Abstract
Purpose
To determine whether G6PDH-activity measured by Brilliant Cresyl Blue known as BCB dye, predicts developmental competence within cohorts of ovine oocytes.
Methods
Ovine oocytes were exposed to BCB staining and categorized into two groups: BCB+ (blue cytoplasm, low G6PDH-activity) and BCB- (colorless cytoplasm, high G6PDH-activity). After maturation in vitro, oocytes were subjected to fertilization followed by in vitro embryo culture.
Results
We observed a significant difference in oocyte diameter considering BCB+ and BCB- oocytes. BCB+ and Control groups showed significantly higher maturation rates compared to BCB- group. There were significantly more cleaved embryos in BCB+ and control groups than in BCB- group. Blastocyst rate was significantly higher for BCB+ group compared to control and BCB- groups with control group being significantly higher than BCB- group.
Conclusion
G6PDH-activity is a strong predictive marker of oocyte competence and may be useful in identifying oocytes with a good prognosis for further develop.
Keywords: Ovine, Oocyte, Glucose-6-phosphate dehydrogenase, BCB, Developmental competence
Introduction
Evidence from experimental and clinical studies has shown that from a total pool of aspirated gametes, a female has only a limited number of oocytes that are capable of undergoing full development [1–3]. Therefore, establishment of criteria for selecting the most viable oocytes/embryos may rectify this limitation and be of considerable benefit for the advancement of assisted reproductive technologies (ARTs). These advantages are even more important for human reproduction, where selection of embryos to be transferred based on in vitro development up to progressed stages of embryonic development is not allowed in a wide range of countries [4]. In this regard, the ability to preserve oocyte/embryo viability is the ultimate request for predictive markers of oocyte competence. To evaluate this concern, we aimed to use an ovine model of oocyte competence that can be extrapolated and to some extent give a picture of what results from human ARTs could be like.
Traditionally, morphological parameters, such as homogeneity of cytoplasm, number of cell layers, and compactness of cumulus cells are utilized as markers of oocyte developmental competence [1]. However, the predictive accuracy of the morphological features is subjective and interobserver variance is known to be great [5]. Attempts to characterize intrinsic attributes associated with oocyte development have been of particular attention over the last decades [6–9], mainly due to their more precise specification and objective value [7, 10–13].
Glucose-6-phosphate dehydrogenase (G6PDH), as an important protein in developmental competence, is synthesized in oocytes during oogenesis while they are in their growth phase. This enzyme is involved in generation of ribose 5-phosphate and NADPH through the pentose phosphate pathway and also plays an important role in a wide range of cellular processes, such as reduction of intracellular glutathione, and nucleotide synthesis [14].
Intracellular activity of G6PDH in oocytes prior to in vitro maturation can be measured using an electron acceptor, namely Brilliant Cresyl Blue (BCB) dye [15]. The BCB test is based on the capability of G6PDH to convert the dye from blue to a colorless state. Due to sufficient concentrations of the enzyme in oocytes undergoing growth, the dye is reduced to colorless, while fully-grown oocytes remain blue due to the low amount of cytoplasmic G6PDH-activity.
In spite of contrary reports [16, 17], our experimental works [8, 12, 18] and other numerous reports [13, 14, 19–23] demonstrate that there is no deleterious effect of the staining procedure on viability of the oocytes. This approach has successfully been used to identify oocytes that possess superior developmental capacity in mice [13] and some domestic animal species [14, 20, 23]. However, its predictive value for developmental competence has not been tested in sheep oocytes.
Therefore, the aim of the present study was to investigate the predictive accuracy of G6PDH-activity through the BCB test by assessing oocyte diameter, meiotic competence and subsequent blastocyst formation in ovine model. Evaluation of such criteria is needed and may lead to the development of an efficient approach which can be exploited to assess the viability of oocytes in ART programs. Taken together, our present work aimed to generate knowledge which could point a way to assess developmental competence of human oocytes being also prognostic for early embryo quality.
Material and methods
Unless otherwise stated, all chemicals used in this study were purchased from sigma chemical Co. (St. Louis, Mo, USA) and Gibco (Grand Island, NY, USA).
