Brilliant Cresyl Blue Staining Research Articles

CX43 and oxidative stress are the targets of BCB staining to predict the developmental potential of buffalo oocytes.

This study used the brilliant cresyl blue (BCB) staining method to group buffalo oocytes (BCB+ and BCB-) and perform invitro maturation, invitro fertilization and embryo culture. At the same time, molecular biology techniques were used to detect gap junction protein expression and oxidative stress-related indicators to explore the molecular mechanism of BCB staining to predict oocyte developmental potential. The techniques of buffalo oocytes to analyse their developmental potential and used immunofluorescence staining to detect the expression level of CX43 protein, DCFH-DA probe staining to detect ROS levels and qPCR to detect the expression levels of the antioxidant-related genes SOD2 and GPX1. Our results showed that the invitro maturation rate, embryo cleavage rate and blastocyst rate of buffalo oocytes in the BCB+ group were significantly higher than those in the BCB- group and the control group (p < .05). The expression level of CX43 protein in the BCB+ group was higher than that in the BCB- group both before and after maturation (p < .05). The intensity of ROS in the BCB+ group was significantly lower than that in the BCB- group (p < .05), and the expression levels of the antioxidant-related genes SOD2 and GPX1 in the BCB+ group were significantly higher than those in the BCB- group (p < .05). Brilliant cresyl blue staining could effectively predict the developmental potential of buffalo oocytes. The results of BCB staining were positively correlated with the expression of gap junction protein and antioxidant-related genes and negatively correlated with the reactive oxygen species level, suggesting that the mechanism of BCB staining in predicting the developmental potential of buffalo oocytes might be closely related to antioxidant activity.

The impact of cumulus cell viability and pre-culture with the healthy cell mass on brilliant cresyl blue (BCB) staining assessment and meiotic competence of suboptimal porcine oocytes

Objectives of the present study were to investigate the characteristics including glucose-6-phosphate dehydrogenase activity, as determined by Brilliant Cresyl Blue (BCB) staining, of suboptimal porcine oocytes and to enhance the meiotic competence of those through pre-culture with cumulus cell masses (CCMs). Percentage of oocyte-cumulus complexes (OCCs) derived from small follicles (SF; <3 mm in diameter) containing the oocytes that were assessed as BCB-negative (BCB-) was significantly higher than those derived from medium follicles (MF; 3–6 mm in diameter). Degrees of dead cumulus cells were significantly higher in OCCs containing BCB- oocytes, regardless of the origin of OCCs (MF vs. SF), than those containing BCB-positive (BCB+) ones. Exposing OCCs containing BCB+ oocytes to the apoptosis inducer, carbonyl cyanide m-chlorophenylhydrazone, for 20 h significantly induced the transition to BCB- and meiotic progression of exposed OCCs were significantly reduced in both SF and MF derived ones. Transit of BCB- oocytes to BCB+ was induced when OCCs were pre-cultured with CCMs of MF derived OCCs containing BCB+ oocytes for 20 h before IVM. This pre-culture also significantly increased the meiotic competence of BCB- oocytes, particularly in SF derived ones. However, reactive oxygen species levels were significantly higher in BCB+ oocytes as compared with BCB- ones, regardless of pre-culture with CCMs, whereas no significant differences were found in the ATP contents among the treatment groups. In conclusion, the BCB result of oocytes could be regulated by the healthy status and content of surrounding cumulus cells and the meiotic competence of suboptimal BCB- porcine oocytes is improved by pre-culture with healthy CCMs.

Transient suppression of Wnt signaling in poor-quality buffalo oocytes improves their developmental competence.

