bluuebunny/crossref_metadata_2025 huggingface dataset:
244 INVOLVEMENT OF Na+/H+ ANTIPORTER 1 IN EMBRYONIC DEVELOPMENT OF RAT OOCYTES ACTIVATED BY ETHANOL
Authors: Ito J. , Shudo K. , Sano D. , Kashiwazaki N.
Published: August 2011

In mammalian oocytes, artificial activation is essential for further embryonic development of somatic cell nuclear transferred or round spermatid-injected oocytes. Unlike the protocol for mice, the optimal activation protocol for rat oocytes is not well established. Recently, we showed for the first time that a combined treatment with ethanol and 6-dimethylaminopurine (6-DMAP) efficiently induced activation of rat oocytes, resulted in a higher proportion of blastocyst formation as compared with other combined treatments or with the 6-DMAP treatment alone (Sano et al. 2009 Zygote 17, 29–36). Although the ethanol treatment has been used for oocyte activation not only in rats, but also in other species to date, it is unclear why the ethanol treatment is effective, especially in activating rat oocytes. Because it has been reported that alcohol alters intracellular pH via exchangers, transporters, or both on the membrane surface in the myocardium, it leads us to the possibility that the ethanol treatment regulates such exchangers, transporters, or both in rat oocytes. The aim of the present study was to clarify the role of Na+/H+ exchanges (NHE) in activating rat oocytes treated with ethanol. First, we examined which types of NHE are expressed in rat ovaries, oocytes, and parthenogenetic embryos. Expression of the NHE1 protein was also confirmed in both oocytes and parthenogenetic embryos, but not in NHE2 and NHE3. Treatment with 5-(N-ethyl-N-isopropyl)amiloride hydrochloride, an NHE inhibitor, did not affect the development of ethanol-activated oocytes to the 2-cell stage; however, it decreased the rate of blastocyst formation in a dose-dependent manner. The 5-(N-ethyl-N-isopropyl)amiloride hydrochloride treatment did not decrease development to either the 2-cell or the blastocyst stage of oocytes activated by 6-DMAP alone. Taken together, we conclude that ethanol treatment was involved in NHE1 activation, resulting in high developmental ability of rat oocytes.

This work was supported in part by a Grant-in-Aid for Scientific Research from JSPS (KAKENHI; 21780253) to J. I. The work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan, Grant-in-Aid for Matching Fund Subsidy for Private Universities, to J. I. and N. K.
248 PRODUCTION OF FLAGGED RECOMBINANT BOVINE BMP15 TO IMPROVE BOVINE IN VITRO EMBRYO PRODUCTION SYSTEMS
Authors: Burns G. , Suchodolski P. F. , Pearks Wilkerson A. J. , Long C.
Published: August 2011

Current in vitro systems for bovine embryo production are inefficient and produce embryos with lower viability than their in vivo-derived counterparts. Recent reports demonstrate that in vitro bovine oocyte maturation systems could benefit from the addition of oocyte-secreted factors, specifically GDF9 and BMP15 (Gilchrist et al. 2007 Theriogenology 67, 6–15). The long-term goal of this work is to produce species-specific recombinant oocyte-secreted factors capable of improving bovine embryo production in vitro. In the current project, the objective was to produce a cell line that expresses recombinant bovine BMP15. This protein is first translated as a large precursor peptide consisting of propeptide and mature regions, which are enzymatically cleaved to form the active mature protein. The wild-type BMP15 gene was cloned using reverse transcriptase PCR with RNA obtained from bovine ovarian tissue. For improved detection and purification of the active form of the recombinant protein, a detectable FLAG tag sequence (DYKDDDDK) was incorporated into the wild-type BMP15 gene by PCR and cloned into pCDNA expression vector. The FLAG tag was introduced immediately 3′ of the cleavage site at the N-terminal portion of the mature protein to produce recombinant FLAG-tagged BMP15 (rbFL-BMP15). To ensure efficient production of the mature protein, a Kozak sequence was inserted 5′ of the start ATG and the cleavage site altered to be recognised by PACE/furin enzymes, which are endogenously expressed in most mammalian cells including HEK-293 cells (Li et al. 2009 Mol. Hum. Reprod. 15, 779–788). Following sequencing to verify transcript fidelity, pCDNA-rbFL-BMP15 was transfected into HEK-293 cells, and mature protein production was detected by Western blot analysis. Cells plated at 85% confluency were transfected with Lipofectamine 2000, and lysates were harvested 48 h post-transfection. The presence of bovine rbFL-BMP-15 in cell lysates was confirmed by Western blot using the anti-FLAG antibody. Ongoing experiments will test the bioactivity of the purified rbFL-BMP15 by evaluating activation of the SMAD 1/5 pathway via Western blot for phosphorylated SMAD 1/5. After a biologically active protein is confirmed, purified protein will be collected for testing during in vitro maturation of bovine oocytes. We anticipate the species-specific form of oocyte-secreted factors will further enhance in vitro embryo production systems beyond that reported using heterologous factors.
25 EFFECT OF REPRODUCTIVE TRACT SIZE ON CONCEPTION RATES IN LACTATING DAIRY COWS UTILIZING A REPRODUCTIVE TRACT SCORING SYSTEM
Authors: Young C. , Di Croce F. A. , Roper D. , Harris J. , Rohrbach N. , Wilkerson J. , Schrick F. N.
Published: August 2011

