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Effects of single and double exposure to brilliant cresyl blue on the selection of porcine oocytes for in vitro production of embryos
Theriogenology 66 (2006) 366–372 www.journals.elsevierhealth.com/periodicals/the
Effects of single and double exposure to brilliant cresyl blue on the selection of porcine oocytes for in vitro production of embryos Pimprapar Wongsrikeao a, Takeshige Otoi a,*, Hirofumi Yamasaki a, Budiyanto Agung a, Masayasu Taniguchi a, Hideaki Naoi a, Ryohei Shimizu a,b, Takashi Nagai b a
Laboratory of Animal Reproduction, Department of Veterinary Science, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan b Department of Research Planning and Coordination, National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305-0901, Japan Received 3 September 2005; accepted 1 December 2005
Abstract The aim of this study was to evaluate the effectiveness and toxicity of single and double application of the brilliant cresyl blue (BCB) test on the selection of porcine oocytes as an indirect measure of oocyte growth for in vitro fertilization (IVF) and nuclear transfer. In the first experiment, oocytes were exposed to BCB before and after maturation culture and classified according to their cytoplasmic coloration: blue coloration and colorless. The classified oocytes were fertilized with spermatozoa and then cultured for 7 days. The percentages of maturation to metaphase II in blue oocytes at the start of maturation culture were higher than those of colorless oocytes (68.7–70.1% versus 0.8–3.6%, P < 0.05). However, double application of BCB test before and after maturation culture had a toxic effect on fertilization and embryonic development. No significant differences in the blastocyst formation were found between blue oocytes without double application of BCB test and control oocytes without any application of BCB test, whereas the total cell number per blastocyst from the blue oocytes was higher than that from the control oocytes (48.0 versus 34.2, P < 0.05). In the second experiment, oocytes were exposed to the BCB test before or after maturation culture, and then the matured oocytes were activated to evaluate the ability of parthenogenetic development. The proportion of blastocyst formation of blue oocytes classified after maturation culture was lower than that of blue oocytes classified before maturation culture (10.0% versus 27.0%, P < 0.05). Therefore, double application of the BCB test before and after maturation culture impaired fertilization and embryonic development, whereas a single application before maturation culture was efficient to select oocytes for IVF and nuclear transfer. # 2005 Elsevier Inc. All rights reserved. Keywords: Brilliant cresyl blue (BCB) test; Porcine oocyte; IVF; Nuclear transfer
1. Introduction
* Corresponding author. Tel.: +81 83 933 5904; fax: +81 83 933 5904. E-mail address: [email protected] (T. Otoi). 0093-691X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2005.12.001
For in vitro production (IVP) of porcine embryos, cumulus–oocyte complexes (COCs) with uniform ooplasm and a compact cumulus cell mass have been collected from antral follicles of ovaries. Cumulus– oocyte complexes are collected from antral follicles, but
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the size of follicles that are selected for collection of COCs varies both between and within investigations. Moreover, an asynchronous progression of meiotic maturation has been observed during in vitro maturation (IVM) [1,2]. Recently, the brilliant cresyl blue (BCB) test has been used successfully to select homologous oocytes for IVP [3–10]. The BCB test permits assessment of the intracellular activity of glucose-6-phosphate dehydrogenase (G6PD), an enzyme synthesized in growing oocytes, but with decreased activity in oocytes that have finished their growth phase. The BCB is a blue compound; it is rendered colorless by G6PD activity. Therefore, oocytes that have finished their growth have a blue-colored cytoplasm, because G6PD activity decreased [7]. In previous studies, the BCB test has been evaluated in pigs [3,4], heifers [5,6] and goats [7–10], with the aim of establishing a non-invasive and non-perturbing means for selecting immature oocytes before in vitro maturation (IVM). Pujol et al. [6] suggested that the BCB test permitted the selection of bovine oocytes with higher percentages of oocytes reaching metaphase II (MII) and higher embryo development, as compared with morphological criteria. On the other hand, somatic cell nuclear transfer (SCNT) has been widely used for producing embryos for multiple purposes, and complete maturation of the recipient oocytes is an important factor for achieving success [11]. To date, most studies have been focused on applying the BCB test to improve the IVP performance by selecting oocytes before IVM. Rodriguez-Gonzalez et al. [7] reported that meiotic competence of goat oocytes without a blue cytoplasm (BCB-negative oocytes) at the start of IVM was lower than that of BCB-positive oocytes, but half of BCB-negative oocytes could reach MII after IVM. In heifer and goats, moreover, it has been shown that a half of BCB-negative oocytes can develop to the cleavage stage after in vitro fertilization (IVF) [6,7]. Therefore, we postulated that double application of BCB test before and after IVM can improve the accuracy on the selection of oocytes for porcine IVP. However, the toxicity, accuracy and effectiveness of BCB test before and/ or after IVM of porcine oocytes have never been investigated. The present study was conducted to standardize the BCB test for selecting porcine oocytes by evaluating the effectiveness or toxicity of a single and a double BCB test on the ability to achieve fertilization and parthenogenetic development in vitro of IVM oocytes.
