bluuebunny/crossref_metadata_2025 huggingface dataset:
275 TEMPORAL PATTERN OF STEROID HORMONE CONCENTRATIONS DURING IN VIVO AND IN VITRO MATURATION OF BOVINE OOCYTES
Authors: Blaschka C. , Stinshoff H. , Poppicht F. , Wrenzycki C.
Published: December 2014

Steroid hormone concentration and property can be modulated via different processes. Sulfoconjugation via sulfotransferases (SULT) changes steroids from hydrophobic to hydrophilic, necessitating a transport system such as the sodium-dependent organic anion transporter (SOAT; SLC10A6). Steroid sulfatase (STS) removes the sulfate moiety from conjugated steroids, transforming them to the free active ones. Moreover, present in vitro maturation systems do not completely mimic the in vivo situation resulting in oocytes of reduced quality. The present study investigates the local effects of sulfated steroids during follicular and oocyte development in vivo and in vitro. Follicles of bovine abattoir-derived ovaries were categorized according to their size (3 to 5, 6 to 8, 9 to 14, and >15 mm) after dissection and measurement via a caliper. Only nonatretic follicles were used (Kruip and Dieleman, 1982). Follicular fluid was collected via aspiration and analysed for the presence of steroids and their sulfated counterparts via LC-MS/MS. Moreover, oocytes were in vitro maturated with a standard protocol. The medium was measured via radioimmunoassay after 0, 4, 8, 12, 16, 20, and 24 h to detect 17β-oestradiol (E2) and progesterone (P4). Data were tested using analysis of variance (ANOVA) followed by multiple pairwise comparisons using Tukey's test. A P-value of ≤0.05 was considered significant. It was possible to detect 17β-oestradiol, progesterone, testosterone, 17β-oestradiol sulfate, estrone sulfate, pregnenolone sulfate, cholesterol sulfate (Table 1), and furthermore androstendione, estrone, androsterone, and 17OH-pregnenolone. During IVM, P4 significantly increased in the medium (4 h: 3.3 ± 1.0 ng mL–1; 24 h: 9.8 ± 1.7 ng mL–1), whereas the E2 concentration did not change (4 h: 52.8 ± 12.1 pg mL–1; 12 h: 68.4 ± 3.7 pg mL–1; 24 h: 66.9 ± 19.7 pg mL–1). In addition, preliminary data suggest that transcripts of the steroid metabolizing and transporting enzymes (SULT1E1, STS, SLC10A6) were present in cumulus cells from immature bovine COC. These results indicate for the first time that only small amounts of sulfated steroids are present in bovine follicular fluid. However, the related enzymes are present at the mRNA level. Further studies are underway to analyse the protein level.

Table 1.Steroid hormone concentration in follicular fluid
We thank Prof. Dr Wudy and A. Sánchez Guijo for the LC-MS/MS analysis. Furthermore, we gratefully acknowledge the financial support of the German Research Foundation (DFG; FOR 1369).
290 3-DIMENSIONAL VISUALIZATION OF BOVINE OOCYTE FERTILIZATION BY CONFOCAL LASER SCANNING MICROSCOPY
Authors: De Monte E. , Reichenbach M. , Reichenbach H. , Wolf E. , Habermann F.
Published: December 2014

Cattle can serve as a model organism to resolve central questions in mammalian reproduction that cannot be clarified in the mouse model due to notable species-specific peculiarities of early mouse embryogenesis. As part of a project on structural, molecular, and functional deficiencies of bovine oocytes, we started to systematically investigate fertilization and the onset of embryo development in vitro by 3-dimensional multicolor fluorescence microscopy. We are using 3D visualisation as key approach to clarify the multiple parallel and sequential processes and events of fertilization as well as to identify and classify errors and failures. Moreover, we aim to gain insights into the mechanisms of aberrations by linking processes at the cellular and the molecular level. We studied class I and II oocytes collected from slaughterhouse ovaries and matured for 23 h in vitro. Oocytes were fixed at different times from 4 to 12 h postinsemination with formaldehyde in a microtubule-stabilising buffer containing taxol in such a way that the 3-dimensional cell architecture was maintained, and were stained for DNA, microtubules, and f-actin microfilaments. In addition, serine 10-phosphorylated histone H3 was used as a marker for chromosome condensation and the spindle midbody. For 3-dimensional imaging of the oocytes in toto, confocal serial sections were captured at 1-µm intervals using a 40× objective (NA = 1.3). For imaging details, we used a high spatial sampling density (pixel size: 50 × 50 nm, z-step size: 200 nm) close to the Nyquist criterion and image restoration by maximum likelihood estimation (MLE) deconvolution. A series of more than 500 three-dimensional snapshots of fertilized oocytes at different points in time gives a first detailed view on the spatial and temporal course of the sperm entry, the formation of the paternal pronucleus and the sperm aster, completion of oocyte meiosis and the formation of the maternal pronucleus, as well as dynamic changes of the cytoskeleton. Moreover, we can document a spectrum of abnormalities including spontaneous parthenogenetic oocyte activation, polyspermy, and aberrations of meiosis I and II. The latter include irregular spindle formation and chromosome segregation, the occurrence of chromatin bridges and abnormal spindle positioning and rotation (e.g. leading to nonextrusion of a first or a second polar body or the extrusion of two second polar bodies). Our microscopic investigation in the bovine system contributes to unraveling the origins of irregular cleavage, aneuploidy, and mosaicism in mammals. Three-dimensional high-speed microscopy of oocytes and zygotes in affordable timeframes could be of great value in improving the differential diagnosis of oocyte and sperm dysfunction, as well as in identifying and dissecting problems, limitations, and potential risks of reproductive technologies (ART).This work is supported by the Deutsche Forschungsgemeinschaft (DFG FOR 1041).
301 ZINC IS INVOLVED IN PORCINE OOCYTE MEIOTIC MATURATION
Authors: Zhao M.-H. , Kim T. , Kim N.-H. , Cui X.-S.
Published: December 2014

