bluuebunny/crossref_metadata_2025 huggingface dataset:
155 EMBRYONIC APOPTOSIS AFTER BTV-8 INFECTION IN BOVINE HATCHED IN VITRO PRODUCED BLASTOCYSTS
Authors: Vandaele L. , Wesselingh W. , De Clercq K. , De Leeuw I. , Favoreel H. , Van Soom A. , Nauwynck H.
Published: August 2011

The recent blue tongue virus serotype-8 (BTV-8) epidemic in central Western Europe has been associated with field fertility problems (De Clercq et al. 2008 Transbound. Emerg. Dis. 55, 352–359). Previous research clearly showed that in vitro produced bovine hatched blastocysts are susceptible for BTV-8 infection (Vandaele et al. 2010 Reprod. Fertil. Dev. 22, 254 abst.). The aim of the present study was to investigate the effect of a BTV-8 infection on the occurrence of apoptosis in embryos in order to gain a clear insight into the role BTV-8 might play in early embryonic death. Immature bovine oocytes were matured and fertilized in vitro. Presumed zygotes were denuded 24 h post-insemination and cultured in 50-μL droplets of modified SOF medium with 10% fetal calf serum (tested negative for BTV antibodies) at 38.5°C in 5% CO2, 5% O2, and 90% N2. At 7 days post-insemination (dpi), blastocysts were grouped to enhance hatching. At 8.5 dpi, 4 to 8 hatched embryos were placed in 800 μL of minimum essential medium (MEM), containing 103.8 to 104.9 50% tissue culture infectious doses (TCID50) of BTV-8 (Bel 2006/3, VAR, Brussels, Belgium) and incubated for 1 h at 38.5°C in an atmosphere of 5% CO2 in air. Two groups of hatched control embryos were incubated under the same circumstances in 800 μL of SOF and 800 μL of MEM, respectively. After inoculation, embryos were washed according to IETS guidelines with the exception that they were not ZP-I and cultured in new SOF. At 48 and 72 h post-inoculation (hpi), half of the embryos of each group were fixed in 4% paraformaldehyde for 12 to 24 h and subsequently stained for BTV-8 and apoptosis with a double immunofluorescent staining using a BTV-8 monoclonal antibody (8A3B.6, ID-Vet, Montpellier, France) in combination with TUNEL (Roche Diagnostics, Mannheim, Germany). All mock-inoculated embryos were negative for BTV-8 virus antigen. At 48 and 72 hpi, respectively, 45 and 38% of the embryos were BTV-8 positive in all embryonic cells, and all remaining embryos had at least some BTV-8 blastomeres. The overall apoptotic cell ratio in infected embryos (17.9 ± 1.14% at 48 hpi and 15.3 ± 1.16% at 72 hpi) was significantly higher than in noninfected, mock-inoculated embryos (10.4 ± 0.57% at 48 hpi and 4.3 ± 0.30% at 72 hpi) (P < 0.01). Furthermore, total cell number was substantially lower in infected embryos (103 ± 14.2 at 48 hpi and 120 ± 17.5 at 72 hpi) compared with noninfected, mock-inoculated embryos (258 ± 47.4 at 48 hpi and 348 ± 64.1 at 72 hpi) (P < 0.05). This study indicates that embryonic apoptosis after BTV-8 infection results in embryonic arrest and early embryonic death and thus might be involved in herd fertility problems during a BTV epidemic.

The first author is supported by Research Foundation Flanders (Grant number G.0210.09).
156 MONITORING SALIVARY TESTOSTERONE CONCENTRATIONS FROM CAPTIVE AMAZONIAN MANATEES (TRICHECHUS INUNGUIS): IS THERE SEASONAL PATTERN?
Authors: Amaral R. S. , Rosas F. C. W. , Viau P. , Nichi M. , da Silva V. M. F. , Oliveira C. A.
Published: August 2011

Seasonality of reproduction in many nondomestic animal species appears to represent an accommodation to environmental variables, as food availability, in which influence reproductive success. Males can show a decrease of testicular size, sperm concentration, and serum concentration of testosterone because of food restriction. The Amazonian manatee (Trichechus inunguis, Mammalia: Sirenia) is a threatened aquatic mammal, endemic of the Amazon basin (South America), and is the only sirenian that occurs exclusively in fresh water. Although information is lacking about the reproductive endocrinology of free-ranging T. inunguis, they are considered seasonal breeders, with copulation and births occurring between December and July, coinciding with the increase in Amazon basin water levels and the period of most availability of food for the species (Best 1982 Biotropica 14, 76–78). Salivary steroid measurement is a noninvasive way to monitor steroid concentration, which is well correlated with the level of steroids in the serum. The aim of this study was to verify if testosterone concentrations of male T. inunguis kept in captivity, without variation of food availability, show a seasonal pattern. Were used 4 adult male Amazonian manatees kept in captivity in the Laboratory of Aquatic Mammals of the National Institute of Amazonian Research–LMA/INPA, Brazil. They were fed all year with paragrass and vegetables, at not less than 8% of body weight per day. For sample collection, the pool was drained and saliva was collected from mouth mucosa using a metal spoon. Samples were collected weekly for 12 weeks in 2 periods (March–June and September–November). Salivary testosterone was measured by RIA for total testosterone using an adapted protocol previously validated for the species (Amaral et al. 2009 J. Zoo Wildl. Med. 40, 458–465) The data were analysed in a 3 × 2 factorial design, where the factors were month (each 4 weeks = 1 month) and period, using GLM test for repeated-measures. The means of salivary androgen levels were 28.80 ± 18.56 pg mL–1 and 30.77 ± 16.76 pg mL–1 for first and second periods, respectively. There were no significant main effects of month (P = 0.454), period (P = 0.607), or interaction between factors (P = 0.635). These results suggest that captive male Amazonian manatees apparently do not show reproductive seasonality. However, it is recommended to verify the existence of variation in testicular size and sperm concentration. This finding is important information for the future reproductive management of captive Amazonian manatee.