Reproductive tract scoring (RTS) systems have been effective in evaluating fertility of heifers. A similar RTS system has not been reported in lactating dairy cows pertaining to conception rates following artificial insemination. Objectives of this study include development of a RTS system for lactating dairy cows and determining if RTS had an effect on artificial insemination (AI) success with various types of semen [conventional (CS), sexed (SS)]. During pre-breeding examinations (typically 30–60 DIM) by trained personnel, cows were assigned a RTS (1, 2, or 3) based on size and length of the cervix and uterine horns, but not including status of the ovaries. Tracts for RTS 1 were small, compact uterine horns resting within the pelvic cavity, RTS 2 were intermediate in size with uterine horns resting partially outside the pelvic cavity, and RTS 3 were large, deep tracts resting entirely outside the pelvis into the abdominal cavity. Additionally, cows were evaluated for RTS at a local abattoir before harvest, and tracts collected for volume and length measurements. Volume measurements were collected using Foley catheters and measuring water displaced in the uterine horns at a constant pressure. Length of the vagina, cervix, and uterine horns were measured using a flexible tape. Insemination data analysis was performed using mixed model analysis of variance in Glimmix SAS 9.2 (SAS Institute Inc., Cary, NC, USA). Insemination data included 613, 308, and 572 scores in first, second, and third+ lactation cows, respectively. First-lactation cows had 65.25% with RTS 1, 30.02% RTS 2, and 4.73% RTS 3. Second-lactation cows had 53.25% RTS 1, 39.29% RTS 2, and 7.47% RTS 3. Cows with 3+ lactations had 43.18% RTS 1, 43.88% RTS 2, and 12.94% RTS 3. Cows were artificially inseminated (n = 2401 inseminations) with either CS (n = 1981) or SS (n = 420). Conception rates for CS were 38.65%, 34.26%, and 24.41% for RTS 1, 2, and 3, respectively (P = 0.0154 for RTS 1 v. 3). Conception rates for CS (38.65%, RTS 1; 34.26%, RTS 2) were higher compared to SS (21.37%, RTS 1 (P = 0.0001); 20.72%, RTS 2 (P = 0.0186)). No differences were detected between CS (24.40%) and SS (22.35%) within RTS 3; however, observations were limited for SS inseminations with RTS 3. Preliminary data regarding total length (vagina, cervix, and uterine horns; RTS 1, 2, 3 = 103.1 cm, 114.9 cm, 134 cm, respectively) and volume (RTS 1, 2, 3 = 102 mL, 131 mL, 133.3 mL, respectively) suggest both increase as RTS increases but limited observations prevent conclusions at this time. Conception rates indicate smaller reproductive tracts have improved fertility in lactating dairy cows when using CS. Identification of larger reproductive tracts will allow for more economically efficient AI programs by utilising inexpensive, high fertility semen.
251 SELECTION OF PREPUBERTAL SHEEP OOCYTES USING BRILLIANT CRESYL BLUE TEST
Authors: Catalá M. G. , Izquierdo D. , Romaguera R. , Hammami S. , Roura M. , Paramio M. T.
Published: August 2011

The aim of this study was to evaluate the utility of the brilliant cresyl blue (BCB) test as an indirect measure of oocyte growth to select competent prepubertal sheep oocytes for in vitro embryo production. The BCB stain allows the determination of glucose–6-phosphate dehydrogenase (G6PD) activity, an enzyme with decreased activity in oocytes that have finished their growth phase. Oocytes were obtained after slicing the surface of lamb ovaries (2–5 months old) obtained from a local abattoir. Oocytes with more than 3 compact cumulus layers and homogeny cytoplasm were selected and stained with different concentrations of BCB diluted in PBS (13-, 26-, 39-, and 52-μM BCB) during 60 min at 38.5°C in a humidified air atmosphere. Oocytes were classified into groups depending on their cytoplasm coloration: oocytes with blue cytoplasm or grown oocytes (BCB+) and oocytes without blue coloration or growing oocytes (BCB–). Oocytes were matured in an enriched TCM-199 medium for 24 h at 38.5°C and 5% CO2 in a humidified atmosphere. Oocyte diameter was also measured. Matured oocytes were partially denuded and transferred to fertilization medium (SOF) supplemented with 10% of oestrous sheep serum. Fresh semen was kept at room temperature (25°C) for 1 h. Highly motile spermatozoa were selected by using Ovipure density gradient (Nidacon EVB S.L.), and oocytes were fertilized with 1 × 106 sp mL–1. After 20 h postinsemination, presumptive zygotes were cultured for 8 days in SOF with 10% of fetal bovine serum at 38.5°C, 5% CO2, and 90% N2. Data was analysed by performing Fisher’s exact test for blastocyst production and ANOVA with Tukey’s post-test for oocyte diameter. Table 1 shows the percentage of BCB-stained oocytes and their embryo development. In this study oocytes exposed during 60 min to 13-μM BCB showed a higher percentage of embryos reaching blastocyst stage than did those in the control group (≤0.01). In other species such as goats (Rodriguez-Gonzalez et al. 2002 Theriogenology 57(5), 1397–1409) and cattle (Alm et al. 2005 Theriogenology 63(8), 2194–2205), the best protocol for the oocyte selection was the use of 26-μM BCB during 90 min. Oocyte diameter showed significant differences between BCB– with BCB+ and control group (110 μm, 134 μm, and 121 μm, respectively, ≤0.001). In conclusion, using 13 μM of BCB during 60 min is a suitable technique for increasing embryo blastocyst rates using lamb oocytes.