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2. Materials and methods 2.1. Recovery and IVM of oocytes Ovaries from prepubertal-crossbred gilts, approximately 6 months old, were collected at a local abbatoir and transported to the laboratory in physiological saline (0.85% [w/v] NaCl) at 35 8C. Cumulus–oocytes complexes (COCs) from follicles (3–6 mm in diameter) were aspirated using an 18-guage needle attached to a 5mL syringe. After being washed twice with modified phosphate-buffered saline (mPBS) (Embryoteck; Nihonzenyaku, Fukushima, Japan), only COCs with uniform ooplasma and compact cumulus cells were used. The selected COCs were transferred into a maturation medium, a modified North Carolina State University (NCSU)-37 solution [12] supplemented with 0.6 mM cysteine, 1 mM dibutyryl cyclic AMP (dbcAMP; Sigma, St. Louis, MO, USA), 10 IU/mL equine chorionic gonadotropin (eCG; Teikokuzoki, Tokyo, Japan), 10 IU/mL human chorionic gonadotropin (hCG; Teikokuzoki, Tokyo, Japan), 50 mg/mL gentamicin (Sigma), and 10% (v/v) porcine follicular fluid (pFF). About 50 COCs were cultured (for 22 h) in each 500 mL of the maturation medium covered with a layer of mineral oil (Sigma) in a four-well dish (Nunc A/ S, Roskilde, Denmark). They were then transferred to the maturation medium without hormones and dbcAMP, and cultured for an additional 22 h under the same conditions described above. All cultures were performed in a 38.5 8C humidified incubator containing 5% CO2 in air. 2.2. IVF of IVM oocytes The IVF was carried out according to the method described by Kikuchi et al. [13]. In brief, after IVM culture, COCs were washed three times with preincubated IVF medium that consists of 90 mM NaCl, 12 mM KCl, 25 mM NaHCO3, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 10 mM sodium lactate, 3 mg/mL bovine serum albumin (BSA; fatty acid free, Sigma), 5 mM caffeine (Sigma) and 50 mg/mL gentamicin. After washing, batches of 10–15 COCs were placed in 90 mL drops of the same medium that had been covered with warm mineral oil in 35 mm 10 mm petri dish. The dishes were kept in the incubator for about 30 min before spermatozoa were added (for fertilization). The frozen semen from a boar was used throughout the experiments. The frozen straw with boar spermatozoa was thawed in 35 8C water bath, and the frozen– thawed spermatozoa were diluted in a tissue culture
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medium (TCM) 199 with Earle’s salts (Gibco, Grand Island, NY, USA) supplemented with 3 mM calcium DLlactate pentahydrate (Wako Purechemical Co., Osaka, Japan), 3 mM D-glucose (Wako), 12% fetal bovine serum (Gibco) and 50 mg/mL gentamicin, adjusted to pH 7.8, before centrifugation at 200 g for 2 min. After centrifugation, the supernatant was removed and the sperm pellet was preincubated for 15 min at 38.5 8C before fertilization. A portion (10 mL) of preincubated spermatozoa was introduced into a 90 mL of fertilization medium drop containing matured COCs to give a final concentration of 1 106 cells/mL. 2.3. Parthenogenetic activation of oocytes After IVM, some oocytes were stripped from cumulus cells in Dulbecco’s phosphate-buffered saline (PBS; Gibco) supplemented with 1 mg/mL hyaluronidase (Sigma) and then washed several times in NCSU-37. Oocytes with a visible polar body, indicating successful nuclear maturation, were selected for oocyte activation. The oocytes were washed in mannitol medium (0.25 M mannitol solution supplemented with 0.01% polyvinylalcohol, 0.5 mM Hepes, 0.1 mM CaCl2, and 0.1 mM MgCl2 with pH 7.2) and then equilibrated for 3 min at room temperature. After equilibration, the oocytes were transferred to a chamber consisting of two electrodes 1 mm apart, that was overlaid with mannitol medium. Oocytes were exposed to double direct current pulses of 2.5 kV/cm for 15 ms using a BTX 2000 Electro-cell Manipulator (BTX, San Diego, CA, USA). All activated oocytes were subsequently cultured for 5 h in postactivation medium (NCSU-37 containing 10 mg/mL of cycloheximide and 10 mg/mL cytochalasin B). 2.4. In vitro development of embryos At 5 h after co-incubation with spermatozoa, cumulus cells surrounding presumptive zygotes were removed by pipetting using a small-bore pipette. The denuded putative zygotes were cultured in 500 mL NCSU-37 solution, supplemented with 4 mg/mL BSA, 0.17 mM sodium pyruvate, 2.73 mM sodium lactate and 50 mg/mL gentamicin. After activation treatment, activated oocytes were washed and then cultured in the same conditions described above. At 72 h after insemination or activation, all cleaved embryos were transferred into fresh culture medium; NCSU-37 solution supplemented with 4 mg/mL BSA, 5.55 mM D-glucose, and 50 mg/mL gentamicin. The cleaved embryos were cultured for an additional 4 days to evaluate their ability to develop to the blastocyst stage.
2.5. Assessment of nuclear status, fertilization and embryo development At the end of IVM culture for 44 h (including exposure time to BCB), oocytes were mechanically denuded from cumulus cells in PBS supplemented with 1 mg/mL hyaluronidase. Denuded oocytes were then mounted on a glass slide, and fixed with acetic acid:ethanol (1:3 v/v) for 48–72 h. The fixed oocytes were stained with acetic–orcein (1% orcein in 45% acetic acid) and examined under a phase-contrast microscope. Oocytes were examined if they underwent the germinal vesicle break down (GVBD), specially highlighted on metaphase II (MII) stage. Oocytes showing an abnormal chromatin configuration or no chromatin at all after staining were considered degenerated. At 16 h after IVF, presumptive zygotes were mounted on a slide, fixed and stained as described above. Oocytes containing both female and male pronuclei were considered as fertilized and categorized as normal or polyspermic, according to the number of swollen sperm head(s) and pronucleus(ei) in the cytoplasm. To evaluate their ability to develop to the cleavage and blastocyst stages, on day 7 (day 0 = insemination or activation) all embryos were fixed and permeabilized for 15 min at room temperature in PBS containing 3.7% (w/v) paraformaldehyde and 1% (v/v) Triton X-100 (Sigma), and then placed in PBS containing 0.3% (w/v) polyvinylpyrrolidone for 15 min at room temperature. Embryos were then placed in the drop of mounting medium on a slide. The mounting medium consisted of 90% (v/v) glycerol containing 1.9 mM Hoechst 33342 (Sigma). Subsequently, the embryos were overlaid with a coverslip supported by four droplets of a vaseline/ paraffin and incubated for one night at 4 8C. The embryos were examined under a fluorescence microscope with a 355 nm wavelength excitation filter. Embryos with a clear blastocoele and more than 20 cells were defined as blastocysts. The numbers of cleaved embryos and blastocysts, and their cell numbers were recorded. 2.6. Experimental design In the first experiment, immediately after oocytes collection, COCs were incubated in mPBS supplemented with 26 mM of BCB (B-5388, Sigma) for 90 min at 38.5 8C in a humidified air atmosphere. After incubation, oocytes were washed three times in mPBS and classified into two groups, depending on the coloration
