Brilliant Cresyl Blue Test Research Articles

New markers for regulation of transcription and macromolecule metabolic process in porcine oocytes during in vitro maturation.

Oocyte maturation is essential for proper fertilization, embryo implantation and early development. While the physiological conditions of these processes are relatively well-known, its exact molecular mechanisms remain widely undiscovered. Oocyte growth, differentiation and maturation are therefore the subject of scientific debate. Precious literature has indicated that the oocyte itself serves a regulatory role in the mechanisms underlying these processes. Hence, the present study performed expression microarrays to analyze the complete transcriptome of porcine oocytes during their in vitro maturation (IVM). Pig material was used for experimentation, as it possesses similarities to the reproductive processes and general genetic proximities of Sus scrofa to human. Oocytes, isolated from the ovaries of slaughtered animals were assessed via the Brilliant Cresyl Blue test and directed to IVM. A number of oocytes were left to be analyzed as the ‘before IVM’ group. Oocyte mRNA was isolated and used for microarray analysis, which was subsequently validated via RT-qPCR. The current study particularly focused on genes belonging to ‘positive regulation of transcription, DNA-dependent’, ‘positive regulation of gene expression’, ‘positive regulation of macromolecule metabolic process’ and ‘positive regulation of transcription from RNA polymerase II promoter’ ontologies. FOS, VEGFA, ESR1, AR, CCND2, EGR2, ENDRA, GJA1, INHBA, IHH, INSR, APP, WWTR1, SMARCA1, NFAT5, SMAD4, MAP3K1, EGR1, RORA, ECE1, NR5A1, KIT, IKZF2, MEF2C, SH3D19, MITF and PSMB4 were all determined to be significantly altered (fold change, >|2|; P<0.05) among these groups, with their downregulation being observed after IVM. Genes with the most altered expressions were analyzed and considered to be potential markers of maturation associated with transcription regulation and macromolecule metabolism process.

Apoptosis of somatic cells in follicules and the functional status of bos taurus oocytes

In the present study, the analysis of apoptotic changes in the somatic cells of the follicle (granulosa and cumulus), depending on the diameter of the follicle (<3 mm, 3-5 mm, 6-8 mm) and the functional status of the oocyte (growing oocytes or oocytes that have finished growth phase) is presented. The functional status of the oocytes was assessed by BCB test based on the difference in the color of the ooplasm after exposure by the vital dye of brilliant cresyl blue (BCB)..Apoptosis in granulosa and cumulus cells isolated from follicles containing growing oocytes or oocytes that have finished growth phase was determined by the TUNEL (Feng W., Pan Zhifang Z. 2014).It is shown that granulosa from Bos Taurus folliclesare characterized by different levels of apoptosis depending on the status of oocytes (growing oocytes or oocytes that have finished growth phase), isolated from these follicles.The proportion of apoptotic granulosa cells in follicles containing the oocytes that have finishedgrowth phase, regardless of diameter of the follicle, was significantly higher than in follicles containing growing oocytes. In cumulus cells, on the contrary, there is no such trend. The level of apoptotic in the cumulus cells of oocytes in a different functional state did not have significantdifferences in all experimental groups. This data allow us to assess the level of apoptosis in granulosa of ovarian follicles as an indicator of the functional status of the developing oocyte and suggest to use this indicator as prediction of the quality of donor’s Bos Taurus oocytes

Single-cell transcriptome sequencing reveals that cell division cycle 5-like protein is essential for porcine oocyte maturation

The brilliant cresyl blue (BCB) test is used in both basic biological research and assisted reproduction to identify oocytes likely to be developmentally competent. However, the underlying molecular mechanism targeted by the BCB test is still unclear. To explore this question, we first confirmed that BCB-positive porcine oocytes had higher rates of meiotic maturation, better rates of cleavage and development into blastocysts, and lower death rates. Subsequent single-cell transcriptome sequencing on porcine germinal vesicle (GV)-stage oocytes identified 155 genes that were significantly differentially expressed between BCB-negative and BCB-positive oocytes. These included genes such as cdc5l, ldha, spata22, rgs2, paip1, wee1b, and hsp27, which are enriched in functionally important signaling pathways including cell cycle regulation, oocyte meiosis, spliceosome formation, and nucleotide excision repair. In BCB-positive GV oocytes that additionally had a lower frequency of DNA double-strand breaks, the CDC5L protein was significantly more abundant. cdc5l/CDC5L inhibition by short interference (si)RNA or antibody microinjection significantly impaired porcine oocyte meiotic maturation and subsequent parthenote development. Taken together, our single-oocyte sequencing data point to a potential new role for CDC5L in porcine oocyte meiosis and early embryo development, and supports further analysis of this protein in the context of the BCB test.

