bluuebunny/crossref_metadata_2025 huggingface dataset:
Spatial and temporal variability in snow accumulation at the West Antarctic Ice Sheet Divide over recent centuries
Authors: Banta J. Ryan , McConnell Joseph R. , Frey Markus M. , Bales Roger C. , Taylor Kendrick
Published: December 2008

Ice cores collected in 2000 (ITASE 00‐1) and 2005 (WDC05A, WDC05Q) from the West Antarctic Ice Sheet Divide (WAIS Divide) project site were used to investigate the spatial and temporal variability in accumulation. The ice cores were dated based on annual layer counting of multiple glaciochemical measurements resulting in bottom depth ages for WDC05A, WDC05Q, and ITASE 00‐1 of 1775, 1521, and 1653 A.D., with mean annual accumulation rates of 0.200, 0.204, and 0.221 mweq a−1, respectively. Small‐scale spatial variability (SSV) was determined using an analysis of variance of accumulation in the ice core array, thereby quantifying the uncertainty in individual accumulation records. Results indicate that the spatial variability was 0.030 mweq a−1, or approximately 15% of the average annual accumulation. An accumulation record representative of the WAIS Divide local area over recent centuries was developed using a principal component analysis to identify the coherent accumulation signal. The WAIS Divide local record exhibited 14% interannual variability (1 standard deviation of the mean) with the SSV reduced to 0.017 mweq a−1. Correlations of the WAIS Divide local accumulation record with atmospheric indices (e.g., Antarctic Oscillation) exhibited periods when the records oscillate in and out of phase. Thus, reconstructing local and global atmospheric indices from WAIS Divide accumulation records over recent centuries may prove problematic.
A fast MoM approach for analyzing large arrays in a grounded multilayered medium
Authors: Mahachoklertwattana Pongsak , Pathak Prabhakar H. , Burkholder Robert J.
Published: December 2008

A fast approach is developed here to analyze large finite planar periodic arrays of elements in a grounded multilayered medium with relatively arbitrary element truncation boundaries. An array shape matrix is introduced which contains information on the array element truncation boundary, and it can be easily incorporated into preconditioned iterative method of moments matrix solvers without significantly increasing computational resources. The performance of the present approach is illustrated through numerical results.
A Comparison of Video Versus Conventional Visual Reinforcement in 7- to 16-Month-Old Infants
Authors: Lowery Kristy J. , von Hapsburg Deborah , Plyler Erin L. , Johnstone Patti
Published: December 2008

Purpose

To compare response patterns to video visual reinforcement audiometry (VVRA) and conventional visual reinforcement audiometry (CVRA) in infants 7–16 months of age.

Method

Fourteen normal-hearing infants aged 7–16 months (8 male, 6 female) participated. A repeated measures design was used. Each infant was tested with VVRA and CVRA over 2 different sessions. The total number of head turns prior to habituation, hit rate (response consistency), false alarm rate, and sensitivity for each reinforcement condition were evaluated.

Results

No significant differences were found between the 2 reinforcement methods for total number of head turns, hit rate, false alarm rate, or sensitivity. Overall, results showed no difference between the 2 reinforcer conditions in infants 7–16 months of age.

Conclusion

The results of the present study suggest that infants in the 7- to16-month-old age range respond similarly to VVRA and CVRA as measured by response consistency and false alarm rate. VVRA is, therefore, a viable option for testing hearing in infants. However, prior to clinical implementation, the effectiveness of VVRA should be explored in infants with hearing loss.
Nuclear transfer and oocyte cryopreservation
Authors: Chang Ching-Chien , Sung Li-Ying , Amano Tomokazu , Tian X. Cindy , Yang Xiangzhong , Nagy Zsolt Peter
Published: December 2008

Somatic cells can be reprogrammed to a totipotent state through nuclear transfer or cloning, because it has been demonstrated that the oocyte has the ability to reprogramme an adult nucleus into an embryonic state that can initiate the development of a new organism. Therapeutic cloning, whereby nuclear transfer is used to derive patient-specific embryonic stem cells, embraces an entire new opportunity for regenerative medicine. However, a key obstacle for human therapeutic cloning is that the source of fresh human oocytes is extremely limited. In the present review, we propose prospective sources of human oocytes by using oocyte cryopreservation, such as an oocyte bank and immature oocytes. We also address some potential issues associated with nuclear transfer when using cryopreserved oocytes. In the future, if the efficacy and efficiency of cryopreserved oocytes are comparable to those of fresh oocytes in human therapeutic cloning, the use of cryopreserved oocytes would be invaluable and generate a great impact to regenerative medicine.
Transcriptional heterogeneity in mouse embryonic stem cells
Authors: Tanaka Tetsuya S.
Published: December 2008

The embryonic stem (ES) cell is a stem cell derived from early embryos that can indefinitely repeat self-renewing cell division cycles as an undifferentiated cell in vitro and give rise to all specialised cell types in the body. However, manipulating ES cell differentiation in vitro is a challenge due to, at least in part, heterogeneous gene induction. Recent experimental evidence has demonstrated that undifferentiated mouse ES cells maintained in culture exhibit heterogeneous expression of Dppa3, Nanog, Rex1, Pecam1 and Zscan4 as well as genes (Brachyury/T, Rhox6/9 and Twist2) normally expressed in specialised cell types. The Nanog-negative, Rex1-negative or T-positive ES cell subpopulation has a unique differentiation potential. Thus, studying the mechanism that generates ES cell subpopulations will improve manipulation of ES cell fate and help our understanding of the nature of embryonic development.
104 ESTROUS CYCLE-DEPENDENT CHANGES IN THE BOVINE ENDOMETRIUM PROTEOME
Authors: Fröhlich T. , Kashirin R. , Bolbrinker P. , Reichenbach H.-D. , Wolf E. , Arnold G. J.
Published: December 2008

