Reached Blastocyst Stage Research Articles

Chemical activation of in vitro matured dromedary camel (Camelus dromedarius) oocytes: Optimization of protocols

Experiments were conducted to study the efficiency of sequential treatments of ionomycine and ethanol combined with phosphorylation inhibitor (6-dimethylaminopurine) or the specific maturation promoting factor inhibitor (roscovitine) in inducing artificial activation in dromedary M-II oocytes. Cumulus oocyte complexes (COCs), collected from slaughterhouse ovaries were cultured at 38.5 degrees C in an atmosphere of 5% CO2 in air for 24-48 h. In experiment 1, the COCs were either fertilized in vitro or activated with 5 microM ionomycine for 5 min or 7% ethanol for 7 min, both followed by exposure to 6-diethylaminopurine or roscovitine for 4h. After 14-15 h of in vitro culture, the oocytes were fixed and stained with 1% aceto-orcein to evaluate their nuclear status. In experiment 2, the oocytes were activated in the same manner as in experiment 1 but were cultured for 7 days to evaluate their post-parthenogenetic development. In experiment 3, oocytes were exposed to the ionomycine for 2, 3, 4 or 5 min to evaluate the better exposure time while as in experiment 4, the oocytes matured for 28-48 h were activated to see the effect of aging on post-parthenogenetic development. Higher proportion (P<0.01) of oocytes was activated in ionomycine/6-DMAP and ionomycine/roscovitine groups when compared with ethanol/6-DMAP, ethanol/roscovitine and in vitro fertilized groups. However, there was no difference (P>0.05) in the proportion of oocytes activated with ethanol when compared with in vitro fertilized group. No significant difference was seen on the proportion of morula on day 7 of culture, however the development to blastocyst stage was higher (P<0.01) in ionomycine/6-DMAP and ionomycine/roscovitine when compared with ethanol/6-DMAP and ethanol/roscovitine treated oocytes. A higher proportion of oocytes reached blastocyst stage when they were exposed to ionomycine for 3 min but they were not significantly different from the others (P>0.05). The proportion of blastocysts obtained was higher (P<0.05) in oocytes activated after 28 h of maturation when compared with oocytes activated after 32, 36, 40, 44 and 48 h of maturation. In conclusion, a protocol for chemical activation of dromedary camel oocytes with ionomycine/6-DMAP is demonstrated and optimized in the present study for further use in the development of assisted reproductive techniques in this species.

132 POST-HATCHING BOVINE EMBRYO DEVELOPMENT IN VITRO: RELATIONSHIP BETWEEN SEX AND SIZE

In vitro post-hatching embryos culture is a procedure that allows the establishment of more accurate tools for evaluating embryo developmental potential without the need of transferring them to recipient animals (Vejlsted et al. 2006 Theriogenology, 65, 153–165). It is well established that in the in vitro embryo production (IVP) technique, the sex ratio is imbalanced in favor of male embryos. The difference in sex ratio observed in the blastocyst stage at day 7 may be attributed to a variety of factors including developmental speed. However, whether or not this difference in sex ratio and speed of development continues after hatching is not known. The objective of this study was to evaluate post-hatching embryonic development until day 11 after in vitro fertilization (day 0) associating embryo size and gender. A total of 468 oocytes, obtained from abattoir-derived ovaries, were used. They were matured, fertilized, and cultured in vitro for 8 days in synthetic oviduct fluid medium (SOF Nutricell�) and incubated at 39�C in 5% CO2 in air. Degenerated embryos on day 8 and non-hatching embryos on day 9 were removed from culture droplets, and only hatched blastocysts were kept. Then, embryos were measured using a graduated ocular and post-hatching development (PHD) medium (Brand�o et al. 2005 Biol. Reprod. 71, 2048–2055) was added in each well, being the final medium 1:1 of SOF:PHD. On day 11, the embryos were evaluated under stereomicroscope and only morphologically normal blastocysts were measured and frozen at –80�C, for gender diagnosis. The DNA from frozen samples was extracted with trizol reagent, sodium citrate solution (0.1 m), and ethanol. Sex embryos determination was performed by PCR and visualized in 2% agarose gel. Data were analyzed using the Mann-Whitney test. The results show that the majority (69%) of the embryos that reached blastocyst stage at day 7 developed in the PHD system until day 11. From the initial oocytes, 144 embryos (30.1%) and 146 (31.1%) embryos had reached the blastocyst stage at days 7 and 8, respectively. At day 9, 89 (19%) embryos were hatched and 65 embryos (13.9%) developed until day 11, of which only 48 embryos (73.8%) had a clear trophoblast. No difference (P &gt; 0.05) in the percentage of male and female embryos was observed when embryos were evaluated at day 11 of culture. In addition the mean size was similar (P &gt; 0.05) for female (467.24 � µm, n = 19) and male (478.84 � 190.21 µm, n = 29) embryos. The results suggest that after post-hatching culture the differences in sex ration and in gender development in IVP bovine embryos are not evident. The development until day 11 showed that post-hatching in vitro culture of bovine blastocyst can be used for embryo evaluation in later phase of development However, several questions still remain to be investigated regarding post-hatching culture of bovine blastocysts before it can be used as a tool to evaluate in vitro embryos. Supported by Embrapa and UnB, Brazil.