Effects of Roscovitine on In Vitro Development of Porcine Oocyte Using Brilliant Cresyl Blue

The objective of this experiment was to explore the effects of Roscovitine (Rosco) prior to in vitro maturation (IVM) of immature pig oocyte. Brilliant cresyl blue test has been used to select the good quality of oocyte. Specifically, the effects of Rosco exposure on nuclear and cytoplasmic maturation, diameter, intracellular glutathione (GSH) and reactive oxygen species (ROS), and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT), and gene expression levels in SCNT embryos have been measured. Cumulus oocyte complexes (COCs) have been exposed in 75 μM of Rosco for 22 and 44 h. The COCs that were matured in the IVM for 44 h without Rosco used as control group. Diameter of matured porcine oocytes 44 h culture with Rosco was significantly lower than 22 h culture with Rosco and control groups. GSH was higher in control group than 22 h and 44 h with Rosco but reduction of ROS in 22 h than 44 h with Rosco. In PA, exposure with Rosco 44 h oocytes group has been significantly lower than 22 h and control group in rates of maturation, cleavage and blastocyst formation. Similarly, in SCNT embryos rates of maturation, cleavage and formation of blastocyst have been also significantly lower in 44 h Rosco treated group than other two groups. SCNT embryos treated with Rosco 22 h showed greater expression levels of POU5F1, DPPA2 and NDP52Il mRNA compared with other two groups. Our results demonstrate that Rosco treatment with 22 h prior to IVM improves the development competence of porcine oocyte.

Selection of developmentally competent human oocytes aspirated during cesarean section

Purpose: To evaluate the efficiency/safety of Brilliant Cresyl Blue (BCB) staining as a selection method of developmentally competent immature human oocytes.Materials and methods: Immature oocytes of 32 pregnant women were recovered during cesarean section (CS). After retrieval, 92 oocytes were randomly divided into two groups: control (directly disposed to in vitro maturation – IVM) and treated – exposed to BCB 26 μM during 60 min. After staining, the treated group was classified as cytoplasm coloration, BCB positive (blue) or negative (colorless), and then disposed to IVM. Nuclear status was checked after 24 and 48 h of IVM. Nuclear maturation (polar body extrusion), meiosis resumption (absence of germinal vesicle) and degeneration rates were evaluated among the three groups (control, BCB positive and BCB negative) using Generalized Estimating Equations, followed by Bonferroni’s correction for multiple comparisons.Results: Nuclear maturation was higher in BCB positive compared to BCB negative, after 24 and 48 h of IVM (p = .004 and p = .032). The control group was equal to BCB positive. There was no difference among groups analyzing meiosis resumption and degeneration rates.Conclusion: The BCB test can be a good marker in pre-selection procedures of developmentally competent human oocytes aspirated during CS.

In vivo and in vitro study of the function of the left and right bovine ovaries

Inequality in function of the left and right bovine ovaries and uterine horns was evaluated in two separate experiments. In the first experiment (in vivo), the relationship between the left and right ovarian activities and reproductive indices was evaluated. Therefore, the total number of 1284 randomly chosen lactating dairy cows were examined from Day 50 to 60 postpartum, and according to the presence of an active CL on the ovaries, they were divided into 502 LCL3-cows and 782 RCL3-cows (cows with an active CL on the left [L] or right [R] ovary, respectively). To induce estrus synchronization and investigate the effects of PGF2α administration on the incidence of estrus in both LCL3-cows and RCL3-cows, the cows were treated with one luteolytic dose of PGF2α and were inseminated after observed estrus (via visual observation lasting at least 30 minutes three times a day). To investigate the effects of side of ovulation at the time of PGF2α administration on reproductive parameters, pregnancy diagnosis was performed 28 days after insemination (using ultrasound) and 42 days after insemination (using transrectal palpation). The results showed that the percentage of the RCL3-cows was greater than the LCL3-cows (60.9% vs. 39.1%, respectively). Furthermore, ovulations switching from the left to right ovary in two successive ovulations were greater than those that switched from the right to left ovary. On the other hand, the sex ratio (male percentage) in the right uterine horn was greater than that of the left one. In the second experiment (in vitro), the developmental potential of bovine oocytes derived from the left (L-oocytes) and right (R-oocytes) ovaries after in vitro embryo production and heterogeneity in the developmental competence of L-oocytes and R-oocytes using the brilliant cresyl blue staining test as a selection criterion were evaluated. Results of the in vitro experiment showed that the percentage of cleavage and blastocyst rate of R-oocytes were greater (P < 0.001) than those of L-oocytes. Moreover, it appears that the side of ovaries had greater effects on the developmental competence of oocytes than other factors associated with heterogeneity in the developmental competence of oocytes, which can be detected by the brilliant cresyl blue test. In conclusion, the results of the in vivo study confirmed the observations in previous studies in which the right ovarian response (distribution of ovulation) was superior to that of the left ones. Interestingly, the in vitro experiments for the first time clearly showed that more ovulation on the right side is not the only reason for this unequal activity. In fact, in cattle, the greater developmental potential of oocytes originating from right ovaries may cause superior activity of the right side, and the effect is even higher than the differences in ovulation response between the left and right ovaries.