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of cytoplasm: oocytes with blue cytoplasm coloration (BCB+ group) and oocytes without blue cytoplasm coloration (BCB group). After classification, the oocytes were cultured in the maturation medium until reaching 22 h of culture and then incubated in the maturation medium without hormones and dbcAMP for 22 h in the same conditions described above. To assess the toxicity of double application of BCB test, after maturation culture for 44 h in total, the COCs in BCB+ group were further incubated for 90 min in mPBS supplemented with (BCB++ group) or without BCB (BCB+/ group), as described above. In the BCB group, all COCs were exposed to BCB after 44 h of maturation culture and classified as blue coloration (BCB + group) and colorless (BCB group). The COCs incubated in mPBS without BCB for 90 min at 38.5 8C in a humidified air atmosphere before and after IVM were used as a control group. After exposure to BCB, some COCs were fixed to assess the nuclear maturation and the rest of COCs were fertilized with spermatozoa and then cultured for 7 days. The fertilization, cleavage and development to the blastocyst stage of IVM oocytes were evaluated at 16 h and 7 days after insemination, respectively, as described above. In the second experiment, to evaluate the developmental competence of parthenogenetically activated oocytes selected by single BCB test, oocytes were exposed to BCB before (BCB+/ group) or after maturation culture (BCB/+ group). Only oocytes with blue cytoplasm coloration were selected and used in this experiment. The oocytes incubated in mPBS without BCB for 90 min before and after IVM were used as a control group. After maturation culture, including exposure to BCB (90 min), only oocytes with a visible polar body were subjected to parthenogenetic activation
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treatment and subsequently cultured for 7 days. The cleavage and blastocyst formation of the activated oocytes were evaluated at 7 days after activation. 2.7. Statistical analysis Four or five replicate trials were carried out. Data are expressed as means S.E.Ms. The percentages of oocytes reaching MII stage, oocytes fertilized, embryos cleaved and embryos developed to the blastocyst stage were subjected to arc sine transformation before analysis of variance (ANOVA). The transformed data and total cell number of blastocysts were tested by ANOVA followed by a post hoc, Fisher’s protected least significant difference test (PLSD test) using the Statview program (Abacus Concepts Inc., Berkeley, CA, USA). Differences with P < 0.05 were considered significant. 3. Results In total, 2069 oocytes were used to investigate their capability of nuclear maturation, fertilization and embryonic development after an exposure(s) to BCB before IVM (single exposure) or before and after IVM (double exposures). All oocytes in the BCB-positive group at the start of IVM also exhibited blue cytoplasm coloration after the second exposure to BCB at the end of IVM. As shown in Table 1, significantly more oocytes in the BCB+/ and BCB++ groups reached MII (68.7 and 70.1%, respectively) than oocytes in control and the other groups (0.8–42.8%; P < 0.05). There were no significant differences in the percentages of oocytes undergoing GVBD and reaching MII between the
Table 1 Nuclear maturation of porcine oocytes treated with the brilliant cresyl blue (BCB) test before and after in vitro maturationa Treatment
Control BCB (+/) BCB (++) BCB ( +) BCB ( )
No. of oocytes examined 129 132 119 84 92
No. of oocytesb (%) GVBD 88 121 105 19 8
(68.2 0.6) a (90.2 3.1) b (89.0 2.0) b (22.7 7.5) c (7.5 4.6) c
MII 54 92 83 3 1
(42.8 1.4) a (68.7 1.3) b (70.1 1.2) b (3.6 3.6) c (0.8 0.8) c
No. of degenerated oocytes (%) 12 6 10 11 15
(9.3 0.3) a (4.9 1.1) b (8.6 1.6) b (13.3 3.9) c (15.5 6.2) c
Control: oocytes were incubated in mPBS, without BCB, before and after IVM; BCB (+/): oocytes were classified into blue coloration before IVM and incubated in mPBS without BCB after IVM; BCB (++): oocytes were classified into blue coloration before and after IVM; BCB ( +): oocytes were classified into blue colorless before IVM, but into blue coloration after IVM; BCB ( ): oocytes were classified into blue colorless before and after IVM; Percentages are presented as mean S.E.M.; GVBD, germinal vesicle breakdown; MII, metaphase II; Within a column, values with different letters (a–c) are different (P < 0.05). a Five replicated trials were conducted. b Oocytes were fixed after 44 h of IVM (including exposure time to BCB).
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Table 2 In vitro fertilization of porcine oocytes treated with the brilliant cresyl blue (BCB) test before and after in vitro maturationa Treatment
Control BCB (+/) BCB (++) BCB ( +) BCB ( )
No. of oocytes examined 121 134 105 47 57
No. of oocytes fertilizedc (%)
No. of oocytes matured beyond MIIb 69 109 91 2 0
Total
(55.4 9.7) a (81.7 2.6) b (86.6 2.1) b (5.6 5.6) c (0) c
73 72 22 0 1
Monospermic
(58.5 9.0) a (54.2 3.2) a (21.1 3.9) b (0) c (3.0 3.0) c
32 47 15 0 1
(26.0 1.1) a (36.3 7.1) a (14.4 2.1) b (0) c (3.0 3.0) c
No. of degenerated oocytes (%)
Polyspermic 41 25 7 0 0
(32.5 9.7) a (17.9 4.1) a,b (6.7 2.0) b,c (0) c (0) c
9 8 5 1 3
(7.6 4.2) (6.0 0.5) (4.9 2.1) (1.9 1.9) (4.2 4.2)
Control: oocytes were incubated in mPBS, without BCB, before and after IVM; BCB (+/): oocytes were classified into blue coloration before IVM and incubated in mPBS without BCB after IVM; BCB (++): oocytes were classified into blue coloration before and after IVM; BCB ( +): oocytes were classified into blue colorless before IVM but into blue coloration after IVM; BCB ( ): oocytes were classified into blue colorless before and after IVM; Percentages are presented as mean S.E.M.; GVBD, germinal vesicle breakdown; MII, metaphase II; Within a column, values with different letters (a–c) are different (P < 0.05). a Four replicated trials were conducted. b MII: oocytes having two polar bodies. c Oocytes were fixed at 16 h after IVF. Oocytes containing both female and male pronuclei were considered as fertilized and categorized as normal or polyspermic, according to the number of swollen sperm heads and pronuclei in the cytoplasm.
BCB + and BCB groups, but these two groups had lower percentages (P < 0.05) of nuclear maturation, compared with the other groups. As shown in Table 2, the percentages of total and monospermic fertilization of oocytes in the BCB++ group were significantly lower than those in the BCB+/ and control groups. Moreover, the percentages of total and monospermic fertilization in BCB + and BCB groups significantly decreased as compared with the other groups. The percentage of cleaved embryos in the BCB+/ group was higher (P < 0.05) than in all other groups (Table 3). Although no significant differences in the blastocyst formation were found between BCB+/ and control groups, the total cell number of blastocyst in the BCB+/ group increased as compared with that in the control group (48.0 versus 34.2, P < 0.05). None of the
embryos in the BCB++, BCB + and BCB groups developed to the blastocyst stage. As shown in Table 4, the percentages of cleavage and development to the blastocyst stage of activated oocytes were lower (P < 0.05) in the BCB/+ group than in the BCB+/ and control groups. However, no significant differences in the total cell number of blastocyst were found among the groups. 4. Discussion In the present study, we observed that the percentages of oocytes reaching MII in the BCB-positive groups at the time of oocyte collection increased as compared with those in the BCB-negative and control groups. Our observations were in agreement with those of previous studies, in which the beneficial effects of
Table 3 In vitro development of porcine oocytes treated with brilliant cresyl blue (BCB) test before and after in vitro maturation and fertilized in vitroa Treatment
No. oocytes examined
No. of embryosb (%) Cleaved
Control BCB (+/) BCB (++) BCB (S+) BCB ( )
151 160 120 71 72
86 146 11 3 0
(56.6 5.3) a (91.4 2.4) b (9.0 2.8) c (4.3 1.5) c (0) c
Developed to blastocyst 17 19 0 0 0
(11.3 1.4) a (11.9 1.8) a (0) b (0) b (0) b
Mean S.E.M. of the total cell number in blastocyst 34.2 3.2 a 48.0 1.4 b 0c 0c 0c
Control: oocytes were incubated in mPBS, without BCB, before and after IVM; BCB (+/): oocytes were classified into blue coloration before IVM and incubated in mPBS without BCB after IVM; BCB (++): oocytes were classified into blue coloration before and after IVM; BCB ( +): oocytes were classified into blue colorless before IVM, but into blue coloration after IVM; BCB ( ): oocytes were classified into blue colorless before and after IVM; Percentages are presented as mean S.E.M.; Within a column, values with different letters (a–c) are different (P < 0.05). a Four replicated trials were conducted. b Embryos with a clear blastocoele and more than 20 cells were defined as blastocysts.