190 SUPPRESSION OF A TRANSCRIPTION FACTOR MSX1 GENE IN BOVINE PREIMPLANTATION EMBRYOS AND ITS EFFECT ON mRNA, PROTEIN, AND EXPRESSION OF DOWNSTREAM GENES

MSX1 is a transcription factor gene that orchestrates gene expression and regulates cell growth, proliferation, differentiation, cell-to-cell communication, and the apoptotic pathway during pattern formation in vertebrate embryogenesis. However, its role in bovine preimplantation embryo is not known. Here we aim to investigate the effects of suppressing MSX1 transcript on the development of in vitro-produced bovine embryos, study the expression of mRNA and protein products of the gene, and identify downstream genes using microarray analysis. In the first experiment, IVP zygotes were injected with 341 bp-long dsRNA (LdsRNA) (n = 384), 19 bp small interfering RNA (siRNA) (n = 374), and scrambled sequence RNA (scRNA) (n = 388). Uninjected zygotes (n = 313) were used as control. Developmental phenotype data were collected during culture until Day 8. The mRNA and protein expression levels of the different treatment groups were validated at the 8-cell and blastocyst stages using quantitative real-time PCR and immunohistochemistry, respectively. Developmental phenotype and mRNA data were analyzed using ANOVA under statistical package SPSS (SPSS, Inc., Chicago, IL, USA). In the second experiment, custom SMARTpool siRNA (Dharmacon Inc., Chicago, IL, USA) targeting bovine MSX1 (NM_174798) was used for microinjection together with siRNA and uninjected control. Following treatment at zygote stage, 8-cell embryos were used for mRNA isolation and subsequent array hybridization using bovine cDNA array containing 2000 clones. Array data analysis was performed using statistical analysis of microarray (SAM) procedure. While 33% and 29% of the zygotes from the control and scRNA treatment groups, respectively, reached blastocyst stage, only 20% and 19% of the zygotes from the LdsRNA and siRNA treatment groups, respectively, reached the same stage. Injection of LdsRNA and siRNA at the zygote stage reduced the mRNA expression level by 52% and 33% at the 8-cell stage and by 77% and 87%, respectively, at the blastocyst stage as compared to the control. Similarly, cellular protein expression levels in LdsRNA- and siRNA-injected treatment groups were found to be lower than the control groups at each stage. In all cases, injection of scRNA had no effect on mRNA and protein levels. SAM analysis revealed that, of the total 2000 clones, 3.5% and 5.4% were found to be differentially expressed in embryos injected with SMARTpool and siRNA, respectively, compared to the control. Genes involved in various activities including transcription factors (ALF), cell growth (BMP-15), metabolism (RIOK3), and cytokinasis (AURKA) were found to be down-regulated in 8-cell embryos treated with SMARTpool siRNA compared to the controls. On the other hand, genes involved in protein synthesis (RPL23), energy metabolism (COQ1), cell growth (MNS1) and skeletal development (LGALS3) were found to be upregulated in the same samples. In conclusion, suppression of MSX1 at the mRNA and protein level significantly affected the development of bovine embryos, and our study revealed list of downstream genes regulated by the activity of MSX1.

235 DEVELOPMENT OF CAMEL (CAMELUS DROMEDARIUS) OOCYTES AFTER CHEMICAL ACTIVATION

Identification of an optimal protocol for activation of the MII oocytes in a species like camel not only allows us to evaluate the quality of oocytes after their in vitro maturation, but also is required for the success of advanced technologies like cloning. The present study was aimed to determine activation of in vitro-matured dromedary (Camelus dromedarius) oocytes using ionomycin or ethanol followed by sequential culture in phosphorylation inhibitor (6-dimethylaminopurine) or the specific maturation promoting factor inhibitor (roscovitine). Cumulus–oocyte complexes (COCs), collected from slaughterhouse ovaries, were randomly distributed to 4-well culture plates (20–25 COCs/well) containing 500 µL of the maturation medium. The maturation medium consisted of TCM-199 supplemented with 0.15 mg mL–1 L-glutamine, 2.1 mg mL–1 sodium bicarbonate, 0.22 mg mL–1 pyruvate, 20 ng mL–1 epidermal growth factor, 50 µg mL–1 gentamycin, 10 µg mL–1 bFSH, 10 µg mL–1 bLH, 1 µg mL–1 estradiol, and 10% estrous dromedary serum (EDS). The COCs were cultured at 38.5�C in an atmosphere of 5% CO2 in air for 36–40 h. The COCs were either fertilized in vitro (positive control) using epididymal spermatozoa collected from slaughtered males or activated with 5 µm ionomycin for 5 min or 7% ethanol for 7 min, both followed by exposure to 2 mm 6-DMAP or 50 µm roscovitine for 4 h. After being washed thoroughly in embryo culture medium, they were cultured for a period of 7 days at 38.5�C in an atmosphere of 5% CO2, 5% O2, and 90% N2 in air. The embryo culture medium consisted of TCM-199 supplemented with 0.15 mg mL–1 L-glutamine, 2.1 mg mL–1 sodium bicarbonate, 0.22 mg mL–1 pyruvate, 50 µg mL–1 gentamicin, 1% insulin-transferrin-selenium (ITS) media supplement, and 10% EDS. First cleavage was recorded on Day 2 and the number of embryos developing to morulas and blastocysts was recorded on Day 7 of culture. The proportions of oocytes cleaved were 58.6 � 4.4, 55.9 � 4.5, 49.1 � 5.3, 43.2 � 6.05, and 54.1 � 3.3%, while the proportions of cleaved oocytes reaching blastocyst stage were 22.5 � 0.9, 19.1 � 2.8, 9.04 � 3.3, 8.2 � 3.8, and 15.2 � 2.3%, and those at morula stage were 61.1 � 4.9, 54.6 � 6.2, 67.1 � 7.2, 57.8 � 4.6, and 53.6 � 5.6% in the ionomycin/ 6-diethylaminopurine, ionomycin/roscovitine, ethanol/6-diethylaminopurine, ethanol/roscovitine, and IVF groups, respectively. The proportions of blastocysts obtained in the ionomycin/6-diethylaminopurine and ionomycin/roscovitine groups were higher (P &lt; 0.05) when compared with the ethanol/6-diethylaminopurine and ethanol/roscovitine groups. Also, the proportion of blastocysts obtained in the ionomycin/6-diethylaminopurine group was higher than that in the in vitro-fertilized group. In summary, methods for oocyte or cytoplast activation in dromedary camel incorporating ionomycin/6-diethylaminopurine and ionomycin/roscovitine giving better results than those incorporating ethanol/6-diethylaminopurine and ethanol/roscovitine.