Among the reproductive tissues, the endometrium plays a central role in the context of embryo-maternal communication and pregnancy recognition. During the estrous cycle, characteristic morphological and functional changes occur in the bovine endometrium, being crucial for uterine receptivity. These changes are mainly regulated by the hormones progesterone, estradiol, and oxytocin. The bovine estrous cycle, with a length of 21 days, can be divided into 4 stages: i) estrus (considered as Day 0, low progesterone level and time of ovulation); ii) metestrus (Days 1 to 5, corpus luteum formation, rising progesterone level); iii) diestrus (Days 6 to 17, high progesterone); and iv) proestrus (Days 18 to 20, corpus luteum degeneration; declining progesterone). The principles of hormonal regulation during the estrous cycle are well understood; however, in-depth knowledge of the detailed molecular mechanisms is still incomplete. To elucidate the underlying biochemical processes, the proteomes of bovine endometrial samples of all 4 stages (cycle Day 0, Day 3.5, Day 12, and Day 18) were compared in a quantitative manner. To maximize the accuracy of protein quantification, sophisticated 2-dimensional (2D) fluorescence difference gel electrophoresis (2D-DIGE) experiments were performed, using internal pooled standards for inter-gel normalization. To enhance the resolution of 2D-polyacrylamide gel electrophoresis separation, the proteins were analyzed by 2 overlapping pH gradients. In total, 28 individual DIGE experiments (14 2D gels × 2 pH gradients) were performed, corresponding to 84 gel images. With a refined statistical analysis of spot intensities, we were able to identify a total of 91 spots altered by at least a factor of ±2 (P < 0.05) in intensity between at least 2 of the 4 stages. Matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) identification of these spots showed that they originated from 66 different proteins. Moreover, for 14 of these proteins, several polymorphic variants could be identified. Gene ontology analysis of the protein IDs revealed a broad diversity of biological and biochemical functions as well as cellular localizations of these proteins. Several proteins detected (e.g. FK506 and 20 alpha-HSD) are crucial components for uterine receptivity and represent interesting targets for further functional studies.

This study was supported by the Deutsche Forschungsgemeinschaft (FOR 478).
107 EARLY EMBRYONIC SURVIVAL IN THE PIG IS HIGHER THAN CONVENTIONALLY REPORTED
Authors: Østrup E. , Vejlsted M. , Nielsen M. B. , Madsen M. T. , Maddox-Hyttel P.
Published: December 2008

It is generally accepted that 30% of the embryos in a porcine litter die within the first 40 days of pregnancy (Pope WP and First NL 1985 Theriogenology 23, 91–105). The aim of the study was to investigate the dynamics of embryonic mortality from the 2nd to the 7th week of pregnancy in a homogeneous pig population in order to test whether this dogma holds true. A total of 141 pregnant Danish Landrace × Yorkshire gilts were divided into three groups dependent on gestational length: Group 1 (Days 9 to 24 post insemination (p.i.)): At Days 9 to 18 p.i., embryos were collected by flushing the uterine horns with PBS containing 1% serum. At Days 19 to 24, embryos were identified in situ by opening of the horns along the anti-mesometrial side. All embryos were staged according to the morphological appearance of embryo proper. Pre-somite stage embryos were categorized as either: Hatched blastocysts, pre-streak 1, pre-streak 2, primitive streak, or neural groove stage embryos (Vejlsted M et al. 2006 Mol. Reprod. Dev. 73, 709–718). Somite stage embryos were staged according to the number of somites. All embryos in Group 2 (Days 24.5 to 33 p.i.) and Group 3 (Days 40.5 to 47 p.i.) were identified in situ by opening the uterine horns as described above. The localization in the uterus and the Crown Rump Length (CRL) was recorded for all embryos in these groups. The average embryo recovery rate, (i.e. the ratio between the numbers of recovered embryos and the CL numbers) was 82%. Moreover, there were no significant differences between the groups with respect to the embryo recovery rate, signaling the absence of continued embryonic mortality. No significant correlations were obtained between the location of the embryos in the uterus and the CRL (only measured for Groups 2 and 3). Our data indicate that (1) the level of embryonic mortality was less than 10 to 15% and (2) there was no continued embryonic mortality occurring between Days 9 to 47 p.i. This is in great contrast to previous reports. Furthermore, there is no evidence that the location in the uterine horn has any influence on the embryonic development.

Table 1.The average numbers of corpora lutea (CL), embryos, and the embryo recovery rates in gilts at different time points after insemination

The Danish Research Council for Technology and Production Sciences.
115 APOPTOSIS EVENT OF PREIMPLANTATION DEVELOPMENT STAGES IN PORCINE IN VITRO-FERTILIZED EMBRYOS
Authors: Hong S. M. , Jeong S. H. , Hyun S. H.
Published: December 2008

Little is known about apoptosis events in porcine preimplantation embryos. In this study, we aimed to determine whether the evaluated markers of cell death could be found at particular developmental stages of normal porcine in vitro-fertilized (IVF) embryos. We investigated the characteristics of spontaneous and induced apoptosis during preimplantation development stages of porcine IVF embryos. In experiment 1, to induce apoptosis of porcine IVF embryos, porcine IVF embryos at 22 h postinsemination were treated at different concentrations of actinomycin D (0, 5, 50 and 500 ng mL–1 in NCSU medium). Four groups were incubated at 37°C in 5% CO2, 5%O2 for 8 h, and then washed to NCSU medium and incubated until blastocyst (BL) stage. We examined cleavage rate at 2 days and BL development rate at 7 days after in vitro culture (IVC). A significantly less rate of cleavage was found in the 500 ng mL–1 group compared with others (500 ng mL–1 v. 0, 5, 50 ng mL–1; 15.4% v. 48.6%, 40%, 32%). In the results of BL formation rate, there was a significantly less BL formation rate in 500 ng mL–1 compared with others (500 ng mL–1 v. 0, 5, 50 ng mL–1; 0% v. 10%, 8.8%, 9%). In experiment 2, to evaluate apoptotic cells at different stage (2-cell, 4-cell, 8-cell and BL stage) of all groups, we conducted TUNEL assay based on morphological assessment of nuclei and on detection of specific DNA degradation under fluorescence microscope. This result showed that apoptosis is a normal event during preimplantation development in control group (0 ng mL–1 actinomycin D). A high number of the BL derived control group contained at least one apoptotic cell. Actinomycin D treated BL responded to the presence of apoptotic inductor by a significant decrease in the average number of blastomeres and a significant increase in the incidence of apoptotic cell death. In the 500 ng mL–1 group, the incidence of apoptosis increased at the 4-cell stage and later. This result suggested that apoptosis is a process of normal embryonic development and actinomycin D is a useful tool for the apoptosis study of porcine preimplantation embryos.