Oocyte collection
Sheep ovaries were obtained from an abattoir and transported to the laboratory in saline at 32–35°C, within 1–2 h after collection. Upon arrival, the ovaries were washed three times with pre-warmed (37°C) fresh saline. Cumulus-oocyte complexes (COCs) were collected from follicles by slicing method and classified as healthy according to the method of Thompson et al. [24].
Brilliant cresyl blue staining
The COCs were subjected to 26 μM BCB diluted in modified PBS, and then incubated for 90 min at 38.5°C in humidified air atmosphere [8]. After washing, the COCs were examined under stereomicroscope and categorized into two groups: COCs with any degree of blue coloration in cytoplasm were considered as BCB+ (low G6PDH-activity) as shown in Fig. 1 (Black arrows) and COCs without visual blue coloration were identified as BCB- (high G6PDH-activity) as presented in Fig. 1 (White arrows). A number of COCs were not exposed to the BCB dye and were assigned to control groups to associate the G6PDH-activity of the oocytes with meiotic and developmental competences.
Fig. 1.
Oocytes with different G6PDH activities; BCB+ oocytes (blue cytoplasm due to low G6PDH-activity) indicated by black arrows and BCB- oocytes (colorless cytoplasm due to high G6PDH-activity) illustrated by white arrows
Measurement of oocyte diameter
Diameters were measured for a number of oocytes immediately after BCB incubation and removal of the cumulus cells using hyaluronidase (600 IU/ml) in TCM-199. Oocytes were examined at 200× magnification on an inverted microscope. The values for oocyte diameter were the average of two independent measurements. These cumulus-free oocytes were frozen to be used in another study.
In vitro maturation
The COCs were washed three times in maturation medium consisted of bicarbonate-buffered TCM-199 with 2 mM L-glutamine supplemented with 10% fetal bovine serum, 5.5 mg/ml sodium pyrovate, 25 μg/ml gentamycin sulphate, 5.0 μg/ml LH, 0.5 μg/ml FSH and 1 μg/ml Estradiol. In vitro maturation (IVM) was performed by culturing approximately 10 COCs for 24 h in 50 μl maturation medium droplet under mineral oil at 39°C, 5% CO2 in air with maximum humidity.
Nuclear chromatin evaluation
After IVM, a number of COCs were freed from surrounding cumulus cells, stained with 2.5 mg/mL Hoechst 33258 in 3:1 (v/v) glycerol/PBS, and kept until observation. Oocytes were evaluated in relation to their meiotic stage under an epifluorescence microscope (Nikon Eclipse-600) as previously described [25]. Oocytes in which condensed or slightly diffused chromatin could be identified, were classified as being in germinal vesicle (GV) stage. Oocytes possessing slightly condensed or clumped chromatin were classified as being in germinal vesicle breakdown (GVBD) stage. Oocytes with strongly condensed chromatin or a metaphase plate but no polar body, were classified as being in metaphase-I (MI) stage. Oocytes with either extruded first polar body or two chromatin masses were classified as being in metaphase-II (MII) stage of the maturation process.
In vitro fertilization
Following IVM, COCs were washed twice in HEPES-buffered synthetic oviductal fluid (HSOF) and groups of 10 oocytes were transferred to 44 μl fertilization drops overlaid with equilibrated mineral oil. The fertilization medium was SOF supplemented with 2% (v/v) estrous sheep serum. A straw of frozen sperm was thawed at 37°C for 1 min, and then carefully layered on top of the Percoll gradient system. Sperm was diluted to 50 × 106 sperm cells/ml in HSOF, which would produce a 2 × 106 spermatozoa/ml in fertilization drops. Fertilization of matured oocytes was completed by adding 2 μl diluted sperm, 4 IU/ml heparin, PHE (20 μM penicillamine, 10 μM hypotaurine, 1 μM epinephrine) into the 44 μl fertilization drops [26]. Oocytes and sperm were co-cultured for 18 h in incubator under the same conditions as described for IVM.
In vitro culture and data collection
Eighteen hours post-insemination, cumulus cells were removed and presumptive zygotes were washed three times in HSOF. Thereafter, 15 to 20 putative zygotes placed into 30 μl culture drops consisting of SOFaa (2% BME-essential amino acids, 1% MEM-nonessential amino acids) supplemented with 8 mg/ml bovine serum albumin, 1 mM glutamine, 0.34 mM tri-sodium citrate and 2.77 mM myo-inositol under equilibrated mineral oil [26]. Developmental data were recorded for cleavage- and blastocyst-stage embryos at day 3 and day 9 post-insemination respectively. The day of insemination was considered as day 0.