In vitro tagging of embryos with nanoparticles

To develop an in vitro method for tagging embryos and to compare the development of the embryos after nanoparticles injection versus externally-applied nanoparticles derived from either polystyrene or polyacrylonitrile. Each mouse 1-cell embryo (the selected test-model) was either: (a) injected by intracytoplasmic injection or (b) co-incubated with different nanoparticles at 37 degrees C, 5% CO2 in air. The embryos were assessed after 2 and 6 days of culture. Embryo development was similar for externally-applied polystyrene nanoparticles and control (97.6 +/- 2.7 versus 100.0 +/- 0%) but different for polyacrylonitrile nanoparticles (90.0 +/- 2.8 %) on day 2. However, the results were similar on Day 6. Injected embryos were linked to lower percent development on Day 2. Few injected embryos reached blastocyst stage on Day 6 after a brief UV-fluorescence exposure. Tagging embryos by external polystyrene-based nanoparticles was the better method when compared with injected nanoparticles. Larger nanoparticles in microsphere range were easier to qualitate. Inhibited hatching limited their use beyond the blastocyst stage.

P-930: Ploidy of day 5 and 6 embryos reaching blastocyst and classified as chromosomally abnormal after single cell biopsy and PGD on day 3

To assess D5 and 6 ploidy and cell numbers in some embryos donated to research that reached post-compaction and were diagnosed as abnormal by Day 3 FISH results. The purpose of this study was not to determine the error rate of PGD, which can only be accomplished by reanalysis of all abnormal embryos, and not only those reaching blastocyst, but to determine if morphology can help detect those errors. Cross-sectional analysis. Some embryos reported as abnormal by Day 3 single blastomere FISH, which reached various post-compaction developmental stages by days 5-6, were frozen. Those which survived freeze-thaw and underwent uneventful nuclear fixation were analysed (N=23). FISH analyses involved chromosomes X,Y,13,15,16,17,18,21,22 and were performed at Reprogenetics, N.J. Day 5 or 6 embryos with <28% abnormal cells were classified as minimal mosaics; mean cell number/embryo was 79.5±44.6 (range:10-230 cells). Of the embryos reported by Day 3 PGD as abnormal, 23 reached day 5 or 6 and survived thawing. Of those, 8 developed into blastocysts with ≥90cells. For embryos classified by PGD as monosomies other than X and 21, the proportion forming blastocysts with ≥90 cells was 2/5. Of those two, one was a minimal mosaic and the other an extensive mosaic with most cells abnormal. Average number of cells for Day 5/6 Extensive Mosaic, Minimal Mosaic, Monosomy X or 21, other monosomies and Trisomy embryos was 78, 87.3, 75, 10, and 64.5, respectively. The frequency of chromosome anomalies and number of Day 5/6 embryos reaching ≥90cells as compared to Day 3 FISH findings is shown in the Table.Tabled 1 This approach does not provide the error rate of PGD since only blastocysts were reanalysed and not arrested embryos. It is well known (Sandalinas et al. 2001) that some abnormalities survive less often to blastocyst than normal embryos, thus the population here has been enriched with normal embryos.These findings indicate that the majority of embryos reaching blastocyst stage previously classified by Day 3 PGD as complex, trisomy or monosomy X or 21 were abnormal. Their average numbers of cells did not differ from normal embryos. In contrast, the few monosomies other than X and 21 reaching blastocyst were not monosomies but extensive mosaics or minimal mosaics. We suggest that for those embryos, a blastocyst biopsy could be considered and if confirmed normal, replaced.