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Table 4 In vitro development of porcine oocytes treated with brilliant cresyl blue (BCB) test before or after in vitro maturation and activated parthenogeneticallya Treatment
No. oocytes examined
No. of embryosb (%) Cleaved
Developed to blastocyst
Control BCB (+/) BCB (/+)
150 149 176
127 (83.9 4.7) a,b 133 (91.2 3.7) a 112 (65.3 7.8) b
38 (26.6 2.1) a 38 (27.0 3.4) a 18 (10.0 1.8) b
Mean S.E.M. of the total cell number in blastocyst 40.6 2.5 40.3 1.9 33.6 1.9
Control: oocytes were incubated in mPBS without BCB before and after IVM; BCB (+/): oocytes were classified into blue coloration before IVM and incubated in mPBS without BCB after IVM; BCB (/+): oocytes were incubated in mPBS without BCB before IVM and classified into blue coloration after IVM; Percentages are presented as mean S.E.M.; Within a column, values with different letters (a,b) are different (P < 0.05). a Four replicated trials were conducted. b Embryos with a clear blastocoele and more than 20 cells were defined as blastocysts.
BCB test on the improvement of oocyte selection for IVM have been shown in pigs [3,4], cows [5,6] and goats [7–10]. All oocytes in the BCB-positive group at the start of IVM exhibited blue cytoplasm coloration at the end of IVM, and the maturation rate of oocytes reaching MII after IVM and beyond MII at 16 h after IVF in the BCB+/ group was similar to that in the BCB++ group. Therefore, the efficacy of a single BCB test on the selection of oocytes for IVM was similar to that of double BCB test. For successful in vitro development of IVM/IVF porcine oocytes, it is necessary for the oocytes to complete nuclear maturation during IVM and to have the ability to be fertilized normally [14]. In the present study, porcine oocytes with double BCB test (BCB++) had decreased ability of fertilization and development after IVF as compared with the oocytes with single BCB test (BCB+/) and control oocytes. Moreover, none of the double-stained oocytes were able to develop to the blastocyst stage, irrespective of positive/negative coloration. Rodriguez-Gonzalez et al. [7] reported a significant decrease on the fertilization rate in goat oocytes that were incubated with a high concentration (52 mM) of BCB (28.6%) as compared with the control oocytes (54.1%). Therefore, excessive exposure of oocytes to BCB decreased oocyte viability and impaired their abilities to undergo fertilization and embryonic development after IVF or when matured, porcine oocytes became very sensitive to BCB. Pujol et al. [6] reported that the percentage of IVF blastocyst formation from BCB+ oocytes increased as compared with BCB oocytes (12.3% versus 1.6%). Similarly, in the present study, BCB+/ oocytes exhibited a higher ability of cleavage and blastocyst formation than BCB-negative oocytes (BCB + and BCB groups). Moreover, none of the BCB-negative oocytes developed to the blastocyst stage. Although no
significant differences in the rates of blastocyst formation after IVF were found between BCB+/ and control groups, the total cell number per blastocyst obtained from BCB+/ oocytes increased as compared with that of control oocytes. The cell number in the embryos is an important indicator of embryonic development and health [15]. It has been suggested that embryos with a large number of cells are more likely to implant and give rise to live offspring [16]. Therefore, our results confirmed that a single BCB test enhanced the reliability and efficiency of selecting for high-quality homologous oocytes for porcine IVF. On the other hand, SCNT has been widely used to clone several mammalian species including sheep, cattle, goats, mice, and pigs for many purposes [11]. One of the most important factors for the highly successful SCNT embryo production seems to be the quality of recipient oocytes used for cloning. When the parthenogenetic development of MII oocytes that had been exposed BCB before or after IVM was examined, no further improvement in the blastocyst formation and total cell number per blastocyst were found in the BCB treatment groups. Moreover, exposure of oocytes to BCB at the end of maturation culture (BCB/+) significantly impaired their developmental ability to the blastocyst stage. Several changes in the morphology and physiology of the oocyte and surrounding cumulus cells occur during oocyte development, including dynamic changes in gap junction [17], cytokine release [18], morphology [19] and membrane physiology [20]. It has been suggested that the permeability characteristic of plasma membrane to both non-permeable and permeable solutions in oocytes at the GV stage is different from that in oocytes at the MII stage [21,22]. Therefore, the difference of the effects of the BCB test between before and after maturation culture may result from the differences in oocyte morphology and
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physiology. Moreover, the BCB test had a detrimental effect on the development of MII oocytes, but not GV oocytes. In conclusion, the selection of oocytes using the single BCB test improved the rates of nuclear maturation, monospermic fertilization of porcine oocytes, and subsequent embryonic development after IVF, as well as enhancement of embryo quality (increasing the total number of cells per blastocyst). However, the excessive exposure of oocytes to the BCB test impaired their ability to undergo fertilization and development after IVF. The BCB selection had no beneficial effects on the development of parthenogenetically activated oocytes, and the application of BCB at the end of maturation impaired the developmental ability of oocytes to the blastocyst stage. Acknowledgements The authors thank the staffs of the Meat Inspection Office of Kitakyushu city, Japan, for supplying us with pig ovaries. This study was supported in part by the Sasakawa Scientific Research Grant from The Japan Science Society. References [1] Funahashi H, Cantley TC, Day BN. Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic adenosine monophosphate improves developmental competence following in vitro fertilization. Biol Reprod 1997;57:49–53. [2] Ye J, Flint AP, Campbell KH, Luck MR. Synchronization of porcine oocyte meiosis using cycloheximide and its application to the study of regulation by cumulus cells. Reprod Fertil Dev 2002;14:433–42. [3] Ericsson SA, Boyce ML, Funahashi H, Day BN. Assessment of porcine oocytes using brilliant cresyl blue. Theriogenology 1993;39:214. [4] 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. [5] 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 brilliant cresyl blue staining before IVM as indicator for glucose-6-phosphate dehydrogenase activity. Theriogenology 2005;63:2194–205. [6] 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.