The authors thank PREVET staff for help with the samples collection, and FAPESP (2008/05760-5), CNPq (Universal 475596/2009-9), Petrobras Ambiental (Projeto Mamíferos Aquáticos da Amazônia: Conservação e Pesquisa), and AMPA for the financial support.
166 THE DIFFERENTIAL TRANSCRIPTOME AND ONTOLOGY PROFILES OF MURAL AND CUMULUS GRANULOSA CELLS IN STIMULATED HUMAN ANTRAL FOLLICLES
Authors: Kõks S. , Velthut A. , Sarapik A. , Altmäe S. , Reinmaa E. , Schalkwyk L. C. , Fernandes C. , Lad H. , Soomets U. , Jaakma Ü.W. , Salumets A.
Published: August 2011

Communication between various cell types in the ovary is a prerequisite for successful folliculogenesis and ovulation. In human antral follicles, granulosa cells divide into 2 distinct cell populations: mural (enveloping the antrum, MGC) and cumulus granulosa cells (surrounding the oocyte, CGC). During infertility treatment using in vitro fertilization (IVF), granulosa cells can be retrieved during puncture of stimulated follicles offering an excellent opportunity for analysing their functional properties. The aim of this study was to compare the transcriptomes of MGC and CGC. Twenty infertile women undergoing IVF-ICSI treatment were enrolled. The MGC were obtained from follicular fluid and CGC were acquired after oocyte denudation before micromanipulation. Gene expression of both cell populations was analysed using a genome-wide transcription array. The expression profile of the 2 granulosa cell populations varied significantly: out of 28 869 transcripts, 4480 were differentially expressed (q-value < 10–4); 623 transcripts differed in their expression levels by at least 2-fold. The transcriptome of MGC showed higher expression of genes involved in immune regulation (toll-like receptors, IL18, IL17R). In CGC, pathways participating in intercellular interactions, tissue remodelling and protein degradation were more clearly distinguished (tenascin C, IGFBP5). Among the identified differentially expressed genes, several are involved in follicle development, oocyte maturation, or ovulatory processes. Our findings fit well with previously published data. The results provide a basis for future studies on intra- and intercellular signaling in the preovulatory follicle leading towards identifying methods for improving oocyte health, embryo selection, and ultimately IVF success rate.

This work was supported by the Estonian Science Foundation (6498, 6585, and 7479); the Estonian Ministry of Education and Science (SF0182641s04, SF0180142Cs08, SF0180044s09, and PBGMR07903); the Estonian University of Life Sciences (P8001VLVL); the European Union Sixth Framework Programme (FP6) (LSHB-CT-2004-503243); the European Cooperation 22 in Science and Technology (COST-STSM-FA0702-03734), and the European Union through the European Regional Development Fund through the Centre of Excellence in Genomics, Estonian Biocentre, and University of Tartu. The authors acknowledge our colleagues from the Nova Vita Clinic for their assistance in sample collection and all patient volunteers for their participation in the study.
167 VISUALISATION OF FAT ACCUMULATION IN BOVINE PREANTRAL AND ANTRAL OOCYTES USING 2-PHOTON MICROSCOPY
Authors: Langbeen A. , Leroy J. L. M. R. , Pintelon I. , Bols P. E. J.
Published: August 2011

Fat metabolism is important in oocyte development (Kim et al. 2001; Sturmey et al. 2009). Our main objective was to develop a straightforward method that allows us to describe fat distribution and quantify fat accumulation in bovine oocytes throughout folliculogenesis. Fat accumulation dynamics can be studied by making microscopically repeated cross-sections or Z-stacks through oocytes of different developmental stages. The most important limiting factor in oocyte 3D visualisation studies is the size of the oocyte (between 110 and 120 μm in diameter), making it impossible to visualise it as a whole. If visualisation of the bottommost hemisphere is desirable, overlying Z-stacks will disperse the excitation and emission light, creating artefacts that will distort the image and trouble the results (indicated by former unpublished results). Therefore, we investigated the use of an alternative visualisation protocol staining the oocytes with Nile Red as an intracellular, triglyceride-specific (when emission is captured at 590 nm), fluorescent dye (Greenspan et al. 1985; Leroy et al. 2005), this combined with 2-photon excitation technology. In total, ovaries from 10 cows were collected at slaughter. Ovaries were pooled per cow. Only cows with apparent follicular activity on both ovaries were selected. Antral follicles with a diameter of <3 mm, between 3 and 6 mm, and >6 mm were aspirated. Apart from oocytes collected from each of these 3 follicle classes, preantral follicles were harvested from ovarian cortex tissue through mechanical isolation and enzymatic digestion by collagenase type IA. In total, 3 oocytes per follicle class were collected for each pair of ovaries. They were fixed in 2% glutaraldehyde and 2% formaldehyde and stained with Nile Red (1 μg mL–1). After mounting, images were acquired with a Zeiss LSM 510 reversed 2-photon microscope (Carl Zeiss GmbH, Jena, Germany) using an excitation wavelength of 807 nm, with an emission spectrum covered between 580 and 591 nm. Our results show that even with biphoton excitation, it remains very difficult to visualise fat droplet distribution in >100-μm oocytes. However, accurate images can be obtained of the upper hemisphere of the oocyte. These images can be used in future research on the dynamics of the distribution and the accumulation of lipid. Preliminary descriptive results clearly show that the relative amount of lipid droplets is lower and their size is smaller in oocytes from preantral follicles compared with antral counterparts. We can preliminary conclude that fat accumulation and the aggregation in fat droplets might take place preceding or even during antral development. We can visualise preantral oocytes as a whole in contrast to antral oocytes where only the upper hemisphere is visible without distortions. Image analysis software is currently applied to allow for a more quantitative interpretation.