Dishevelled proteins regulate cell adhesion in mouse blastocyst and serve to monitor changes in Wnt signaling

Wnt signaling is essential for the regulation of cell polarity and cell fate in the early embryogenesis of many animal species. Multiple Wnt genes and its pathway members are expressed in the mouse early embryo, raising the question whether they play any roles in preimplantation development. Dishevelled is an important transducer of divergent Wnt pathways. Here we show that three of the mouse Dishevelled proteins are not only expressed in oocytes and during preimplantation development, but also display distinct spatio-temporal localization. Interestingly, as embryos reach blastocyst stage, Dishevelled 2 becomes increasingly associated with cell membrane in trophectoderm cells, while at E4.5, Dishevelled 3 is highly enriched in the cytoplasm of ICM cells. These changes are coincident with an increase in the active form of β-catenin, p120catenin transcription and decrease of Kaiso expression, indicating an upregulation of Wnt signaling activity before implantation. When Dishevelled-GFP fusion proteins are overexpressed in single blastomeres of the 4-cell stage embryo, the progeny of this cell show reduction in cell adhesiveness and a rounded shape at the blastocyst stage. This suggests that perturbing Dvl function interferes with cell–cell adhesion through the non-canonical Wnt pathway in blastocysts.

Blastocyst development after assisted reproduction using spermatozoa obtained after testicular stem cell transplantation in mice

Since its introduction in 1994, testicular stem cell transplantation (TSCT) has been widely used for research. This technique may also become important for preserving fertility in pre-pubertal cancer patients. Therefore, it is necessary to investigate the safety aspects of reproduction using spermatozoa obtained after TSCT. In this study, preimplantation development of mouse embryos, using spermatozoa obtained after TSCT, was examined. TSCT-derived spermatozoa were used for IVF and ICSI. Embryos were cultured for five days until they reached blastocyst stage and were evaluated by differential staining. IVF revealed significantly lower fertilization and development rates after TSCT-IVF compared to control-IVF. Blastocysts derived from TSCT-IVF had significantly lower inner cell mass numbers (ICMs) and lower ICM/trophectoderm (TE) ratios compared to control-IVF blastocysts. No differences in fertilization and development rates were observed between TSCT-ICSI and control-ICSI, and blastocyst quality in the transplanted group was similar to that of the control blastocysts. Our study showed that after TSCT-IVF, fertilization and preimplantation development were disturbed and blastocysts showed reduced ICM and ICM/TE ratio. However, after TSCT-ICSI, both fertilization and preimplantation development were normal and blastocyst formation was comparable to control-ICSI.

Comparison Between Two Methods of Zona Drilling for Human Embryo Biopsy

Comparison Between Two Methods of Zona Drilling for Human Embryo Biopsy

Morphological Analysis of Giant Oocytes to Assess Their Relevance for the Origin of Triploid Zygote Formation

We studied the frequency, morphological characteristics, and clinical significance of giant oocytes. The union of gametes during normal fertilization is characterized by the formation of two pronuclei (2PN) at syngamy. Development of triploidy (3PN) zygotes is considered to be abnormal fertilization mechanisms. Main explanations for the occurrence of 3PN include the failure of the second polar body extrusion, dispermic penetration, or the entry of one diploid sperm. Here we report the observation of giant oocytes, which can offer an additional explanation for the origin of 3PN. Observational Study This study includes 10 giant oocytes obtained from 10 patients who underwent conventional IVF or ICSI. For IVF cases, giant oocytes were identified during the fertilization assessment. As for ICSI cases, unfertilized giant oocytes could be identified during a course of cumulus cell removal just before the ICSI procedure. They were examined for the number of the pronucleus and polar bodies(PB)18-20 hours after IVF/ICSI. Fertilized giant oocytes were further cultured to assess the developmental potential. Results: A total of 2564 oocytes were collected during the study period. 10 of them (10/2564 oocytes: 0.39%) were classified as giant oocytes. When considering only those patients having giant oocytes, the frequency of giant oocytes was found to be 9.6%(10/104 oocytes). Diameter of all giant oocytes was measured to be around 150 μm. The volume of giant oocytes was calculated to be about twice as large as that of a normal sized oocyte. For ICSI cases(n=4), all giant oocytes had apparently the two distinct first-PBs at the 9 o’clock and 12 o’clock position in perivitelline space in relation to the holding pipette. Afterward 3PN were confirmed and the second-PB extrusion was clearly recognized adjacent to the respective first-PB. All 3PN zygotes showed subsequent early cleavage to reach blastocyst stage. For IVF cases, half of giant oocytes(n=3) showed 3PN formation with extrusion of two second-PBs, and the rest of them(n=3) remained unfertilized. 1) An additional explanation for the occurrence of 3PN zygote after ICSI/IVF can be drawn from this observation. Most giant oocytes seems to have two sets of metaphase II(MII)/PB structures. It is possible that chromosomal content in these giant oocytes with two first-polar bodies is diploid, so that giant oocytes can cause digynic triploidy. 2) Considering the volume of a giant oocyte, one mechanism of its formation might be the fusion of two oocytes. 3) The early recognition of 3PN embryo is of considerable clinical significance. Majority of 3PN embryos arising from giant oocytes are thought to be composed of all triploid blastomeres. No risks should be taken and these embryos are always excluded from transfer.