This work was supported by a grant (#20070301034040) from BioGreen 21 program, Rural Development Administration, Republic of Korea.
119 LEPTIN ENHANCED THE DEVELOPMENT OF BUFFALO (BUBALUS BUBALIS) EMBRYO CULTURED IN VITRO
Authors: Lu Y. Q. , Ye D. N. , Zhang M. , Lu S. S. , Lu K. H.
Published: December 2008

Buffalo is an important livestock resource in many Asian and Mediterranean countries. In vitro embryo production (IVEP) and transfer of the embryos to produce calves with high genetic merit would be of great interest in buffalo species. The efficiency of the IVEP in buffalo is low compared to that in bovine. It may be due to the reproductive physiology of buffalo or the technical factors in IVEP procedures. Recent research revealed that supplementation of leptin in the in vitro culture (IVC) medium could significantly increase embryo development (2005 Mol. Cell Endocrinol. 229, 141–147; 2006 Reproduction 132, 247–256). In this study, the effect of leptin on buffalo embryo development in vitro was assessed by supplementation of the leptin into the IVC medium. Methods: Buffalo oocytes were aspirated form 2 to 6 mm follicles from slaughterhouse ovaries and washed in TCM199 and once more with in vitro maturation (IVM) medium (TCM199, 5% ECS, 15 μg mL–1 FSH). Oocytes with compact cumulus cells were matured in IVM medium at 38.5°C, 5% CO2 for 22–22 h. The frozen–thawed buffalo sperm underwent a centrifugation in Percoll gradient to remove the dead sperm. Ten to 15 matured oocytes were added to a drop of 40 μL modified Tyrode’s medium supplemented with 0.6% BSA, 2.0 mm caffeine and 20 μg mL–1 Heparin. Concentration of sperm added into the fertilization medium was 1 to 2 million per mL. Eight to 10 h after insemination, the presumptive zygotes were transferred to IVC medium (TCM199, 10% newborn cow serum) supplemented with 0 ng mL–1 (control), 10 ng mL–1, 100 ng mL–1 or 500 ng mL–1 of leptin. Cleavage and blastocyst development rate was recorded on Day 2 and Day 6 to 8 after insemination. The experiment was repeated 10 times, and a total of 831 oocytes were used with the IVF procedures. The results revealed that the cleavage rates in the group of 0 ng mL–1, 10 ng mL–1, 100 ng mL–1 and 500 ng mL–1 of leptin were 50.1 ± 3.5%, 55.0 ± 1.3%, 50.0 ± 1.8% and 52.9 ± 2.2%, respectively. No statistical difference was observed regarding cleavage rates between treatments (P > 0.05). The percentage of oocytes developing to blastocysts in the group of 10 ng mL–1 and 100 ng mL–1 leptin were 26.1 ± 1.5% and 23.5 ± 1.2%, respectively, significantly higher than that of 17.5 ± 2.1% in the control (P < 0.05). The blastocyst development rate in the group of 500 ng mL–1 leptin was 20.9 ± 1.4%, less than that of 10 ng mL–1 (P < 0.05). In conclusion, the results of this study indicated that supplementation of leptin in the IVC medium could enhance the blastocyst development in buffalo species and the optimal concentration of leptin in the present procedures was 10 ng mL–1.

This work was jointly supported by National Science and Technology Supporting Program (No. 2006BAD04A18), Guangxi Science Foundation (0832012) and Guangxi University Key Research Program (No. 2005ZD05).
120 EFFECT OF INDIVIDUAL CULTURE SYSTEM USING WOW OR CELL-TAK ON DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS
Authors: Matoba S. , Lonergan P.
Published: December 2008

The culture of embryos individually in vitro is generally associated with poorer developmental rates. However, the ability to do this successfully would greatly facilitate studies where identification of individual embryos, or the embryos from a particular donor, is necessary. The objective of this study was to examine the effect of culture system on the development of individual IVP bovine embryos. Presumptive zygotes (n = 1301, 6 replicates), produced by IVM/IVF, were used. The aim of Experiment 1 was to compare development of bovine embryos in SOF or CR1aa supplemented with 5% FCS. Zygotes were cultured in droplets under oil as follows: (i) 20/25 μL, (ii) 20/100 μL or (iii) 20/100 μL individually in the Well of the Well (WOW) system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 254–264). Twenty WOW were prepared in a 100 μL droplet of medium under oil using a sterile rod. The aim of Experiment 2 was to compare development of embryos cultured in groups but individually identifiable on the cell adhesive Cell-Tak (Stokes et al. 2005 Dev. Biol. 284, 62–71) or in the WOW system. Zygotes were cultured as follows: (i) 20/20 μL, (ii) 20/20 μL with Cell-Tak, (iii) 20/100 μL with Cell-Tak or (iv) 20/100 μL in WOW. A drop of Cell-Tak (1 μL/20 μL medium) was placed on the base of the dish, dried for 20 min, washed with sterile water and dried completely. Once dried, the area was covered with 10 μL of FCS-free medium and groups of 20 zygotes were placed on the Cell-Tak in a 5 × 4 grid formation a maximum of 160 μm apart. Then, an additional 10 μL or 90 μL medium supplemented with FCS was added to give a final volume of 20 or 100 μL. Cleavage and blastocyst rates were assessed on Day 2 and Days 7–9, respectively. Data (means ± SE) were analyzed by one way ANOVA. In Experiment 1, there were no differences between SOF and CR1aa with respect to culture of embryos individually in WOW (P > 0.05); therefore, SOF was used as the basal medium for Experiment 2. There were no differences (P > 0.05) between the cleavage and blastocyst rate among drop sizes and individual culture systems; individual culture, irrespective of the system used (Cell-Tak or WOW), resulted in the similar developmental rates to the control. In conclusion, individual embryo culture offers the opportunity to study embryo development in a more powerful manner. Furthermore, the use of the cell adhesive Cell-Tak may be more practical because it removes the potential variability associated with well dimensions in the WOW system and may improve any potential paracrine effects during embryo culture. Further studies are required to establish the viability of such embryos after transfer.