D. De Rijck.
178 DISTINCT EXPRESSION AND REGULATION OF ECTONUCLEOTIDE PYROPHOSPHATASE/PHOSPHODIESTRASE 2 AND ITS POTENTIAL ROLE(S) IN THE UTERUS OF RATS DURING ESTROUS CYCLE
Authors: Ahn H.-J. , Jeung E.-B.
Published: August 2011

Ectonucleotide pyrophosphatase/phosphodiestrase 2 (ENPP2) is a recently discovered phospholipase D that transforms lysophosphatidylycholine into lysophospatidic acid. Previously, multiple analyses have been carried out to identify oestrogen response genes, such as ENPP2, in the hypothalamus. However, little is known about the role(s) of the ENPP2 gene in the uterus during the oestrous cycle and the mechanism(s) of its regulation. In this study, the ENPP2 gene was investigated using RT-PCR, Western blot, and immunohistochemistry analyses in the uterine of rats. Quantitative PCR analysis demonstrated that the levels of uterine ENPP2 mRNA decreased at oestrus compared with those at proestrus and diestrus. To investigate whether ENPP2 expression is affected by sex steroid hormone in the uterus of rats, we treated rats with 17β-oestradiol (E2), progesterone (P4), or E2 plus P4 from postnatal Day 14 to 16 in an immature rat model. Interestingly, ENPP2 mRNA and protein level expression was down-regulated by E2 in the uterus at oestrus, but not in the proestrus and diestrus, suggesting that the ENPP2 gene may be involved in uterine function during the oestrous cycle. Using immunohistochemistry with a specific antibody, we demonstrated the expression of ENPP2 protein localization in the uterus of rats during the oestrous cycle. In particular, ENPP2 was mainly located in the stromal cells in the uterine endometrium at the proestrus and oestrus cycles. The diestrous stage showed greater expression, and ENPP2 was mainly expressed in decidual cells. In conclusion, these results suggest that uterine ENPP2 induction is caused by steroid hormones during the oestrous cycle in rats, and this gene may be involved in reproductive function during a critical developmental stage of female rats.
179 EFFECT OF SERUM DURING CULTURE ON DAY 14 ELONGATED BOVINE EMBRYOS
Authors: Angulo J. , Gentry G. T. , Godke R. A. , Bondioli K. R.
Published: August 2011

It has been reported that the addition of serum to embryo culture media alters gene expression and triggers the development of large offspring syndrome. The objectives of this study were to determine gene expression levels in embryos cultured in the absence or presence of 5% calf serum and in vivo-derived (IVD) embryos and to determine the effects of serum on the length of elongated embryos. Abattoir-derived oocytes were obtained from a commercial provider and fertilized at 24 h of maturation with semen from a bull previously used for IVF. At 18 h post-insemination (hpi), embryos were denuded and groups of 15 presumptive zygotes were cultured in 30-μL drops of modified SOF medium with amino acids and 6 mg mL–1 of BSA (mSOFaa). At 72 hpi, cleavage rate was assessed and embryos were randomly allocated into 2 treatments: mSOFaa without and with 5% calf serum. Embryos were then cultured to 168 hpi and blastocyst rates were assessed and recorded. Blastocysts (n = 5 to 10) from each treatment were transferred into synchronized recipients, and Day 14 embryos were recovered 7 days post-transfer. Embryos were photographed, measured, and immediately stored at –80°C in a minimal volume of PBS + 0.1% polyvinyl alcohol. Messenger RNA was isolated using a Dynabeads mRNA Direct Kit™ (Invitrogen, Carlsbad, CA), and reverse transcription was performed using an iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., CA). Quantitative PCR was performed to determine the transcript abundance for COX6A, IFNT1a, PLAC8, IGF2R, and GAPDH for each sample. The GAPDH was used as a reference gene, and gene expression was calculated as a ratio of expression levels between each gene of interest and GAPDH. Expression levels for each gene were determined from standard curves generated by serial dilutions of PCR amplicons starting with 0.4 pg/reaction. Blastocyst development rates were higher in embryos cultured with serum compared with the nonserum treatment (14.9 and 7.4% respectively; chi-square, P < 0.001). Lengths of elongated embryos from the serum (3395.3 ± 414.7 μm) and nonserum (2784 ± 741.8 μm) culture treatments differed from the IVD (6297.7 ± 677.2 μm) treatment (mean ± SE; ANOVA, P < 0.0052). There were no differences in the mean expression levels for COX6A, IFNT1a, PLAC8, and IGF2R across treatment groups, but in the serum treatment, 3 out 11 overexpressed IFNT1a, 4 out of 11 overexpressed IGF2R, and 2 out of 11 overexpressed PLAC8, defined as being 2 standard deviations above the mean of the IVD treatment for each respective gene. In the in vitro-produced nonserum and IVD treatments, overexpression by this definition was not observed. Although mean expression levels were not affected by culture with serum under these conditions, very high expression of IFNT1a, IGF2R, and PLAC8 was observed in some embryos cultured with serum, but not in embryos cultured without serum or IVD embryos.
18 EFFECTS OF SKIM MILK ON THE QUALITY AND FERTILITY OF BOAR SEMEN FOLLOWING LIQUID PRESERVATION AT 5°C AND 15°C
Authors: Namula Z. , Kodama R. , Kaedei Y. , Tanihara F. , Vien V. L. , Otoi T.
Published: August 2011