Table 1.Effect of BCB1 concentration on embryo development of lamb oocytes

The grant sponsor was the Spanish Ministry of Science and Innovation, Code: AGL2007-60227-CO2-01.
258 ROLE OF PROGESTERONE AND ITS RECEPTORS ON DEVELOPMENTAL COMPETENCE OF OOCYTES IN CATTLE
Authors: Aparicio I. M. , Garcia-Herreros M. , O'Shea L. C. , Hensey C. , Lonergan P. , Fair T.
Published: August 2011

Several studies have demonstrated the importance of progesterone (P4) through its receptors (PR) in the regulation of the ovulatory cycle, but its participation in oocyte maturation in mammals has not yet been clarified. Previous results in our group showed changes in the protein expression of genomic (nPR-A/B) and nongenomic (mPRα/β) PR in bovine cumulus–oocyte complexes (COC) during in vitro maturation (IVM), indicating a possible function for these receptors on bovine oocyte maturation. Therefore, we aimed to study the role of P4 and PR in oocyte developmental competence. Good-quality immature COC were placed in maturation medium [TCM-199 supplemented with 10% (vol/vol) FCS and 10 ng mL–1 of epidermal growth factor] and cultured at 39°C for 22 h in a humidified atmosphere containing 5% CO2, in the presence or absence of trilostane (0.001, 0.1, and 10 μM), P4 (50 and 100 ng mL–1), promegestone (50 and 100 ng mL–1), RU 486 (0.1, 1, and 10 μM), or antibodies against mPRα or mPRβ. Matured COC were washed and placed in wells containing 250 μL of fertilization medium (25 mM bicarbonate, 22 mM Na-lactate, 1 mM Na-pyruvate, 6 mg mL–1 fatty acid-free BSA, and 10 mg mL–1 heparin). In vitro fertilization was performed with 250 μL of frozen–thawed semen at a final concentration of 1 × 106 spermatozoa mL–1 at 39°C under 5% CO2 during 20 h. Presumptive zygotes were denuded, washed, and transferred to 25-μL culture droplets (SOF + 5% FCS) at 39°C under 5% CO2, 90% of N2, and 5% O2 atmosphere with maximum humidity. Number of embryos was recorded at day 2, 3, 7, and 8 (Day 0 = day of IVF). Data were analysed using ANOVA analysis using SPSS v. 15.0 software package. Inhibition of P4 production by 10 μM of trilostane during IVM reduced progesterone production in the media, cumulus cells expansion, but had no effect on meiotic resumption in Day 2 and Day 3 embryos; however, there was a significant decrease in the percentage of blastocysts at Day 7 (12 ± 3.5) and Day 8 (14 ± 3.3) compared with the control (30 ± 4.5 and 42 ± 2.5). This reduction on embryo development was overcome by the addition of 50 or 100 ng mL–1 of P4 at Day 8 (33 ± 5.7 and 32 ± 4.1). The same results were obtained with nonmetabolizable P4, where the reduction on blastocysts with trilostane at Day 8 (20 ± 2.0) was completely overcome by 50 or 100 ng mL–1 of promegestone (41 ± 5.1 and 40 ± 6.7). Specific inhibition nPR-A/B with 10 μM of RU 486 produced a significant reduction on blastocysts at Day 7 (24 ± 4.3) and Day 8 (29 ± 5.5) compared with the control (44 ± 2.6 and 47 ± 3.0). Inhibition of mPRα reduced cleavage rate and Day 3 embryos, whereas the inhibition of mPRβ had no effect on meiotic resumption or embryo development. In conclusion, the intracellular signaling of P4 on developmental competence of oocytes in cattle seems to be mainly mediated by nPR-A/B receptors and could be associated with cytoplasmic maturation.
259 Cdk7 AND CYCLIN H, BUT NOT Mat1, ARE INVOLVED IN MEIOTIC RESUMPTION OF PORCINE IMMATURE OOCYTE
Authors: Fujii W. , Nishimura T. , Kano K. , Naito K.
Published: August 2011

The complex kinase Cdk-activating kinase (CAK) consists of the catalytic subunit Cdk7, regulatory subunit Cyclin H, and assembly factor Mat1. The CAK is essential for maturation-promoting factor (MPF) activation by phosphorylating threonine 161 (T161) of Cdc2 in mitosis. Although it is known that meiotic resumption of oocytes is regulated by MPF activity, the role of CAK in meiosis is still unclear. In the present study, we attempted to confirm the involvement of CAK in meiotic resumption of porcine immature oocyte. Cumulus–oocyte complexes (COC) were collected from antral follicles of gilts and cultured up to 48 h in TYH medium containing 20% porcine follicular fluid, 3.2 mg/mL of BSA, and 1.0 IU mL–1 of pregnant mare serum gonadotropin. The T161 phosphorylation level of Cdc2 in cultured oocytes was analysed by Western blot analysis. The transcripts were collected from noncultured or cultured oocytes, and Cdk7, Cyclin H, and Mat1 expression were detected by RT-PCR. Overexpression of Cdc2 or inhibition of Cdk7, Cyclin H, and Mat1 during oocyte maturation was performed by microinjection of mRNA or antisense RNA into ooplasm of immature COC and verified by Western blot or semiquantitative RT-PCR. Maturation-promoting factor kinase activity was assayed by Histone H1 kinase activity assay. Statistical analyses in this study were carried out by Student’s t-test. The T161 phosphorylation of Cdc2 was found during the culture period from 18 h to 48h, which was after germinal vesicle breakdown (GVB). Overexpression of Cdc2 increased the incidence of GVB at 18 h, but overexpression of mutant Cdc2 (replaced T161 by alanine) had no influence on GVB. These results indicate that T161 phosphorylation of Cdc2 is important for meiotic resumption. Next, we attempted to confirm the CAK function during oocyte maturation. Transcripts of Cdk7, Cyclin H, and Mat1 were detectable throughout the culture period. Inhibition of Cdk7 and Cyclin H caused a decrease in T161 phosphorylation and MPF activity, and the incidence of GVB was significantly lower than in nontreated oocytes. In contrast, Mat1-inhibited oocytes resumed meiosis and developed to the metaphase II stage, and the incidence was not different between Mat1-inhibited oocytes and nontreated oocytes. These results suggest that Cdk7 and Cyclin H are working as CAK and activate Cdc2 by T161 phosphorylation, although Mat1 is dispensable during oocyte maturation.
264 MITOCHONDRIAL DNA COPY NUMBER IN OOCYTES OF PREPUBERTAL AND CYCLIC GILTS
Authors: Pawlak P. , Pers-Kamczyc E. , Lechniak-Cieslak D.
Published: August 2011