Bovine non-competent oocytes (BCB-) negatively impact the capacity of competent (BCB+) oocytes to undergo in vitro maturation, fertilisation and embryonic development.

Competent oocyte selection remains a bottleneck in the in vitro production (IVP) of mammalian embryos. Among the vital assays described for selecting competent oocytes for IVP, the brilliant cresyl blue (BCB) test has shown consistent results. The aim of the first experiment was to observe if oocytes directly submitted to IVM show similar cleavage and blastocyst rates as those obtained with oocytes maintained under the same in vitro conditions as the oocytes that undergo the BCB test. Bovine cumulus-oocyte complexes (COCs) were recovered from slaughterhouse-derived ovaries and, after morphological evaluation, were randomised grouped into three groups: (1) directly submitted to IVM; (2) oocytes submitted to the BCB test without the addition of BCB stain (BCB control group); and (3) submitted to the BCB test. The results showed that oocytes directly submitted to IVM reached similar cleavage (48/80 - 60%) and embryonic development rates to the blastocyst stage (10/48 - 21%) as the results obtained with the BCB control group oocytes (45/77 - 58% and 08/45 - 18%, respectively). The aim of the second experiment was to determine the cleavage and blastocyst rates obtained from BCB+ oocytes undergoing IVM in the presence of BCB- oocytes at a ratio of 10:1. COCs were recovered from slaughterhouse-derived ovaries and, after morphological evaluation, were randomised into two groups that were submitted to IVM either directly (1: control group) or submitted to the BCB test prior to IVM. After the BCB test, the COCs were classified as either BCB+ (blue cytoplasm) or BCB- (colourless cytoplasm) and then divided into four experimental groups: (2) BCB+; (3) BCB-; and (4) BCB+ matured in same IVM medium drop as (5) BCB- at a ratio of 10:1. After IVM (24 h), oocytes from the different experimental groups were submitted to in vitro fertilisation (IVF) and in vitro culture (IVC) under the same culture conditions until they reached the blastocyst stage (D7). With regards to the cleavage rate (48 h after IVF), only group 3 (102/229 - 44%) differed (P < 0.05) from the other groups [1 (145/241 - 60%); 2 (150/225 - 67%); 4 (201/318 - 63%) and 5 (21/33 - 63%)]. On day 7, the embryos from group 2 (BCB+) achieved the highest blastocyst rate (46/150 - 31%) (P < 0.05) when compared with the embryo development capacity of the other experimental groups (1: 31/145 - 21%; group 3: 17/102 - 17%; group 4: 46/201 - 23%; and group 5: 2/21 - 10%). In conclusion, submitting BCB+ oocytes that were separated from BCB- oocytes to IVM increases the rate of embryonic development to the blastocyst stage when compared to the control group, BCB- oocyte group, BCB+ paracrine group and BCB- paracrine group. The presence of non-competent oocytes during IVM, even in low proportion (1:10), reduces the capacity of competent oocytes to undergo embryo development and achieve blastocyst stage during IVC.

Intracellular glutathione content, developmental competence and expression of apoptosis-related genes associated with G6PDH-activity in goat oocyte

To associate glucose-6-phosphate dehydrogenase (G6PDH) activity in goat oocytes with intracellular glutathione (GSH) content, meiotic competence, developmental potential, and relative abundance of Bax and Bcl-2 genes transcripts. Goat oocytes were exposed to brilliant cresyl blue (BCB) staining test and categorized into BCB(+) (blue-cytoplasm), and BCB(-) (colorless-cytoplasm) groups. A group of oocytes were not exposed to BCB test and was considered as a control group. After maturation in vitro, a group of oocytes were used for determination of nuclear status and intracellular GSH content while another group was subjected to parthenogenetic activation followed by in vitro embryo culture. We found that BCB(+) oocytes not only yielded higher rate of maturation, but also showed an increased level of intracellular GSH content than BCB(-) and control oocytes. Furthermore, BCB(+) oocytes produced more blastocysts than BCB(-) and control oocytes. Our data revealed that the expression of anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) genes were interacted with G6PDH-activity in mature oocyte, their surrounding cumulus cells, and blastocyst-stage embryos. The results of this study demonstrate that selection of goat oocytes based on G6PDH-activity through the BCB test improves their developmental competence, increases intracellular GSH content, and affects the expression of the apoptosis-related genes.

