%) and normal fertilization (55.1%), whereas the use of caffeine (44.6 and 31.2%, total and normal fertilization rate, respectively) and PHE (31.8 and 20.6%, total and normal fertilization rate, respectively) as motility enhancers did not improve the results obtained in the control group (48.7% and 37.2%, total and normal fertilization rate, respectively). These were no differences for the results of morulae and blastocysts.     

In vitro maturation and fertilization of prepubertal goat oocytes

The aim of this work was to study the IVM-IVF of prepubertal goat oocytes collected from a slaughterhouse as an alternative source of oocytes to those of FSH-primed adult goats. In Experiment 1, IVM of prepubertal goat oocytes in co-culture with granulosa cells were compared with IVM in 50 microl microdrops of medium. There was no significant difference in the percentage of maturation (72.0 vs 76.9%) between the 2 groups. In Experiment 2, a low percentage of normal fertilization (24.4%) was observed for prepubertal goat oocytes matured with granulosa cells from prepubertal goats. This result was significantly lower than that obtained for ovulated (62.2%) or in vitro-matured (48.7%) oocytes from adult goats. There were no significant differences with respect to the oocytes from adult goats matured in vitro when prepubertal goat oocytes were cultured with adult goat granulosa cells (33.3%) or in microdrops (29.7%). No differences were observed among the treatments in the percentage of oocytes showing evidence of fertilization (normal fertilization + abnormal fertilization + polyspermy). In Experiment 3, it was shown that there were no differences in the percentage of normally fertilized oocytes after in vitro maturation in microdrops containing oocytes with 1 to 2 and 3 or more complete layers of cumulus cells (32.1 and 33.3% respectively). In conclusion, the ovaries of prepubertal slaughterhouse goats were found to be an economical alternative for an abundant source of oocytes for IVM-IVF research. In vitro maturation of oocytes in microdrops yielded maturation and fertilization rates comparable to those obtained with oocytes from FSH-primed adult goats. Moreover, similar maturation and fertilization rates were obtained using oocytes with 1 to 2 layers or 3 or more layers of cumulus cells.     

Effects of steroidal glycoalkaloids from potatoes (Solanum tuberosum) on in vitro bovine embryo development

alpha-Solanine and alpha-chaconine are two naturally occurring steroidal glycoalkaloids in potatoes (Solanum tuberosum), and solanidine-N-oxide is a corresponding steroidal aglycone. The objective of this research was to screen potential cyto-toxicity of these potato glycoalkaloids using bovine oocyte maturation, in vitro fertilization techniques and subsequent embryonic development as the in vitro model. A randomized complete block design with four in vitro oocyte maturation (IVM) treatments (Experiment 1) and four in vitro embryo culture (IVC) treatments (Experiment 2) was used. In Experiment 1, bovine oocytes (n=2506) were matured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVM medium only. The in vitro matured oocytes were then subject to routine IVF and IVC procedures. Results indicated that exposure of bovine oocytes to the steroidal glycoalkaloids during in vitro maturation inhibited subsequent pre-implantation embryo development. Potency of the embryo-toxicity varied between these steroidal glycoalkaloids. In Experiment 2, IVM/IVF derived bovine embryos (n=2370) were cultured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVC medium only. The results showed that the pre-implantation embryo development is inhibited by exposure to these glycoalkaloids. This effect is significant during the later pre-implantation embryo development period as indicated by fewer numbers of expanded and hatched blastocysts produced in the media containing these alkaloids. Therefore, we conclude that in vitro exposure of oocytes and fertilized ova to the steroidal glycoalkaloids from potatoes inhibits pre-implantation embryo development. Furthermore, we suggest that ingestion of Solanum species containing toxic amounts of glycoalkaloids may have negative effects on pre-implantation embryonic survival.     