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 < 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).
265 INHIBITION OF CLASS III PHOSPHATIDYLINOSITOL-3-KINASE, BY 3-METHYLADENINE, REVERSIBLY ARRESTS PORCINE OOCYTES AT GERMINAL VESICLE STAGE
Authors: Park M. R. , Gupta M. K. , Uhm S. J. , Shin S. T. , Han Y. M. , Lee H. T.
Published: August 2011

Phosphatidylinositol-3-kinases (PI3K) play pivotal roles in the meiotic progression of oocytes from metaphase I to metaphase II stage. This study evaluated the effect of 3-methyladenine (3MA), a specific inhibitor of Class III PI3K, on the meiotic progression of porcine oocytes. Immature porcine oocytes (n = 4744) retrieved from abattoir-derived oocytes were cultured in the absence or presence (10 mM) of 3MA for 22 h and evaluated for meiotic progression by florescent Hoechst 33342 staining. Data were analysed by chi-square test or ANOVA using SPSS software, and differences at P < 0.05 were considered significant. Results showed that 3MA treatment arrested the immature porcine oocytes at germinal vesicle (GV) stage in the presence (99.2 ± 0.8 v. 54.0 ± 10.1%) or absence (96.5 ± 1.8 v. 41.0 ± 17.6%) of cumulus cells. Furthermore, a significantly high proportion (60.9 ± 13.8%) of 3MA-treated oocytes acquired a nucleolus completely surrounded by a rim of highly condensed chromatin (GV-II stage). When immature oocytes, arrested at GV stage for 22 h by 3MA, were further cultured for 22 h in the absence of 3MA, 96.1 ± 1.5% of oocytes reached metaphase II stage at 42 h of in vitro maturation and did not differ (P > 0.05) from nontreated control oocytes with respect to their ability to fertilize, cleave (74.1 ± 1.2 v. 72.7 ± 2.8%), and form blastocyst (15.4 ± 1.5 v. 12.7 ± 0.6%) upon IVF or parthenogenetic activation (cleavage rate: 89.8 ± 1.7 v. 84.6 ± 5.1%; blastocyst rate: 44.3 ± 12.4 v. 45.1 ± 7.6%). These data suggest that 3MA reversibly blocks and synchronizes the meiotic progression of porcine oocytes at GV stage without affecting their ooplasmic maturation in terms of post-fertilization/activation in vitro embryonic development. Our data also provide indirect evidence for the likely participation of Class III PI3K in meiotic maturation of porcine oocytes beyond GV stage.

This work was supported by grants (Code #200901FHT010305191 and #20070401034017) from BioGreen 21 program of RDA, Republic of Korea.
268 INFLUENCE OF DONOR AGE ON DEVELOPMENTAL COMPETENCE OF IN VITRO v. IN VIVO MATURED BOVINE OOCYTES OBTAINED BY REPEATED OVUM PICKUP FROM FOLLICLE-STIMULATING-HORMONE-STIMULATED AND NONSTIMULATED ANIMALS
Authors: Matthiesen M. , Reichenbach H. D. , Habermann F. A. , Reichenbach M. , Arnold G. J. , Wolf E.
Published: August 2011

Recent findings on oogenesis, folliculogenesis, and ovarian aging in cows make the bovine system an attractive model for elucidating ovarian function and dysfunction as well as reproductive aging in women. The aim of the present study was to investigate the influence of donor age on the developmental competence of in vitro v. in vivo matured bovine cumulus–oocyte complexes (COC) obtained by ultrasound-guided repeated ovum pickup (OPU). Two groups (G1 and G2) of German Simmental heifers (14 months old at the beginning of the experiment, n = 5 and n = 7), first-lactation young cows (2–4 y old, n = 5 and n = 3), and old cows (10–15 y old, n = 5 and n = 3) were subjected to twice-weekly OPU without hormonal prestimulation 32 (G1) and 6 times (G2). Afterward, animals in G1 were punctured at 5-week intervals 9 times after FSH superstimulation to obtain in vivo matured COC at the metaphase II stage. Data were analysed using a mixed model (SAS). In the twice-weekly OPU for G1 and G2 combined, significantly (P < 0.05) more COC per animal and OPU session were obtained from the old cows (9.9 ± 1.0) compared with heifers and young cows (6.0 ± 0.8 and 7.0 ± 1.0, respectively). When G1 and G2 were regarded separately, lower numbers of COC (P < 0.01) were obtained in G1 than in G2 (2.7 ± 0.8, 4.4 ± 0.8, 7.0 ± 0.8 and 9.2 ± 1.5, 9.4 ± 2.3, 12.9 ± 2.3 for heifers, young cows, and old cows of G1 and G2, respectively). Cleavage rates (CR) on day 3 after IVF (day 0) were not affected by donor age and were not different between groups. Cultivation of COC from young cows in G1 led to higher blastocyst rates (BR) on day 7 (P < 0.05) compared with old cows and heifers. No differences in BR were observed between animals of G2. Significantly more COC (P < 0.01) were obtained in all age groups from FSH superstimulated donors (10.6 ± 0.8, 9.0 ± 0.9, and 11.7 ± 0.9 for heifers, young cows, and old cows, respectively). Cleavage rates and BR were significantly higher (P < 0.05) in all age groups after FSH superstimulation compared with those of nonstimulated donors. However, there were no differences in CR and BR between age groups (CR: 82.8 ± 7.0, 89.9 ± 7.0, 77.1 ± 6.2%; BR: 34.4 ± 7.2, 44.6 ± 7.2, 36.7 ± 7.2%). We conclude that although the numbers of COC obtained per animal and session were significantly different between G1 and G2, in vitro results were highly repeatable after OPU without hormonal prestimulation. Higher CR and BR were obtained after IVF of in vivo matured COC obtained from FSH superstimulated donors, regardless of animal age.