Comparison of embryo quality in high-yielding dairy cows, in dairy heifers and in beef cows

The purpose of this study was to compare embryo quality of lactating Holstein Friesian cows (LHFC), non-lactating Holstein Friesian heifers (NLHFH) and Belgian Blue beef cows (BB) and to identify factors that are associated with embryo quality in LHFC and NLHFH. After superovulation and embryo recovery at Day 7, embryos ( n = 727 from 47 LHFC, 27 NLHFH and 50 BB) were scored morphologically for quality, colour and developmental stage. Blood samples and data concerning parity, age, milk production and management were collected. Data were compared univariably between the three groups. A multivariable regression model was built with quality and colour of the LHFC and NLHFH embryos as dependent variables. Only 13.1% of LHFC embryos were categorized as excellent compared to 62.5% and 55.0% of the embryos in NLHFH and BB, respectively. Almost none of the NLHFH or BB embryos displayed a dark appearance of the cytoplasm compared to 24.1% of the LHFC embryos. Only 4% of all LHFC embryos reached blastocyst stage compared to 23.2% and 17.3% in NLHFH and BB. Based on the multivariable regression analysis, “physiological status” (lactating or not) together with the serum total protein concentration of LHFC and NLHFH, was significantly associated with embryo quality and colour. Thus, LHFC display an inferior embryo quality compared to NLHFH and BB. Producing milk or not seems to be significantly associated with embryo quality. Therefore, reduced embryo quality on Day 7 following AI, could be an important factor in the subfertility problem in modern high-yielding dairy cows.

Occurrence and developmental consequences of vacuoles throughout preimplantation development

Since little is known about the actual incidence and fate of vacuoles at different stages of development this preliminary study was set up to accurately measure vacuoles and track them to day 5. Prospective study. Women's General Hospital in Austria. A total of 223 consecutive IVF and intracytoplasmic sperm injection (ICSI) cycles (206 patients). Accurate measurement of vacuoles. Affected gametes and embryos were cultured individually and tracked until day 5. Size and number of vacuoles, fertilization rate, blastocyst formation rate, blastocyst quality. There was a significant relationship between size of the vacuole (cut-off value 14 microm) and fertilization (P<.05). At zygote stage the incidence of vacuoles was higher (P<.01) in ICSI (11.6%) than in IVF (5.3%). Only 32.2% of affected ICSI-embryos reached blastocyst stage on day 5 compared with 53.0% of the normal ones (P<.001). In terms of blastocyst formation vacuolization on day 4 (P<.001) turned out to be the most severe one. At blastocyst stage inner cell mass was affected less frequently than the trophectoderm (P<.05). Three types of vacuoles could be identified: (1) those already present at oocyte collection, which develop during maturation (day 0); (2) those artificially created by ICSI (day 1); and (3) those accompanied with developmental arrest (day 4). The later that vacuoles arose, the more detrimental their effect on blastocyst formation.

Eight years' experience with an IVF surrogate gestational pregnancy programme

The aim of this study was to evaluate the effect of sperm DNA damage and protamine deficiency on fertilization and embryo development post-intracytoplasmic sperm injection (ICSI), and also to assess the effect of protamine deficiency on DNA damage. Semen samples were collected from 28 patients participating in the ICSI programme. Following sperm preparation and ICSI, the remaining processed semen samples were used to assess protamine deficiency and DNA damage employing chromomycin A3 (CMA3) staining and comet assay, respectively. Comet parameters, CMA3 percentage positivity, fertilization rate, embryo cleavage score and embryo quality score were assessed. Except for CMA3, none of the comet parameters showed significant correlation with fertilization rate. However, among comet parameters, head area and head intensity showed positive correlation with the embryo cleavage score, while comet mean intensity and head mean intensity showed a significant negative correlation with CMA3 positivity. Results of this study demonstrate that DNA fragmentation is more frequent in protamine-deficient spermatozoa. Unlike protamine deficiency, sperm DNA fragmentation does not preclude fertilization. Nonetheless, embryos derived from spermatozoa with high DNA damage have a lower potential to reach blastocyst stage.

233. Oxygen concentration during in vitro maturation of murine oocytes affects blastocyst cell lineage

Follicular antral oxygen tension is thought to influence subsequent oocyte developmental competence. Despite this, in vitro maturation (IVM) is routinely performed in either 5 or 20% O2 and while low O2 has been shown to be beneficial to embryo development in many species, the effect of altering O2 concentration during IVM has not been adequately investigated. Here we investigated the effects of a range of O2 concentrations during IVM on meiotic maturation and subsequent embryo development after IVF. Ovaries from eCG-stimulated CBA F1 female mice (21 days) were collected and intact cumulus oocyte complexes (COCs) cultured for 17–18 h under 2, 5, 10 or 20% O2 (6% CO2 and balance of N2). Matured COCs were denuded of cumulus cells, fixed and stained (1% aceto-orcein) for visualisation of maturation status. No significant difference in maturation rates between treatment groups was observed. Following IVF (performed under 5% O2, 6% CO2 and balance of N2), no difference in fertilisation rates between treatment groups was observed in a randomly selected cohort 7 h post-fertilisation. There was also no significant difference in cleavage rates after 24 h or ability to reach blastocyst stage after 96 h, with a tendency (P = 0.079) for more blastocysts in 2% O2. However there was a significant increase in the number of trophectoderm cells present in the resulting blastocysts (P &lt; 0.05) in the 2% O2 group (35 ± 2.1) compared to 20% O2 (25 ± 2.8). Our data suggests that O2 concentration during IVM does not influence nuclear maturation or subsequent fertilisation, cleavage and blastocyst development rates. However, maturation in 2% O2 significantly alters subsequent cell lineage within blastocysts to favour trophectoderm development. Such skewed trophectoderm cell number may influence embryo viability. Funded by NHMRC and NIH.