Table 1.Effect of individual culture system on development of IVP bovine embryos

Supported by Science Foundation Ireland.
121 DIFFERENTIAL mRNA EXPRESSION BETWEEN IN VIVO AND IN VITRO-DERIVED BOS INDICUS AND BOS TAURUS EMBRYOS
Authors: Nasser L. , Stranieri P. , Gutiérrez-Adán A. , Clemente M. , Jorge de Souza L. , Palasz A. T.
Published: December 2008

Brazil is a leading country in the world of commercial use of in vitro-produced bovine embryos with 200 000 transfers per year. The majority of in vitro-produced embryos are pure breed Nelore and are transferred fresh with 40% pregnancy rate. However, pregnancies are drastically reduced with frozen in vitro embryos. This experiment is part of our effort to learn more about molecular composition and morphology of in vitro-derived embryos that may be responsible for such discrepancy. We examined molecular expression of mRNA transcripts of 6 selected genes; apoptosis Bax,TP53(p53), SHC1SHC(p66), insulin growth factor receptor (IGF2R), stabilization of the plasma membrane PLAC8 and glucose conversion H6PD in in-vivo (control) and in-vitro Nelore and Bos taurus embryos. In vivo embryos were collected from superovulated cows at Day 7. In vitro embryo was produced from oocytes aspirated from live cows. A total of 284 oocytes (4 replicates) were matured and fertilized by standard IVF procedures. Presumptive zygotes were cultured in CR2 medium with 5% BSA in 50 μL drops (25 zygotes per drop) at 39°C under paraffin oil and 5% CO2 in humidified air. Embryos that developed on Days 7 to blastocyst were transferred to recipients, and 10 blastocysts from each replicate were frozen for evaluation of gene expression patterns. Poly(A) mRNA was prepared from 3 groups of pools of 10 in vitro embryos and 10 of control in vivo-derived embryos. The quantification of all gene transcripts was carried out by real-time quantitative RT-PCR using the comparative CT method. Data on mRNA expression were normalized to the endogenous H2a.z and was analyzed by one-way repeated-measures ANOVA. The cleavage rates at Day 2 and number of blastocysts developed at Day 7 were 80.3 ± 3.2 and 42.2 ± 6.4, respectively. The level of expression of IGF2R was significantly (P < 0.05) higher in in vivo-derived embryos than in both groups of in vitro embryos. The expression of all 3 apoptosis genes were lower (P < 0.05) in in vivo than in vitro embryos with exception of p53 gene that was not different between Nelore in vitro and in vivo embryos but was significantly higher (P < 0.05) in Bos taurus in vitro embryos. There was no difference in expression of PLAC8 gene among any tested group of embryos and in expression of H6PD gene between Nelore in vitro and in vivo embryos. We concluded that significant differences in molecular makeup between in vitro and in vivo-derived Nelore embryos exist. Of particular importance seems to be pattern of expression of IGF2R receptor gene known as a good indicator of embryo quality, which promotes proliferation and differentiation. Similarly, higher expression of 2 BAX and p66 genes of apoptosis in in vitro embryos seems to be a further indication of inferior quality of Nelore in vitro-derived embryos that showed to be more profound in Bos taurus in vitro-derived embryos.
122 GLYCOSAMINOGLYCANS IMPROVE THE NUMBER OF BLASTOMERES IN ZONA-FREE RAT AND MOUSE EMBRYOS
Authors: Okuyama M. , Funahashi H.
Published: December 2008

In general, early mammalian embryos are enclosed by zona pellucida until the blastocyst stage. The possible role of zona pellucida could be to maintain the three-dimensional structure of cleaving embryos, as well as preventing blastomeres from attack by immune cells and miscellaneous germs. However, beneficial roles of the zona on early development of blastomeres are still unknown. The present study was undertaken to examine if the absence of zona pellucida of rat and mouse embryos at the 8-cell stage affected the early development to the blastocyst stage. Furthermore, we examined whether supplementation of culture medium with glycosaminoglycans, such as hyaluronic acid and heparan sulfate, improved the early development or not. In the first experiment, embryos at the 8-cell stage were collected from mated Wistar rats or ICR mice and then, directly or after removing zona pellucida by using acidity Tyrode’s solution, cultured in modified R1ECM or kSOM medium for 24 h in an atmosphere of 5% CO2 in air. Following culture, the incidence of blastocyst formation and the cell number of blastocysts were examined. In the second experiment, intact or zona-free 8-cell embryos were cultured in various concentration of hyaluronic acid (0, 65, 125, 250 μg mL–1) or heparin sulfate (0 or 15 μg mL–1) for 24 h. After culture, blastocyst formation and cell number of blastocyst were similarly examined. Statistical analyses was performed by one-way ANOVA with Bonferroni/Dunn’s post hoc test (significance, P < 0.05). All percentage data were subjected to arc-sine transformation before statistical analysis. Percentage of blastocyst formation and the mean cell number of the blastocyst were less when zona-free 8-cell rat and mouse embryos were cultured (72.1 ± 2.9% and 22.8 ± 0.7), as compared with control intact embryos (94.3 ± 3.5% and 30.6 ± 1.1). Supplementation with hyaluronic acid (250 μg mL–1) improved the blastocyst formation rate of rat embryos (86.4 ± 5.0%) and the cell number of blastocysts (28.8 ± 0.5) to the same level of zona-intact embryos. In mouse embryos, the same concentration of hyaluronic acid improved only the cell number of blastocysts (from 21.7 ± 0.7 to 28.9 ± 0.7) to the same level of zona-intact embryos (30.9 ± 0.7). When heparin sulfate was supplemented, the incidence of blastocyst formation of rat embryos did not improve, but the cell number of the blastocyst (31.1 ± 0.7) did improve to the similar level with zona-intact embryos (30.6 ± 1.1). These results indicate that the zona pellucida has a beneficial effect on the early development of rat and mouse embryos and suggest that glycosaminoglycans, such as hyaluronic acid and heparan sulfate, contribute to the beneficial effect.
125 A COMPARISON OF SEQUENTIAL MEDIUM WITH NON-SEQUENTIAL MEDIUM: DO SOME PATIENTS' EMBRYOS CULTURE BETTER IN ONE THAN IN THE OTHER?
Authors: Pomeroy K. O. , Foley S. , Faber B. , Moffitt D. V. , Johnson M. D.
Published: December 2008