This work was supported by the Deutsche Forschungsgemeinschaft (FOR 1041).
27 CLONING OF ADULT PIGS USING SCRIPTAID TREATMENT AND PREOVULATORY EMBRYO TRANSFER
Authors: Albornoz M. , Colato C. , El-Beyrouthi N. , Mellano F. , Mellano A. , Mellano P. H. , Mellano M. L. , Mellano M. A. , Mellano J. C. , Baldassarre H. , Bordignon V.
Published: August 2011

There is growing interest in the use of swine in biomedical research. Cloning from cultured somatic cells (SCNT) has been the preferred method to generate genetically modified swine models. In a recent report, swine cloning efficiency was increased by treatment of reconstructed embryos with the inhibitor of deacetylase enzymes Scriptaid (Zhao et al. 2010 Cel. Reprog. 12, 75). Also, the timing of SCNT-embryo transfer with respect to the recipient’s expected time of ovulation was shown to affect cloning efficiency, whereas preovulatory embryo transfer resulted in a higher rate of cloned piglets born compared to postovulatory embryo transfer (Petersen et al. 2008 Cloning Stem Cells 10, 355). Therefore, our objective was to combine Scriptaid treatment and preovulatory embryo transfer in the same protocol for swine cloning. Cumulus–oocyte complexes aspirated from 3- to 6-mm diameter follicles were matured in vitro under standard conditions (Martinez Diaz et al. 2010 Cel. Reprog. 12, 85) and used as host oocytes for SCNT. Fibroblast cell lines were established from skin biopsies collected from 2 adult boars and cultured in DMEM supplemented with 10% FBS and 1% antibiotics. Oocytes were micromanipulated in Tyrode’s lactate-pyruvate-HEPES medium supplemented with 7.5 μg mL–1 cytochalasin B (CB) and electrically fused using a single DC pulse of 1.6 kV cm–1 for 70 μs. Activation was performed using ionomycin (15 μM/5 min) followed by exposure to CB (7.5 μg mL–1) and cyclohexemide (10 μg mL–1) for 5 h in porcine zygote medium (PZM-3; Yoshioka et al. 2002 Biol. Reprod. 66, 112). Reconstructed embryos were exposed to 500 nM Scriptaid for 10 to 12 h starting after ionomycin treatment. Oocytes were then washed and cultured in PZM-3 medium until transfer. Peripubertal recipient gilts were synchronized by oral administration of altrenogest (Regu-Mate®; 20 mg day–1) for 12 days, followed by 1.000 IU eCG injected on the last day of altrenogest treatment and 500 IU hCG 72 h later. 1-cell stage embryos were transferred into the oviduct after ∼20 h from hCG injection or 22 h before the expected ovulation time. Pregnancy was confirmed and monitored by ultrasonography and parturition was induced by injecting PGF2α at Day 115 of pregnancy. A total of 840 reconstructed embryos were transferred into 10 gilts [average 84 (range 60–110) embryos/gilt]. 4 gilts (40%) were detected to be pregnant 4 weeks after transfer, and 2 (20%) delivered 1 (1100 g) and 2 (950 and 850 g) healthy cloned piglets. The number of embryos transferred to these 2 gilts was 85 and 70. These results confirm that Scriptaid treatment and preovulatory embryo transfer can be applied in the same cloning protocol to produce cloned piglets from adult cell lines. To our knowledge, these are the first cloned pigs produced in Latin America.
272 INTRACELLULAR NITRIC OXIDE LEVEL OF PORCINE OOCYTES IS NEGATIVELY CORRELATED WITH OOCYTE MATURATION RATE AND CUMULUS EXPANSION INDEX IN A CHEMICALLY DEFINED MEDIUM
Authors: Uozumi T. , Funahashi H.
Published: August 2011

Nitric oxide (NO) has been known to inhibit nuclear maturation in cumulus–enclosed oocytes in rodents. The objective of this study was to examine if meiotic stimulators, such as dibutyryl cAMP and epidermal growth factor (EGF), influence intracellular NO level of oocytes and if the level is correlated with oocyte maturation rate and cumulus expansion in a chemically defined medium. Oocyte–cumulus complexes (OCC) were aspirated from mid-size follicles (3–6 mm in diameter) of prepuberal porcine ovaries. The OCC were cultured in modified porcine oocyte medium with various supplements – gonadotropins plus dibutyryl cAMP (Gn + cAMP), EGF plus dibutyryl cAMP (EGF + cAMP), dibutyryl cAMP alone (cAMP), EGF alone (EGF), and non-supplements (none) – for a first 20-h period and then in fresh porcine oocyte medium (without those supplements) for another 24 h in an atmosphere of 5% CO2 in air at 39°C. Following in vitro maturation culture, OCC were assessed for the degree of cumulus expansion (scored from 0 as cumulus free to 5 as full expansion) and then additionally cultured with DAF2-DA, an indicator of NO, for an additional 1-h period in the same condition. The oocytes were denuded with 0.1% hyaluronidase, and the intensity of fluorescence was measured. The oocytes were also fixed, stained with acetic orcein, and observed for meiotic stage. Statistical analysis was performed by ANOVA with a Bonferroni-Dunn post hoc test (significance, P < 0.05). Maturation rates and cumulus expansion indexes were significantly affected by various supplement conditions (Table 1). The intensity of fluorescence showing intracellular NO level was also different among experimental groups (Table 1). A negative correlation was found between intracellular NO intensity and maturation rate (r2 = 0.71) or cumulus expansion index (r2 = 0.70). From these results, we conclude that there is a synergistic effect of cAMP and EGF on cumulus expansion and oocyte maturation and the reduction of oocyte NO levels in a chemically defined medium. Furthermore, a reduction of oocyte NO level seems to be included in the induction of cumulus expansion and oocyte maturation.