One of the most evolutionary conserved communication systems, the Wnt signaling pathway is a major gene regulatory pathway that affects the developmental competence of oocytes and regulates most embryonic developmental processes. The present study was undertaken to modulate the canonical Wnt (Wingless/integration) signaling pathway in the poor-quality (colorless cytoplasm after Brilliant Cresyl Blue staining, BCB-) buffalo cumulus-oocyte complexes (COCs) to improve their in vitro maturation (IVM) and embryo production (IVEP) rates. The expression of key Wnt pathway genes was initially assessed in the good (blue cytoplasm after Brilliant Cresyl Blue staining, BCB+) and poor quality (BCB-) buffalo COCs to establish a differential activity of the Wnt pathway. The BCB- COCs were supplemented with the Wnt pathway inhibitor, Dickkopf-related protein 1 (DKK1) and later subjected to IVM and IVEP along with the BCB+ and BCB- controls. The cumulus expansion index (CEI), rate of nuclear maturation (mean percentage of oocytes in the MII stage) and embryo production, and the expression of developmentally important genes were evaluated to assess the effect of Wnt pathway inhibition on the development competence of these poor-quality oocytes. The Wnt pathway genes exhibited a significantly higher expression (p < 0.05) in the poor-quality BCB- oocytes compared to the good-quality BCB+ oocytes during the early maturation stages. The supplementation of BCB- COCs with 100 ng/mL DKK1 effectively inhibited the expression of the key mediators of the Wnt pathway (β-catenin and dishevelled homolog 1, DVL1). DKK1 supplemented BCB- COCs exhibited significantly improved cytoplasmic and nuclear maturation indices, development rates and significantly elevated expression (p < 0.05) of genes implicated in germinal vesicle breakdown (GVBD) and embryonic genome activation (EGA) vis-à-vis BCB- control COCs. These data indicate that inhibition of the Wnt pathway during the initial course of oocyte maturation can improve the development competence of poor-quality buffalo oocytes.

Modification of Natt-Herrick’s solution using Hibiscus sabdariffa red calyx powder for erythrocyte and leukocyte evaluation in Gallus gallus domesticus

Background: The Natt-Herrick method is commonly used in veterinary laboratories for quantitative white blood cell counting in birds, as standard automated cell counting is unreliable due to the nucleation of all avian blood cells; as a result, quantitative white blood cell counting in birds is still done by hand. Aim: The research work was designed to compare the staining capacity and suitability of Hibiscus sabdariffa red calyx powder modified Natt-Herrick’s stain and as a natural staining substitute to commonly used standard avian dilution fluids, Natt-Herricks stain and brilliant cresyl blue stain. Methodology: One hundred apparently healthy local chickens (Gallus gallus domesticus) within Maiduguri metropolitan city were used. The chickens were kept in Poultry House of the Department of Veterinary Pathology, University of Maiduguri and were fed ad libitum. The total erythrocyte and total leucocyte counts were determined and compared between modified Natt-Herrick and two other avian standard dilution fluids. Result: There were no statistically significant differences between Hibiscus sabdariffa red calyx powder modified Natt-Herricks’ and the two standard stains when total erythrocyte and leucocytes counts were conducted. The total erythrocyte and leucocyte counts were found to be higher in males than the females which still confirmed the clinical assessment and evaluation. Hibiscus extract revealed distinct and satisfactory staining of the erythrocytes and leucocytes comparable to the two standard stains. Conclusion: This study therefore, established that Hibiscus sabdariffa red calyx powder modified Natt-Herrick’s stain can be used as dilution fluid for RBC and WBC counts. The Hibiscus sabdariffa red calyx are domestically available, easy to prepare and showed excellent blood cells colouration. However, there is need to improve the staining duration of this local extract as an avian dilution fluid.

Effect of Brilliant cresyl blue (BCB) staining in oocyte’s maturation and parthenogenetic activation in alpaca (Vicugna pacos) oocytes