Liquid preservation of semen can be an alternative to frozen–thawed semen for artificial insemination. The success of a selection of boar semen extenders has been studied over storage periods of 5 to 7 days. The objective of this study was to evaluate the effects of skim milk on the viability and in vitro fertility of boar spermatozoa preserved in Modena-based extenders at 5°C and 15°C for 2 weeks. A total of 7 ejaculates were collected from one boar. The sperm-rich fraction of each ejaculate was centrifuged and diluted in Modena extenders supplemented with 0 (control), 7.5, and 15 mg mL–1 of dry skim milk. The final sperm concentration was adjusted to 1 × 108 cells mL–1, and then the semen was stored at 5°C and 15°C for 2 weeks. In the first experiment, the motility, viability (live/dead fluorescence viability assay), plasma membrane integrity (hypoosmotic swelling test; HOST), and acrosome integrity (FITC-labelled peanut agglutinin staining) of semen stored for 2 weeks were assessed. In the second experiment, the fertilization of stored semen after 20 h of co-incubation with in vitro matured oocytes and their development were examined. Data were analysed using ANOVA. When the semen was stored at 5°C for 2 weeks, the mean total sperm motility of semen stored with 7.5 and 15 mg mL–1 of dry skim milk was significantly higher than that of semen in the control group (41.4% and 41.5% v. 17.4%; P < 0.05). However, the beneficial effects of skim milk on the sperm motility were not observed in the semen stored at 15°C. Moreover, there were no significant differences in the other parameters of semen quality among the groups in each storage temperature. Significantly higher penetration rates of semen stored with 7.5 and 15 mg mL–1 of dry skim milk were observed in the storage at 5°C (41.1% and 34.8% v. 19.8%; P < 0.05) but not at 15°C (38.9% and 26.0% v. 30.0%; P > 0.05) when compared with the control group. When the semen was stored at 5°C, the development rate to the blastocyst stage of oocytes fertilized with semen stored with 7.5 mg mL–1 of dry skim milk was significantly higher than that with control and 15 mg mL–1 of dry skim milk (15.4% v. 1.1% and 7.8%; P < 0.01). However, there were no significant differences in the development rates of oocytes fertilized with semen stored at 15°C among the groups (9.6–11.9%). In conclusion, our results indicate that the effect of skim milk on the viability and in vitro fertility of liquid-stored boar spermatozoa is dependent on the storage temperature. The addition of 7.5 mg mL–1 of dry skim milk may be effective for the improvement of viability and fertility of semen stored at 5°C but not at 15°C.
182 CALBINDIN-D28k IS PREDOMINANTLY EXPRESSED AND REGULATED BY ESTROGEN IN HUMAN ENDOMETRIUM DURING MENSTRUAL CYCLE
Authors: Choi K.-C. , Yang H. , Jeung E.-B.
Published: August 2011

The human endometrium resists embryo implantation except during the window of receptivity. A change in endometrial gene expression is required for the development of receptivity. Uterine calbindin-D28k (CaBP-28k) has been shown to be involved in the regulation of endometrial receptivity by intracellular Ca2+. Nowadays, this protein has been mainly linked to the brain, kidneys, and pancreas, but potential role(s) of CaBP-28k remain to be clarified in the uterus of humans during the menstrual cycle. Thus, we demonstrated in this study that the expression of CaBP-28k in the human endometrium in more divided in the menstrual phases. During the menstrual cycle of humans, uterine expression levels of CaBP-28k mRNA and protein increased at the proliferative phase and fluctuated in these tissues, compared with other phases. We assessed the effects of the sex-steroid hormones E2 and P4 on the expression of CaBP-28k in the Ishikawa cell line. A significant increase in the expression of CaBP-9k mRNA was observed at the concentration of 17β-oestradiol (E2; 10–9 to 10–7 M). In addition, spatial expression of CaBP-28k was detected by immunohistochemistry. CaBP-28k is abundantly localised in the cytoplasm of the luminal and glandular epithelial cells during the menstrual cycle. Taken together, these results indicate that CaBP-28k, a uterine calcium-binding protein, is abundantly expressed in the human uterus, suggesting that uterine expression of CaBP-28k may be involved in reproductive functions during the menstrual cycle of humans.
184 EXPRESSION OF GENES RELATED TO DNA METHYLATION AND GLUCOSE METABOLISM DURING THE PRE-IMPLANTATIONAL STAGE OF BOVINE EMBRYOS
Authors: Ferreira A. R. , Machado G. M. , Azevedo J. M. , Sartori R. , Dode M. A. N. , Franco M. M.
Published: August 2011

During pre-implantation embryonic development in mammals, extensive epigenetic reprogramming occurs; patterns of DNA methylation are erased during the first cell divisions and are restored at the 8- to 16-cell stage (Dean et al. 2001). An important step during this developmental phase, which is controlled by epigenetic factors such as DNA methylation, is inactivation of the X chromosome (XCI). It is established in the morula stage, where the paternal X is inactivated, with immediate reactivation in the blastocyst (Ferreira et al. 2010). The aim of this study was to evaluate the mRNA expression profile of the DNMT3a and DNMT3b genes, which are related to DNA methylation, and the G6PD and PGK1 genes, which are related to glucose metabolism and are subject to XCI. Cumulus–oocyte complexes (COC) were isolated from slaughterhouse ovaries from Bos taurus indicus Nellore cows. Oocytes were matured in vitro and inseminated with X-sorted sperm from a Holstein bull. Embryos of each developmental stage [4-cell (44 hpi), 8- to 16-cell (72 hpi), morula (144 hpi), blastocyst (156 hpi), and expanded blastocyst (168 hpi)] were produced. Three pools of 17 embryos for each stage were frozen at –80°C until RNA extraction. Total RNA was isolated using the Invisorb RNA Spin Cell Mini Kit (Invitek, Berlin, Germany) according to the manufacturer’s protocol. Complementary DNA was done using Oligo dT primers (Invitrogen, São Paulo, Brazil) and SuperScript III reverse transcriptase (Invitrogen). Relative expression of target genes was assessed by quantitative PCR using a Power Sybr®Green PCR Master Mix (Applied Biosystems, Foster City, CA) in an ABI Prism 7500 Fast Sequence Detection System (Applied Biosystems). The cyclophilin-A gene was used as an endogenous control. To compare gene expression between treatments, we used the ΔΔCt method. The identity of PCR products was confirmed by the amplicon size in agarose gel and by the melt curve in quantitative PCR. Gene quantification was compared among treatments using one-way ANOVA and Tukey’s test, in the Prophet program, version 5.0 (1996, BBN Systems and Technologies, Cambridge, MA). The 4 genes showed higher expression (P ≤ 0.05) in 4-cell stage embryos than in morulae, blastocysts, or expanded blastocysts. The 8- to 16-cell embryos showed no differences for any gene compared with other embryos, except for the DNMT3b gene, which was not tested in these embryos. We conclude that these genes have a greater quantity of mRNA molecules because of the presence of maternal mRNA and that this stock was being spent during embryo development until the expanded blastocyst stage.