Zinc is an extremely important trace element that plays important roles in several biological processes. In this study, we investigated the role of zinc during meiotic resumption and metaphase arrest in in vitro-matured porcine oocytes. Oocytes at either germinal vesicle (GV) or MII stage were treated with TPEN, a Zn2+ chelator. Meiotic resumption and activation were assayed. Effect of PMA, a PKC activator, on GV breakdown (GVBD) and oocytes activation after TPEN treatment were checked. Results showed that depletion of zinc with 3 µM TPEN-blocked oocytes at GV stage (60.85 ± 5.15 v. 15.60 ± 0.20%; P < 0.05) after 25 h of maturation. The 10-µM TPEN treatment at MII stage significantly (P < 0.05) increased pronucleus formation (90.61 ± 9.10 v. 5.56 ± 9.62%; P < 0.05) and the second polar body extrusion (93.64 ± 5.53 v. 8.59 ± 8.34%; P < 0.05). The p34cdc2 activity was decreased in both MII and GVBD oocytes that were treated with TPEN as compared to control. Phosphorylated MAPK measured by Western blot was also decreased in GVBD oocytes when zinc was depleted. This might be explained by the low expression of C-mos Cyclin B1 and Cdc2 at this stage. Treatment of the oocytes with PKC agonist PMA (100 nM) rescued the meiotic resumption arrest observed after TPEN treatment (GV stage: 26.91 ± 3.10 v. 83.89 ± 11.94%; P < 0.05). The level of phosphorylation of MAPK and p34cdc2 activity were rescued when PMA were used. Treatment oocytes with 100 nM PMA in the GV stage also increased the signal of zinc indicator, fluozin-3-a.m., by about 4-fold in cytoplasm (P < 0.05). These results showed that zinc regulates meiotic resumption probably through PKC. However, although the TPEN treatment reduced phosphorylation of PKC substrates in both meiotic resumption and the MII stage, rescue of PKC substrates phosphorylation with PMA did not prevent the activation of oocytes caused by zinc depletion. These data demonstrate that zinc regulates meiotic resumption via a PKC-dependent pathway, but independent of that in maintaining of metaphase arrest in porcine oocytes.This work was supported by the Bio-industry Technology Development Program, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea, and by a grant from the Next-Generation BioGreen 21 Program (No. PJ009601 and PJ009098), Rural Development Administration, Republic of Korea.
325 CHARACTERIZATION OF PRIMED PORCINE PLURIPOTENT STEM CELL LINES DERIVED FROM VARIOUS ORIGINS INCLUDING iPS-NT
Authors: Kim E. , Lee C.-K. , Hyun S.-H.
Published: December 2014

Pigs are significant as a disease model in translational research. However, authentic porcine embryonic stem cells (ESC) have not yet been established showing limited capacities until now. In this study, a total of 7 primed ESC lines were derived from porcine embryos of various origins, including in vitro-fertilized (IVF), parthenogenetic activation (PA), and nuclear transfer (iPS-NT) from a donor cell with induced pluripotent stem cells (iPSC). We observed typical morphology, intensive alkaline phosphatase activity, and normal karyotype in all pESC lines. Also, the expression of pluripotency markers such as OCT4, Sox2, NANOG, SSEA4, TRA 1–60, and TRA 1–81 was shown in our pESC. We investigated expression of key markers of lineage commitment to confirm the differentiation potentials of the 7 cell lines to formation of EB and all 3 germ layers, such as AFP (endoderm), DESMIN (mesoderm), and CRABP2 (ectoderm) by RT-PCR and Cytokeratin 17 (endoderm), Desmin (mesoderm), and Vimentin (ectoderm) by immunofluorescence analysis. We also examined the XIST gene expression and nuclear H3K27me3 foci from all female cell lines for analysing epigenetic characteristics. Furthermore, we classified 2 colony types (normal and transformed colony) and 3 subpopulations of ES cells composed of transformed colonies with intrinsic morphological characteristics: petaloid rapidly self-renewing cells, small spindle-shaped cells, and large flattened cells. This result will help to approach the goal for establishing authentic naive pluripotent stem cells in pigs and it will make possible sophisticated genetic manipulation to create ideal animal models for preclinical research and studies of human diseases.This work was supported, in part, by a grant from the National Research Foundation of Korea Grant Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.
33 TELOMERASE ACTIVITIES IN CLONED BEAGLE DOGS
Authors: Kim G. A. , Oh H. J. , Kim M. J. , Jo Y. K. , Setyawan E. M. N. , Choi Y. B. , Lee S. H. , Lee B. C.
Published: December 2014