Differences in Cytoplasmic Maturation Between the BCB+ and Control Porcine Oocytes Do Not Justify Application of the BCB Test for a Standard IVM Protocol

The Brilliant Cresyl Blue (BCB) test relies on G6PDH activity and a simple protocol for the selection of higher quality oocytes. Although the BCB+ oocytes of all the species that have been investigated are characterized by superior quality when compared to BCB- counterparts, application of the test for embryo production still remains an open issue. The aim of our study was to compare BCB+ and the control oocytes (not subjected to the BCB test) in terms of selected aspects of cytoplasmic maturation (mtDNA copy number, mitochondria distribution, relative transcript abundance of six marker genes). The results of our study revealed more relevant differences within the BCB+ and the control oocytes (before and after IVM) than between the two categories of oocytes. There was no difference in the transcript abundance of the BCB+ and the control oocytes in 5 out of 6 analyzed genes (BMP15, GDF9, ATP5A1, EEF1A, ZAR1) and in mtDNA content (pre-IVM 179609 vs. 176595 and post-IVM 187243 vs. 246984, respectively). With regard to mitochondria distribution in pre- and post-IVM oocytes, there was nonsignificant tendency for a more frequent occurrence of the expected patterns in the BCB+ group. The results of the present study do not support the application of BCB staining in a routine IVM protocol due to relatively high similarity in selected parameters characterizing cytoplasmic maturation of BCB+ and control oocytes. This high similarity may results from the limited amount of less competent BCB- oocytes (10%) still present among nonselected oocytes of proper morphology.

Selection of developmentally competent immature equine oocytes with brilliant cresyl blue stain prior to in vitro maturation with equine growth hormone

Immature oocytes synthesize a variety of proteins that include the enzyme glucose-6-phosphate dehydrogenase (G6PDH). Brilliant cresyl blue (BCB) is a vital blue dye that assesses intracellular activity of G6PDH, an indirect measure of oocyte maturation. The objective was to evaluate the BCB test as a criterion to assess developmental competence of equine oocytes and to determine if equine growth hormone (eGH) enhanced in vitro maturation (IVM) of equine oocyte. Cumulus-oocytes complexes (COCs) were recovered by aspirating follicles <30 mm in diameter from abattoir-derived ovaries and were evaluated morphologically. Thereafter, COCs were exposed to BCB (26 μM) for 90 min at 39°C and selected based on the colour of their cytoplasm (BCB positive/BCB+ or BCB negative/BCB-). The COCs were allocated as follows: (a) IVM medium; (b) eGH group; (c) BCB-/IVM; (d) BCB+/IVM; (e) BCB-/eGH; and (f) BCB+/eGH. Then, COCs were cultured in vitro for 30 h, at 39°C in a 5%CO2 humidified air atmosphere. Cumulus-free oocytes were incubated in 10 μg/ml of bis-benzamide for 20 min at 39°C and nuclear maturation was evaluated with epifluorescence microscopy. Of the 39 COCs selected morphologically and subjected to BCB staining, 18/39 (46.2%) were classified as BCB+ and 21/39 (53.8%) as BCB- (P > 0.05). Maturation was not affected significantly by BCB classification, but the maturation rate was higher for oocytes that had been exposed to exogenous eGH versus controls (16/28, 57.1% versus 8/26, 30.8%, P < 0.05). In the present study, the BCB test was not useful for predicting competent equine oocytes prior to IVM. However, eGH enhanced equine oocyte maturation in vitro.

Modulation of Glycolysis and the Pentose Phosphate Pathway Influences Porcine Oocyte In Vitro Maturation

Glycolytic and pentose phosphate pathway (PPP) activities were modulated in porcine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) by the addition of inhibitors or stimulators of key enzymes of the pathways to elucidate their relative participation in oocyte maturation. The activities of glycolysis and PPP were evaluated by lactate production per COC and by the brilliant cresyl blue test, respectively. Glucose uptake per COC and the oocyte maturation rate were also evaluated. Lactate production, glucose uptake and the percentage of oocytes reaching metaphase II decreased in a dose-dependent manner in the presence of the pharmacological (NaF) or the physiological (ATP) inhibitors of glycolysis (p<0.05). The addition of the physiological stimulator of glycolysis (AMP) caused no effect on lactate production, glucose uptake or the meiotic maturation rate. The pharmacological (6-AN) and the physiological (NADPH) inhibitors of PPP induced a dose-dependent decrease in the percentage of oocytes with high PPP activity and in the nuclear maturation rate (p<0.05). The physiological stimulator of PPP (NADP) caused no effect on the percentage of oocytes with high PPP activity. The glycolytic and PPP activities of porcine COCs and maturational competence of oocytes seem to be closely related events. This study shows for the first time the regulatory effect of ATP and NADPH as physiological inhibitors of glycolysis and PPP in porcine COCs, respectively. Besides, these pathways seem to reach their maximum activities in porcine COCs during IVM because no further increases were achieved by the presence of AMP or NADP.