[7] 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. [8] 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. [9] Urdaneta A, Jimenez AR, Izquierdo D, Paramio MT. Effect of the addition of glutathione and glucose to the culture medium on embryo development of IVM–IVF prepubertal goat oocytes. Zygote 2003;11:131–8. [10] Urdaneta A, Jimenez-Macedo AR, Izquierdo D, Paramio MT. Supplementation with cysteamine during maturation and embryo culture on embryo development of prepubertal goat oocytes selected by the brilliant cresyl blue test. Zygote 2003;11:347–54. [11] Westhusin ME, Long CR, Shin T, Hill JR, Looney CR, Pryor JH, et al. Cloning to reproduce desired genotypes. Theriogenology 2001;55:35–49. [12] Petters RM, Wells KD. Culture of pig embryos. J Reprod Fertil Suppl 1993;48:61–73. [13] Kikuchi K, Onishi A, Kashiwazaki N, Iwamoto M, Noguchi J, Kaneko H, et al. Successful piglet production after transfer of blastocysts produced by a modified in vitro system. Biol Reprod 2002;66:1033–41. [14] Nagai T. In vitro maturation and fertilization of pig oocytes. Anim Reprod Sci 1996;42:153–63. [15] Brison DR, Schultz RM. Apoptosis during mouse blastocyst formation: evidence for a role for survival factors including transforming growth factor alpha. Biol Reprod 1997;56:1088–96. [16] Van Soom A, Ysebaert MT, de Kruif A. Relationship between timing of development, morula morphology, and cell allocation to inner cell mass and trophectoderm in in vitro-produced bovine embryos. Mol Reprod Dev 1997;47:47–56. [17] Wright CS, Becker DL, Lin JS, Warner AE, Hardy K. Stagespecific and differential expression of gap junctions in the mouse ovary: connexin-specific roles in follicular regulation. Reproduction 2001;121:77–88. [18] Kol S, Adashi EY. Intraovarian factors regulating ovarian function. Curr Opin Obstet Gynecol 1995;7:209–13. [19] Zeilmaker GH, Vermeiden JP, Verhamme CM, van Vliet AC. Observations on rat and mouse oocyte maturation in vivo and in vitro: morphology and physiology. Eur J Obstet Gynecol Reprod Biol 1974;4:15–24. [20] Okamoto H, Takahashi K, Yamashita N. Ionic currents through the membrane of the mammalian oocyte and their comparison with those in the tunicate and sea urchin. J Physiol 1977;267:465–95. [21] Ruffing N, Steponkus P, Pitt R, Parks J. Osmometric behavior, hydraulic conductivity, and incidence of intracellular ice formation in bovine oocytes at different developmental stages. Cryobiology 1993;30:562–80. [22] Agca Y, Liu J, Peter AT, Critser ES, Critser JK. Effect of developmental stage on bovine oocyte plasma membrane water and cryoprotectant permeability characteristics. Mol Reprod Dev 1998;49:408–15.
Effects of single and double exposure to brilliant cresyl blue on the selection of porcine oocytes for in vitro production of embryos Pimprapar Wongsrikeao a, Takeshige Otoi a,*, Hirofumi Yamasaki a, Budiyanto Agung a, Masayasu Taniguchi a, Hideaki Naoi a, Ryohei Shimizu a,b, Takashi Nagai b a
Laboratory of Animal Reproduction, Department of Veterinary Science, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan b Department of Research Planning and Coordination, National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305-0901, Japan Received 3 September 2005; accepted 1 December 2005
Abstract The aim of this study was to evaluate the effectiveness and toxicity of single and double application of the brilliant cresyl blue (BCB) test on the selection of porcine oocytes as an indirect measure of oocyte growth for in vitro fertilization (IVF) and nuclear transfer. In the first experiment, oocytes were exposed to BCB before and after maturation culture and classified according to their cytoplasmic coloration: blue coloration and colorless. The classified oocytes were fertilized with spermatozoa and then cultured for 7 days. The percentages of maturation to metaphase II in blue oocytes at the start of maturation culture were higher than those of colorless oocytes (68.7–70.1% versus 0.8–3.6%, P < 0.05). However, double application of BCB test before and after maturation culture had a toxic effect on fertilization and embryonic development. No significant differences in the blastocyst formation were found between blue oocytes without double application of BCB test and control oocytes without any application of BCB test, whereas the total cell number per blastocyst from the blue oocytes was higher than that from the control oocytes (48.0 versus 34.2, P < 0.05). In the second experiment, oocytes were exposed to the BCB test before or after maturation culture, and then the matured oocytes were activated to evaluate the ability of parthenogenetic development. The proportion of blastocyst formation of blue oocytes classified after maturation culture was lower than that of blue oocytes classified before maturation culture (10.0% versus 27.0%, P < 0.05). Therefore, double application of the BCB test before and after maturation culture impaired fertilization and embryonic development, whereas a single application before maturation culture was efficient to select oocytes for IVF and nuclear transfer. # 2005 Elsevier Inc. All rights reserved. Keywords: Brilliant cresyl blue (BCB) test; Porcine oocyte; IVF; Nuclear transfer
1. Introduction
* Corresponding author. Tel.: +81 83 933 5904; fax: +81 83 933 5904. E-mail address: [email protected] (T. Otoi). 0093-691X/$ – see front matter # 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2005.12.001
For in vitro production (IVP) of porcine embryos, cumulus–oocyte complexes (COCs) with uniform ooplasm and a compact cumulus cell mass have been collected from antral follicles of ovaries. Cumulus– oocyte complexes are collected from antral follicles, but
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the size of follicles that are selected for collection of COCs varies both between and within investigations. Moreover, an asynchronous progression of meiotic maturation has been observed during in vitro maturation (IVM) [1,2]. Recently, the brilliant cresyl blue (BCB) test has been used successfully to select homologous oocytes for IVP [3–10]. The BCB test permits assessment of the intracellular activity of glucose-6-phosphate dehydrogenase (G6PD), an enzyme synthesized in growing oocytes, but with decreased activity in oocytes that have finished their growth phase. The BCB is a blue compound; it is rendered colorless by G6PD activity. Therefore, oocytes that have finished their growth have a blue-colored cytoplasm, because G6PD activity decreased [7]. In previous studies, the BCB test has been evaluated in pigs [3,4], heifers [5,6] and goats [7–10], with the aim of establishing a non-invasive and non-perturbing means for selecting immature oocytes before in vitro maturation (IVM). Pujol et al. [6] suggested that the BCB test permitted the selection of bovine oocytes with higher percentages of oocytes reaching metaphase II (MII) and higher embryo development, as compared with morphological criteria. On the other hand, somatic cell nuclear transfer (SCNT) has been widely used for producing embryos for multiple purposes, and complete maturation of the recipient oocytes is an important factor for achieving success [11]. To date, most studies have been focused on applying the BCB test to improve the IVP performance by selecting oocytes before IVM. Rodriguez-Gonzalez et al. [7] reported that meiotic competence of goat oocytes without a blue cytoplasm (BCB-negative oocytes) at the start of IVM was lower than that of BCB-positive oocytes, but half of BCB-negative oocytes could reach MII after IVM. In heifer and goats, moreover, it has been shown that a half of BCB-negative oocytes can develop to the cleavage stage after in vitro fertilization (IVF) [6,7]. Therefore, we postulated that double application of BCB test before and after IVM can improve the accuracy on the selection of oocytes for porcine IVP. However, the toxicity, accuracy and effectiveness of BCB test before and/ or after IVM of porcine oocytes have never been investigated. The present study was conducted to standardize the BCB test for selecting porcine oocytes by evaluating the effectiveness or toxicity of a single and a double BCB test on the ability to achieve fertilization and parthenogenetic development in vitro of IVM oocytes.