The development of sequential media is often reported as one of the reasons why human IVF pregnancy rates have increased over the last 10 years. The objective was to determine if a single culture medium (global) is as effective in human embryo culture as a sequential medium (G series) and whether embryos of some patients prefer one medium over the other. Patients (n = 144) were placed into one of three treatment arms. Treatment 1, sequential: culture in G Series medium. Treatment 2, nonsequential: culture in global medium. Treatment 3, split: After fertilization, a patient’s embryos are split between the above two treatments. All patients with embryos from April 2007 to January of 2008 (PGD patients were excluded) were allocated to one of the three treatments. Eggs were collected in and fertilized in vitro Life media (GMOPS and GFert, respectively). In treatment 1, patients’ embryos (n = 346) were cultured in sequential media. In treatment 2, patients’ embryos (n = 482) are cultured in nonsequential media. In treatment 3, embryos (n = 420) from each patient are split between sequential and nonsequential media after rinsing in the treatment medium. Patients whose embryos met the criteria (at least 2 embryos, >6 cells, grade excellent to good) were transferred on day 5. All embryos remaining after embryo transfer were frozen on day 5. A maximum of 2 embryos was transferred per patient. Frequencies were compared with a chi square test. Results: The percentage of cases that achieved transfers on Day 5 (an indication of embryo quality) were 44.0%, 50.0% and 68.4% for sequential, nonsequential and split, respectively. Split was significantly different from the sequential (P < 0.05). The percentage of transferrable blastocysts was 27% for sequential, 43% for nonsequential and 39% for split (difference between sequential and the other two was significant at P < 0.0005). Pregnancy rates and implantation rates were 32%/19.0%, 36%/22.5% and 50%/32.2% for sequential, nonsequential and split. (Only implantation rates between split and sequential were significant P < 0.05). For embryos in the split treatment, development to transferable blastocysts was 37.0% in sequential media v. 41.6% in nonsequential medium (not significant). In conclusion, a single medium (nonsequential) for culture to blastocyst yields results similar to embryos cultured in the more complex sequential medium. There is some evidence that a benefit can be gained by splitting embryos between two culture media to increase implantation rates. Further studies need to be done to see if this will result in increased pregnancy rates.
131 APOPTOSIS AND DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS EXPOSED TO SUPRAPHYSIOLOGICAL CONCENTRATIONS OF INSULIN-LIKE GROWTH FACTOR-1 (IGF-1)
Authors: Velazquez M. A. , Niemann H.
Published: December 2008

It has been hypothesized that high non-physiological IGF-1 levels are partially responsible for the recurrent pregnancy loss observed in women with the polycystic ovary syndrome (Eng GS et al. 2007 Diabetes 56, 2228–2234). The aim of this study was to determine the effect of supraphysiological concentrations of IGF-1 on blastocyst production and the occurrence of apoptosis in bovine embryos, which are a good model for human embryo development (Baumann CG et al. 2007 Mol. Reprod. Dev. 74, 1345–1353). COC obtained by slicing from abattoir ovaries were matured (TCM-199, Sigma) for 24 h and fertilized (Fert-TALP) for 18 h (Day 0) in vitro. Two different IGF-1 (Recombinant human IGF-1, R&D Systems GmbH, Wiesbaden, Germany) concentrations (supraphysiological = 1000 ng mL–1 and physiological = 100 ng mL–1) were added to the culture media (Synthetic oviduct fluid/BSA) and compared with a control group (no IGF-1 supplementation). On Day 8, blastocyst rates (22 replicates) were recorded and DNA degradation was detected in blastocyst nuclei using a cell death detection kit (Roche Diagnostics GmbH, Mannheim, Germany) based on the terminal deoxinucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) principle. Embryos (n = 27 [control], n = 29 [both IGF-1 groups]) from 4 replicates were examined by confocal laser scanning microscopy. Data were analyzed by ANOVA and the Fisher exact test using the SigmaStat 2.0 software package (Jandel Scientific, San Rafael, CA). Cleavage was numerically improved by both, 1000 (59.1 ± 1.8) and 100 (58.2 ± 2.8) ng IGF-1 over controls (53.5 ± 2.2), but the differences did not reach statistical significance (P = 0.22). The proportion of hatched blastocysts was enhanced by 100 (5.8 ± 1.0, P = 0.03) and 1000 (5.1 ± 0.7, P = 0.03) ng IGF-1 compared to controls (2.8 ± 0.6). Total blastocyst rate was increased by 100 ng IGF-1 (34.4 ± 1.9, P = 0.02) over controls (28.3 ± 1.7), but not by 1000 ng IGF-1 (29.1 ± 1.6 P = 0.75). The 100 ng IGF-1 group (38.5 ± 3.7) had fewer degenerated embryos (P = 0.01) compared to 1000 ng IGF-1 (49.7 ± 3.3). The proportion of embryos displaying at least one apoptotic cell was greater in the 1000 ng IGF-1 group over controls (96% v. 77% P = 0.04). The number of blastomeres with TUNEL-positive nuclei per embryo was higher in the supraphysiological group (5.5 ± 0.6, P < 0.001) compared with the control (2.3 ± 0.4) and the physiological group (2.5 ± 0.3). There were no significant differences between the control and the 100 ng IGF-1 group in this regard (P = 0.49). In conclusion, supraphysiological concentrations of IGF-1 do not increase blastocyst production but increase levels of apoptosis in bovine embryos produced in vitro.