Table 1.Effects of supplements on nuclear maturation, cumulus expansion, and intracellular NO level of porcine oocytes1
275 EFFECT OF REACTIVE OXYGEN SPECIES DURING IN VITRO MATURATION ON PORCINE OOCYTE NUCLEAR MATURATION AND DEVELOPMENTAL COMPETENCE
Authors: Yuan Y. , Krisher R.
Published: August 2011

The generation of excessive reactive oxygen species (ROS) may contribute to the decreased competence of in vitro matured (IVM) oocytes. However, ROS are also generated in normal cellular metabolism and can be important regulators of cellular functions. The objective of this study was to examine the effect of ROS during IVM on porcine oocyte nuclear maturation and subsequent embryonic development. Oocytes were matured in different redox environments for 40 h in 7% CO2 in air at 38.7°C. The basic maturation medium was defined PPM supplemented with 1 mM hypoxanthine. Reactive oxygen species were generated by the hypoxanthine–xanthine oxidase (XOD) system at 3 different concentrations: XOD0 (0 mU), XOD1 (1 mU), and XOD10 (10 mU). In each XOD treatment, 2 different concentrations of cysteine (Cys) were added as an antioxidant: Cys1 (0.57 mM) and Cys2 (1.14 mM). This resulted in 6 experimental treatments in a 3 × 2 factorial design; XOD0-Cys1 was considered the control. For fertilization, gametes were co-incubated in modified Tween medium B with milk powder for 5 h and then cultured in NCSU-23 medium in 5% CO2, 10% O2 for 6 days, at which point cleavage, blastocyst development, and blastocyst cell number were determined (30–50 per treatment per replicate; 4 replicates). Data were analysed by two-way ANOVA, and differences were determined by Fisher’s least significant difference multiple-comparison test; percentage data were arcsin transformed (significance, P < 0.05). Results are shown in Table 1. Percentage of mature oocytes was not different between any XOD0 and XOD1 treatments, but maturation was decreased in both XOD10 treatments. Embryonic cleavage was also decreased in both XOD10 treatments compared with the control. Blastocyst development was decreased in XOD0-Cys2 and XOD10-Cys1 when compared with the control. Blastocyst total cell number was not different between any treatments (P > 0.05). In conclusion, 10-mU XOD during IVM resulted in decreased nuclear maturation, embryonic cleavage, and blastocyst development, possibly due to excessive ROS generated by XOD. The negative effect of high levels of XOD on blastocyst formation could be reversed by adding additional antioxidant capacity to the environment (Cys2). This result suggests that adequate ROS balance is important for oocyte quality. Interestingly, adding extra antioxidant capacity alone (XOD0-Cys2) was detrimental to blastocyst formation, possibly due to the creation of an environment that was too reduced. These results demonstrate the importance of keeping the redox environment balanced during oocyte maturation. Excessive oxidative or reducing environments both appear to be detrimental to oocyte developmental competence.

Table 1.Nuclear maturation and developmental competence of oocytes matured in different redox environments
277 EFFECTS OF SEMINAL PLASMA ON SEX-SORTING BOVINE SPERM
Authors: Burroughs C. A. , Lenz R. W. , Evans K. M. , Graham J. K. , Seidel G. E.
Published: August 2011

This experiment evaluated how characteristics of bovine ejaculates affect the sortability of X- and Y-bearing spermatozoa. Ejaculates were collected by artificial vagina, 2 each from 10 bulls with an average of 1 h between collections. Only ejaculates with at least 60% motile and 70% normal sperm were used. Semen was centrifuged at 1 000 × g for 15 min to separate sperm from seminal plasma; seminal plasma was clarified by 10 min of additional centrifugation at 2 000 × g. Sperm were rediluted to 160 million sperm/mL with TALP (pH 7.4) and 0, 5, 10, or 20% seminal plasma, from the same ejaculate or reciprocally from first/second ejaculates. Following incubation with Hoechst 33342 for 45 min, an equal volume of TALP (pH 5.5) containing red food dye was added, and sperm were analysed on a MoFlo (Dako, Glostrup, Denmark) flow cytometer for percentage live-oriented cells, X sort rate, coincidence rate, percentage dead or dying (sperm membrane permeable to red dye), and splitability (peaks to valley ratio). The percentage live-oriented sperm was higher for treatments with 0% seminal plasma (64.4%) than for 5 (59.6%), 10 (59.0%), and 20% (57.8%) seminal plasma (P < 0.01). The percentage live-oriented sperm was higher for second (63.0%) than for first ejaculates (56.2%). Sort rate was higher for 0% seminal plasma and second ejaculates (P < 0.05). Dead/dying rates were lower for 0% (16.5%) than for 5 (21.9%), 10 (23.6%), or 20% (23.4%) seminal plasma (P < 0.003); and for first ejaculates (25.9%) compared with second ejaculates (18.2%). Seminal plasma percentage and ejaculate had no effect on splitability, and there was no difference in sorting parameters whether the seminal plasma was from the first or second ejaculate. The initial sperm concentration of the ejaculate (range, 1.3 to 3.4 billion sperm/mL) did not affect any sperm sorting parameter. Thus, effects of sperm concentration on sortability need to be reconsidered, as current practice is to select ejaculates to sort based on initial sperm concentration. The initial pH, percentage morphological abnormalities, initial seminal plasma pH, and age of bull did not affect sorting parameters. In conclusion, the presence of seminal plasma during staining and sorting may decrease sort rate and percentage of live-oriented cells, as well as increase death rate. In addition, sorting second ejaculates may be more advantageous than sorting first ejaculates. Future studies are needed to determine if the results hold true if sperm are stored for several hours and how the various factors affect sperm post-thaw viability.
279 IN VITRO DEVELOPMENT IN THREE CULTURE SYSTEMS OF BOVINE OOCYTES FERTILIZED WITH SEX-SORTED SPERM
Authors: Trigal B. , Gomez E. , Muñoz M. , Caamaño J.N. , Carrocera S. , Martin D. , Moreno J. , Diez C.
Published: August 2011