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2. Materials and methods 2.1. Recovery and IVM of oocytes Ovaries from prepubertal-crossbred gilts, approximately 6 months old, were collected at a local abbatoir and transported to the laboratory in physiological saline (0.85% [w/v] NaCl) at 35 8C. Cumulus–oocytes complexes (COCs) from follicles (3–6 mm in diameter) were aspirated using an 18-guage needle attached to a 5mL syringe. After being washed twice with modified phosphate-buffered saline (mPBS) (Embryoteck; Nihonzenyaku, Fukushima, Japan), only COCs with uniform ooplasma and compact cumulus cells were used. The selected COCs were transferred into a maturation medium, a modified North Carolina State University (NCSU)-37 solution [12] supplemented with 0.6 mM cysteine, 1 mM dibutyryl cyclic AMP (dbcAMP; Sigma, St. Louis, MO, USA), 10 IU/mL equine chorionic gonadotropin (eCG; Teikokuzoki, Tokyo, Japan), 10 IU/mL human chorionic gonadotropin (hCG; Teikokuzoki, Tokyo, Japan), 50 mg/mL gentamicin (Sigma), and 10% (v/v) porcine follicular fluid (pFF). About 50 COCs were cultured (for 22 h) in each 500 mL of the maturation medium covered with a layer of mineral oil (Sigma) in a four-well dish (Nunc A/ S, Roskilde, Denmark). They were then transferred to the maturation medium without hormones and dbcAMP, and cultured for an additional 22 h under the same conditions described above. All cultures were performed in a 38.5 8C humidified incubator containing 5% CO2 in air. 2.2. IVF of IVM oocytes The IVF was carried out according to the method described by Kikuchi et al. [13]. In brief, after IVM culture, COCs were washed three times with preincubated IVF medium that consists of 90 mM NaCl, 12 mM KCl, 25 mM NaHCO3, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 10 mM sodium lactate, 3 mg/mL bovine serum albumin (BSA; fatty acid free, Sigma), 5 mM caffeine (Sigma) and 50 mg/mL gentamicin. After washing, batches of 10–15 COCs were placed in 90 mL drops of the same medium that had been covered with warm mineral oil in 35 mm 10 mm petri dish. The dishes were kept in the incubator for about 30 min before spermatozoa were added (for fertilization). The frozen semen from a boar was used throughout the experiments. The frozen straw with boar spermatozoa was thawed in 35 8C water bath, and the frozen– thawed spermatozoa were diluted in a tissue culture
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medium (TCM) 199 with Earle’s salts (Gibco, Grand Island, NY, USA) supplemented with 3 mM calcium DLlactate pentahydrate (Wako Purechemical Co., Osaka, Japan), 3 mM D-glucose (Wako), 12% fetal bovine serum (Gibco) and 50 mg/mL gentamicin, adjusted to pH 7.8, before centrifugation at 200 g for 2 min. After centrifugation, the supernatant was removed and the sperm pellet was preincubated for 15 min at 38.5 8C before fertilization. A portion (10 mL) of preincubated spermatozoa was introduced into a 90 mL of fertilization medium drop containing matured COCs to give a final concentration of 1 106 cells/mL. 2.3. Parthenogenetic activation of oocytes After IVM, some oocytes were stripped from cumulus cells in Dulbecco’s phosphate-buffered saline (PBS; Gibco) supplemented with 1 mg/mL hyaluronidase (Sigma) and then washed several times in NCSU-37. Oocytes with a visible polar body, indicating successful nuclear maturation, were selected for oocyte activation. The oocytes were washed in mannitol medium (0.25 M mannitol solution supplemented with 0.01% polyvinylalcohol, 0.5 mM Hepes, 0.1 mM CaCl2, and 0.1 mM MgCl2 with pH 7.2) and then equilibrated for 3 min at room temperature. After equilibration, the oocytes were transferred to a chamber consisting of two electrodes 1 mm apart, that was overlaid with mannitol medium. Oocytes were exposed to double direct current pulses of 2.5 kV/cm for 15 ms using a BTX 2000 Electro-cell Manipulator (BTX, San Diego, CA, USA). All activated oocytes were subsequently cultured for 5 h in postactivation medium (NCSU-37 containing 10 mg/mL of cycloheximide and 10 mg/mL cytochalasin B). 2.4. In vitro development of embryos At 5 h after co-incubation with spermatozoa, cumulus cells surrounding presumptive zygotes were removed by pipetting using a small-bore pipette. The denuded putative zygotes were cultured in 500 mL NCSU-37 solution, supplemented with 4 mg/mL BSA, 0.17 mM sodium pyruvate, 2.73 mM sodium lactate and 50 mg/mL gentamicin. After activation treatment, activated oocytes were washed and then cultured in the same conditions described above. At 72 h after insemination or activation, all cleaved embryos were transferred into fresh culture medium; NCSU-37 solution supplemented with 4 mg/mL BSA, 5.55 mM D-glucose, and 50 mg/mL gentamicin. The cleaved embryos were cultured for an additional 4 days to evaluate their ability to develop to the blastocyst stage.
2.5. Assessment of nuclear status, fertilization and embryo development At the end of IVM culture for 44 h (including exposure time to BCB), oocytes were mechanically denuded from cumulus cells in PBS supplemented with 1 mg/mL hyaluronidase. Denuded oocytes were then mounted on a glass slide, and fixed with acetic acid:ethanol (1:3 v/v) for 48–72 h. The fixed oocytes were stained with acetic–orcein (1% orcein in 45% acetic acid) and examined under a phase-contrast microscope. Oocytes were examined if they underwent the germinal vesicle break down (GVBD), specially highlighted on metaphase II (MII) stage. Oocytes showing an abnormal chromatin configuration or no chromatin at all after staining were considered degenerated. At 16 h after IVF, presumptive zygotes were mounted on a slide, fixed and stained as described above. Oocytes containing both female and male pronuclei were considered as fertilized and categorized as normal or polyspermic, according to the number of swollen sperm head(s) and pronucleus(ei) in the cytoplasm. To evaluate their ability to develop to the cleavage and blastocyst stages, on day 7 (day 0 = insemination or activation) all embryos were fixed and permeabilized for 15 min at room temperature in PBS containing 3.7% (w/v) paraformaldehyde and 1% (v/v) Triton X-100 (Sigma), and then placed in PBS containing 0.3% (w/v) polyvinylpyrrolidone for 15 min at room temperature. Embryos were then placed in the drop of mounting medium on a slide. The mounting medium consisted of 90% (v/v) glycerol containing 1.9 mM Hoechst 33342 (Sigma). Subsequently, the embryos were overlaid with a coverslip supported by four droplets of a vaseline/ paraffin and incubated for one night at 4 8C. The embryos were examined under a fluorescence microscope with a 355 nm wavelength excitation filter. Embryos with a clear blastocoele and more than 20 cells were defined as blastocysts. The numbers of cleaved embryos and blastocysts, and their cell numbers were recorded. 2.6. Experimental design In the first experiment, immediately after oocytes collection, COCs were incubated in mPBS supplemented with 26 mM of BCB (B-5388, Sigma) for 90 min at 38.5 8C in a humidified air atmosphere. After incubation, oocytes were washed three times in mPBS and classified into two groups, depending on the coloration