Combination of oocyte and zygote selection by brilliant cresyl blue (BCB) test enhanced prediction of developmental potential to the blastocyst in cattle

The cumulus oocyte complexes (COCs) were obtained from local abattoir. After aspiration, the COCs were allotted into four treatments to evaluation of brilliant cresyl blue (BCB) test. Control treatment (C): oocytes were cultured directly (without exposure to BCB) after recovery in in vitro production (IVP) process. Oocyte treatment (OBCB): immediately after aspiration, COCs were incubated in modified Dulbecco's phosphate-buffered saline (mDPBS) supplemented with 26μM of BCB for 90min and classified into two classes: oocytes with blue cytoplasm coloration (OBCB+: more competent oocytes) and oocytes without blue cytoplasm coloration (OBCB−: low competent oocytes). Directly after classification, the oocytes were maintained undisrupted in the IVP process. Zygote treatment (ZBCB): After oocyte collection, maturation and fertilization, zygotes were stained with BCB for 10min and categorized into three ways, according to whether they were highly stained (ZBCB++: low competent zygotes), moderately stained (ZBCB+: moderate competent zygotes) and unstained (ZBCB−: more competent zygotes). Directly after classification, the zygotes were maintained undisrupted in the culture process. Oocyte and zygote treatments (OBCB/ZBCB): COCs were stained with BCB after recovery and classified into two classes (OBCB+ and OBCB−). After fertilization, the zygotes produced from OBCB+ and OBCB− oocytes were further stained with BCB for 10min and categorized six ways (OBCB+/ZBCB++, OBCB+/ZBCB+, OBCB+/ZBCB−, OBCB−/ZBCB++, OBCB−/ZBCB+ and OBCB−/ZBCB−). Directly after classification, the zygotes were maintained undisrupted in the culture process. The selection rate produced from OBCB treatment (OBCB+; 54.3%) was greater (P<0.05) than ZBCB treatment (ZBCB−; 44.3%). In addition, the selection rate produced from double application (combination of oocyte and zygote selection) of BCB test (OBCB+/ZBCB−: 28.8%) was less (P<0.01) than single application of BCB test (ZBCB−: 44.3%or OBCB+: 54.3%). The percentage of blastocyst production from OBCB+ oocytes (35.7%) and ZBCB− zygotes (36.6%) were greater (P<0.05) than that from C oocytes (25.7%), OBCB− oocytes (16.5%), ZBCB++ (13.5%) and ZBCB+ zygotes (21.3%). However, there were no significant differences (P>0.05) in the percentages of blastocyst production between OBCB+ oocytes (35.7%) and ZBCB− zygotes (36.6%). The proportion of blastocyst production from double application of BCB test (OBCB+/ZBCB−: 48.0%) was greater (P<0.05) than that from single application of BCB test (OBCB+: 35.7% or ZBCB−: 36.6%). In conclusion, current results confirmed that combination of oocyte and zygote selection by BCB test enhanced the efficiency of selecting for high quality embryos, compared to the single BCB test.

Selection of developmentally competent sheep zygotes using the Brilliant Cresyl Blue (BCB) test, after IVF

Selecting oocytes and competent zygotes likely to develop, is crucial for in vitro sheep embryo production. Ovine oocyte and zygote selection, based on their morphology, is often influenced by personal preference and the lack of universal standards. Therefore, the objective of this study was to identify a colorimetric assay associated in evaluating zygote developmental competence – with selection based on the use of the Brilliant Cresyl Blue (BCB) test. The BCB test allows researchers to determine the activity of glucose-6-phosphate dehydrogenase (G6PDH), an enzyme synthesized by fertilized oocytes. After in vitro fertilization (IVF), presumptive zygotes were exposed to BCB, diluted in mDPBS (DPBS with 0.4% BSA) for 10min at 38.5°C in a humidified air atmosphere. Zygotes were categorized into 3 categories, according to whether they were stained (category 1), moderately stained (category 2) or unstained (category 3). Category 1, 2 and 3 zygotes were considered to have a low, mid or high G6PDH activity, respectively. Control zygotes were incubated directly after IVF (without exposure to BCB). After BCB categorization, zygotes were maintained in the culture medium for 7 days. The category 3 zygotes yielded a higher blastocyst developmental rate (31.1%), than the control (not tested; 18.8%; P<0.01), category 2 (17.1%; P<0.01) and category 1 (11.6%; P<0.01) zygotes. In addition, the total number of cells per blastocyst obtained from the category 3 zygotes was higher, compared to the other groups. In conclusion, it would seem as if the BCB staining, facilitates sheep zygote categorization for high developmental potential.

Selection of developmentally competent sheep oocytes using the brilliant cresyl blue test and the relationship to follicle size and oocyte diameter