Telomerase is important ribonucleoprotein for restoring telomere length from its own RNA template. Regarding cloned animals derived from somatic cell nuclear transfer (SCNT), interesting questions have been raised about whether the cloning process restores cellular telomerase activity undergone by their donor cells. The present study was conducted to determine the effects of cloning on telomerase activity in the dog and normality of telomerase activity in cloned dogs. Focusing our attention on differences in telomerase activity depending on the age, we analysed telomerase activity in dogs produced by natural breeding of various ages. Comparison of the telomerase activities of cloned dogs and those of dogs produced by natural breeding was also performed. For SCNT, 2 cell donors, 7- and 9-year-old beagles, were used and donor cells were isolated from ear skin. After establishing donor cell lines, the enucleated canine in vivo-matured oocytes and the cells were injected and fused by electrofusion. After 30 days from embryo transfer, pregnancy diagnosis was performed and 7 cloned dogs were produced on the due date. For standardization of telomerase activity in beagles produced by natural breeding, blood of total 14 dogs at each age (10 months, 20 months, 5, 7, and 8 years old) were collected and telomerase activity was measured by the telomeric repeat amplification protocol (TRAP) assay. Telomerase activity measurements of at least 6 replications in each dog were performed. For statistical analysis, one-way ANOVA with Dunn's Multiple Comparison Test was used. Significant differences in telomerase activity were observed between the blood of cloned and donor dogs. It was shown that mean telomerase activities were decreased according to biological aging with significances. Mean telomerase activities in 10 cloned dogs were higher than those of a donor dog. Cloned dogs also showed similar levels of telomerase activities as their age-matched natural bred dogs, suggesting that they are within the variation in normal dogs. These observations indicate that the cloning process restores the telomerase activity in the dog. Thus, complex regulation of telomerase activity during nuclear reprogramming may regulate and be involved in telomerase activity in cloned dogs. It remains to be determined whether telomere length is correlated with telomerase activity and if it accurately reflects the physiological age of cloned dogs.This study was supported by IPET (#311062–04–2-SB010), RDA (PJ008975022013), Research Institute for Veterinary Science, the BK21 program, Nestle Purina Korea, and TS Corporation.
343 DIRECT EXPRESSION OF PLURIPOTENCY MARKERS IN CULTURED SOMATIC CELLS BY SMALL REPROGRAMMING MOLECULES
Authors: Toschi P. , Iuso D. , Anzalone D. A. , Czernik M. , Ptak G. , Loi P.
Published: December 2014

The differentiate state of the cell may be reversed by a process called reprogramming. To date, a totipotent status is conferred to a somatic cell by nuclear transfer (SCNT) and a condition of pluripotency is conferred by induced expression of defined factors (iPSC). While the restoration of full totipotency by SCNT is rarely achieved, pluripotency by Yamanaka's factors (Oct4, c-myc, Sox-2, Klf4) is inducible, although with low efficiency, in a large set of cell in different animal models. However, the isolation of iPSC requires complex technical skills and time-consuming protocols. In our laboratory we have observed that the simple expansion of fibroblasts in culture switches on pluripotency markers such as Oct4 and Nanog (Anzalone et al. 2015 Reprod. Fertil. Dev. IETS Abstract 344). CHIR99021 is a small molecule, targeting the Wnt/β-catenin signalling pathway, which is used for stem cell culture (Li et al. 2009). CHIR99021 acts as selective inhibitor of both isoform of GSK3 α/β regulating cellular proliferation and differentiation. In this work we tested the hypothesis that the exposure to a small reprogramming molecule (CHIR99021) induces pluripotency marker expression in primary cultures of somatic cells. Sheep and mouse primary fibroblasts cultured in low oxygen and induced to enter GO (low serum, 0.5% FBS for 5 days, <3% cell proliferation in our conditions) were treated with different CHIR concentrations (from 2.5 to 5 µM) for different time periods (from 1 to 5 days) in order to test the proper concentration and to exclude any cytotoxic effects. Nuclear reprogramming was assessed in treated and control cells by analysing β-catenin and oct4, nanog, sox2, klf4, and c-myc expression by immuno-detection and PCR. We found that CHIR interferes with β-catenin pathway in both sheep and mouse fibroblast in a time- and dose-dependent manner; the best results were obtained using 3 µM of CHIR for 3 days. Western blot analysis confirmed that CHIR treatment leads to an increased cellular level of β-catenin; furthermore, pluripotency marker expression (protein and mRNA) was increased (P = 0.023 nonparametric Mann-Whitney test) in CHIR-treated cells compared to controls. These observations, confirmed in both the experimental models, indicate that treatment with a small molecule inhibitor interfering with glucose metabolism induces the expression of pluripotency marker in somatic cells.
346 EXPRESSION OF IMPRINTED NONCODING RNA FROM THE DLK1-DIO3 LOCUS IN HUMAN EMBRYONIC STEM CELLS ADVANTAGES NEURAL LINEAGE DIFFERENTIATION
Authors: Mo C.-F. , Wu F.-C. , Tai K.-Y. T. , Chang W.-C. , Chang K.-W. , Kuo H.-C. , Ho H.-N. , Chen H.-F. , Lin S.-P.
Published: December 2014

Pluripotent stem cells are increasingly used for therapeutic models, including transplantation of neural progenitors derived from human embryonic stem cells (hESC). Recently, long noncoding RNA (lncRNA), including Maternally Expressed Gene 3 (MEG3) derived from the DLK1-DIO3-imprinted locus, were found to be expressed during neural developmental events. Their deregulations are associated with various neurological diseases. The DLK1-DIO3-imprinted locus encodes abundant noncoding RNA (ncRNA) that are regulated by differential methylation on the locus. The aim of our research was to study the correlation between the DLK1-DIO3-derived ncRNA and the capacity of hESC neural lineage differentiation. We classified hESC into MEG3-ON and MEG3-OFF based on the expression levels of MEG3 as well as its downstream microRNA by qRT-PCR. Initial embryoid body (EB) formation was conducted to examine the 3 germ layer's differentiation ability. Complementary DNA microarray was used to analyse the gene expression profiles of hESC. Directed neural lineage differentiation was performed, followed by analysis of neural lineage marker expression levels and neurite formation via qRT-PCR and immunocytochemistry methods to investigate the capacity of neural differentiation in MEG3-ON and MEG3-OFF hESC. As for statistics, error bars indicate standard error of the mean. Student's t-test was used for calculating P-values, and a P-value of less than 0.05 was considered to be significant. Our results showed that MEG3-ON and MEG3-OFF hESC differed greatly in DLK1-DIO3-derived ncRNA expression levels, but had comparable pluripotency gene expression profiles. Genes related to nervous system development and neural cancers were differentially expressed in MEG3-OFF hESC, where DLK1-DIO3-derived ncRNA were repressed compared to MEG3-ON ones before differentiation. In neural lineage-like cells derived from MEG3-OFF hESC, lower expression levels of neural lineage markers and impaired neurite formation were observed compared to MEG3-ON hESC at the same time points after differentiation. We suggest that the expression of DLK1-DIO3-derived lncRNA, MEG3, can be used as a simple and effective screening criterion for identifying MEG3-ON hESC with activation of DLK1-DIO3-imprinted ncRNA as starting materials to benefit neural lineage-associated studies.
363 EXTENDING THE IN VIVO MATURATION TIME TO PERMIT FLEXIBLE TIMING OF OOCYTE COLLECTION IN SUPERSTIMULATED WOOD BISON (BISON BISON ATHABASCAE)
Authors: von der Porten M. W. , Cervantes M. P. , Palomino J. M. , Adams G. P.
Published: December 2014