Oocyte quality is a limiting factor for in vitro embryo production (IVEP). This study aimed to evaluate the brilliant cresyl blue (BCB) staining as a marker for selecting competent oocytes in alpacas, in vitro maturation (IVM), and get blastocyst embryos after parthenogenetic activation (PA). Ovaries from a slaughterhouse were collected and transferred to the laboratory at either 10 °C or 23 °C during 21 h. Oocytes from large follicles (3–7 mm) were classified into BCB+ and BCB- after exposure to BCB (26 μM) for 90 min; then cultured in Medium 199. Oocyte quality was assessed by blastocyst rates after PA in Potassium supplemented simplex optimized medium (KSOM) with 5 % CO2 (condition 1) or in Global Total (GT) medium with 5 % CO2, 5 % O2, and 90 % N2 (condition 2). Mann-Whitney U non-parametric statistics test was used to compare the BCB status and the control group (BCB-free). BCB-free and BCB+ oocytes had higher maturated oocyte than BCB- oocytes after 48 h of IVM (34.7 %, 29.1 % vs.11.7 %); and after 36 h of IVM, BCB-free vs. BCB+ oocytes (55.1 % vs. 51.5 %). In addition, BCB-free group had a higher cleavage rate. Under condition 2, only blastocysts were obtained in both BCB-free (10.6 %) and BCB+ (6.3 %). Therefore, we conclude BCB is a tool for identifying the developmental competence after PA in oocytes BCB stained previously to IVM. Moreover, our findings demonstrate that BCB status does not affect the developmental competence of alpaca oocytes. However, transport temperature, IVM time, culture medium, and culture conditions significantly impact their developmental competence.

Brilliant Cresyl Blue Negative Oocytes Show a Reduced Competence for Embryo Development after In Vitro Fertilisation with Sperm Exposed to Oxidative Stress.

The extent of oxidative damage transferred by the damaged sperm to the progeny is likely to be limited by the oocyte's repair and antioxidative capacity. We aimed to assess the association between Brilliant Cresyl Blue (BCB) staining in oocytes and their competence for embryo development after in vitro fertilisation (IVF) with damaged sperm. For this purpose, bovine sperm were incubated without (non-oxidised sperm, NOX S) or with 100 µM H2O2 (oxidised sperm, OX S) and were used to fertilise in-vitro-matured bovine oocytes (BCB-pos./BCB-neg.). Unstained oocytes served as controls (US). Development was assessed at 30, 46, 60 h and on Days (D) 7 and 8 after IVF. Total cell number and apoptotic index were analysed in D7 blastocysts. BCB-neg. oocytes showed lower cleavage rates and blastocyst rates than unstained oocytes after IVF with NOX S (p < 0.05). They showed the highest reduction in D7 blastocyst rate upon fertilisation with OX S and showed a delayed embryo development at 46 and 60 h after IVF compared to embryos produced with NOX S (p < 0.05). Total cell number in blastocysts produced with BCB-neg. oocytes was lower (p < 0.05) in the embryos produced with OX S than in embryos after IVF with NOX S. In conclusion, BCB-neg. oocytes have a lower competence to support embryo development after in vitro fertilisation with oxidised sperm.

Selection of Rattus norvegicus cumulus-oocyte complex for vitrification by brilliant cresyl blue.

The influence of the method of evaluating developmentally competent oocytes on their viability after cryopreservation still needs to be better understood. The objective of this study was to determine the cleavage and embryo developmental rates after parthenogenetic activation of cumulus-oocyte complexes (COCs) selected by different concentrations of brilliant cresyl blue (BCB) and cryopreservation. In the first experiment, COCs were separated into groups and incubated for 1 h in medium containing BCB (13 μM, 16 μM, or 20 μM). The control group was not exposed to BCB staining. In the second experiment, COCs were divided into four groups: 13 μM BCB(+), 13 μM BCB(-), fresh control (selected by morphologic observation and immediately in vitro matured) and vitrified control (selected by morphologic evaluation, vitrified, and in vitro matured). In the first experiment, the 13 μM BCB group displayed greater development rates at the morula stage (65.45%, 36/55) when compared with the other groups. In the second experiment, cleavage (47.05%, 72/153) and morula development (33.55%, 51/153) of the control group of fresh COCs were increased compared with the other groups. However, when comparing morula rates between vitrified COC control and BCB(+) groups, the BCB(+) group had better results (19.23%, 5/26 and 64.7%, 11/17, respectively). Our best result in rat COC selection by BCB staining was obtained using a concentration of 13 μM. This selection could be a valuable tool to improve vitrification outcomes, as observed by the BCB(+) group that demonstrated better results compared with the vitrified COC control.