Financial support was provided by Embrapa and CNPq.
192 INDUCIBLE TRANSGENE EXPRESSION IN PIGS
Authors: Klymiuk N. , Boecker W. , Baehr A. , Radic T. , Wuensch A. , Schilling E. , Kurome M. , Kessler B. , Nagashima H. , Mutschler W. , Schieker M. , Wolf E.
Published: August 2011

Genetically modified pigs are important models for biomedical research. For certain applications, tightly controlled transgene expression is mandatory. For instance, expression of soluble receptor activator of the NFκB ligand (RANKL) is an established principle for inducing clinically relevant osteoporosis (Mizuno et al. 2002 J. Bone Miner. Metab. 20, 337–344). However, constitutive overexpression of RANKL is lethal; thus, an inducible system is required. A second example is the expression of porcine CTLA-4Ig to prevent co-stimulation of T-cells in pig-to-primate xenotransplantation. Because constitutive expression of porcine CTLA-4Ig causes immunosuppression in pigs (Phelps et al. 2009 Xenotransplantation 16, 477–485), an inducible expression system is desirable. The Tet-On system is used most widely. It is based on a tetracycline transactivator (TA) and a transactivator response element (TARE), which drives expression of the coding sequence for the transgene product of choice. Association of doxycyclin with TA allows binding to TARE, resulting in activation of transgene expression. Although widely used in mice, this system has not yet been established in pigs. Because consistently high expression of TA is required for a functional system, we first cloned an expression vector for TA under the control of the CMV enhancer/chicken β-actin promoter (CAG). A floxed neomycin resistance cassette was linked to this vector to facilitate selection of stable clones after transfection of primary porcine kidney cells. The latter were used for nuclear transfer, and cloned embryos were transferred to synchronized recipient gilts, resulting in the birth of 5 CAG-TA transgenic piglets. A spectrum of organs and tissues was investigated by RT-PCR and Western blot for TA mRNA and protein expression. Primary kidney cells from all 5 TA transgenic piglets were transfected with TARE-RANKL and TARE-CTLA-4Ig constructs linked with a blasticidin resistance cassette to facilitate selection of double transgenic/transfected cell clones. These cell clones were individually stimulated with doxycyclin to identify cell clones with low background and high inducibility of expression of RANKL or CTLA-4Ig. For both combinations, several clones meeting these criteria were identified. Such cell clones were used for nuclear transfer to produce double-transgenic pigs. Application of doxycyclin (25 to 50 mg kg–1 of body weight/day) to a TA/TARE-RANKL transgenic pig resulted in a marked increase in plasma RANKL concentrations. In addition, 4 TA/TARE-CTLA-4Ig transgenic piglets have been born, which will undergo doxycyclin stimulation experiments in the near future. In summary, CAG-TA transgenic pigs serve as a reliable basis for inducible transgene expression, as shown in the present study for RANKL and CTLA-4Ig. Studies to show the biological functionality of the transgene are currently underway.

Supported by DFG (FOR793) and BFS (FORZebRA).
193 BOVINE EMBRYO GENOTYPING USING A 50K SINGLE NUCLEOTIDE POLYMORPHISM CHIP
Authors: D. Le Bourhis A. , Mullaart E. , Humblot P. , Coppieters W. , Ponsart C.
Published: August 2011

Genomic tools are now available for most livestock species and are used routinely for marker-assisted selection (MAS) and genomic selection (GS) in cattle. Recently, multiple-marker detection has been achieved from biopsies of preimplantation stage embryos, thus allowing embryos to be selected before transfer (Le Bourhis et al. 2009 Reprod. Fertil. Dev. 21, 192 abst). This strategy provides the opportunity to estimate some traits of particular interest, the presence of genetic abnormalities, or both. The present work aimed to assess the efficiency of MAS/GS evaluation from biopsied bovine embryos by using the bovine 50K single nucleotide polymorphism (SNP) Illumina chip. A biopsy of 5 to 10 cells was obtained under laboratory conditions, using a microblade under a stereomicroscope, from 29 in vitro-cultured morulae and blastocysts. Biopsies were transferred individually as dry samples in tubes and sent frozen (n = 13) or at room temperature (n = 16) to the genotyping laboratory. The genomic DNA of each biopsy was amplified using a whole-genome amplification (WGA) kit according to the manufacturer’s instructions (Qiagen REPLI-g® Mini Kit, Qiagen, Valencia, CA). Following WGA, DNA concentration was determined by using PicoGreen. For subsequent genotyping, a custom CRV 50K Illumina chip was used. Call rates were calculated from 50 905 SNP. Percentage of allele drop-out (%ADO), which was estimated from the number of heterozygous markers [%ADO = (calculated hetero – observed hetero)/calculated hetero]. Parentage error was estimated from 12 embryos by using the genotypes of the parents of the embryos. Both groups of transport conditions were compared using Student’s t-test. Results are presented as mean ± SEM. A greater quantity of DNA was obtained after amplification of biopsies that were sent frozen to the laboratory when compared with those at room temperature (P < 0.05). However, the SNP call rate, %ADO, and parentage error did not differ between groups. These results indicate that genotyping from embryo biopsies following WGA can be achieved with good efficiency when using high-density marker chips. To validate the use of MAS/GS from early embryos in breeding schemes, a larger number of in vivo embryos are currently genotyped under field conditions. This will allow the reliability of this method to be assessed and the correlation between embryo and calf genetic evaluation to be quantified with the current WGA efficiency.