Wood bison are a species threatened by endemic brucellosis and tuberculosis. Reproductive technologies are being developed in an effort to ensure the genetic diversity of wild wood bison, and to prevent disease transmission to healthy bison, livestock, and humans. For the purposes of IVF, recent results revealed that cumulus cell expansion was more extensive in in vivo- v. in vitro-matured cumulus-oocyte complexes (COC), and more oocytes reached maturity after 30 v. 24 h of in vivo maturation following hCG treatment (Cervantes et al. 2013 Reprod. Fert. Develop. 25, 283). An experiment was designed to determine the effects of an additional 4 h of in vivo maturation on follicle development, unwanted ovulation, and COC collection efficiency. Wood bison cows (n = 28) underwent transvaginal ultrasound-guided follicle ablation to induce emergence of a new follicular wave (Day 0 = day of wave emergence, 1 day after ablation) during the non-breeding season. Bison were given FSH diluted in hyaluronan IM on Days 0 (300 mg) and 2 (100 mg), and 2500 IU hCG IM on Day 4. Bison were then assigned randomly to 2 groups (n = 14 per group) in which transvaginal oocyte collection was done at either 30 or 34 h after hCG treatment. The number and size of follicles available for aspiration (i.e. = 5 mm) was compared between groups by Student's t-test. Binomial data (COC collection rate and ovulation rate) were compared by chi-square, and the proportion of cows that ovulated was compared using a Fisher's exact test. Ovulation was defined as the sudden disappearance of follicles ≥10 mm from the hCG treatment to the time of COC collection. The numbers of follicles ≥5 mm and ≥10 mm at the time of COC collection were not different between the 30 and 34 h groups (19.0 ± 1.4 v. 17.4 ± 2.4, and 9.5 ± 1.2 v. 7.7 ± 1.8), nor was the average size of follicles = 5 mm (9.9 ± 0.2 v. 9.8 ± 0.2 mm). The number of follicles aspirated was similar between the 30 and 34 h groups (16.4 ± 1.4 v. 13.4 ± 2.1), but the pre-collection ovulation rate was lower in the 30 h group (12/89 [13.5%] v. 47/147, [32.0%]; P = 0.003), as was the proportion of bison that ovulated (3/14 v. 10/14, P = 0.02). The COC collection rate was lower in the 30 v. 34 h group (64.3% v. 78.2%; P = 0.003), but the total number of COC collected per bison was similar (10.6 ± 1.7 v. 10.5 ± 1.5). Although waiting for 34 h before COC collection resulted in a larger proportion of unwanted ovulations, a greater collection efficiency in the 34 h group resulted in a similar number of COC collected per bison. We conclude that the 30 to 34 h in vivo maturation window provides flexibility for the purposes of oocyte collection and immediate in vitro fertilization in wood bison.We thank Bioniche Animal Health for providing FSH (Folltropin-V) and hyaluronan (MAP-5), and Merck Animal Health for hCG (Chorulon).
4 DEVELOPMENTAL OUTCOMES AND EFFICIENCY OF TWO CRISPR/Cas9 MICROINJECTION METHODS IN BOVINE ZYGOTES
Authors: Bogliotti Y. S. , Vilariño M. , Chitwood J. L. , Wu J. , Mutto A. , Mucci N. , Belmonte J. C. , Ross P. J.
Published: December 2014

The CRISPR/Cas9 system is a fast, effective, and easy method for gene disruption, allowing generation of knockout animals by direct zygote injection. To date there is no report on the efficiency of this microinjection system in bovine zygotes and its effects on early development. The aim of this study was to compare 2 microinjection methods on developmental rates and efficiency to induce gene disruption. Microinjection effects on embryo development were evaluated by blastocyst (BL) formation rates at Day 8 of culture and by the proportion of lysed embryos (damaged during injection); while the efficiency of CRISPR/Cas9 RNA to create targeted mutations was studied by sequencing resulting blastocysts. Three groups were evaluated: (1) noninjected (control), (2) direct intracytoplasmic injection (direct-ICI), and (3) laser-assisted ICI (laser-ICI). Direct-ICI was performed with a beveled spiked glass needle (5 μm ID; Origio, Måløv, Denmark) to pass the zona pellucida (ZP) and deliver CRISPR/Cas9 RNA as earlier described (Ross et al. 2008 BMC Dev. Biol. 8, 16). For laser-ICI, a Research Instruments (RI) Saturn 5 Active™ laser system (Research Instruments Ltd., Falmouth, United Kingdom) was used to perforate the ZP, and a blunt-end glass needle (5–6 μm ID) used to deliver CRISPR/Cas9 RNA. In both cases, cytoplasm was aspirated into the pipette to disrupt the plasma membrane and the aspirated cytoplasm and CRISPR/Cas9 injected back into the embryo. Embryos were obtained by IVF of in vitro-matured oocytes aspirated from abattoir ovaries. At 18 h post-IVF (hpf), zygotes were denuded from cumulus cells and cultured in groups of 25 in 50-μL drops of KSOM (Evolve, Zenith Biotech, Guilford, CT, USA) with 4 mg mL–1 of BSA. Zygotes were injected at 20 to 22 hpf. Four biological replicates were assessed for BL rates (258 embryos total). A 2-tailed t-test was used to evaluate statistically significant differences (P ≤ 0.05) between groups. Direct-ICI had a greater proportion of lysed embryos (29.5 ± 10.6%) compared with laser-ICI (15.6 ± 5%; P = 0.056). BL development was significantly lower on direct-ICI (15.5 ± 8%) compared with laser-ICI (31.4 ± 5.9%; P = 0.02) and control groups (32.8 ± 6.6%; P = 0.01). These results indicate that laser-ICI causes less damage and results in normal BL rates after microinjection (P = 0.24). Because laser-ICI had normal BL rates and less embryo lysis, we used this method to evaluate efficiency of the CRISPR/Cas9 system to induce genomic mutations. Twelve BL were sequenced. Analysis of the CRISPR targeted region showed that 50% of the embryos had biallelic mutations, 33% monoallelic mutations, and 17% were wild type. These results show that laser-ICI method was very efficient for injecting CRISPR/Cas9 RNA into zygotes, resulting in normal developmental rates and a high amount of mutations in BL.
The authors thank Research Instruments Ltd. for providing the laser used in this study.
45 SPINDLE CONFIGURATION AND DNA FRAGMENTATION OF VITRIFIED BOVINE OOCYTES AFTER IN VITRO MATURATION WITH L-CARNITINE AND/OR RESVERATROL
Authors: Spricigo J. F. W. , Arcarons N. , Mogas T. , Dode M. A. N. , Morato R.
Published: December 2014