Brilliant Cresyl Blue Staining Enhances the Morphological Quality Evaluation of Porcine Oocytes-a Preliminary Study

Abstract Brilliant cresyl blue (BCB) staining is a method used for selection of developmentally competent porcine oocytes, in addition to the standard morphological classification. The aim of the current study was to assess the relationship between BCB staining in porcine oocytes with high and low morphological characteristics and its maturation rate. In the first part of the study, cumulus-oocytes complexes (COCs) (n=434) were aspirated from 60 ovaries. According to the morphological classification and BCB staining, they were divided in 4 groups: COC+/BCB+, COC−/BCB+, COC+/BCB−, and COC−/BCB−. In the second experiment, COCs (n=445) were categorized into 3 groups: control, BCB+, and BCB−. Significant differences in the maturation rate were observed between COC+/BCB+ and COC−/BCB− (66% vs. 23%), and between COC+/BCB+ and COC−/BCB+ (66% vs. 38%) (p&lt;0.0001). Significant differences were also registered between COC−/BCB+ and COC−/BCB-(38% vs. 23%) and between COC+/BCB− and COC−/BCB− (53% vs. 23%) (p&lt;0.01). Results from the second experiment showed that only BCB− oocytes had a significantly lower maturation rate (28%) compared to the control (63%) and BCB+ oocytes (59%) (p&lt;0.001). These findings showed that COCs with high morphological characteristics had higher developmental ability compared to COCs with low morphology. The BCB-staining in high-quality oocytes did not have a significant impact on their maturation ability compared to a control group, but it might be useful for selecting developmentally competent oocytes with low morphology characteristics.

Luteinizing hormone secretion, ovulatory capacity, and oocyte quality in peripubertal Gir heifers.

Induction of puberty in cattle breeds that attain puberty in later stages, such as Gir, allows the earlier beginning of reproductive life and it might increase oocyte quality. Here, the ovulatory capacity of prepuberal Gir heifers was studied and its relationship to follicular growth, luteinizing hormone (LH) secretion and oocyte quality was evaluated. Peripubertal Gir heifers were treated with a progesterone-based protocol and according to ovulatory response were separated into groups: not-ovulated (N-OV) and ovulated (OV). Serial blood samples were taken 24 h after estradiol treatment on day 12 to evaluate LH secretion. Cumulus-oocyte complexes (COCs) were collected using ovum pick-up and assessed for brilliant cresyl blue (BCB) staining rate, IVF-grade oocytes rate, and mean oocyte diameter, in comparison with cow oocytes. Gene expression of developmental competence markers (ZAR1, MATER, and IGF2R) was also analyzed. The largest follicle diameters were similar between N-OV and OV groups on the day of estradiol treatment (d12) and the next day and decreased (P = 0.04) in the N-OV group thereafter. LH pulse secretion was different between groups (N-OV = 3.61 ± 0.34 vs OV = 2.83 ± 0.21 ng/ ml; P = 0.04). COC assessment showed that the number of recovered oocytes, BCB+ rate, IVF-grade oocytes and oocyte size was similar (P > 0.05) among groups, resembling adult cow patterns. ZAR1, MATER and IGF2R gene expression in oocytes were also similar (P > 0.05) in N-OV and OV groups. In conclusion, our results demonstrate a lower LH secretion profile in peripubertal Gir heifers prone to ovulate after induction protocol, and that oocyte quality is not affected on a short-term basis by ovulation itself.

Restoration of Developmental Competence in Low-Quality Porcine Cumulus–Oocyte Complexes through the Supplementation of Sonic Hedgehog Protein during In Vitro Maturation