Table 1.Amount of DNA after WGA and genotyping results
198 THE EFFECT OF SOURCE AND IN VITRO MATURATION ON THE ABUNDANCE OF MATERNAL mRNA OF SELECTED GENES IN FOLLICULAR BOVINE OOCYTES AND THEIR INFLUENCE ON IN VITRO DEVELOPMENT
Authors: Somfai T. , Imai K. , Kaneda M. , Akagi S. , Haraguchi S. , Watanabe S. , Mizutani E. , Dang-Nguyen T. Q. , Inaba Y. , Geshi M. , Nagai T.
Published: August 2011

The aim of the present study was to investigate the effect of oocyte source and in vitro maturation (IVM) on the expression of selected genes in bovine oocytes and their contribution to in vitro embryo development. Follicular oocytes were collected either by ovum pick-up from live cows or by the aspiration of ovaries of slaughtered cows following storage in Dulbecco’s PBS at 15°C for overnight. In vitro maturation was performed according to the method of (Imai et al. 2006 J. Reprod. Dev. 52, 19–29 suppl.). Gene expression was assessed before and after IVM by real-time PCR. The following genes were investigated: GAPDH, G6PDH, ACTB, H2A, CCNB1, MnSOD, OCT4, SOX2, CX43, HSP70, GLUT8, PAP, GDF9, COX1, ATP1A1, CDH1, CTNNB1, AQP3, DYNLL1, DYNC 1/1, and PMSB1. In brief, mRNA was extracted from 20 oocytes per sample using a Qiagen RNeasy Micro Kit (Qiagen, Valencia, CA). Gene expression was analysed by a Roche Light Cycler 480 device and software (Roche, Indianapolis, IN). Relative expression of each gene was normalized to CCNB1, which in preliminary experiments appeared the most stably expressed irrespective of oocyte source and meiotic stage. Three replications were performed. Data were analysed by paired t-test. In immature ovum pick-up oocytes, genes related to metabolism (GAPDH, G6PDH, GLUT8) and stress (MnSOD, HSP70), and also OCT4, ATP1A1, and DYNC1/1 showed significantly (P < 0.05) higher expression compared with immature oocytes collected from slaughtered-stored ovaries. The expression of GDF9, GLUT8, CTNNB1, and PMSB1 was significantly (P < 0.05) reduced during IVM irrespective of the oocyte source. In a second experiment, IVF IVM oocytes showing an early (at 22 to 25 h after IVF) or late (at 27 to 30 h after IVF) first cleavage were either cultured in vitro or analysed for gene expression at the 2-cell stage. A higher (P < 0.05) rate of early-cleaving oocytes developed to the blastocyst stage compared with the rate of late-cleaving ones (46.2% v. 15.6%, respectively). Nevertheless, only ATP1A1 showed significantly reduced (P < 0.05) expression in late-cleaving embryos compared with early-cleaving ones. Our results suggest that although removal and storage of ovaries and IVM caused a reduction in the relative abundance of several genes in oocytes, in most cases, this did not affect embryo development. Among the genes studied, only ATP1A1 was correlated with in vitro development.
20 FACTORS THAT AFFECT PURITY AND YIELD OF BOVINE SEX-SORTED SPERM
Authors: Sakamoto Y. , Ueda M. , Toda S. , Kimura H.
Published: August 2011

The purposes of these studies were to examine the influence on the yield of sex-sorted sperm by the different size of sorting gate and to evaluate factors that affect the purity of sex-sorted sperm. As the sorting gate was expanded, so the yield of bovine sex-sorted sperm using flow cytometry was increased. At the same time, the purity of sex-sorted sperm became low. In addition, though the sorting gates were the same size, the purity of sex-sorted sperm differed among trials. These observations indicated the existence of factors that affect purity besides the size of sorting gate. To examine the yield of sex-sorted sperm, X-bearing sex-sorted sperm from 4 Holstein bulls were produced repeatedly 34 times by 3 flow cytometers. The sizes of sorting gates were fixed at 40–42%, 44%, and 46%. Each yield of sorting gate at 40–42%, at 44% and at 46% was compared. To evaluate factors that affect purity, X- or Y-bearing sex-sorted sperm were produced by one flow cytometer. These trials were repeated 160 times for the sorting of X-bearing sperm and 45 times for the sorting of Y-bearing sperm. Stepwise multiple regression analysis was used to analyse the relationships between the purity of sex-sorted sperm and the following sorting conditions, the percentage of oriented sperm, the percentage of dead sperm, degree of separation between X-bearing and Y-bearing sperm, the size of sorting gate, event rate, drop drive frequency, drop delay value and drop delay accuracy. The highest yield was acquired by sorting gate at 44%. The number of sex-sorted sperm was increased as sorting gate was expanded, however, the purity became low. The purities of the sperm by some trials using sorting gate at 46% were less than our acceptable lowest purity that was 90%. So that those sperm must be discarded. Therefore the yield of sorting gate at 44% was greater than sorting gate at 46%. Stepwise multiple regression analysis revealed that the factors for increasing purity of X-bearing sex-sorted sperm were the percentage of oriented sperm (P < 0.001), the degree of separation between X-bearing sperm and Y-bearing sperm (P < 0.001), the drop delay accuracy (P < 0.001), the event rate and the drop drive frequency, and the factor for decreasing purity of X-bearing sex-sorted sperm was the size of sorting gate (P < 0.001). On the other hand, the factors for increasing purity of Y-bearing sex-sorted sperm were the percentage of oriented sperm (P < 0.01), the degree of separation between X-bearing sperm and Y-bearing sperm (P < 0.01) and the event rate (P < 0.05), and the factor for decreasing purity of Y-bearing sex-sorted sperm was the size of sorting gate (P < 0.01). From these results, it can be concluded that the purity of sex-sorted sperm was not depend on simply by size of sorting gate but was more completely explained by other sorting conditions.
204 ALTERED GENE EXPRESSION FOLLOWING EXPOSURE TO BISPHENOL A IN HUMAN OVARIAN CANCER CELLS EXPRESSING ESTROGEN RECEPTORS BY MICROARRAY
Authors: Yi B.-R. , Jeung E.-B. , Choi K.-C.
Published: August 2011