Experimental design
Experiment I. G6PDH-activity in association with oocyte diameter at retrieval time
In order to determine the association between G6PDH-activity in ovine oocyte and diameter of the oocyte at retrieval time, oocytes diameters were measured immediately after BCB incubation and removal of the cumulus cells.
Experiment II. G6PDH-activity in relation to meiotic competence after IVM
The relation between G6PDH-activity of the oocytes and meiotic competence was determined after IVM. Nuclear status was determined following maturation in vitro for each BCB+, BCB-, and Control oocytes after fixing and staining.
Experiment III. G6PDH-activity in association with developmental competence after IVF
To determine whether G6PDH-activity in oocyte has a positive predictive value for developmental competence, matured COCs obtained from different groups were fertilized in vitro, and subsequent developmental capacity in terms of cleavage and development to the blastocyst stage was recorded.
Statistical analysis
The diameter of BCB+ and BCB- oocytes were analyzed by one-way ANOVA followed by Tukey’s pairwise comparisons. The nuclear status of oocytes after IVM, as well as cleavage and blastocyst rate after IVF/IVC was compared between groups of oocytes by Chi-square analysis.
Results
Overall, a total of 1,238 ovine COCs were collected, of which 242 COCs were used as control groups and 996 COCs were exposed to BCB test. After staining with BCB, 552 (55.4%) COCs were classified as BCB+ group and 444 (44.6%) COCs were classified as BCB- group.
Experiment I. G6PDH-activity in association with oocyte diameter at retrieval time
All together, 380 oocytes were measured for their diameters. A significant difference was observed in oocyte diameter considering BCB+ and BCB- oocytes. Diameter of BCB+ oocytes (n = 212; 147.5 ± 7.5 μm) were significantly greater (P < 0.05) than those of BCB- oocytes (n = 168; 130.1 ± 8.8 μm).
Experiment II. G6PDH-activity in relation to meiotic competence after IVM
At 24 h after IVM, 227, 185 and 141 oocytes were used to determine nuclear status of BCB+, BCB- and Control groups respectively. Significantly more oocytes of BCB+ (78.9%) and Control group (73.8%) reached MII stage compared to BCB- group (52.4%, P < 0.05). Accordingly, the rate of oocytes with MI nuclear status observed after culture was significantly lower in BCB+ (18.9%) and Control group (19.9%) compared to BCB- group (38.4%, P < 0.05). However, there was no difference among the groups in terms of the rate of oocytes with GV and GVBD nuclear status (Table 1).
Table 1.
Relationship between G6PDH-activity and nuclear status in ovine oocytes after in vitro maturation (cumulative results of five replicates)
| Group | n | Nuclear status | |||
|---|---|---|---|---|---|
| GV n (%) | GVBD n (%) | MI n (%) | MII n (%) | ||
| BCB+ | 227 | 0 (0) | 5 (2.2) | 43 (18.9)a | 179 (78.9)a |
| BCB- | 185 | 5 (2.7) | 12 (6.5) | 71 (38.4)b | 97 (52.4)b |
| Control | 141 | 2 (1.4) | 7 (4.9) | 28 (19.9)a | 104 (73.8)a |
a, bDifferent superscripts within the columns indicate a significant difference (P < 0.05)
Experiment III. G6PDH-activity in association with developmental competence after IVF/IVC
After maturation in vitro, 113, 91 and 101 oocytes were fertilized for BCB+, BCB- and Control groups respectively. Proportional data for cleavage and development to blastocysts are summarized in Table 2. Cleavage rate differs significantly (P < 0.05) among BCB+ (71.7%) and BCB- (50.5%) groups whereas no difference was found between Control group (67.3%) and BCB+ group. The blastocyst rate of BCB+ oocytes was significantly (P < 0.05) higher (34.5%), as compared to both the Control (20.8%) and BCB- (4.4%) groups. Also, the rate of blastocyst development in Control group was significantly (P < 0.05) higher compared to BCB- group.