Following in vitro fertilization and culture, the timing of both embryonic cleavage and appearance of blastocysts has been reported to be altered when using sorted spermatozoa (Lu et al. 1999 Theriogenology 52, 1393–1405). To optimize an in vitro culture system for bovine embryos produced with sexed sperm, sorted and unsorted (control) semen samples of 5 bulls (semen samples provided by XY Inc., Fort Collins, CO, USA) were separated using a Percoll gradient followed by a washing step with Fert-TALP medium (Trigal et al. 2010 Reprod. Domest. Anim. 45, 83). The obtained pellet was used for fertilizing in vitro matured cumulus–oocyte complexes from slaughterhouse ovaries. Zygotes were cultured in 1) Vero cells monolayer + B2 + 10% FCS; 2) SOFaaci + 10% FCS; or 3) SOFaaci + 6 g L–1 BSA. A total of 1980 oocytes were in vitro matured and fertilized. Development was recorded on Days 3 and Days 6 to 8, and analysed by ANOVA and Duncan test. Bull, replicate, and culture effects were nonsignificant, and consequently were not considered in the final analysis. Data are percentages of development related to the zygotes cultured, and are expressed as LSM ± SE (see Table 1). The use of sexed sperm significantly reduced development rates compared with unsorted sperm. Thus, sorted sperm showed reduced 5- to 8-cell, Day-6 morulae, and Day-8 blastocyst rates over unsorted sperm within the 3 culture systems analysed. Culture in SOF + BSA was the least efficient embryo culture system in terms of Day-7 blastocyst rates, both with unsorted and sorted sperm. Day-7 blastocyst rates produced with unsorted sperm after culture in Vero cells or SOF + FCS were significantly higher than those obtained after culture in SOF + BSA. Day-7 blastocyst rates obtained with sorted sperm and cultured in Vero cells or SOF + FCS were comparable to those obtained with unsorted sperm in SOF + BSA. No differences were detected between male- and female-sorted sperm (data not shown). Sorted sperm is an effective tool for producing sex-known embryos. Quality assessment (differential cell counts and cryosurvival) of embryos produced with sorted sperm under different conditions are being undertaken.

Table 1.In vitro development of bovine embryos fertilized with either untreated or sorted sperm and cultured in Vero cells, SOF + FCS, or SOF + BSA

Supported by INIA RTA2008.#0082. M. Muñoz, B. Trigal and D. Martin are sponsored by RYC08-03454, Cajastur and PTA2007-#0268-I. We acknowledge Sexing Technologies for collaboration.
287 DEVELOPMENT OF DOMESTIC CAT EMBRYOS GENERATED BY INTRACYTOPLASMIC SPERM INJECTION EXPOSED TO IONOMYCIN ACTIVATION AND DIFFERENT CULTURE CONDITIONS
Authors: Moro L. N. , Salamone D. F.
Published: August 2011

Assisted reproduction techniques in the domestic cat could be successfully applied in endangered wild felids for sustaining genetic biodiversity. One technique with great potential is intracytoplasmic sperm injection (ICSI). The objective of this study was to evaluate the development of domestic cat embryos after ICSI exposed to ionomycin activation, using a free-radical reducer (insulin-transferrin-selenium, ITS) in oocyte maturation and low oxygen tension in culture. Domestic cat ovaries were recovered from cats subjected to ovariectomy and transported to the laboratory within 2 h. They were washed in TALP-H and the oocytes were released from the follicle by repeatedly puncturing and scraping the ovaries. The cumulus–oocyte-complexes selected were in vitro matured in TCM 199 containing 1 IU mL–1 HCG, 10 ng mL–1 ECG, 2.2 mM calcium lactate, 0.3 mM pyruvate, 0.3% BSA, and 3% antibiotic-antimycotic. Matured oocytes were denuded of cumulus cells after 22 h of culture and individually injected with a frozen–thawed epididymal spermatozoon. After ICSI, one group of presumptive zygotes was immediately cultured (ICSI-control group) and another group was activated by incubation with 5 μM ionomycin in TALP-H for 4 min before culture (ICSI-Io group). In both experiments culture conditions were 5% CO2 in air at 39°C. A third group was matured using ITS supplementation, activated with ionomycin after ICSI and then cultured in 5% O2, 5% CO2, and 90% N2 at 39° (ITS-ICSI-Io-O2 group). Control SHAM groups were done for each ICSI treatment: SHAM-control, SHAM-Io, and ITS-SHAM-Io-O2 groups. The culture medium used was SOF and all treatments were analysed by Fisher test (P < 0.05). Non statistical differences were observed among the three ICSI groups in cleavage (day 2), 61.2% (n = 85); 70.7% (n = 58), and 65.5% (n = 58), neither in blastocyst rates (day 8) 10.6%, 10.3%, and 17.2%, for ICSI-control, ICSI-Io, and ITS-ICSI-Io-O2, respectively. In SHAM controls, cleavage rate was low 38.3% (n = 60) and no blastocysts were obtained when no ITS supplementation nor artificial activation was applied (SHAM-control group). However, cleavage improved to 70% (n = 20) and 81% (n = 37) in SHAM-Io and ITS-SHAM-Io-O 2 groups, respectively. Moreover, no statistical differences were observed in these both groups respect to ICSI groups in cleavage neither in blastocyst rates (0% and 5.4%). Our results showed that ITS supplementation during maturation, ionomycin exposure after ICSI, and low oxygen tension in culture did not improve embryo development after ICSI, though allowed parthenogenetic progression to blastocyst stage after SHAM. In conclusion, ICSI without external assistance is a good reproductive technique to be applied in the domestic cat, potentially applicable in wild cat conservation.