Although endocrine-disrupting chemicals (EDC) may interfere with the endocrine system(s) of animals and humans and have an estrogenicity or androgenicity, the exact mechanism(s) underlying their detrimental effects is not clearly understood. Among them, bisphenol A (BPA) is widespread in the environment and is commonly ingested by animals because it is used in the manufacture of polycarbonate plastics, food-storage containers, and other plastics. Thus, in this study, we evaluated altered gene expression following exposure to BPA by microarry in human ovarian cancer cells, which highly express oestrogen receptors (ER). Treatment with BPA and endogenous oestrogen (E2) for 24 h resulted in an increase in cell proliferation and enhanced the oestrogen response element (ERE) activity in human BG-1 ovarian cancer cells with ER. Bisphenol A-induced cell growth and the activation of ERE were reversed by an oestrogen receptor antagonist, ICI 182 780, suggesting that ER appear to be involved in BPA-induced cell growth and ERE activation in these ovarian cells. Following BPA treatment, the expression levels of representative genes, namely, apoptosis inhibitor 4 (survivin), RAB31 ras oncogene family, v-myc, v-myb, cyclin A2, cyclin B1, amphiregulin, insulin-like growth factor binding protein 4, chemokine-like factor 3, fibroblast growth factor, and E2F transcription factor 4, were subsequently confirmed in these ovarian cells by real-time PCR. Taken together, these results indicate distinctly altered expression of responsive genes following exposure to BPA, and implicate distinct effects of endogenous E2 and environmental EDC in human ovarian cancer cells expressing ER.

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST; No. 2010-0003093).
21 THE INJECTION OF CORTISOL TO UTERUS INCREASES THE IMPLANTATION RATE AND LITTER SIZE IN PIG ARTIFICIAL INSEMINATION USING CYROPRESERVED SPERMATOZOA
Authors: Shimada M. , Okazaki T.
Published: August 2011

Cryopreserved boar spermatozoa are not routinely available to swine artificial insemination (AI) because conception and farrowing rates, along with litter size, have remained low. We have reported the positive roles of seminal plasma in frozen–thawed sperm functions (Okazaki et al. 2009 Theriogenology 71, 491–498). Moreover, the injection of seminal plasma to uterus with frozen–thawed spermatozoa significantly increased the implantation rate. Thus, the factors in seminal plasma act not only on sperm but also on uterus to induce successful fertilization and implantation in pig AI using cryopreserved spermatozoa. To test this hypothesis, we identified the factors in seminal plasma and then developed novel pig AI method using cryopreserved spermatozoa. The sperm-rich fraction was collected weekly from each boar using the gloved-hand technique. The seminal plasma was removed just after collection by centrifuge and then was frozen as described in our previous study (Okazaki et al. 2009 Theriogenology 71, 491–498). When the frozen–thawed sperm was incubated with Fluo-3/AM to determine the level of intercellular Ca2+, the level of Ca2+ was increased in a time-dependent manner, and spontaneous capacitation that was judged by tyrosine phosphorylation of sperm protein by Western blotting (Shimada et al. 2008 Development 135, 2001–2011), was also induced in post-thawed sperm. The addition of EGTA to thawing solution significantly suppressed the Ca2+-induced capacitation. Moreover, the treatment increased fertilization rate in in vitro fertilization and in vivo in artificial insemination as similar as those in sperm with seminal plasma. The same number of blastocyst was collected from uterus by AI using post-thawed sperm with EGTA. However, the pregnancy rate remained low, and the number of leukocytes in the uterus was increased. In the next experiment, we examined in seminal plasma, the level of cortisol that has been known to play an important role in controlling immune function. The results showed that cortisol (1.0 ng mL–1) was detected in seminal plasma. When the sows of natural oestrus were twice artificial inseminated with or without cortisol, the injection of cortisol (5 μg/50 mL) to uterus with sperm significantly decreased the number of leukocytes in the uterus or endometrium at 24 to 36 h after AI. The low number of leukocytes in the uterus was similar to that in uterus injected fresh semen. The cortisol injection significantly increased the implantation rate and litter size of sows as compared to AI without cortisol (implantation rate; 83% v. 51%, litter size; 10.6 v. 7.3). From these results, we concluded that the injection of cortisol with frozen–thawed spermatozoa by EGTA-containing solution was a novel method of pig AI using cryopreserved spermatozoa.