Table 2.
Relationship between G6PDH-activity and embryo development in ovine oocytes matured in vitro and subjected to in vitro fertilization (cumulative results of five replicates)
| Group | n | Embryo development | |
|---|---|---|---|
| Cleaved n (%) | Blastocyst n (%) | ||
| BCB+ | 113 | 81 (71.7)a | 39 (34.5)a |
| BCB- | 91 | 46 (50.5)b | 4 (4.4)b |
| Control | 101 | 68 (67.3)a | 21 (20.8)c |
a, b, cDifferent superscripts within the columns indicate a significant difference (P < 0.05)
Discussion
Improvements in oocyte screening have been achieved in several species by application of BCB test [13, 14, 20, 27–29], a concept that we have successfully incorporated into the design of our study. To determine the validity of BCB test for in vitro ovine oocyte selection, G6PDH-activity was associated with oocyte diameter, meiotic competence and subsequent pre-implantation development.
The oocyte diameter has often been used as a marker for oocyte maturity or meiotic competence [30]. One association, evidenced in a variety of species, is the inverse relationship between G6PDH-activity and oocyte diameter [13, 21, 23, 28, 30] as observed in our study. Moreover, either oocyte volume [7], or size of originating follicle [31], associated with G6PDH-activity. In this study COCs were isolated from antral follicles by slicing methods. Thus, COCs may have been recovered not only from antral follicles on the surface, but also from smaller antral follicles from inner parts of the ovary which might have caused rather lower diameter of BCB- oocytes compared to BCB+ oocytes [19].
Nuclear maturation is characterized by forming the second metaphase plate and extruding the first polar body [32]. The lower oocyte diameter recorded for BCB- oocytes compared to those of BCB+ oocytes have raised the question whether differences in G6PDH-activity might have been reflected in nuclear maturation. Our data support that low G6PDH-activity in ovine oocytes is strongly associated with elevated nuclear maturation after IVM. The lower meiotic competence of BCB- oocytes may be attributed, in part, to their smaller follicles of origin, which in turn is likely to be correlated with smaller size of these oocytes. Our finding is in accordance with a recent study by Egerszegi et al. [33] who demonstrated a higher maturation rate in BCB+ pig oocyte compared to BCB- oocytes. Further evidence is provided by a study in mice, which shows that BCB+ oocytes gained better cytoplasmic maturity determined as the intracellular glutathione level, pattern of mitochondrial distribution and higher developmental competence after IVM compared to BCB- oocytes [13]. Of note, in contrast to ours and findings of others, Shirazi et al. [25] reported no correlation between the size of sheep oocytes and their ability to reach MII stage. However, oocyte selection in Shirazi’s work was based on morphological characteristics and not on G6PDH-activity which could explain these contradictory observations on meiotic competence regarding smaller ovine oocytes prior to IVM. Another explanation for the discrepancy may be that the duration of maturation period was longer (26–27 h) in Shirazi’s study, compared to our work (24 h). If so, it could be expected that BCB- oocytes might mature more slowly compared to BCB+ oocytes. Further investigation is required on the kinetics of meiotic resumption and IVM conditions for BCB- oocytes.
In order to elucidate if G6PDH-activity of ovine oocytes is related to subsequent developmental competence, COCs were classified due to their G6PDH-activity, followed by maturation, fertilization, and subsequent culture of the resultant embryos up to the blastocyst stage. Table 2 shows that the rate of cleaved embryos 3 days post-insemination was significantly lower in the BCB- group than in either Control group or in oocytes from BCB+ group. At day 9 post-insemination, the blastocyst rate in BCB+ oocytes was significantly higher compared to the Control and BCB- groups. This is consistent with previous reports in goat [23, 29], heifer [22], cattle [14, 19], and buffalo [20]; thus confirming the inverse relationship between G6PDH-activity in oocyte prior to IVM and blastocyst formation following fertilization in vitro. To the best of our knowledge, current report is the first to document this relationship in sheep embryos. According to Alm et. al [14], the better performance of BCB+ oocytes compared to BCB- oocytes can be related to their better cytoplasmic maturation during the final phases of folliculogenesis [14]. An oocyte that has not completed cytoplasmic maturation is of insufficient quality, and thus unable to complete normal developmental processes successfully [34]. In further support of the prognostic value of the BCB test, previous studies also indicated that differences in developmental competence of oocytes derived from high and low G6PDH-activity were also accompanied by differences in the relative abundance of transcripts related to the various molecular events and processes regulating oocyte competence [12, 18, 35].