The aim of the present study was to evaluate the relationship between follicle size and oocyte diameter and the use of the brilliant cresyl blue (BCB) test prior to in vitro maturation (IVM) in the selection of competent sheep oocytes for in vitro embryo production (IVEP). Sheep oocytes were exposed to the BCB stain, diluted in Dulbecco's phosphate-buffered saline, modified with 0.4% BSA (mDPBS) for 90min. Oocytes with or without blue coloration of the cytoplasm were designated as BCB+ and BCB−, respectively. In Experiment 1, the percentage of selected oocytes following the BCB test and oocyte diameter were evaluated (13, 26 or 39μM BCB in mDPBS). In Experiment 2, the relationship between follicle size (small: <3mm, medium: 3–5mm and large: >5mm), oocyte diameter and percentage of selected BCB+ oocytes were evaluated. In Experiment 3, the effectiveness of different concentrations BCB to select the oocytes most competent for IVEP, were evaluated. Oocytes were tested in 3 groups: a control (immediately cultured); holding-control (kept in mDPBS for 90min before being cultured); and a treatment-incubation (incubated with BCB). In Experiment 1, the percentage and diameter of BCB+ oocytes obtained after exposure to the different BCB concentrations were similar. The diameter of the BCB+ oocytes was significantly greater than the diameter of the BCB− oocytes for each concentration (P<0.01). In Experiment 2, the oocyte diameter and percentage of fully grown (BCB+) oocytes increased with follicular size. The percentage of the BCB+ oocytes from the small (37.9%), medium (53.5%) and large (72.9%) ovarian follicles was respectively lower (P<0.001), similar (P>0.05) and higher (P<0.0001) than those of the BCB− oocytes (62.1, 46.5 and 27.1%, respectively). In addition, the diameter of the BCB+ oocytes recovered from the medium (154.3±5.3μm) and small follicles (150.1±5.3μm) was larger (P<0.01), than those of the BCB− oocytes (148.1±6.0 and 145.9±5.7μm, respectively). In Experiment 3, BCB+ oocytes yielded more blastocysts than BCB− oocytes, control and holding-control oocytes. However, cleavage and blastocyst (development) rates was not significantly affected by BCB concentration. In conclusion, it can be said that the BCB test may be a useful way of selecting developmentally competent sheep oocytes for increased IVEP success.

The quality of porcine oocytes is affected by sexual maturity of the donor gilt

Although differences in the quality of oocytes derived from young gilts and adult sows are well documented, evidence concerning gametes of pre-pubertal and cycling gilts is scarce and inconsistent. The aim of this work was to establish whether sexual maturity of gilts affects the quality of their oocytes with the use of the brilliant cresyl blue (BCB) test, oocyte diameter and apoptosis. Ovarian morphology was evaluated, and gonads with corpus luteum or albicans were recognized as originating form cycling gilts (C) and those with follicles as originating form pre-pubertal females (P). Altogether 952 cumulus-oocyte complexes (COCs; group P: 554; group C: 398) were examined, whereas 149 COCs, not subjected to BCB test, served as a control for TUNEL. COCs of proper morphology were evaluated by the BCB test which differentiated two categories of gametes: more competent, BCB+, and less competent BCB- oocytes. The control group comprised oocytes of proper morphology aspirated from ovaries of P and C gilts not subjected to BCB test. Finally five groups of COCs were matured in vitro: 1/P-BCB+, 2/P-BCB-, 3/C-BCB+, 4/ C-BCB- and 5/ control. Significantly more large oocytes (≥ 120 µm), more BCB+ oocytes and more high quality (both BCB+ and ≥ 120 µm) oocytes originated from ovaries of cycling gilts than pre-pubertal gilts (p<0.001). The rate of mature oocytes at the MII stage differed significantly between C-BCB+ (68.5%) and P-BCB+ (32.9%) oocytes. The incidence of apoptosis among BCB-treated oocytes after in vitro maturation was 21.4% and was similar to that observed in control oocytes (17.4%). BCB+ oocytes from cycling gilts showed significantly higher (28.7%) incidence of apoptosis than that of the group P (16.2%). Interestingly, high quality oocytes displayed a similar level of apoptosis regardless of the donor puberty. We demonstrated that C gilts provided more BCB+ oocytes as well as more large oocytes than P gilts, although C-BCB+ oocytes showed higher apoptotic rate. In conclusion, high incidence of apoptosis and a big variation in the diameter of more competent BCB+ oocytes make the BCB test a less effective selection tool than previously reported.

Zona pellucida glycoprotein 3 (pZP3) and integrin β2 (ITGB2) mRNA and protein expression in porcine oocytes after single and double exposure to brilliant cresyl blue test