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of cytoplasm: oocytes with blue cytoplasm coloration (BCB+ group) and oocytes without blue cytoplasm coloration (BCB group). After classification, the oocytes were cultured in the maturation medium until reaching 22 h of culture and then incubated in the maturation medium without hormones and dbcAMP for 22 h in the same conditions described above. To assess the toxicity of double application of BCB test, after maturation culture for 44 h in total, the COCs in BCB+ group were further incubated for 90 min in mPBS supplemented with (BCB++ group) or without BCB (BCB+/ group), as described above. In the BCB group, all COCs were exposed to BCB after 44 h of maturation culture and classified as blue coloration (BCB + group) and colorless (BCB group). The COCs incubated in mPBS without BCB for 90 min at 38.5 8C in a humidified air atmosphere before and after IVM were used as a control group. After exposure to BCB, some COCs were fixed to assess the nuclear maturation and the rest of COCs were fertilized with spermatozoa and then cultured for 7 days. The fertilization, cleavage and development to the blastocyst stage of IVM oocytes were evaluated at 16 h and 7 days after insemination, respectively, as described above. In the second experiment, to evaluate the developmental competence of parthenogenetically activated oocytes selected by single BCB test, oocytes were exposed to BCB before (BCB+/ group) or after maturation culture (BCB/+ group). Only oocytes with blue cytoplasm coloration were selected and used in this experiment. The oocytes incubated in mPBS without BCB for 90 min before and after IVM were used as a control group. After maturation culture, including exposure to BCB (90 min), only oocytes with a visible polar body were subjected to parthenogenetic activation
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treatment and subsequently cultured for 7 days. The cleavage and blastocyst formation of the activated oocytes were evaluated at 7 days after activation. 2.7. Statistical analysis Four or five replicate trials were carried out. Data are expressed as means S.E.Ms. The percentages of oocytes reaching MII stage, oocytes fertilized, embryos cleaved and embryos developed to the blastocyst stage were subjected to arc sine transformation before analysis of variance (ANOVA). The transformed data and total cell number of blastocysts were tested by ANOVA followed by a post hoc, Fisher’s protected least significant difference test (PLSD test) using the Statview program (Abacus Concepts Inc., Berkeley, CA, USA). Differences with P < 0.05 were considered significant. 3. Results In total, 2069 oocytes were used to investigate their capability of nuclear maturation, fertilization and embryonic development after an exposure(s) to BCB before IVM (single exposure) or before and after IVM (double exposures). All oocytes in the BCB-positive group at the start of IVM also exhibited blue cytoplasm coloration after the second exposure to BCB at the end of IVM. As shown in Table 1, significantly more oocytes in the BCB+/ and BCB++ groups reached MII (68.7 and 70.1%, respectively) than oocytes in control and the other groups (0.8–42.8%; P < 0.05). There were no significant differences in the percentages of oocytes undergoing GVBD and reaching MII between the
Table 1 Nuclear maturation of porcine oocytes treated with the brilliant cresyl blue (BCB) test before and after in vitro maturationa Treatment
Control BCB (+/) BCB (++) BCB ( +) BCB ( )
No. of oocytes examined 129 132 119 84 92
No. of oocytesb (%) GVBD 88 121 105 19 8
(68.2 0.6) a (90.2 3.1) b (89.0 2.0) b (22.7 7.5) c (7.5 4.6) c
MII 54 92 83 3 1
(42.8 1.4) a (68.7 1.3) b (70.1 1.2) b (3.6 3.6) c (0.8 0.8) c
No. of degenerated oocytes (%) 12 6 10 11 15
(9.3 0.3) a (4.9 1.1) b (8.6 1.6) b (13.3 3.9) c (15.5 6.2) c
Control: oocytes were incubated in mPBS, without BCB, before and after IVM; BCB (+/): oocytes were classified into blue coloration before IVM and incubated in mPBS without BCB after IVM; BCB (++): oocytes were classified into blue coloration before and after IVM; BCB ( +): oocytes were classified into blue colorless before IVM, but into blue coloration after IVM; BCB ( ): oocytes were classified into blue colorless before and after IVM; Percentages are presented as mean S.E.M.; GVBD, germinal vesicle breakdown; MII, metaphase II; Within a column, values with different letters (a–c) are different (P < 0.05). a Five replicated trials were conducted. b Oocytes were fixed after 44 h of IVM (including exposure time to BCB).
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Table 2 In vitro fertilization of porcine oocytes treated with the brilliant cresyl blue (BCB) test before and after in vitro maturationa Treatment
Control BCB (+/) BCB (++) BCB ( +) BCB ( )
No. of oocytes examined 121 134 105 47 57
No. of oocytes fertilizedc (%)
No. of oocytes matured beyond MIIb 69 109 91 2 0
Total
(55.4 9.7) a (81.7 2.6) b (86.6 2.1) b (5.6 5.6) c (0) c
73 72 22 0 1
Monospermic
(58.5 9.0) a (54.2 3.2) a (21.1 3.9) b (0) c (3.0 3.0) c
32 47 15 0 1
(26.0 1.1) a (36.3 7.1) a (14.4 2.1) b (0) c (3.0 3.0) c
No. of degenerated oocytes (%)
Polyspermic 41 25 7 0 0
(32.5 9.7) a (17.9 4.1) a,b (6.7 2.0) b,c (0) c (0) c
9 8 5 1 3
(7.6 4.2) (6.0 0.5) (4.9 2.1) (1.9 1.9) (4.2 4.2)
Control: oocytes were incubated in mPBS, without BCB, before and after IVM; BCB (+/): oocytes were classified into blue coloration before IVM and incubated in mPBS without BCB after IVM; BCB (++): oocytes were classified into blue coloration before and after IVM; BCB ( +): oocytes were classified into blue colorless before IVM but into blue coloration after IVM; BCB ( ): oocytes were classified into blue colorless before and after IVM; Percentages are presented as mean S.E.M.; GVBD, germinal vesicle breakdown; MII, metaphase II; Within a column, values with different letters (a–c) are different (P < 0.05). a Four replicated trials were conducted. b MII: oocytes having two polar bodies. c Oocytes were fixed at 16 h after IVF. Oocytes containing both female and male pronuclei were considered as fertilized and categorized as normal or polyspermic, according to the number of swollen sperm heads and pronuclei in the cytoplasm.
BCB + and BCB groups, but these two groups had lower percentages (P < 0.05) of nuclear maturation, compared with the other groups. As shown in Table 2, the percentages of total and monospermic fertilization of oocytes in the BCB++ group were significantly lower than those in the BCB+/ and control groups. Moreover, the percentages of total and monospermic fertilization in BCB + and BCB groups significantly decreased as compared with the other groups. The percentage of cleaved embryos in the BCB+/ group was higher (P < 0.05) than in all other groups (Table 3). Although no significant differences in the blastocyst formation were found between BCB+/ and control groups, the total cell number of blastocyst in the BCB+/ group increased as compared with that in the control group (48.0 versus 34.2, P < 0.05). None of the
embryos in the BCB++, BCB + and BCB groups developed to the blastocyst stage. As shown in Table 4, the percentages of cleavage and development to the blastocyst stage of activated oocytes were lower (P < 0.05) in the BCB/+ group than in the BCB+/ and control groups. However, no significant differences in the total cell number of blastocyst were found among the groups. 4. Discussion In the present study, we observed that the percentages of oocytes reaching MII in the BCB-positive groups at the time of oocyte collection increased as compared with those in the BCB-negative and control groups. Our observations were in agreement with those of previous studies, in which the beneficial effects of
Table 3 In vitro development of porcine oocytes treated with brilliant cresyl blue (BCB) test before and after in vitro maturation and fertilized in vitroa Treatment
No. oocytes examined
No. of embryosb (%) Cleaved
Control BCB (+/) BCB (++) BCB (S+) BCB ( )
151 160 120 71 72
86 146 11 3 0
(56.6 5.3) a (91.4 2.4) b (9.0 2.8) c (4.3 1.5) c (0) c
Developed to blastocyst 17 19 0 0 0
(11.3 1.4) a (11.9 1.8) a (0) b (0) b (0) b
Mean S.E.M. of the total cell number in blastocyst 34.2 3.2 a 48.0 1.4 b 0c 0c 0c
Control: oocytes were incubated in mPBS, without BCB, before and after IVM; BCB (+/): oocytes were classified into blue coloration before IVM and incubated in mPBS without BCB after IVM; BCB (++): oocytes were classified into blue coloration before and after IVM; BCB ( +): oocytes were classified into blue colorless before IVM, but into blue coloration after IVM; BCB ( ): oocytes were classified into blue colorless before and after IVM; Percentages are presented as mean S.E.M.; Within a column, values with different letters (a–c) are different (P < 0.05). a Four replicated trials were conducted. b Embryos with a clear blastocoele and more than 20 cells were defined as blastocysts.