166 EFFECTS OF LEPTIN AND IGF-1 ON PRE-IMPLANTATION DEVELOPMENT, DNA FRAGMENTATION, AND GENE EXPRESSION OF BOVINE EMBRYOS CULTURED IN VITRO

Adequate regulatory proteins, growth factors, and hormones in in vitro embryo culture systems are important for improving the quality of embryos to a level similar to that in vivo conditions. The objective of this study was to define the effects of leptin, insulin-like growth factor-1 (IGF-1), and their combination on embryonic development, apoptosis, and expression profiles of a panel of developmentally important genes. Presumptive zygotes (16–18 h post-insemination) were randomly assigned and cultured in control (no supplementation), 5 ng/mL leptin (Group I), 100 ng/mL IGF-1 (Group II), and 5 ng/mL leptin and 100 ng/mL IGF-1 (Group III), all supplemented with 10% FCS on Day 4. On Day 8, the embryos reaching blastocyst stage were randomly either fixed for determination of DNA-fragmented nuclei by using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) or frozen for real-time relative quantitative RT-PCR analysis. The RT-PCR was performed to assess gene transcripts of glucose transporter-1 (Glut-1), heat shock protein 70.1 (Hsp70.1), interferon tau (IF-tau), insulin-like growth factor II receptor (IGF-IIr), desmosomal glycoprotein desmocollin III (DcIII), and DNA methyltransferase 3a (Dnmt3a). A total of 349, 322, 347, and 360 zygotes were used for the control group and Groups I, II, and III, respectively. Data were analyzed with a randomized complete block design and arcsine square root transformation of the dependent variables consisting of four treatments and six replicates. Cleavage rates were 79.5, 84.2, 87.3, and 82.4% for the control group and Groups I, II, and III, respectively, and only Group II was different from the control (P &lt; 0.05). The percentages of embryos developed beyond the 8–16 cell stage were 44.2, 48.2, 49.0, and 50.7 for the control group and Groups I, II, and III, respectively, and Group III was different from the control (P &lt; 0.05). Percentages of blastocyst development were 26.7, 29.6, 31.5, and 29.8, and the mean blastocyst cell numbers were 96.6, 98.6, 104.4, and 104.1 for the control group and Groups I, II, and III, respectively. The percentage of nuclei with fragmented DNA were 4.2, 3.3, 2.5, and 1.9 for the control group and Groups I, II, and III, respectively. Addition of IGF-1 and/or combination with leptin (Groups II and III) decreased the number of nuclei with fragmented DNA (P &lt; 0.01) as compared to the control group. Although the expression of Glut1, DcIII, and Igf2r did not change among the groups, IF-tau and Dnmt3a were down-regulated in Group II. Hsp70 and IF-tau were up regulated in Group III. Results indicate that addition of IGF-I in culture media improved the cleavage rate; combination with leptin also improved the development rates to 8–16-cell-stage embryos, decreased the TUNEL-positive nuclei, and altered expression of some of the developmentally important genes.

Effect of sperm DNA damage and sperm protamine deficiency on fertilization and embryo development post-ICSI

The aim of this study was to evaluate the effect of sperm DNA damage and protamine deficiency on fertilization and embryo development post-intracytoplasmic sperm injection (ICSI), and also to assess the effect of protamine deficiency on DNA damage. Semen samples were collected from 28 patients participating in the ICSI programme. Following sperm preparation and ICSI, the remaining processed semen samples were used to assess protamine deficiency and DNA damage employing chromomycin A3 (CMA3) staining and comet assay, respectively. Comet parameters, CMA3 percentage positivity, fertilization rate, embryo cleavage score and embryo quality score were assessed. Except for CMA3, none of the comet parameters showed significant correlation with fertilization rate. However, among comet parameters, head area and head intensity showed positive correlation with the embryo cleavage score, while comet mean intensity and head mean intensity showed a significant negative correlation with CMA3 positivity. Results of this study demonstrate that DNA fragmentation is more frequent in protamine-deficient spermatozoa. Unlike protamine deficiency, sperm DNA fragmentation does not preclude fertilization. Nonetheless, embryos derived from spermatozoa with high DNA damage have a lower potential to reach blastocyst stage.