M. A. V. is in the PhD program of the University of Veterinary medicine, Hannover, Germany, and is supported by the German Academic Exchange Service (DAAD)
135 PREGNANCY RATES AFTER SINGLE DIRECT TRANSFER OF BIOPSIED FROZEN-THAWED BOVINE EMBRYOS ACCORDING TO QUALITY
Authors: Guyader-Joly C. , Gonzalez C. , Le Bourhis D. , Moulin B. , Heyman Y. , Humblot P.
Published: December 2008

In cattle, reliable methods for whole genome amplification (WGA) have been implemented for DNA pre-amplification and subsequent genotyping from embryo biopsy containing 5 cells or more. In France, these methods are now tested under field conditions. Several studies report pregnancy rates after direct transfer of biopsied frozen–thawed grade 1 embryos similar to that intact frozen ones. Even so, grade 2 and 3 embryos represent 25.5% of the transferable embryos (AETE data, 2007) and may limit the use of the above mentioned techniques if results are not satisfactory. The objective of this study was to investigate the impact of the embryo quality on the pregnancy rates after single direct transfer of biopsied frozen–thawed embryos. Embryos were collected on 12 donor cows after 15 sessions of superovulation treatment using FSH injected twice daily in decreasing doses over 4 days. Cows were inseminated at 12 and 24 hours after onset of estrus. Embryos were recovered 7 days post-insemination and evaluated according to IETS standards. Biopsies were performed on stage 4 to 7 grade 1 to 3 embryos using a microblade. Embryonic cells from the biopsy were dry deposited in microtubes and frozen before WGA (QIAGEN REPLI-g® Mini kit, Valencia, CA, USA) and multi-genotyping (GeneMapper software® – Applied Biosystems Europe). Each biopsied embryo was equilibrated for 10 min in 1.5 m Ethylene Glycol and then loaded into straw containing two columns of F1 medium separated by a central column of 1.5 m EG with the embryo. The freezing sequence was: –7°C directly; seeding; held for 10 min; 0.5°C min–1 until –35°C before plunging into liquid nitrogen. Embryos were thawed (straws 10 s in air and 20 s in water at 20°C) and directly transferred into synchronized recipient heifers. Pregnancy was diagnosed by ultrasonography at 35 and 90 days. Effect of embryonic stage and quality on pregnancy rates were analysed by log linear models (Proc CATMOD, SAS Institute Inc). A total of 58 embryos were micromanipulated. All grade 1 and 2 embryos were successfully biopsied and frozen and, 0.8% of grade 3 (2/25) were discarded due to their low quality after biopsy. No significant effect of embryo stage and quality on pregnancy rates was found after direct transfer (Table 1). These preliminary results suggest that high pregnancy rates can be achieved after direct transfer of biopsied frozen–thawed embryos of Grade 1, 2 and 3 allowing most of the embryos to be involved in the genotyping process.

Table 1.Pregnancy rates following single direct transfer of biopsied frozen–thawed G1 to 3 bovine embryos

This work has been performed through the programme TYPAGENAE (GENANIMAL 4-03) supported by FRT/ANR and Apis-Genes.
137 DIFFERENCES IN EMBRYO PRODUCTION BETWEEN LACTATING AND NON-LACTATING HOLSTEIN DONOR COWS
Authors: Demetrio D. G. B. , Magalhaes A. , King K. , Demetrio C. G. B.
Published: December 2008

Embryo transfer is a common practice in reproductive management of large dairy herds. Data from 2 dairies in the San Joaquin Valley in California (5000 lactating Holstein producing 27 500 pounds of milk in 365 days) was used to evaluate the differences in embryo production between lactating (105.3 ± 24.6 pounds of milk/day, 161 ± 85 days in milk and 3 ± 1 lactations) and nonlactating donor cows, from July 2006 to June 2007. Cows were observed daily for estrous signs and superovulation treatment was initiated 10 d later. Cows received FSH twice a day during 4 days in decreasing doses. On the fifth treatment of FSH, cows received an injection of PGF2α and 48 h later an injection of GnRH. Cows were inseminated 12 and 24 h after the GnRH injection. Uterine flush was performed 7 d after the first AI only in cows that had 2 or more CL. The total number of ova/embryos, viable embryos (transferred fresh or frozen), degenerated, and unfertilized structures were recorded (classified according to the guidelines of IETS). A Bernoulli regression model was fitted using the R software (www.r-project.org) for the variable superovulation response and the covariates were cow condition (lactating or nonlactating), season, and interactions. From 719 superovulated cows, 668 were flushed (92.9%): 86.8% (309/356) of the lactating cows and 98.9% (359/363) of the nonlactating cows (P < 0.01). There was evidence of season effect (P = 0.09). Cows tended to respond less to superovulation treatment during summer and fall. A negative binomial regression model was fitted to the response variables total number of ova/embryo and viable embryos and the covariates were cow condition, season, and interactions. Lactating cows produced fewer (6.77) ova/embryos than nonlactating cows (14.14; P < 0.01). The adjusted means were 5.93 v. 12.53 for fall, 6.30 v. 13.32 for winter, 8.24 v. 17.40 for spring and 6.32 v. 13.34 for summer, for lactating and nonlactating cows, respectively. Cows produced more ova/embryos during spring (P < 0.01). Less viable embryos were recovered from lactating cows (3.62) than nonlactating cows (5.44; P < 0.01). The adjusted means were 2.52 v. 3.98 for fall, 3.61 v. 5.69 for winter, 4.51 v. 7.12 for spring and 3.37 v. 5.31 for summer, for lactating and non lactating cows, respectively. The production of viable embryos in fall was different from winter and spring (P < 0.01). Lactating cows responded less to superovulation treatment and produced less viable embryos than nonlactating cows, probably because of the increased metabolism due to milk production. Warmer seasons were associated with lower viable embryo production. It is important to have a group of nonlactating donors to increase embryo production in large ET programs.
140 INTRAUTERINE INOCULATION OF CATTLE WITH BOVINE VIRAL DIARRHEA VIRUS SIMULTANEOUS TO TRANSFER OF IN VIVO-DERIVED BOVINE EMBRYOS
Authors: Gard J. A. , Givens M. D. , Galik P. K. , Marley M. S. D. , Riddell K. P. , Edmondson M. A.
Published: December 2008