Dr. Carlos Benaglia, Departamento de Zoonosis, San Martín.
290 GENERATION OF INDUCED PLURIPOTENT STEM CELLS FROM BOVINE FETAL FIBROBLAST CELLS BY DEFINED FACTORS
Authors: Cao H. G. , Liu Y. , Yin H. Q. , Sun X. P. , Li Y. S. , Tao Y. , Zhang Y. H. , Zhang X. R.
Published: August 2011

Induced pluripotent stem cells (iPS) have broad potential applications in drug screening, regenerative medicine, and basic biology, and using iPS as starting materials that have identical properties to embryonic stem cell would probably revolutionize the production of gene-targeted livestock. However, iPS in livestock is still lacking except for in pigs. Instructed by Yamanaka’s idea, in the current study we attempted to generate bovine iPS from fetal fibroblast cells (bFF; from a fetus 2.5 months old after gestation) by using 4 defined transcriptional factors: Oct-4, Sox2, Klf4, and c-Myc. A lentivirus haboring the 4 factors acted as a vehicle to transfect the bFF. One bFF cell line was infected for at least 3 replicates. After transfection, the treated bFF were then cultured in DMEM supplemented with 4 ng mL–1 of basic fibroblast growth factor and 1000 IU mL–1 of leukemia inhibitory factor at 37.5°C, 5% CO2, on the mouse embryonic feeder cells pretreatd with mitomycin C. From Day 16 after the onset of induction, morphology of a few typical spindle-like bFF gradually changed into round, ball-like cells and grew into colonies. Afterward, when these colonies were harvested and subcultured in stem cell medium supplemented with 1000 IU mL–1 of leukemia inhibitory factor and 4 ng mL–1 of basic fibroblast growth factor, new colonies with clear-cut, round edge emerged, and the cells in the colonies had increased nuclear:cytoplasm ratio, kept normal karyotype up to 10 passages, and were alkaline phosphatase staining positive. We also found that the cells exhibited part of the stem cell markers, as evidenced by being Nanog, SSEA1 positive but SSEA3 and TRA-60 negative. Moreover, embryoid bodies could be formed in vitro, and terotoma formation after injection into nude mice also displayed 3 layers. Taken together, we found that 4 lentivirus-mediated, defined transcriptional factors could successfully induce bFF into iPS-like cells.

H. G. Cao and Y. Liu contributed equally to this work. The corresponding authors are Y. H. Zhang and X. R. Zhang. This work was supported by NSFC (30800784/c120103, 30700574), 973 (2009CB941004).
291 ISOLATION AND CHARACTERIZATION OF MESENCHYMAL STEM CELLS DERIVED FROM HUMAN AMNIOTIC FLUID
Authors: Choi S.-A. , Lee J.-H. , Kim K.-J. , Kim E.-Y. , Park K.-S. , Park Y.-B. , Li X. , Ha Y.-N. , Park J.-Y. , Kim M.-K.
Published: August 2011

Adult stem cells have the capacity to differentiate into several different cell types, although their differentiation potential is limited compared with that of embryonic stem cells. Thus, adult stem cells are regarded as an exciting source for new cell therapies. Recent observations also indicate that stem cells derived from second-trimester amniocentesis are pluripotent – capable of differentiating into multiple lineages, including representatives of all 3 embryonic germ layers. In addition, amniotic fluid stem cells can be used in the generation of disease- or patient-specific stem cells, and amniotic fluid stem cells could be an ideal source for autologous cell replacement therapy in the later life of the fetus. The aim of the present study was to investigate isolation and characterisation of human amniotic fluid-derived mesenchymal stem cells (hAFS). We successfully isolated and characterised hAFS. Amniotic fluid samples were collected in the second trimester (median gestational age: 16 weeks, range: 15–17 weeks) for prenatal diagnosis. Specimens (2 mL) were centrifuged and incubated in low-glucose DMEM supplemented with 10% FBS, 25 ng of basic fibroblast growth factor, and 10 ng of epidermal growth factor at 37°C with 5% CO2. Human amniotic fluid cell (passage 6) expression of stem cell specific markers OCT-4, SOX2, Rex1, FGF4, and NANOG was confirmed by RT-PCR. Flow cytometric analysis showed that hAFS (passage 10) were positive for CD44, CD29, CD146, STRO1, and CD90 but negative for CD19. Immunocytochemical analysis of hAFS (passage 11) also showed the expression of OCT-4, SSEA-1, CD44, CD29, CD146, STRO1, and CD90, but hAFS were negative for CD19 and CD14. In conclusion, according to the previous studies on other mammalians, hAFS are an appropriate source of pluripotent stem cells. Here, we demonstrated that hAFS have a high expression of stem cell specific marker, including embryonic stem cell marker and mesenchymal stem cell marker. Therefore, amniotic fluid may be a suitable alternative source of multipotent stem cells.