In vitro developmental competence of prepubertal goat oocytes cultured with recombinant activin-A

The present study was designed to evaluate the effect of activin-A during the in vitro oocyte maturation (IVM) and in vitro embryo culture (IVC) on nuclear maturation, blastocyst yield and blastocyst quality of prepubertal goat oocytes. In Experiment 1, three groups of oocytes were used during the IVM of prepubertal goat oocytes to determine the optimal concentration of recombinant human activin-A added to the maturation medium. Cumulus–oocyte complexes were matured in an IVM medium containing 0, 10 and 100 ng/ml (groups A0, A10 and A100), fertilized and in vitro cultured using standard procedures. In Experiment 2, the addition of 10 ng/ml activin-A at IVM (A10A0), IVC (A0A10) or IVM+IVC (A10A10) was studied and compared with the control group (A0A0). Results of the first experiment demonstrated that the addition of activin-A yielded similar percentages of maturation (⩽71.0%) and blastocyst formation rates (⩽24.9%) than the control group (A0). Experiment 2 showed that exposure of prepubertal goat oocytes to an IVC medium containing 10 ng/ml activin-A (A0A10) significantly increased the rates of development to the blastocyst stage, as compared with the control group (A0A0) (19.5±2.21% v. 13.1±2.37%, respectively; P<0.05). With regard to the blastocyst quality, total number of cells, inner cell mass (ICM) and trophectoderm of prepubertal goat embryos produced in the presence of activin-A did not differ significantly among experimental groups. In summary, these results indicate that supplementation of the IVC medium with activin-A enhances embryo development of prepubertal goat oocytes.     

Effect of cysteamine during in vitro maturation on buffalo embryo development

The purpose of this study was to evaluate whether the addition of cysteamine during in vitro maturation (IVM) of buffalo oocytes enhances embryo development. Cumulus-oocyte complexes (COC) from slaughterhouse ovaries were matured in vitro in TCM 199 supplemented with 10% fetal calf serum (FCS), 0.5 microg mL(-1) FSH, 5 microg mL(-1) LH, 1 microg mL(-1) 17 beta estradiol and 0 (control), 50, 100 or 200 micromol L(-1) of cysteamine for 24 hours. The matured oocytes then were fertilized and cultured for 7 days. No beneficial effect on maturation and cleavage rate was related to the addition of cysteamine. However, the percentage of embryos that developed to compact morula and blastocyst stage was significantly higher (P < or = 0.01) for oocytes matured in medium containing 50 micromol L(-1) of cysteamine than it was for oocytes matured with 0, 100 and 200 micromol L(-1) cysteamine (22.6% vs 14.9%, 15.7% and 13% respectively); moreover, the addition of 50 micromol L(-1) of cysteamine during IVM significantly (P < or = 0.01) increased the proportion of transferable quality (Grades 1 and 2) embryos (19.3% vs 11.3%, 11.6% and 11.2% respectively). The present study showed that adding a thiol compound (such as cysteamine) to the IVM medium improves buffalo in vitro embryo production (IVEP) efficiency, which so far has been unsatisfactory.     

Oocytes selected using BCB staining enhance nuclear reprogramming and the in vivo development of SCNT embryos in cattle

The selection of good quality oocytes is crucial for in vitro fertilization and somatic cloning. Brilliant cresyl blue (BCB) staining has been used for selection of oocytes from several mammalian species. However, the effects of differential oocyte selection by BCB staining on nuclear reprogramming and in vivo development of SCNT embryos are not well understood. Immature compact cumulus-oocyte complexes (COCs) were divided into control (not exposed to BCB), BCB+ (blue cytoplasm) and BCB- (colorless cytoplasm) groups. We found that BCB+ oocytes yielded a significantly higher somatic cell nuclear transfer (SCNT) blastocyst rate and full term development rate of bovine SCNT embryos than the BCB- and control oocytes. BCB+ embryos (embryos developed from BCB+ oocytes) showed increased acetylation levels of histone H3 at K9 and K18 (AcH3K9, AcH3K18), and methylation levels of histone H3 at K4 (H3K4me2) than BCB- embryos (embryos developed from BCB- oocytes) at the two-cell stage. Furthermore, BCB+ embryos generated more total cells, trophectoderm (TE) cells, and inner cell mass (ICM) cells, and fewer apoptotic cells than BCB- embryos. The expression of SOX2, CDX2, and anti-apoptotic microRNA-21 were up-regulated in the BCB+ blastocysts compared with BCB- blastocysts, whereas the expression of pro-apoptotic gene Bax was down-regulated in BCB+ blastocysts. These results strongly suggest that BCB+ oocytes have a higher nuclear reprogramming capacity, and that BCB staining can be used to select developmentally competent oocytes for nuclear transfer.