Conclusion
The results of this study confirm a strong relationship between G6PDH-activity, oocyte diameter, nuclear maturation, and subsequent blastocyst yield in ovine oocytes.
Using BCB test, we successfully incorporated the G6PDH-activity into selecting a subset of oocytes with superior developmental capacity. Collectively the present work points a way to assess oocyte’s developmental competence in the ovine model which could also be helpful for human ARTs. Further studies, however, are needed to fully address that issue.
Acknowledgments
The authors thank the members of their own laboratories for their helpful discussions. We are also indebted to Miss Lida Langroudi for editing the manuscript.
Footnotes
Capsule Glucose-6-phosphate dehydrogenase activity in ovine oocytes prior to in vitro maturation is associated with oocyte diameter, meiotic competence and developmental capacity to the blastocyst stage in vitro.
References
- 1.Ebner T, Moser M, Sommergruber M, Tews G. Selection based on morphological assessment of oocytes and embryos at different stages of preimplantation development: a review. Hum Reprod Update. 2003;9:251–62. doi: 10.1093/humupd/dmg021. [DOI] [PubMed] [Google Scholar]
- 2.Hammadeh ME, Fischer-Hammadeh C, Ali KR. Assisted hatching in assisted reproduction: a state of the art. J Assist Reprod Genet. 2011;28:119–28. doi: 10.1007/s10815-010-9495-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Rhodes TL, McCoy TP, Higdon HL, 3rd, Boone WR. Factors affecting assisted reproductive technology (ART) pregnancy rates: a multivariate analysis. J Assist Reprod Genet. 2005;22:335–46. doi: 10.1007/s10815-005-6794-1. [DOI] [PubMed] [Google Scholar]
- 4.Borini A, Cattoli M, Bulletti C, Coticchio G. Clinical efficiency of oocyte and embryo cryopreservation. Ann N Y Acad Sci. 2008;1127:49–58. doi: 10.1196/annals.1434.012. [DOI] [PubMed] [Google Scholar]
- 5.Wang Q, Sun QY. Evaluation of oocyte quality: morphological, cellular and molecular predictors. Reprod Fertil Dev. 2007;19:1–12. doi: 10.1071/RD06103. [DOI] [PubMed] [Google Scholar]
- 6.Matos DG, Gasparrini B, Pasqualini SR, Thompson JG. Effect of glutathione synthesis stimulation during in vitro maturation of ovine oocytes on embryo development and intracellular peroxide content. Theriogenology. 2002;57:1443–51. doi: 10.1016/S0093-691X(02)00643-X. [DOI] [PubMed] [Google Scholar]
- 7.Shourbagy SH, Spikings EC, Freitas M, St John JC. Mitochondria directly influence fertilisation outcome in the pig. Reproduction. 2006;131:233–45. doi: 10.1530/rep.1.00551. [DOI] [PubMed] [Google Scholar]
- 8.Koester M, Mohammadi-Sangcheshmeh A, Montag M, Rings F, Schimming T, Tesfaye D, et al. Evaluation of bovine zona pellucida characteristics in polarized light as a prognostic marker for embryonic developmental potential. Reproduction. 2011;141:779–87. doi: 10.1530/REP-10-0471. [DOI] [PubMed] [Google Scholar]
- 9.Wang WH, Day BN. Development of porcine embryos produced by IVM/IVF in a medium with or without protein supplementation: effects of extracellular glutathione. Zygote. 2002;10:109–15. doi: 10.1017/S0967199402002150. [DOI] [PubMed] [Google Scholar]
- 10.Ambruosi B, Lacalandra GM, Iorga AI, Santis T, Mugnier S, Matarrese R, et al. Cytoplasmic lipid droplets and mitochondrial distribution in equine oocytes: implications on oocyte maturation, fertilization and developmental competence after ICSI. Theriogenology. 2009;71:1093–104. doi: 10.1016/j.theriogenology.2008.12.002. [DOI] [PubMed] [Google Scholar]