After cryopreservation, oocytes may suffer morphological and functional damage, due to the high cytoplasm lipid content and to the reactive oxygen species formation. The aim of this work was to evaluate the spindle configuration and the DNA fragmentation of vitrified/warmed oocytes after in vitro maturation (IVM) in a media supplemented with l-carnitine and/or resveratrol, a lipolytic and antioxidant agent, respectively. The IVM viable COC with at least 3 cumulus cell layers and homogenous cytoplasm were randomly distributed into 4 groups: (1) control: conventional IVM media with TCM-199, epidermal growth factor, and 10% FCS; (2) L-CAR: control media supplemented with 0.6 mg mL–1 of l-carnitine; (3) RES: control media supplemented with 1 μM mL–1 of resveratrol; and 4) L+R: control media supplemented with 0.6 mg mL–1 of l-carnitine and 1 μM mL–1 of resveratrol. After 22 h of IVM, half of the COC from each group were vitrified and warmed, using the cryotop methodology. After warming, the oocytes were allowed to recover in their respective media for 2 additional hours. After 24 h of IVM, oocytes from all treatments were completely denuded and fixed and stained using specific fluorescent probes. The microtubule/chromosome configuration and the DNA fragmentation were analysed by immunocytochemistry under a fluorescent microscope (A.40FL, Carl Zeiss, Oberkochen, Germany). All statistical analyses were conducted with IBM SPSS 19 (IBM; Chicago, IL, USA). ANOVA was performed to analyse differences in meiotic spindle configuration, and the Chi-squared test was used for DNA fragmentation. The significance level was 5%. Although vitrification may cause severe oocyte damage, IVM with l-carnitine alone or in association with resveratrol was able to reduce the percentage of abnormal spindle configurations (Table 1), whereas the addition of resveratrol alone or its association with l-carnitine reduced DNA fragmentation of IVM oocytes after a vitrification/warming process. These results indicate the IVM supplementation with RES and/or L-CAR could modify oocyte composition, increasing its cryotolerance. However further studies are required to confirm the beneficial effect of these molecular interactions.

Table 1.Evaluation of spindle configuration (Experiment 1) and apoptotic cell status (Experiment 2) of fresh or vitrified/warmed oocytes matured with RES and/or L-CAR
48 GLOBAL GENE EXPRESSION PATTERN OF BOS INDICUS AND BOS TAURUS VITRIFIED EMBRYOS
Authors: Cancian R. , Macelai M. , Tavares G. , Valente R. S. , Caixeta E. S. , Martins A. , Machado R. , Buratini J. , Mesquita F. S. , Landim-Alvarenga F. C. , Sudano M. J.
Published: December 2014

The cryopreservation of in vitro-produced (IVP) bovine embryos is one of the most challenging areas of the assisted reproductive biotechnologies. The aim of the present study was to evaluate the global gene expression pattern of Bos indicus (Nellore) and Bos taurus (Simmental) IVP embryos after vitrification. Follicular aspiration was performed on Nellore (n = 14) and Simmental (n = 14) cows, and oocytes (n = 840 and 450; respectively) were submitted to in vitro maturation and in vitro fertilization. Presumptive zygotes were denuded and cultured in SOFaa with 0.5% BSA and 2.5% FCS during 7 days under standard culture conditions. Blastocysts (grade 1 and 2) were vitrified, warmed, and cultured for an additional 12 h under the same conditions. Nellore (n = 8) and Simmental (n = 8) IVP blastocysts considered viable after vitrification, with re-expanded blastocoel, were submitted to total RNA extraction (PicoPure, Arcturus, Applied Biosystems®, Foster Dity, CA, USA), DNAse I treatment (Qiagen®, Valencia, CA, USA), and amplification (RiboAmp, Applied Biosystems®). Fragmented cRNA were obtained through 3′IVT Express Kit (Affymetrix®, Santa Clara, CA, USA) to perform the hybridization using GeneChip Bovine Genome Array (Affymetrix®). Microarray data analysis was performed using the FlexArray 1.6.1.1 software. Genes with at least a 1.5-fold change and a P-value of less than 0.05 were considered differentially expressed. Of the 1278 genes differentially expressed between Bos taurus and Bos indicus vitrified embryos, 1108 were annotated, with 1193 genes up-regulated and 85 genes down-regulated in Bos taurus compared with Bos indicus IVP vitrified embryos. Differentially expressed genes were associated with the functional networks of cell cycle, cellular movement and DNA replication, recombination and repair; RNA post-transcriptional modifications; gene expression, protein synthesis; RNA damage and repair; cellular function and maintenance; and cell death and survival. The top 6 canonical pathways generated by Ingenuity Pathway Analysis® with the differentially expressed genes were ELF2 signalling, oxidative phosphorylation, tricarboxylic acid cycle, protein ubiquitination pathway, mTOR signalling, and IGF-1 signalling. In conclusion, Bos taurus IVP embryos seem to trigger different cellular response mechanisms to the vitrification stress in comparison with Bos indicus IVP embryos. Differential response is mainly represented by different expression profiles of genes regulating important canonical pathways involved in cellular response to stress that could be related with the higher post-cryopreservation survival capacity observed in Bos taurus embryos.Research was supported by FAPESP, CNPq, FAPERGS, and LNBio – National Laboratory of Biosciences/MCT.
55 EFFECT OF TREHALOSE FOR FROZEN–THAWED RAM SPERM MOTILITY PARAMETERS
Authors: Toishibekov M. M. , Jazkbayev M. T. , Molzhigitov B. B.
Published: December 2014