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Table 4 In vitro development of porcine oocytes treated with brilliant cresyl blue (BCB) test before or after in vitro maturation and activated parthenogeneticallya Treatment
No. oocytes examined
No. of embryosb (%) Cleaved
Developed to blastocyst
Control BCB (+/) BCB (/+)
150 149 176
127 (83.9 4.7) a,b 133 (91.2 3.7) a 112 (65.3 7.8) b
38 (26.6 2.1) a 38 (27.0 3.4) a 18 (10.0 1.8) b
Mean S.E.M. of the total cell number in blastocyst 40.6 2.5 40.3 1.9 33.6 1.9
Control: oocytes were incubated in mPBS without BCB before and after IVM; BCB (+/): oocytes were classified into blue coloration before IVM and incubated in mPBS without BCB after IVM; BCB (/+): oocytes were incubated in mPBS without BCB before IVM and classified into blue coloration after IVM; Percentages are presented as mean S.E.M.; Within a column, values with different letters (a,b) are different (P < 0.05). a Four replicated trials were conducted. b Embryos with a clear blastocoele and more than 20 cells were defined as blastocysts.
BCB test on the improvement of oocyte selection for IVM have been shown in pigs [3,4], cows [5,6] and goats [7–10]. All oocytes in the BCB-positive group at the start of IVM exhibited blue cytoplasm coloration at the end of IVM, and the maturation rate of oocytes reaching MII after IVM and beyond MII at 16 h after IVF in the BCB+/ group was similar to that in the BCB++ group. Therefore, the efficacy of a single BCB test on the selection of oocytes for IVM was similar to that of double BCB test. For successful in vitro development of IVM/IVF porcine oocytes, it is necessary for the oocytes to complete nuclear maturation during IVM and to have the ability to be fertilized normally [14]. In the present study, porcine oocytes with double BCB test (BCB++) had decreased ability of fertilization and development after IVF as compared with the oocytes with single BCB test (BCB+/) and control oocytes. Moreover, none of the double-stained oocytes were able to develop to the blastocyst stage, irrespective of positive/negative coloration. Rodriguez-Gonzalez et al. [7] reported a significant decrease on the fertilization rate in goat oocytes that were incubated with a high concentration (52 mM) of BCB (28.6%) as compared with the control oocytes (54.1%). Therefore, excessive exposure of oocytes to BCB decreased oocyte viability and impaired their abilities to undergo fertilization and embryonic development after IVF or when matured, porcine oocytes became very sensitive to BCB. Pujol et al. [6] reported that the percentage of IVF blastocyst formation from BCB+ oocytes increased as compared with BCB oocytes (12.3% versus 1.6%). Similarly, in the present study, BCB+/ oocytes exhibited a higher ability of cleavage and blastocyst formation than BCB-negative oocytes (BCB + and BCB groups). Moreover, none of the BCB-negative oocytes developed to the blastocyst stage. Although no
significant differences in the rates of blastocyst formation after IVF were found between BCB+/ and control groups, the total cell number per blastocyst obtained from BCB+/ oocytes increased as compared with that of control oocytes. The cell number in the embryos is an important indicator of embryonic development and health [15]. It has been suggested that embryos with a large number of cells are more likely to implant and give rise to live offspring [16]. Therefore, our results confirmed that a single BCB test enhanced the reliability and efficiency of selecting for high-quality homologous oocytes for porcine IVF. On the other hand, SCNT has been widely used to clone several mammalian species including sheep, cattle, goats, mice, and pigs for many purposes [11]. One of the most important factors for the highly successful SCNT embryo production seems to be the quality of recipient oocytes used for cloning. When the parthenogenetic development of MII oocytes that had been exposed BCB before or after IVM was examined, no further improvement in the blastocyst formation and total cell number per blastocyst were found in the BCB treatment groups. Moreover, exposure of oocytes to BCB at the end of maturation culture (BCB/+) significantly impaired their developmental ability to the blastocyst stage. Several changes in the morphology and physiology of the oocyte and surrounding cumulus cells occur during oocyte development, including dynamic changes in gap junction [17], cytokine release [18], morphology [19] and membrane physiology [20]. It has been suggested that the permeability characteristic of plasma membrane to both non-permeable and permeable solutions in oocytes at the GV stage is different from that in oocytes at the MII stage [21,22]. Therefore, the difference of the effects of the BCB test between before and after maturation culture may result from the differences in oocyte morphology and
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physiology. Moreover, the BCB test had a detrimental effect on the development of MII oocytes, but not GV oocytes. In conclusion, the selection of oocytes using the single BCB test improved the rates of nuclear maturation, monospermic fertilization of porcine oocytes, and subsequent embryonic development after IVF, as well as enhancement of embryo quality (increasing the total number of cells per blastocyst). However, the excessive exposure of oocytes to the BCB test impaired their ability to undergo fertilization and development after IVF. The BCB selection had no beneficial effects on the development of parthenogenetically activated oocytes, and the application of BCB at the end of maturation impaired the developmental ability of oocytes to the blastocyst stage. Acknowledgements The authors thank the staffs of the Meat Inspection Office of Kitakyushu city, Japan, for supplying us with pig ovaries. This study was supported in part by the Sasakawa Scientific Research Grant from The Japan Science Society. References [1] Funahashi H, Cantley TC, Day BN. Synchronization of meiosis in porcine oocytes by exposure to dibutyryl cyclic adenosine monophosphate improves developmental competence following in vitro fertilization. Biol Reprod 1997;57:49–53. [2] Ye J, Flint AP, Campbell KH, Luck MR. Synchronization of porcine oocyte meiosis using cycloheximide and its application to the study of regulation by cumulus cells. Reprod Fertil Dev 2002;14:433–42. [3] Ericsson SA, Boyce ML, Funahashi H, Day BN. Assessment of porcine oocytes using brilliant cresyl blue. Theriogenology 1993;39:214. [4] 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. [5] 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 brilliant cresyl blue staining before IVM as indicator for glucose-6-phosphate dehydrogenase activity. Theriogenology 2005;63:2194–205. [6] 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.
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