- 11.Spikings EC, Alderson J, St John JC. Regulated mitochondrial DNA replication during oocyte maturation is essential for successful porcine embryonic development. Biol Reprod. 2007;76:327–35. doi: 10.1095/biolreprod.106.054536. [DOI] [PubMed] [Google Scholar]
- 12.Torner H, Ghanem N, Ambros C, Holker M, Tomek W, Phatsara C, et al. Molecular and subcellular characterisation of oocytes screened for their developmental competence based on glucose-6-phosphate dehydrogenase activity. Reproduction. 2008;135:197–212. doi: 10.1530/REP-07-0348. [DOI] [PubMed] [Google Scholar]
- 13.Wu YG, Liu Y, Zhou P, Lan GC, Han D, Miao DQ, et al. Selection of oocytes for in vitro maturation by brilliant cresyl blue staining: a study using the mouse model. Cell Res. 2007;17:722–31. doi: 10.1038/cr.2007.66. [DOI] [PubMed] [Google Scholar]
- 14.Alm H, Torner H, Lohrke B, Viergutz T, Ghoneim IM, Kanitz W. 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. Theriogenology. 2005;63:2194–205. doi: 10.1016/j.theriogenology.2004.09.050. [DOI] [PubMed] [Google Scholar]
- 15.Tiffin GJ, Rieger D, Betteridge KJ, Yadav BR, King WA. Glucose and glutamine metabolism in pre-attachment cattle embryos in relation to sex and stage of development. J Reprod Fertil. 1991;93:125–32. doi: 10.1530/jrf.0.0930125. [DOI] [PubMed] [Google Scholar]
- 16.Pawlak P, Pers-Kamczyc E, Renska N, Kubickova S, Lechniak D. Disturbances of nuclear maturation in BCB positive oocytes collected from peri-pubertal gilts. Theriogenology. 2011;75:832–40. doi: 10.1016/j.theriogenology.2010.10.025. [DOI] [PubMed] [Google Scholar]
- 17.Pawlak P, Renska N, Pers-Kamczyc E, Warzych E, Lechniak D. The quality of porcine oocytes is affected by sexual maturity of the donor gilt. Reprod Biol. 2011;11:1–18. doi: 10.1016/s1642-431x(12)60060-6. [DOI] [PubMed] [Google Scholar]
- 18.Ghanem N, Holker M, Rings F, Jennen D, Tholen E, Sirard MA, et al. Alterations in transcript abundance of bovine oocytes recovered at growth and dominance phases of the first follicular wave. BMC Dev Biol. 2007;7:90. doi: 10.1186/1471-213X-7-90. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Bhojwani S, Alm H, Torner H, Kanitz W, Poehland R. Selection of developmentally competent oocytes through brilliant cresyl blue stain enhances blastocyst development rate after bovine nuclear transfer. Theriogenology. 2007;67:341–5. doi: 10.1016/j.theriogenology.2006.08.006. [DOI] [PubMed] [Google Scholar]
- 20.Manjunatha BM, Gupta PS, Devaraj M, Ravindra JP, Nandi S. Selection of developmentally competent buffalo oocytes by brilliant cresyl blue staining before IVM. Theriogenology. 2007;68:1299–304. doi: 10.1016/j.theriogenology.2007.08.031. [DOI] [PubMed] [Google Scholar]
- 21.Mota GB, Batista RI, Serapiao RV, Boite MC, Viana JH, Torres CA, et al. Developmental competence and expression of the MATER and ZAR1 genes in immature bovine oocytes selected by brilliant cresyl blue. Zygote.18: 209–16. [DOI] [PubMed]
- 22.Pujol M, Lopez-Bejar M, Paramio MT. Developmental competence of heifer oocytes selected using the brilliant cresyl blue (BCB) test. Theriogenology. 2004;61:735–44. doi: 10.1016/S0093-691X(03)00250-4. [DOI] [PubMed] [Google Scholar]