Bovine viral diarrhea virus (BVDV) has been shown to be associated with single transferable in vivo-derived and in vitro-produced bovine embryos despite washing. Hence, the primary objective of this study was to evaluate the potential of BVDV to be transmitted via the intrauterine route at the time of embryo transfer. A total of 10 in vivo-derived Day 7 bovine embryos were nonsurgically collected from a BVDV negative and seronegative donor cow. After collection, embryos were washed in accordance with the International Embryo Transfer Society (IETS) standards. Following washing, embryos were placed into transfer media containing BVDV (SD-1; type 1a). The embryos were immediately aspirated into 0.25-mL straws and transferred into seronegative recipients (Day 0). The total quantity of virus transferred into the uterus of each recipient was 900 to 1000 cell culture infective dose 50 (CCID50)/straw. This amount of virus was previously shown to be consistent with the average amount of BVDV associated with in vivo-derived and in vitro-produced embryos following standard IETS washing procedures after in vitro exposure to virus. The positive control heifer received 1.5 × 106 CCID50/straw of BVDV without an embryo. The negative control heifer received 1.5 × 106 CCID50/straw of heat-inactivated BVDV without an embryo. Serum and buffy coat samples were drawn from all heifers on Days 0, 3, 4, 6, 7, 8, 9, 10, 12, 15, and 30 after inoculation and analyzed for serum neutralizing antibodies and virus, respectively. The positive control heifer and all recipients of virus-exposed embryos exhibited viremia by Day 6 and seroconverted by Day 15. The negative control heifer did not exhibit viremia or seroconvert. All recipients receiving embryos were assessed for pregnancy using transrectal ultrasonography on Day 30 and 6 of 10 heifers were pregnant. On Day 60 the pregnant heifers were again assessed for pregnancy using transrectal ultrasonography. At this time only 1 of the 6 heifers was still pregnant. However, the fetus was determined to be nonviable and was removed via colpotomy. The fetus, fetal fluids and membranes were determined to be positive for BVDV via immunohistochemistry and PCR. Additionally, 213 base pairs of the 5′ nontranslated region of this PCR product were sequenced and found to be consistent with the inoculated strain. Results demonstrate that the average quantity of BVDV associated with bovine embryos after in vitro exposure and washing can result in viremia and seroconversion of seronegative recipients following transfer into the uterus during diestrus.
143 SUPEROVULATORY RESPONSE AND EMBRYO PRODUCTION INFLUENCED BY THE ADDITION OF LH AND EFFECT OF THE REPEATABILITY IN SANTA INES SHEEP
Authors: Oliveira M. E. F. , Santos I. C. C. , Pieroni J. S. P. , Ferreira R. M. , Cordeiro M. F. , Nascimento P. M. P. , Souza S. F. , Fonseca J. F. , Vicente W. R. R.
Published: December 2008

The aim this study was to evaluate the effect of the addition of LH in superovulatory response and embryo production in Santa Inês sheep. Ten donors with 60.3 ± 10.7 kg and BCS of 3.9 ± 0.3 were superovulated in a cross-over design, with a 60-day interval. Estrus was synchronized with a progesterone-releasing intravaginal device (CIDR™; Pfizer Animal Health, Brazil) inserted on Day 0 and replaced by a new one on Day 7, that was maintained to Day 14. Two doses of 37.5 g of D-cloprostenol (Prolise™, Arsa, Buenos Aires, Argentina) were administered, on Days 7 and 14. Donors also receive 256 mg of pFSH (Folltropin™, Bioniche, Belleville, ON, Canada) in 8 decreasing doses, starting on Day 12. On Day 14, all females received 200 IU of eCG (Novormon ™, Syntex, Argentina). On Day 15, the animals were homogeneously allocated in 1 of 2 groups: Control (GC, n = 10) and treated (G-LH, n = 10). Ewes in GC did not receive exogenous LH, whereas ewes in G-LH were treated with 7.5 mg of LH (Lutropin™, Bioniche), on Day 15. All females were inseminated by laparoscopy, with frozen–thawed semen, 42 and 48 h after CIDR removal. On Day 21, the embryos were surgically collected. The superovulatory response was classified in scores: (0) 4 or fewer CL; (1) between 5 and 10 CL, and (2) 11 or more CL. Means were compared using Kruskal-Wallis test and percentages using chi-square (P < 0.05). Most of donors (70%, 7/10) from G-LH presented a superovulatory response classified as score 2, and the remaining (30%, 3/10) as score 1, whereas, half of the controls were classified as score 2 and half as score 1. Ovulation rate tended to be greater in G-LH (135/158, 85.4% v. 105/135, 77.7%, P = 0.08). The number of CL (mean ± SD) was 10.5 ± 3.8 in GC and 13.5 ± 4.84 in G-LH, but was not statistically different. The number of anovulatory follicles (AF) did not differ between groups (GC: 3.0 ± 3.2; G-LH: 2.3 ± 1.6), but the proportion of AF tended to decrease in G-LH (30/135, 22.2% v. 23/158, 14.5%, P = 0.08). Considering embryo production, there was no difference between GC and G-LH (P > 0.05) related to number of recovered ova/embryos (6.1 ± 4.6 v. 8.4 ± 5.2), viable embryos (3.8 ± 4.3 v. 4.2 ± 5.2), unfertilized (1.7 ± 3.4 v. 2.0 ± 2.9) and degenerated embryos (0.7 ± 0.7 v. 2.2 ± 2.9), respectively. Data showed that the addition of LH tended to increase ovulation rate and to decrease the proportion of AF, but did not affect the number of viable embryos.
147 A PROSTAGLANDIN F2Alpha RECEPTOR ANTAGONIST MAY INHIBIT BAX EXPRESSION IN BOVINE PRE-IMPLANTATION EMBRYOS DURING EMBRYO FLUSHING
Authors: Tsuchiya K. , Ideta A. , Hayama K. , Urakawa M. , Nakamura Y. , Aoyagi Y.
Published: December 2008