244REAL TIME PCR EVALUATION OF EXPRESSION PROFILES OF SOME MATERNAL-SOURCED GENE TRANSCRIPTS IN IN VITRO PRODUCED PRE IMPLANTATION STAGE BOVINE EMBRYOS

Gene expression profiling data collected in a time series and quality related parameters are important for understanding the developmental mechanisms carried out in a developing embryo, and are also a source to enrich the knowledge base of embryo development. However, such data are frequently constrained by limitation and handling of the sample as well as cost associated with generating such data. Cumulatively, these factors have contributed to the existing insufficient data compared to the large need stemming from a drive to control and guide optimum embryo development. In this ongoing study, with objectives to quantify and evaluate gene transcripts identified from certain developmental stages, expression profiles of two ESTs (C256 and C112), derived from an oocyte cDNA library, were analyzed from the above perspectives to understand the change in the level of these gene transcripts throughout the pre-implantation stage of embryo development. For this analysis, pools of oocytes and embryos were prepared by balancing the amount proportional to the number of cells present. mRNA was isolated separately from each pool of matured oocytes, 2-cell, 4-cell, 8-cell, and 16-cell stages, as well as morula and blastocyst stages by using Dynal beads Oligo (dt)25 (Dynabeads, Dynal Biotech, Oslo, Norway) following the manufacturer’s recommendations. These mRNAs were checked for DNA contamination and, when proved free, first-strand cDNA was synthesised by reverse transcribtion at 42°C for 2h following standard laboratory procedures. Transcript quantification was performed by real-time PCR using gene-specific primers, equal amounts of cDNA from each sample and SYBR Green universal master mix. Following this analysis, both transcripts were found to be expressed in a wave-like manner being highly expressed in mature oocytes, declining gradually as the development stage advanced, with the lowest level at the 16-cell stage, and then reviving in level thereafter until it reached blastocyst stage. Taking the 16-cell stage as calibrator for both, C256 was 26.4, 23.2, 8.5, 1.7. 2.4 and 2.7 times more expressed in oocyte, 2-cell, 4-cell, 8-cell, morula and blastocyst stages, respectively. Similarly, C112 was 110.7, 169.2, 9.8, 2.5, 4.1 and 7.3 times more expressed in oocyte, 2-cell, 8-cell, morula and blastocyst stages, respectively. These expression patterns suggest the probable origin of these transcripts initially to be maternal. C256 is strongly similar to human retinoid X receptor beta (RXRb) gene (NM_021976.3), which is involved in transcriptional functions and in increasing DNA binding, whereas C112 is strongly similar to TATA box-binding protein-associated factor gene (AY189986.1), which is also involved in transcriptional functions. As seen from their functions, these transcripts can be vital for developing the embryo and the variations at different developmental stages shows their most probable role as part of genes contributing to developmental competence in pre-implantation development stages.

216.The effects of Viagra on sperm function and early embryo development

In an audit of UK fertility units, we have demonstrated that 42% prescribe Viagra to aid patient semen production. Viagra is a phosphodiesterase inhibitor (PDE5) and as non-specific PDEs have been shown to affect fertility, safety concerns have been raised. The aims of this study are to investigate the effects of Viagra on sperm function and early embryo cleavage. Human semen was incubated with and without Viagra (450�ng/mL sildenafil citrate, equivalent to plasma concentrations after 100�mg oral dose; Pfizer, UK). Aliquots were also prepared by a 90/45% density centrifugation gradient to separate good and poor subpopulations. All samples were analysed by computer assisted semen analysis (HTM-IVOS) up to 60 and 120�min. Prepared samples were also labelled with fluorescein isothiocyanate–peanut agglutinin to determine acrosome status. Male mice were gavaged with Viagra (equivalent dose/body wt) and mated with superovulating females. Twenty females were sacrificed 12�h later, their oviducts flushed and viable fertilized oocytes counted. Another 20 females were sacrificed 4�days after mating and their embryo numbers and cleavage stages determined. Viagra increased % progressive motility in semen (n�=�22) by 38%, VAP by 21%, VSL by 21% and VCL by 16% at 60�min (all P values &lt;0.001). These effects were sustained at 120�min. Sperm isolated from 90% (n�=�57) and 45% (n�=�15) fractions showed similar increases. Viagra also increased the proportion of acrosome reacted sperm in the 90% (+79%, P�&lt;�0.001) and 45% (+77%, P�&lt;�0.001) fractions. Further, Viagra caused a reduction in both the numbers of fertilised oocytes (–35%, P�&lt;�0.001) and those reaching blastocyst stage (85%, P�&lt;�0.001). This study demonstrates that Viagra increases human sperm motility. However, Viagra induces human premature acrosome reactions and impairs mouse fertilisation and embryo cleavage. This study raises significant concerns for its use in assisted reproduction.