Brilliant cresyl blues (BCB) staining test is a useful tool in assessing the competence of cumulus-oocyte-complexes (COCs) in several mammalian species. It is mostly used to select gametes after they are recovered from the ovary or before and after IVM to isolate those oocytes that reach developmental competency. However, there is evidence that double exposure to BCB test may lead to impaired fertilization or even have a toxic effect on cells. The aim of the present study was to investigate the expression pattern of sperm-egg interaction molecules in oocytes after single and double exposure to BCB test. Follicles were dissected from porcine ovaries after slaughter and aspirated COCs were cultured in standard porcine IVM culture medium (TCM 199) for 44 h. The BCB test was applied to COCs before and after IVM. In developmentally competent oocytes, assessed by determining the activity of glucose-6-phosphate dehydrogenase (G6PDH; BCB test), real-time quantitative PCR reaction methods, western blot and confocal microscopy analysis were applied to determine the transcript levels of porcine zona pellucida glycoprotein 3 (pZP3), and integrin beta 2 (ITGB2), as well as the levels of pZP3 and ITGB2 proteins. In the control group, assessment of the expression of the investigated genes was performed before and after IVM without BCB test. We observed a significantly higher level of pZP3 mRNA in oocytes after single exposure to BCB test compared to control before and after IVM (P < 0.001), and to double staining (P < 0.05). The level of ITGB2 mRNA was also increased in gametes after single exposure to BCB test as compared to control before and after IVM (P < 0.001, P < 0.01, respectively), and double staining (P < 0.05). Western blot analysis demonstrated a higher level of pZP3 protein in oocytes after single staining with BCB as compared to control both before and after IVM (P < 0.001, P < 0.05, respectively) and double staining (P < 0.05). Confocal microscopic observations have revealed the same pattern of increased level of pZP3 and ITGB2 expression after single exposure to BCB test. In both cases we detected specific cytoplasmic localization of both proteins. The ITGB2 protein has zona pellucida and membrane localization in control oocytes before IVM. After IVM and after single exposure to BCB, ITGB2 was also strongly detected in the cytoplasm. In both cases, after double exposure to BCB both proteins were detected only partially in the cytoplasm. Our results suggest that (i) single exposure to BCB increased the expression of sperm-oocyte interaction genes, (ii) double exposure to BCB leads to only partial expression of pZP3 and ITGB2 in oocyte cytoplasm, (iii) the BCB staining test itself may be a cause of specific pZP3 translocation from the zona pellucida to the cytoplasm, and that (iv) in vitro maturation of oocytes may increase ITGB2 expression and translocation from the zona pellucida to the cytoplasm.

205 GLUCOSE-6-PHOSPHATE DEHYDROGENAZE ACTIVITY IN OVINE OOCYTES IS ASSOCIATED WITH PRE-IMPLANTATION EMBRYONIC DEVELOPMENT IN VITRO

A large proportion of ovine oocytes fail to develop into viable embryos following maturation, fertilization, and culture in vitro. Accurate, fast, and noninvasive predictors of ovine oocyte quality are therefore in urgent need for oocyte selection before in vitro maturation (IVM). Recent studies have shown that oocyte competence can be predicted through the presence of the glucose-6-phosphate dehydrogenase (G6PD) enzyme, as indicated by brilliant cresyl blue (BCB), a dye that can be degraded by G6PD. Thus, oocytes that have completed their growth phase show decreased G6PD activity and exhibit cytoplasm with a blue colouration (BCB+), whereas growing oocytes are expected to have a high level of G6PD, which results in colourless cytoplasm (BCB–). The brilliant cresyl blue staining test, as a noninvasive intrinsic criterion, has been successfully used to identify the more competent oocytes in various species. Therefore, this study aimed to investigate whether BCB staining, as an indicator of G6PD activity, can be used to select developmentally competent ovine oocytes before IVM and thereby increase the efficiency of in vitro embryo production. Ovine ovaries were obtained from a local slaughterhouse and transported to the laboratory, where cumulus–oocyte complexes (COC) were recovered by slicing the ovaries. Only oocytes with one or more complete layers of unexpanded cumulus cells and a homogeneous cytoplasm were used. The COC were exposed to 26 mM BCB diluted in modified Dulbecco’s PBS for 90 min at 39°C in humidified air. After BCB exposure, the COC were examined under a stereomicroscope and divided into 2 groups: BCB+ (blue cytoplasm, low G6PD activity) and BCB– (colourless cytoplasm, high G6PD activity). Cumulus–oocyte complexes in the control group were incubated for IVM directly after selection, without exposure to BCB dye. After IVM, oocytes were subjected to IVF followed by embryo culture for 7 days (5% CO2, 39°C, humidified air). Results were analysed by a chi-square test, and P &lt; 0.05 was considered statistically significant. The proportion of oocytes that cleaved by Day 2 after insemination was significantly (P &lt; 0.05) higher for the control and BCB+ groups [67.3% (68/101) and 71.7% (81/113), respectively] than for the BCB– group [50.5% (46/91)]. Significant differences among groups were also observed on Day 7 after fertilization, when the embryos reached the blastocyst stage of development. The BCB+ group yielded a significantly (P &lt; 0.05) higher proportion of blastocysts [34.5% (39/113)] than both the control [20.8% (21/101)] and BCB– [4.3% (4/93)] groups. In addition, the blastocyst rate of development in the control group was significantly (P &lt; 0.05) higher than that for the BCB– group. In conclusion, results of this study show that selection of ovine oocytes based on G6PD activity through the BCB test can be used as an efficient predictor of in vitro embryonic developmental competence. This positive predictive parameter of oocyte quality may also be useful in increasing the efficiency of blastocyst production during in vitro embryo production procedures in the ovine.