- 23.Rodriguez-Gonzalez E, Lopez-Bejar M, Izquierdo D, Paramio MT. Developmental competence of prepubertal goat oocytes selected with brilliant cresyl blue and matured with cysteamine supplementation. Reprod Nutr Dev. 2003;43:179–87. doi: 10.1051/rnd:2003012. [DOI] [PubMed] [Google Scholar]
- 24.Thompson JG, Gardner DK, Pugh PA, McMillan WH, Tervit HR. Lamb birth weight is affected by culture system utilized during in vitro pre-elongation development of ovine embryos. Biol Reprod. 1995;53:1385–91. doi: 10.1095/biolreprod53.6.1385. [DOI] [PubMed] [Google Scholar]
- 25.Shirazi A, Sadeghi N. The effect of ovine oocyte diameter on nuclear maturation. Small Rumin Res. 2007;69:103–7. doi: 10.1016/j.smallrumres.2005.12.022. [DOI] [Google Scholar]
- 26.Wan PC, Hao ZD, Zhou P, Wu Y, Yang L, Cui MS, et al. Effects of SOF and CR1 media on developmental competence and cell apoptosis of ovine in vitro fertilization embryos. Anim Reprod Sci. 2009;114:279–88. doi: 10.1016/j.anireprosci.2008.09.020. [DOI] [PubMed] [Google Scholar]
- 27.Roca J, Martinez E, Vazquez JM, Lucas X. Selection of immature pig oocytes for homologous in vitro penetration assays with the brilliant cresyl blue test. Reprod Fertil Dev. 1998;10:479–85. doi: 10.1071/RD98060. [DOI] [PubMed] [Google Scholar]
- 28.Rodrigues BA, Rodriguez P, Silva AE, Cavalcante LF, Feltrin C, Rodrigues JL. Preliminary study in immature canine oocytes stained with brilliant cresyl blue and obtained from bitches with low and high progesterone serum profiles. Reprod Domest Anim. 2009;44(Suppl 2):255–8. doi: 10.1111/j.1439-0531.2009.01408.x. [DOI] [PubMed] [Google Scholar]
- 29.Rodriguez-Gonzalez E, Lopez-Bejar M, Velilla E, Paramio MT. Selection of prepubertal goat oocytes using the brilliant cresyl blue test. Theriogenology. 2002;57:1397–409. doi: 10.1016/S0093-691X(02)00645-3. [DOI] [PubMed] [Google Scholar]
- 30.Griffin J, Emery BR, Huang I, Peterson CM, Carrell DT. Comparative analysis of follicle morphology and oocyte diameter in four mammalian species (mouse, hamster, pig, and human) J Exp Clin Assist Reprod. 2006;3:2. doi: 10.1186/1743-1050-3-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Antosik P, Kempisty B, Bukowska D, Jackowska M, Wlodarczyk R, Budna J, et al. Follicular size is associated with the levels of transcripts and proteins of selected molecules responsible for the fertilization ability of oocytes of puberal gilts. J Reprod Dev. 2009;55:588–93. doi: 10.1262/jrd.20252. [DOI] [PubMed] [Google Scholar]
- 32.Sirard MA, Richard F, Blondin P, Robert C. Contribution of the oocyte to embryo quality. Theriogenology. 2006;65:126–36. doi: 10.1016/j.theriogenology.2005.09.020. [DOI] [PubMed] [Google Scholar]
- 33.Egerszegi I, Alm H, Ratky J, Heleil B, Brussow KP, Torner H. Meiotic progression, mitochondrial features and fertilisation characteristics of porcine oocytes with different G6PDH activities. Reprod Fertil Dev. 2010;22:830–8. doi: 10.1071/RD09140. [DOI] [PubMed] [Google Scholar]
- 34.Bettegowda A, Lee KB, Smith GW. Cytoplasmic and nuclear determinants of the maternal-to-embryonic transition. Reprod Fertil Dev. 2008;20:45–53. doi: 10.1071/RD07156. [DOI] [PubMed] [Google Scholar]
- 35.Kempisty B, Jackowska M, Piotrowska H, Antosik P, Wozna M, Bukowska D, et al. Zona pellucida glycoprotein 3 (pZP3) and integrin beta2 (ITGB2) mRNA and protein expression in porcine oocytes after single and double exposure to brilliant cresyl blue test. Theriogenology. 2011;75:1525–35. doi: 10.1016/j.theriogenology.2010.12.016. [DOI] [PubMed] [Google Scholar]