The sonic hedgehog (SHH) pathway is an important signaling pathway for mammalian ovarian folliculogenesis and oocyte maturation. A previous study demonstrated that low-quality porcine cumulus-oocyte complexes (COCs) have low developmental competence, with lower SHH signaling protein expression before and after in vitro maturation (IVM) than high-quality COCs. However, there is no reported evidence on the restorative effects of SHH protein supplementation during the IVM of low-quality porcine COCs. Therefore, this study investigated the effects of SHH protein supplementation on the IVM of low-quality porcine COCs, as assessed by brilliant cresyl blue (BCB) staining. To examine this, we designed four groups: (i) BCB- (low-quality), (ii) BCB- + SHH, (iii) BCB+ (high-quality), and (iv) BCB+ + SHH. While the supplementation of SHH protein with high-quality COCs had no effect, supplementation with low-quality COCs significantly improved cumulus cell expansion, metaphase II rate, and subsequent embryo development following parthenogenetic activation. Our results provide the first evidence that the low developmental competence of low-quality porcine COCs can be improved by supplementation with the SHH protein. These results indicate that an active SHH signaling pathway is required for the acquisition of developmental competence in porcine COCs.

Cleavage kinetics is a better indicator of embryonic developmental competency than brilliant cresyl blue staining of oocytes

In vitro production of embryos (IVP) is a valuable technology to produce embryos of high genetic value. Despite advances in IVP, the efficiency of culture systems remains low. One method to increase IVP success is the early selection of oocytes or embryos that may have greater developmental potential. Here, we investigated two methods of selection, namely BCB staining and cleavage kinetics, both individually and in conjunction, for improved developmental outcomes in vitro. We hypothesized that a synergistic use of both BCB staining and cleavage kinetics would result in identification of embryos of greater developmental potential. The selection of oocytes by BCB staining does select for those oocytes with higher developmental potential, as noted by a greater blastocyst development between BCB positive (32.6%) and BCB negative (22.0%) on day 8 post-fertilization. However, the utilization of BCB staining and cleavage kinetics in tandem resulted in a complete masking of the effect observed when using BCB alone. We obtained the highest proportion of blastocyst development per selection group using cleavage kinetics alone, in which 53.1% of embryos grouped as Fast produced a blastocyst, which was significantly different from the three other groups (Fast+, Slow, not cleaved). We observed, however, that the separation of embryos by cleavage kinetics did not predict their survival to cryopreservation. In conclusion, in standard culture systems, cleavage kinetics is an effective method for the selection of embryos with increased developmental potential to develop blastocysts, however, it may not be effective to select healthy embryos for transfer following cryopreservation.

Impact of Staining Methods and Human Factors on Accuracy of Manual Reticulocyte Enumeration.

Although peripheral blood reticulocyte enumeration reflects bone marrow functional integrity, which is important for differential diagnosis of hematological diseases, the factors affecting its accuracy have not been adequately addressed. Using 100 consecutive venous blood samples being processed with four supravital staining techniques [i.e., brilliant cresyl blue (BCB), new methylene blue (NMB), and BCB/NMB with Liu’s stain] for reticulocyte enumeration, two technologists (senior vs. junior) conducted microscopic counting. The results were compared with those obtained with an automated system (Sysmex XE-5000) that served as the standard. The aims of this study were to identify (1) the technique that gave the most reliable outcome, and (2) possible human factors (i.e., seniority, repeated counting) that may affect the counting results. Analysis showed least bias (i.e., deviation from automated counting) associated with BCB staining, followed by NMB. In addition, the senior observer exhibited a higher bias in counting compared with their junior counterpart. Repeated counting also correlated with a higher rate of bias. Nevertheless, inter-observer consistency was high (intraclass correlation coefficient >0.95) and inter-/intra-observer variations were non-significant (both p > 0.05). Our results supported the use of BCB stain for reticulocyte enumeration and the reliability of manual counting despite the involvement of human factors, which had negligible impacts on the final outcomes.