Computer-assisted sperm analysers have become the standard tool for evaluating sperm motility because they provide objective results for thousands of mammalian spermatozoa. Ram semen was collected using electro-ejaculation from 10 adult rams of Chingizskaya indigenous sheep breed. Motility was determined using computer-automated semen analysis (Hamilton Thorne Motility Analyzer, Beverly, MA, USA). Trehalose solution (0.375 M) was added to Tris-buffered saline solution to give the following trehalose extenders: 25, 50, 75, and 100% (vol:vol), and analysed for motility using computer-automated semen analysis. The sperm pellets were resuspended at 24°C in cooling extender – trehalose extenders of each concentration containing 5% egg yolk. The diluted semen was cooled to 5°C within 2 h. The semen was then further diluted 1 : 1 with freezing extender – each trehalose extender containing 1.5% glycerol to obtain a sperm concentration of 2.0 × 108 cells mL–1 – and then loaded into 0.5-mL straws. Straws were frozen using a programmable freezer with a freezing curve of 5°C to –5°C at 4°C per min, –5°C to –110°C at 25°C per min, and –110°C to –140°C at 35°C per min, and then the straws were plunged into liquid nitrogen for storage. Frozen samples were thawed in a 37°C water bath for 30 s and analysed for motility using computer-automated semen analysis. Statistical analyses were performed with a Student's test. The fresh semen samples showed the next results: motility 88.3 ± 2.4%, progressive motility 26.8 ± 6.9%, and progressive velocity 61.9 ± 4.2 μm s–1. Motility of the frozen-thawed spermatozoa was 63.6 ± 2.9% (25% trehalose), 55.6 ± 5.2% (50%), 32.4 ± 4.7% (75%), and 23.6 ± 3.2 (100%). Progressive motility was 15.6 ± 3.9% (25%), 13.7 ± 3.7% (50%), 4.5 ± 1.3% (75%), and 5.2 ± 1.3% (100%). Progressive velocity was 93.5 ± 8.3 μm s–1 (25%), 85.4 ± 8.1 μm s–1 (50%), 65.7 ± 6.1 μm s–1 (75%), 35.2 ± 3.3 μm s–1 (100%). Motility of the frozen-thawed spermatozoa significantly decreased with increasing concentrations of trehalose in the extender (P < 0.05). These preliminary studies showed that further research is needed of use trehalose for ram spermatozoa cryoconservation.
57 THE EFFECT OF VITRIFICATION FOR SHEEP EMBRYO VIABILITY
Authors: Valieva G. A. , Toishibekov M. M. , Askarov S. M. , Molzhigitov B. B.
Published: December 2014

This work evaluated different methods for sheep embryo cryopreservation by vitrification (V) and super-cooling ultra-rapid vitrification (SCURV). The vitrification method was applied according to the method described by Vajta et al. Both treatments used a vitrification solution (VS) containing 20% ethylene glycol, 20% dimethylsulfoxide (Me2SO), 0.5 mol L–1 sucrose in Dulbecco's phosphate buffered saline (DPBS) with 10% BSA. The super-cooled LN facilitates heat transmission between LN and the cryosolution interface, and this is efficient for bovine semen and blastocyst cryoconservation (Arav et al. 2002). By surgical flushing 25 super-stimulated ewes, 109 transferable morulae were harvested; 35 morulae were transferred fresh to synchronized recipients (control) and the others were cryopreserved by V (n = 36) or SCURV (n = 38), respectively, thawed or warmed, and transferred to recipients. Embryos were vitrified using the HSV Kit. They were first incubated in 50% VS for 2 min and then transferred for 30 s into 100% VS. Each embryo was loaded by HSV Kit, which was immediately submerged into and stored in LN. Warming was done by placing the narrow end of the straw into DPBS + 0.25 M sucrose for 5 min. Embryos were then transferred into DPBS + 0.125 M sucrose for 3 min and finally to DPBS until transfer. The SCURV morulae were then exposed to 50 and 100% VS at 37°C for 2 min and 30 s, respectively. Embryos after saturation in VS were transferred on a surface of a nylon loop (volume 20 μL, diameter 0.5 mm) and using negative pressure of LN in the chamber for freezing with the VIT-Master. Thawing vitrified embryos was accomplished by placing the vitrified embryos in solutions of sucrose 0.25 M and 0.125 M with expositions 2 and 3 min accordingly. After thawing embryos, only good-quality embryos were transferred. Statistical analyses were performed with Student's t-test. The lambing rate following transfer of fresh, frozen-thawed vitrification and SCURV methods were 18, 12, 14 lambs accordingly. No statistical difference was found for the percentage of does lambing following transfer thawed after vitrification (33.4 ± 5.2a%) and SCURV methods (36.8 ± 6.3b%). The survival rate following transfer of fresh embryos (51.4 ± 4.8c) was higher and in line with previous findings using VS. Differences were statistically significant (ac,bc P < 0.05). Importantly, our data suggest that the HSV Kit can be used to produce viable morulae for implantation as the SCURV, and to as vitrification method. Although further work on the developmental competence of embryos cryopreserved with the SCURV method are needed, these data suggest that with SCURV a faster freeze rate and lower level of cryoprotectants is able to minimize ice crystal formation and should be further evaluated as a routine mechanism for cryopreserving sheep embryos.
6 EQUINE SPERM INDUCES PRONUCLEAR FORMATION BY INTRACYTOPLASMIC SPERM INJECTION IN BOVINE, SWINE, AND FELINE OOCYTES INDEPENDENTLY OF CHEMICAL ACTIVATION ASSISTANCE
Authors: Rodríguez M. B. , Gambini A. , Bevacqua R. J. , Salamone D. F.
Published: December 2014