59IN VITRO AND IN VIVO SURVIVAL OF NELORE NUCLEAR TRANSFER EMBRYOS RECONSTRUCTED WITH ADULT AND FETAL FIBROBLASTS

Adult skin and fetal fibroblasts are the most frequently used donor cell types for bovine cloning by nuclear transfer (NT) but there are few reports concerning Nelore cattle. The aim of this study was to evaluate in vitro and in vivo viability of Nelore nuclear transfer embryos reconstructed with Metaphase II oocytes and differentiated somatic cells (adult ear and fetal fibroblasts). Oocytes from ovaries collected at slaughterhouse were matured in vitro for 17h. Enucleation was conducted by aspiration of the first polar body (PB) and small volume of cytoplasm containing metaphase plate. For NT, each nucleus donor cell was inserted under the zona pellucida of each enucleated oocyte and the enucleated oocyte-nuclei donor cell complexes were electrofused (2 pulses of 4KVcm−1 for 20s). After electrical activation, the couplets were incubated in TCM199 plus 7.5% FCS supplemented with cycloheximide (10gmL−1) and cytochalasin D (2.5gmL−1) for 1h and ciclohexemide alone for 4 additional hours. Immediately after activation, reconstructed embryos were co-cultured with granulosa cells in SOF + 5% FCS for 7–9 days. At 7th day of culture, some blastocysts were fixed for counting cells and some transferred into recipients. A total of 377 couplets were reconstructed from fetal and 457 from adult fibroblasts. After electrofusion, 138 (fetal cells) and 166 (adult cells) embryos were incubated, and 24 (17.4%) and 26 (15.7%) reached blastocyst stage, respectively. The blastocyst cell number means were 101.3, 120 and 114.3, respectively, for adult, fetal and IVF (control) embryos. There were no significant differences in the numbers of cells of blastocysts among the groups. After transferring 18 (fetal cells) and 21 (adult cells) blastocysts, pregnancy rates at day 90 were 16.7% (3) and 19% (4). There were no significant differences between pregnancy rates. The first pregnancy from fetal cells delivered a healthy male calf weighing 34kg at Day 290. One of the remaining recipients died with hydrallantois at Day 229 and the other aborted at Day 252. There are four 5-month-pregnancies of adult fibroblast reconstructed embryos. These results indicate that NT embryos produced by fetal and adult fibroblasts of Nelore breed show similar rates of in vitro and in vivo developments. This work was supported by FAPESP (99/07377-3).

139PROSTAGLANDIN F2± COMPROMISES DEVELOPMENT OF PRE-IMPLANTATION BOVINE EMBRYOS DURING COMPACTION

Several studies have implicated prostaglandin F2α (PGF) as a major embryotoxic factor during early embryonic development in cattle. Elevated uterine concentrations of PGF were negatively associated with embryo development, quality and pregnancy rates (Schrick FN et al. 1993 Biol. Reprod. 49, 617–621; Hockett ME et al. 1998 J. Anim. Sci. 76 (Suppl 1), 241 abst; Seals RC et al. 1998 Prostaglandins 56, 377–389). Moreover, addition of PGF to culture medium decreased hatching rates of compacted morulae (Scenna FN et al. 2002 Theriogenology 53, 512 abst) and decreased development of pre-compacted (16–32 cell) bovine embryos to blastocyst stage (Scenna FN et al. 2003 Theriogenology 59, 335 abst). Furthermore, administration of an inhibitor of PGF synthesis at the time of embryo transfer improved pregnancy rates in cattle (Schrick FN et al. 2001 Theriogenology 55, 370 abst). The objective of the current study was to identify the period of time during early embryonic development that is most susceptible to the deleterious effects of PGF. After in vitro maturation and fertilization of bovine oocytes, putative zygotes were cultured in KSOMaa plus 0.3% BSA. On Day 4 post-insemination, pre-compacted (16–32 cell) embryos were removed from culture, evaluated for quality, and randomly assigned to one of the following treatments: 1) Control (KSOMaa plus 0.3% polyvinyl alcohol (KSOM-PVA; n=470) or 2) PGF-1 (1ngmL−1 PGF in KSOM-PVA; n=473; Scenna FN et al. 2003 Theriogenology 59, 335 abst). After 48h of incubation in assigned treatments, assessment of development to compacted morula stage was determined. Thereafter, embryos were kept separate according to treatments, sorted by stage of development and quality, and randomly assigned to receive either Control (CON) or PGF-1 supplemented medium until assessment of blastocyst development on Day 9. This random sorting resulted in the formation of four treatment groups comprising the initial treatments and assigned treatments during Days 6–9 (CON-CON, n=366; PGF-CON, n=226; CON-PGF, n=149; PGF-PGF, n=287). Analyses were performed incorporating a randomized incomplete block design using mixed models of SAS (2000) to determine effects of PGF on Days 4–6, 6–9 and 4–9 of development. Data were also analyzed using chi-square. Addition of 1ngmL−1 of PGF to culture medium on Days 4–9 decreased the percentage of pre-compacted embryos reaching blastocyst stage (CON-CON, 47.8%; PGF-PGF, 36%; P&amp;lt;0.05). Moreover, addition of 1ngmL−1 of PGF to the culture medium of pre-compacted bovine embryos on Days 4–6 of development decreased the percentage of compacted morulae on Day 6 (Control, 68.1%; PGF-1, 60.5%; P=0.01). However, the percentage of embryos developing to blastocyst was not decreased following addition of 1ngmL−1 of PGF on Days 6–9 of development (CON-CON, 47.8%; CON-PGF, 42.6%; P&amp;gt;0.05). Results suggest that morula stage embryos during compaction are most susceptible to deleterious effects of PGF.