Tight gene co-expression in BCB positive cattle oocytes and their surrounding cumulus cells

BackgroundCytoplasmic and nuclear maturation of oocytes, as well as interaction with the surrounding cumulus cells, are important features relevant to the acquisition of developmental competence.MethodsHere, we utilized Brilliant cresyl blue (BCB) to distinguish cattle oocytes with low activity of the enzyme Glucose-6-Phosphate Dehydrogenase, and thus separated fully grown (BCB positive) oocytes from those in the growing phase (BCB negative). We then analyzed the developmental potential of these oocytes, mitochondrial DNA (mtDNA) copy number in single oocytes, and investigated the transcriptome of single oocytes and their surrounding cumulus cells of BCB positive versus BCB negative oocytes.ResultsThe BCB positive oocytes were twice as likely to produce a blastocyst in vitro compared to BCB- oocytes (P < 0.01). We determined that BCB negative oocytes have 1.3-fold more mtDNA copies than BCB positive oocytes (P = 0.004). There was no differential transcript abundance of genes expressed in oocytes, however, 172 genes were identified in cumulus cells with differential transcript abundance (FDR < 0.05) based on the BCB staining of their oocyte. Co-expression analysis between oocytes and their surrounding cumulus cells revealed a subset of genes whose co-expression in BCB positive oocytes (n = 75) and their surrounding cumulus cells (n = 108) compose a unique profile of the cumulus-oocyte complex.ConclusionsIf oocytes transition from BCB negative to BCB positive, there is a greater likelihood of producing a blastocyst, and a reduction of mtDNA copies, but there is no systematic variation of transcript abundance. Cumulus cells present changes in transcript abundance, which reflects in a dynamic co-expression between the oocyte and cumulus cells.

Higher quality rather than superior quantity of oocytes determine the amount of fertilizable oocytes in two outbred Dummerstorf high‐fertility mouse lines

Dummerstorf fertility lines FL1 and FL2 represent two models of enhanced fertility characterized by the doubling of the litter size compared with an unselected control population (ctrl line, Dummerstorf FztDU). Both biodiverse FLs managed to reach this goal by increasing the ovulation rate per cycle, even showing decreased pregnancy rate and irregular oestrous cycle and metabolic hormone levels, compared with ctrl. The aim of the present study was to analyse oocytes in terms of quality and quantity by comparing the entire pool of oocytes per ovary, with those from the antral follicles within the same animal. We performed Brilliant Cresyl Blue staining as a non-invasive marker of oocyte quality in combination with an analysis of additional morphological indicators, e.g. cytoplasm clarity, cumulus cell layers, nuclear anatomy, size and shape. We compared our fertility lines with the unselected control population and with another independent line selected from the same founder population, showing lower litter size (DU6P). Our results suggest that fertility lines show decreased number of oocytes per ovary compared with DU6P but increased number of high-quality oocytes before ovulation. Hence, the raise in the ovulation rate and litter size of those super fertile mouse lines are not associated with an increased number of oocytes per ovary but rather with an increased number of higher quality fertilizable oocytes per cycle. In addition, the most conspicuous method to acquire oocytes with the highest quality in our lines is to assess their morphology, rather than their status after staining. All these discoveries together may be of fundamental importance for further studies in livestock farm animals showing some similar characteristics, e.g. irregular cycle or hormonal misbalances, to improve production while lowering costs, and in humans to increase the possibilities of successful pregnancies for couples undergoing in vitro fertilization (IVF).

Noniron deficiency microcytic anemia, dysmorphic features, and intellectual disability: Diagnostic clues for α-thalassemia/mental retardation associated with chromosome 16 syndrome

Noniron deficiency microcytic anemia, dysmorphic features, and intellectual disability: Diagnostic clues for α-thalassemia/mental retardation associated with chromosome 16 syndrome

Comparison of Two In vitro Maturation Media in Their Efficiency for Production of Competent Alpaca Oocytes