Interspecific intracytoplasmic sperm injection (ICSI) is a valuable tool to study early events of fertilization in species for which oocyte availability is reduced. Equine in vitro fertilization remains unsuccessful and ICSI is the technique of choice for the in vitro production of high-value embryos. Therefore, the objective of this study was to evaluate the rate of pronuclear (PN) formation after ICSI with stallion sperm in bovine, swine and feline oocytes with or without chemical activation assistance. Ovaries from cows and pigs were collected at abattoirs whereas gonads from female domestic cats were obtained from ovariectomized animals at veterinary sterilization centers. Cumulus-oocyte complexes were matured in TCM-199 supplemented following standard protocols for each species. ICSI was performed in 100-μL drops of TALP-HEPES, using frozen-thawed semen from one stallion. Spermatozoa were held separate in 3-μL droplets of 7% (vol/vol) polyvinylpyrrolidone, where one of them was immobilized by swiping the injection pipette across its tail, and then injected into the matured oocyte. After ICSI, some oocytes were chemically activated with 5 μM ionomycin for 4 min (cow and cat) or with an electric pulse (sow) followed by 3 h in culture medium to allow extrusion of the second polar body and then exposure to 1.9 mM 6-DMAP solution for 3 h. Embryos were cultured in SOF medium. After 17 h of culture, embryos were stained with propidium iodide to identify the percentage of oocytes activated and with PN. Haploid and diploid parthenogenetic controls were included. Cleavage (48 h after activation) and blastocyst formation (7–8 days) of the partenogenetic control groups were assessed. There were no statistical differences (chi-squared analysis) in PN formation between the activated and nonactivated groups within species. When the activated group was compared between the different species, no differences were observed. However, for the nonactivated group, significant differences were observed between species. The feline oocyte showed the higher percentage of PN and activation, whereas the bovine oocyte exhibited the lower rate of PN formation (cat: 22/27, 81.48%; swine: 19/39, 71.64%; cow:18/63, 43.07%). Our results suggest that the feline oocyte can be used as model to study fertilization events associated with the stallion sperm due to the higher efficiency in supporting PN formation. Our results indicate that the equine sperm is capable of inducing PN formation in these 3 species without further chemical activation assistance.
68 DIFFERENCE IN THAWING CURVE OF STALLION FROZEN SEMEN DILUTED IN 2 EXTENDERS
Authors: Freitas-Dell'aqua C. P. , Ramires Neto C. , Sancler-Silva Y. F. R. , Papa P. M. , Dell'aqua J. A. , Alvarenga M. A. , Papa F. O.
Published: December 2014

Commercial freeze extenders have different composition and ratio of cryoprotectors; freezing and thawing protocols are different for each extender. The aim of this experiment was to observe the effect of thawing curve in stallion frozen semen with 2 commercial extenders. Two ejaculates from each of 9 stallions of different breeds (Quarter Horses and Mangalarga Marchador) were used. Semen was collected using an artificial vagina, and the ejaculate was divided into 2 groups following the manufacture's protocol: group 1 (INRA), in which the semen was diluted 1 : 1 with the extender INRA 96TM (IMV, Paillette Crista, France) and group 2 (BC), in which the semen was diluted (1 : 1) with the extender Botu-SemenTM (Botupharma, Brazil). The samples of the 2 groups were centrifuged at 600 × g for 10 min, the supernatant was discarded, and the pellet was resuspended with INRA FreezeTM (group INRA, IMV) and with BotucrioTM (group BC, Botupharma) at the concentration of sperm 100 × 106 sperm mL–1. After this, the semen was packaged in 0.5-mL straws. For each group the freezing process was carried out according to the manufacturer's instructions. The straws were thawed in a water bath with 3 different thawing curves: 37°C for 30 s (37/30), 46°C for 20 s (46/20), and 75°C for 7 s (75/7) before analysis. The aim of these rates is to keep the semen in 37°C post-thaw. The sperm kinetic analysis was performed by computerized method (CASA, HTM-IVOS, IMV, USA) and the analysis of plasma membrane integrity by flow cytometer (BD LSR Fortessa, Becton Dickinson, Mountain View, CA, USA). Data of sperm kinetic and of plasma membrane integrity were compared among the 3 thawing curves for one extender using analysis of variance. Differences were considered significant at a probability level of 5%. No differences were observed in total motility (%, BC 37/30 = 72.8 ± 14.4; BC 46/20 = 70.0 ± 14.2; BC 75/7 = 70.3 ± 12.0 v. INRA 37/30 = 57.2 ± 19.1; INRA 46/20 = 50.0 ± 21.9; BC 75/7 = 58.8 ± 20.8), progressive motility (%, BC 37/30 = 36.9 ± 8.2; BC 46/20 = 34.4 ± 10.5; BC 75/7 = 33.6 ± 7.8 v. INRA 37/30 = 25.3 ± 12.7; INRA 46/20 = 21.9 ± 13.9; BC 75/7 = 28.9 ± 14.8), rapid sperm (%, BC 37/30 = 59.7 ± 16.4; BC 46/20 = 56.8 ± 17.1; BC 75/7 = 58.1 ± 14.9 v. INRA 37/30 = 38.3 ± 20.9; INRA 46/20 = 35.3 ± 22.9; BC 75/7 = 44.4 ± 23.8), and plasma membrane integrity (%, BC 37/30 = 49.1 ± 14.8; BC 46/20 = 43.1 ± 13.1; BC 75/7 = 46.7 ± 11.8 v. INRA 37/30 = 32.2 ± 10.7; INRA 46/20 = 29.6 ± 10.1; BC 75/7 = 37.4 ± 9.1) among the 3 thawing curves for INRA and BC groups. In this study, we can conclude there is no influence of the 3 tested thawing curves in sperm quality for stallion frozen semen with INRA Freeze and Botucrio extenders.
70 INCORPORATING MULTIPLE STAGES OF MASS SPECTROMETRY INTO LIPID PROFILING OF OOCYTES AND PRE-IMPLANTATION EMBRYOS
Authors: Pirro V. , Jarmusch A. K. , Ferreira C. R. , González-Serrano A. F. , Hallett J. E. , Houser R. , Niemann H. , Cooks R. G.
Published: December 2014