This work was supported by the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry, and JST-Grant (No. 12-068 and No. 12-104).
212 EFFECT OF CONSECUTIVE SUPERSTIMULATORY TREATMENT-INDUCED FOLLICULAR WAVE SYNCHRONIZATION TO OPTIMIZE OOCYTE RETRIEVAL AND EMBRYO PRODUCTION BY OVUM PICKUP AND IN VITRO FERTILIZATION IN COWS
Authors: Imai K. , Ohtake M. , Aikawa Y. , Sugimura S. , Hirayama M. , Hashiyada Y. , Kobayashi S. , Konishi K.
Published: August 2011

We previously reported that superstimulatory (SS) treatment-induced follicular wave synchronization after ovum pickup (OPU) was effective in enhancing the quality of obtained oocytes and blastocysts derived from in vitro maturation (IVM) and fertilization (IVF; Imai et al. 2010 Reprod. Fertil. Dev. 22, 296). The present study was designed to examine the efficiency of embryo production by 4 sessions of OPU-IVF using a series of the SS treatment-induced follicular wave synchronizations. For the SS protocols, 3 consecutive SS (3CSS) and 2 separated SS (2SSS) were used. In the 3CSS group, the first OPU was performed on random days of the oestrous cycle (Day 0) and all follicles larger than 2 mm in diameter were aspirated. On Day 5, follicles larger than 8 mm in diameter were aspirated and a CIDR (InterAg, Hamilton, New Zealand) was inserted. The cows then received 20 armour units of FSH (Kawasaki-Seiyaku, Kawasaki, Japan) in twice-daily decreasing doses by IM injection from Day 7 to 10. Cloprostenol (PGF; 0.75 mg, Fujita-Pharm, Tokyo, Japan) was administered on the morning of Day 9. The second OPU was performed 48 h after PGF administration on Day 11; the CIDR was removed from the cows just before OPU. After the second OPU, donors were treated consecutively with the SS protocol mentioned above for the third and fourth OPU sessions. In the 2SSS group, donors received 2 sets of the SS treatment mentioned above, with an interval of 11 days between the second and the third OPU session. Four OPU sessions were performed every 11 days on all cows. In this study, 8 Japanese Black cows were divided into the 3CSS and 2SSS groups, and the treatment for each group was reversed after a 65-day interval as crossover trials. After OPU, Grade 1 and 2 oocytes were used for IVM and IVF, and putative zygotes were cultured as described by (Imai et al. 2006 J. Reprod. Dev. 52, S19–S29 suppl.). A part of the zygotes were cultured in a micro-well system. Data were analysed by Student’s t-test and chi-square test. There were differences (P < 0.05) in the mean (±SD) number of follicles, collected oocytes, and cultured oocytes in the 3CSS (35.0 ± 8.6 and 24.4 ± 11.2, respectively) and 2SSS (30.8 ± 10.5 and 20.2 ± 9.0, respectively) groups. There were no differences in mean percentage of blastocyst formation and Grade 1 blastocyst rates between the 3CSS (38.5 and 55.8%, respectively) and 2SSS (34.8 and 54.8%, respectively) groups. However, the mean number of blastocysts produced per OPU session was significantly (P < 0.05) higher in the 3CSS group (8.1 ± 6.3) compared with the 2SSS group (5.8 ± 4.4). These results indicate that a series of 3 consecutive SS treatments had greater efficiency in producing OPU-IVF embryos.

This work was supported in part by the Research and Development Program for New Bio-industry Initiatives.
215 AN IMPROVED SYSTEM FOR THE IN VITRO PRODUCTION OF PORCINE EMBRYOS
Authors: Kelly J. M. , Weaver A. , Kleemann D. O. , Frazer L. M. , Kind K. L. , van Wettere W. H. , Walker S. K.
Published: August 2011

A reliable in vitro system for the production of porcine embryos (IVP) is important for use in basic research and in the reproductive technologies. Despite recent developments, blastocyst rates remain low compared with those obtained for most domestic species. Here, we report a porcine IVP system, based largely on our ovine and bovine systems (Walker et al. 1996 Biol. Reprod. 55, 703–708; Kelly et al. 2007 Reprod. Dom. Anim. 42, 577–582), that gives blastocyst development rates higher than previously reported. Abattoir-sourced ovaries were collected into PBS, and cumulus–oocyte complexes (COC) were aspirated (from 3- to 6-mm follicles) into HEPES–TCM-199 supplemented with 0.4% BSA. The COC (up to 30/well) were matured in 600 μL of maturation medium (TCM-199, 20% (vol/vol) porcine follicular fluid, FSH, LH, epidermal growth factor, cysteamine, and estradiol) for 42 h at 38.6°C in humidified 5% CO2. After 42 h, excess cumulus cells were removed and the COC were placed into modified Tris medium supplemented with BSA and caffeine (500 μL well–1). Sperm from pooled semen were washed in HEPES SOF supplemented with BSA, caffeine, and heparin and incubated for 45 min. Sperm were then centrifuged and the pellet was resuspended; a concentration of 5 × 105 sperm mL–1 was used. Oocytes and sperm were co-cultured for 6 h, cumulus cells were removed, and presumptive zygotes were placed into 600 μL of culture medium (SOF, BSA, and amino acids). Zygotes were cultured in 5% CO2:5% O2:90% N2, and cleavage and embryo development to Day 6 were assessed. Separate studies were conducted with COC from mature sows and from prepubertal gilts. In the latter, COC were exposed to ±dbcAMP for the first 22 h of the maturation period. Blastocyst rates in the sow were similar to those routinely produced in sheep and cows in our laboratory. Cleavage rates, mean blastocyst cell numbers, and incidence of polyspermy (assessed to be 10 to 12% in prepubertal gilts) were improvements on those generally reported (see Nagai et al. 2006 Front. Biosci. 11, 2565–2573 and Gil et al. 2010 Reprod. Dom. Anim. 45, 40–48 for general reviews). Observations indicate that the use of dbcAMP to regulate oocyte maturation had a negative effect on most parameters. Although blastocyst cell numbers are indicative of blastocyst quality, the significance of these findings can be validated only by transfer studies to determine embryo viability.

Table 1.Development rates for in vitro-fertilised oocytes derived from sow and prepubertal gilts1

This work was partly supported by the Australian Cooperative Research Centre for an Internationally Competitive Pork Industry.