Oocyte maturation is the most critical process in in vitro culture, since the oocyte acquires competence for future processes, which involve the resumption of meiosis, epigenetic regulation, polyspermy block, activation of the zygote, exchange of protamines to histones, cleavage, cell polarization, embryonic stem cell proliferation, and implantation. Therefore, the objective of this study was to evaluate the efficiency of two different in vitro maturation media in the production of competent alpaca oocytes matured in vitro. The competency evaluated by morphometry and cytoplasmic and nuclear staining techniques. Alpaca ovaries (n=44) were sourced from slaughterhouse, transported to laboratory in thermoses with saline solution at 33-36ºC and subjected to follicular aspiration and/or dissection. A total of 212 oocytes were obtained. The oocytes were selected, randomized and distributed in one of the two experimental groups: Maturation medium 1 (MM1) and Maturation medium 2 (MM2) with three replicates per experiment. In vitro maturation was carried out in a portable incubator for 40h under 5% CO2 conditions (produced by effervescent granules) and 38.5ºC (by thermostatic bath). Nuclear maturation was assessed using acetate-orcein staining, cytoplasmic maturation using Brilliant Cresyl Blue (BCB) staining and morphometry using ImageJ Software. Nuclear maturation was classified according to the meiotic stage. Cytoplasmic maturation was classified by the presence (BCB+) or absence (BCB-) of BCB staining. Morphometry evaluated the measurements of the diameter and thickness of the zona pellucida and perivitelline space of the oocyte. Nuclear maturation rate was evaluated in Metaphase, observing no significant differences (P=0.088) between MM1 (46.08 ± 5.90 %) and MM2 (54.65 ± 3.00%) in this parameter. The cytoplasmic maturation rate of MM1 (67.78 ± 4.50%) was significantly lower compared to MM2 (77.61 ± 3.58) % (P=0.014). For the diameter of the oocyte, MM1 (133.42 ± 14.56 μm) and MM2 (136.14 ± 15.2 μm) were no different (P=0.397), same for the thickness of the zona pellucida, MM1 (16.84 ± 3.96 μm) and MM2 (16.73 ± 4.98 μm) were no different (P=0.919). For the perivitelline space area, MM1 (11018.91 ± 2465.40 μm2 ) and MM2 (8686.18 ± 3016.88 μm2 ) were significantly different (P=0.0005). In conclusion, the present study shows that the MM2 medium results are better in cytoplasmic maturation and the area of the perivitelline space. The other parameters (nuclear maturation, diameter of the oocyte and thickness of the zona pellucida) were no different between the two maturation media evaluated.

In vitro maturation of goat oocytes selected using Brilliant cresyl blue staining

The present study was conducted to assess the developmental competence of goat oocytes selected using Brilliant cresyl blue (BCB) staining. Goat ovaries were collected from the slaughtered animals with unknown reproductive history. The oocytes retrieved by aspiration technique were selected based on morphology and subjected to BCB staining. Brilliant cresyl blue staining is based on the activity of glucose-6-phospahte dehydrogenase (G6PDH) enzyme synthesised by the oocytes. The cytoplasm remains blue in oocytes that have finished the growth phase (BCB+) while the growing oocytes remain colourless (BCB-). The stained and unstained oocytes were subjected to in vitro maturation separately to assess cumulus cell expansion index and polar body extrusion. A total of 206 culture grade oocytes were subjected to study, out of which, 76.75 ± 2.38 per cent of oocytes showed positive to BCB staining and 23.21 ± 2.38 per cent were negatively stained. Significantly higher maturation rate was observed in BCB+(92.89 ± 2.37%) oocytes than BCB-(29.72 ± 2.46%). The present study concluded that BCB staining can be used for selecting goat oocytes with good cytoplasmic maturation for further in vitro embryo production

Treatment of mouse cumulus-oocyte complexes with L-carnitine during vitrification and in vitro maturation affects maturation and embryonic developmental rate after parthenogenetic activation.

The technique of oocyte vitrification remains a challenge in most animal species. The present study aimed to evaluate the effects of cumulus cell presence and L-carnitine (LC) treatment during vitrification of selected immature oocytes by brilliant cresyl blue (BCB) staining on maturation and embryonic developmental rate after parthenogenetic activation. Immature oocytes were obtained from C57BL/6 female mice ovaries and stained with BCB. The BCB+ cumulus-oocyte complexes (COCs) were then selected and random parts of COCs were denuded from cumulus cells (denuded oocytes: DOs). COCs and DOs were treated with/out LC (0.6mg/ml) during vitrification and in vitro maturation (IVM) procedures. A number of non-vitrified COCs were also treated with LC during the IVM process (fresh group). Maturation rate, intracellular glutathione (GSH) contents, and developmental competence of oocytes were also examined. The GSH levels in vitrified DOs+LC and vitrified COCs+LC groups were significantly higher (p<0.01) than untreated vitrified-warmed COCs and DOs. Maturation rate and blastocyst developmental rate were reduced after the vitrification-warming procedure compared with the fresh group. The vitrified COCs+LC group showed a higher percentage of mature oocytes and the ability to develop to blastocyst stage than the vitrified-warmed DOs group (p<0.01). These data indicated that the presence of cumulus cells around the competent oocyte and LC treatment during vitrification and IVM procedure could improve parthenogenetic developmental competence of vitrified-warmed oocytes by increasing GSH levels and accelerating oocyte maturation.