Disturbances of nuclear maturation in BCB positive oocytes collected from peri-pubertal gilts

The developmental competence (quality) of oocytes is affected by several factors linked to their intrinsic properties and also to growth and maturation environment. Donor puberty and chromosomal complement are one of the main factors influencing oocyte quality. A high rate of porcine oocytes matured in vitro is chromosomally imbalanced. Moreover, there is no published data on chromosomal aberrations in oocytes selected by the brilliant cresyl blue (BCB) test. Therefore, the aim of this study was to analyze whether BCB positive (BCB+) oocytes derived from ovaries of peripubertal gilts (prepubertal NCL and cyclic CL) differ with respect to the incidence of numerical chromosome aberrations. COCs collected from NCL and CL ovaries were selected by the BCB test. Only BCB+ oocytes were matured in vitro and subjected to FISH analysis using molecular probes for chromosome pairs 1 and 10. The rate of BCB+ oocytes was similar for both groups of ovaries (NCL 80%, CL 92%). Altogether 554 oocytes were fixed and 471 oocytes at the MII stage were analyzed cytogenetically. Diploid (2MII) and aneuploid oocytes were detected. The contribution of MII oocytes was similar for NCL (85%) and CL (90%) group. Chromosomally aberrant BCB+ oocytes accounted for 18.0% and ranged from 13.7% for CL and 22.2% for NCL ovaries. Diploidy was a predominant anomaly observed (11.2%) with a significantly higher frequency in BCB+ oocytes of pre-pubertal (16.7%) than cyclic gilts (5.6%, P < 0.05). Aneuploid oocytes occurred with similar rate in NCL (6.7%) and CL (8.5%) females. The majority of aneuploid spreads (72.2%; P < 0.01) concerned the chromosome pair 10. The overall rate of disomy (56%) and nullisomy (44.4%) was similar. We have shown that donor puberty affects the incidence of chromosomal abnormalities in porcine oocytes matured in vitro. Significantly more diploid oocytes was derived from prepubertal ovaries, whereas the frequency of aneuploidy was similar in NCL and CL gilts.

264 MITOCHONDRIAL DNA COPY NUMBER IN OOCYTES OF PREPUBERTAL AND CYCLIC GILTS

In many domestic species (pig, cow, sheep), oocytes from prepubertal females show impaired quality when compared with those from adult animals. Incomplete cytoplasmic maturation is thought to be the main factor responsible for reduced developmental competence of embryos derived from prepubertal oocytes. The status of ooplasm maturation is also reflected by the copy number of mitochondrial DNA (mtDNA). Because replication of mtDNA ceases when oocytes reach their final size and occurs again at the blastocyst stage, the mtDNA copy number is a proved marker of oocyte quality in the pig (El Shourbagy et al. 2006 Reproduction 131, 233–245). The number of mtDNA copies in the grown oocyte is crucial to sustain the first embryonic divisions. To increase the rate of good-quality blastocysts, oocytes of domestic animals have been evaluated by the brilliant cresyl blue test (BCB). According to El Shourbagy et al. (2006), more competent BCB+ oocytes possess higher copy number of mtDNA (on average 222 446) than do their BCB– counterparts (115 352). However, there are no published data on the variation in mtDNA copy number in oocytes derived from ovaries of prepubertal (NCL) and cyclic (CL) gilts. Ovaries of NCL and CL gilts were collected in a local slaughterhouse. Cumulus–oocyte complexes (COC) were aspirated from nonatretic follicles 2 to 6 mm in diameter and evaluated morphologically. Only COC with a proper morphology were subjected to the BCB test. A group of non-BCB-treated COC served as control. Four groups of COC were collected: BCB+ (CL, NCL) and control (CL, NCL). Follicular cells attached to oocytes were removed by pipetting, and completely denuded gametes were individually frozen in liquid nitrogen. Analysis of the mtDNA copy number included isolation of the total DNA followed by amplification of the Cytochrome b (CYTB) gene by real-time PCR (one copy per one mitochondrial genome). Differences in mtDNA copy number among experimental groups were evaluated by Student’s t-test. To date, 30 BCB+ oocytes have been analysed individually (15 CL and 15 NCL). The analysed parameter varied in a wide range from 79 852 to 522 712 copies in CL oocytes and from 52 270 to 287 852 copies in NCL oocytes. Oocytes from cyclic gilts contained significantly more mtDNA copies (on average 267 524) than did gametes of prepubertal females (179 339; P &lt; 0.05). The data on the mtDNA copy number in the control oocytes are currently under investigation. The preliminary results indicate that impaired oocytes quality of prepubertal gilts may be also attributed to the reduced copy number of mtDNA. This project was sponsored by MSHE Poland (grant no. 451/N-COST/2009/0).

Advances in Swine In Vitro Embryo Production Technologies

Recent advances in new technologies to produce cloned and genetically modified pigs involve manipulating oocytes and/or embryos in vitro. Although a great deal of progress has been made, the current IVM-IVF systems still result in major problems: a high rate of polyspermy; and a low development rate and low quality of blastocysts for in vitro compared with the in vivo-produced embryos. This study summarizes recent advancements in IVM-IVF-IVC porcine systems. Recent methods to select monospermic embryos are also discussed. Finally, achievements in vitrification and in somatic cell nuclear transfer are discussed.