Several in vivo studies have shown negative effects of prostaglandin F2α (PGF2α) on embryonic survival in cows. The PGF2α receptor (FPr) was detected in bovine embryos (Scenna FN et al. 2006 Reprod. Fertil. Dev. 18, 180). Recently, it was reported that addition of a FPr antagonist (AL-8810) to the embryo flushing medium prevented the detrimental action of PGF2α on pregnancy (Scenna FN et al. 2008 Reprod. Fertil. Dev. 20, 154). In the present study, we examined the morphological development in utero and gene expression of bovine pre-implantation embryos collected using flushing medium containing AL-8810. Embryos were collected from superovulated donors on Day 7 after artificial insemination with medium containing 1000 nm AL-8810 (n = 4, Sigma-Aldrich, St, Louis, MO, AL embryos) or with vehicle (n = 4, DMSO, Sigma-Aldrich, CON embryos). Following collection, the embryos were classified by quality and stage, and then 2 to 5 grade-1, stage 4 and 5 embryos (IETS classification) were transferred to recipient heifers (AL-embryos: n = 4, CON embryos: n = 4) for transient development in utero up to Day 15 of gestation. Embryos at Day 15 occasionally collapsed after uterine flushing due to breaches in trophectoderm (TE) tissue caused by the collection procedures. Only whole embryos were used and evaluated in this study. Using real-time PCR, the TE cells were examined to quantify the mRNA levels for 3 genes, interferon-τ (maternal recognition of pregnancy, INF-τ), connexin 43 (cell communication, CX 43) and Bcl-2-associated X protein (apoptosis, Bax). Data were analyzed using Student’s t-test and chi-square test. The average number of ova/embryos recovered at Day 7 was 12.0 ± 4.1 (AL embryos) and 8.8 ± 5.8 (CON embryos). There was no significance in the percentages of transferable embryos (AL-embryos; 67.0 ± 14.4%, 35/48 and CON-embryos; 79.6 ± 7.1%, 27/35; P > 0.05) between the 2 groups. The whole-embryo recovery rate on Day 15 for AL embryos (73.3%, 11/15) was >30 percentage points greater than that for CON embryos (41.2%, 7/17). The length of major axis of embryos and the diameter of embryonic disks were the same between AL embryos (59.6 ± 12.3 mm, 0.44 ± 0.03 mm, respectively) and CON embryos (49.3 ± 24.6 mm, 0.43 ± 0.05 mm, respectively). Transcripts of INF-τ and CX 43 in TE cells did not differ significantly between AL embryos and CON embryos (P > 0.05). However, the abundance of Bax transcripts in TE cells tended to be lower in AL embryos than in CON embryos (P = 0.07). These results suggest that addition of an FPr antagonist to the embryo flushing medium may promote the developmental capacity of pre-implantation embryos in utero.
149 RISK ASSESSMENT OF INFECTIOUS DISEASE TRANSMISSION VIA IN VITRO EMBRYO PRODUCTION USING SOMATIC CELL NUCLEAR TRANSFER
Authors: Gregg K. , Chen S. , Sadeghieh S. , Guerra T. , Xiang T. , Meredith J. , Polejaeva I.
Published: December 2008

Somatic cell nuclear transfer (SCNT) technology is a powerful tool for preservation and propagation of superior genetics of livestock animals. Bovine oocytes derived from abattoirs are usually used in SCNT embryo production. The puncture of the zona pellucida during the nuclear transfer process has raised additional concerns about the risk of disease transmission through application of this new technology. The objective of this study was to use bovine viral diarrhea virus (BVDV) as a model to perform a comprehensive risk assessment on infectious disease transmission in the SCNT system. Thirteen batches of cumulus–oocyte complexes (COC; n = 550) were collected from several abattoirs over 6 months. Two hundred were tested for BVDV before cumulus cell removal. Cumulus cells were removed from the other 350 COC by gentle vortexing in 0.1% hyaluronidase in HEPES-M199 with Hanks’ salts. The cumulus-cell-free oocytes (CFO) were then washed 3 times with FBS-free HEPES-M199 with Hanks’ salts containing penicillin-streptomycin solution (5 μg mL–1). Both COC and CFO were pooled in groups (5/group) and tested for presence of BVDV using sensitive real-time PCR method. Only 2.5% of the COC were BVDV positive and all of the CFO were BVDV negative. Additionally, 293 embryos were produced from 14 different cell lines using the previously described SCNT procedure (Zhou et al. 2008 Mol Reprod Dev. 75, 1281–1289). These embryos were generated using in vitro-matured oocytes collected as 23 different batches over 7 months. The embryos were cultured in vitro to blastocyst stage and then tested for BVDV. All of the 293 SCNT embryos (100%) were BVDV negative. In conclusion, under these SCNT embryo production conditions, a small portion of COC were BVDV positive. However, all CFO and SCNT embryos were BVDV negative. Therefore, the risk of disease transmission using abattoir oocytes and SCNT procedure is relatively low and can be eliminated by using a combination of cumulus cell removal and adequate oocyte washing procedures.

F. Arenivas, B. Findeisen, V. Farrar, E. Hwang helped with the SCNT embryo production for this study.