Transcriptional profile of bovine preimplantation development selected based on G6PDH activity

The oocyte is the female gamete that contributes not only half of the genetic material but also all of the cytoplasm to the zygote, supplying the transcripts, proteins, mitochondria and other components necessary for early embryonic development. The intrinsic oocyte quality is one of the main factors affecting the embryo yield, the implantation rate and the rate of healthy offspring. It is obvious that a fertilized oocyte must reach the blastocyst stage within 6–9 days in the proper culture conditions to have a significant chance of inducing a pregnancy and producing an offspring. The ability to sustain the first week of embryonic development is clearly influenced by the follicular status from which the oocyte is obtained indicating that this developmental potential is inherent within certain oocytes. Since most early embryos that do not reach the blastocyst stage are blocked at or close to the maternal to zygotic transition (MZT)-stage, which occurs at the eight-cell stage in cattle, one could speculate that incompetent oocytes fail to appropriately activate the embryonic genome. Oocyte selection based on glucose-6-phosphate dehydrogenase (G6PDH) activity has been successfully used to differentiate between competent and incompetent bovine oocytes. Recently, molecular regulation of genes regulating biological process of Brilliant Cresyl Blue staining (BCB) selected oocytes and embryos was investigated to explain their variation in quality and developmental potentiality. This short review will highlights some of these efforts that have been done in this interesting area of research.

Cellular and molecular alterations of buffalo oocytes cultured under two different levels of oxygen tension during in vitro maturation.

This study was conducted to monitor the cellular and molecular changes of buffalo cumulus-oocytes complexes (COCs) cultured under high or low oxygen levels. Morphologically good quality COCs (n = 1627) were screened using brilliant cresyl blue (BCB) staining and placed into three groups (BCB+, BCB- and control). All groups of COCs were cultured under low (5%) or high (20%) oxygen tensions. Intracellular and molecular changes including oocyte ultrastructure, lipid contents, mitochondrial activity and transcript abundance of genes regulating different pathways were analyzed in the matured oocyte groups. The results revealed that oxygen tension did not affect cumulus expansion rates, however the BCB+ group had a higher (P ≤ 0.05) expansion rate compared with the BCB- group. BCB- oocytes recorded the lowest meiotic progression rate (P ≤ 0.05) under high oxygen levels that was linked with an increased level of reactive oxygen species (ROS) compared with the BCB+ oocytes. Ultrastructure examination indicated that BCB+ oocytes had a higher rate of cortical granules migration compared with BCB- under low oxygen tension. In parallel, our results indicated the upregulation of NFE2L2 in groups of oocytes cultured under high oxygen tension that was coupled with reduced mitochondrial activity. In contrast, the expression levels of MAPK14 and CPT2 genes were increased (P ≤ 0.05) in groups of oocytes cultured under low compared with high oxygen tension that was subsequently associated with increased mitochondrial activity. In conclusion, data from the present investigation indicated that low oxygen tension is a favourable condition for maintaining the mitochondrial activity required for nuclear maturation of buffalo oocytes. However, low-quality oocytes (BCB-) responded negatively to high oxygen tension by reducing the expression of gene-regulating metabolic activity (CPT2). This action was an attempt by BCB- oocytes to reduce the increased levels of endogenously produced ROS that was coupled with decreased expression of the gene controlling meiotic progression (MAPK14) in addition to nuclear maturation rate.