Lipid profiling by mass spectrometry (MS) is increasingly used for the analysis of oocytes, embryos, and other reproductive cells. This analytical approach has several advantages, such as simple preparation (no need to perform extraction or separation), low detection limits (no need of sample pooling), and detection of structurally intact and diverse lipids. Many degrees of freedom are ensured by MS techniques (e.g. with the adoption of diverse ionization sources, mass analyzers, data acquisition systems), and this broadens the classes of lipids that can be detected and identified. Tandem or high-resolution MS experiments are normally performed for chemical characterisation. However, the use of novel approaches is a constant need to obtain deeper structural insights into lipids of biological interest, resulting in an information-rich dataset. Here we propose the use of multiple stages of MS for lipid profiling, specifically MS/MS data domain (i.e. ion mapping) experiments, so that comprehensive structural and relationship information (i.e. classes) can be extracted from a dataset. Indeed, the data generated have 2 dimensions of mass (i.e. precursor and product ions) and one of ion intensity, resulting in a datacube structure. Cutting through the datacube in different ways allows the extrapolation of (i) chemical composition of specific compounds (i.e. product scans) and (ii) pattern recognition for compounds that share identical neutral or charged fragments loss (i.e. neutral loss and precursor scans, respectively). The global chemical information enclosed into the datacube can be also processed by means of multiway statistical analyses to chemically characterise cells and cellular compartments. Preliminary data have been acquired, and the development of statistical tools for data processing is ongoing. Bovine and rat embryos were used for the experiments and analysed by extraction spray ambient MS. Experiments were performed with a Thermo Finnigan LTQ linear ion trap. Dimethylformamide-acetonitrile (1 : 1 v/v) was used as spray solvent. The ion mapping experiment was configured to scan ions of mass-to-charge (m/z) ratio 700 to 900 and perform MS/MS every m/z 1.5 window with a collision energy of 25 (arbitrary units). Fragments were detected in the m/z range of 150 to 900. Chemical differences are present between bovine and rat embryos, of note are palmitic, oleic, and stearic acids. The application of ion mapping to characterise species-specific and developmental dynamics regarding lipid composition is currently under investigation.
79 microRNA EXPRESSION IN BOVINE CUMULUS CELLS
Authors: Uhde K. , van Tol L. T. A. , Stout T. A. E. , Roelen B. A. J.
Published: December 2014

A mammalian oocyte within an ovarian follicle is surrounded by cumulus cells, together this structure is known as the cumulus-oocyte complex (COC). Cumulus cells are important for the development of the oocyte, they support the maturation process of the oocyte within the ovary and aid in sperm recognition. Because it is known that a Dicer knockout leads to infertility, microRNAs (miRNA) are focused to have an important role in oocyte development. MiRNAs are small noncoding RNA sequences that act as transcriptional regulators. Little is known about the expression of miRNA in cumulus cells or how cumulus-derived miRNA may regulate or be used to indicate the developmental competence of the maturing oocyte. Our aim was to investigate miRNA expression in oocytes and to identify and establish how specific miRNA influence the acquisition of developmental competence by bovine oocytes. Normalization of qPCR data requires stable reference genes. To this end, we tested the expression of various miRNA with respect to their ability to be used as reference miRNA for bovine cumulus cells; these included miR-103, miR-93, miR-26, let-7a, miR-191, and the small noncoding nuclear RNA U6. Cumulus-oocyte complexes were recovered from the ovaries of slaughtered cows and matured in vitro. Small samples of cumulus cells were collected from these COC before and after maturation. From the cumulus cell groups recovered at different stages, small RNA were extracted and cDNA was synthesised, followed by qRT-PCR. To identify the optimal combination of reference genes, the geNorm algorithm was used. MiR-26a and let-7a were identified as the most stably expressed miRNAs, whereas U6 showed the most variable expression levels. Future investigations are planned to identify miRNA in cumulus cells that can be used as markers for oocyte developmental competence. Using a single oocyte-embryo culture system will enable us to retrospectively relate cumulus miRNA expression to the developmental capacity of the oocyte.This work was supported by EU FP7 EpiHealthNet (N°317146).