This study was conducted to determine the effect of Nitric oxide (NO) inhibition in bovine in vitro development and expression analysis of the three Nitric oxide synthase (NOS) isoforms: endothelial (eNOS), neuronal (nNOS) and inducible (iNOS), mRNA and protein in bovine oocytes and embryos. Selective inhibitor of NOS, N-omega-nitro-l-arginine methyl ester (l-NAME) was applied at different doses (0, 0.1, 1 and 10 mm) in maturation (experiment 1A), culture medium (experiment 1B) and in both maturation and culture media (experiment 1C). No significant differences were observed in cleavage and blastocyst rates when oocytes were matured in the presence of l-NAME as long as the inhibitor was omitted during fertilization and culture. However, significantly lower blastocyst rates were observed when l-NAME was present at higher level (10 mm) in culture medium alone and in both maturation and culture media. In experiment 2, mRNA isolated from triplicate pools of oocytes and embryos (n = 15-20) was subjected to quantitative real time reverse transcription polymerase chain reaction to investigate the expression of eNOS, iNOS and nNOS mRNA in normal IVP bovine oocytes and embryos. While eNOS and iNOS transcripts were detected at higher level in oocytes (immature and mature), two-cell and four-cell stage embryos, the nNOS was detected only in immature oocyte, two-cell and morula stages. In experiment 3, eNOS and iNOS protein expression analysis was performed in IVP oocytes and embryos and both proteins were detected in the cytoplasm and the nuclei (weak) of oocytes and embryos. These data provide the first evidence for the role of NO production and the presence of mRNA and protein products of NOS isoforms during bovine embryogenesis.

This study was conducted with the aim of optimizing the experimental design of array experiments. We compared two image analysis and normalization procedures prior to data analysis using two experimental designs. For this, RNA samples from Charolais steers Longissimus thoracis muscle and subcutaneous adipose tissues were labeled and hybridized to a bovine 8,400 oligochip either in triplicate or in a dye-swap design. Image analysis and normalization were processed by either GenePix/MadScan or ImaGene/GeneSight. Statistical data analysis was then run using either the SAM method or a Student’s t-test using a multiple test correction run on R 2.1 software. Our results show that image analysis and normalization procedure had an impact whereas the statistical methods much less influenced the outcome of differentially expressed genes. Image analysis and data normalization are thus an important aspect of microarray experiments, having a potentially significant impact on downstream analyses such as the identification of differentially expressed genes. This study provides indications on the choice of raw data preprocessing in microarray technology.

200 Reproduction, Fertility and DevelopmentDevelopmental Biology for the first experiment, loaded into straws, and assigned to one of 4 treatment groups. Half the straws from each bull were exposed to 90 MPa/30 min, 90 MPa/90 min, 30 MPa/30 min, or 30 MPa/90 min, and then cryopreserved. Controls consisted of straws that were cryopreserved without pressure treatment. Cryopreservation steps were 60 min equilibration at 5 • C, followed by 10 min at −110 • C, and then plunging into liquid nitrogen. Straws were thawed in a 35 • C water-bath for 30 s. Each treatment and control group was replicated 8 times (8 samples per bull). The average post-thaw motility was significantly superior with pressure pre-treatment in each of the pressurized groups compared to the samples frozen without previous pressurization (P < 0.001) (Bull I: 2-3% without pressurization vs. 17-33% with pressurization; Bull II: 0% without pressurization vs. 21-35% with pressure pre-treatment). Among the pressure/time parameters used, 30 MPa/90 min proved significantly superior (33 and 35%; P < 0.05) for each of the bulls. Expt. 2 clearly demonstrates the beneficial effect of a previous pressure treatment on post-thaw motility of bull semen cryopreserved in our experiment. Further investigations are needed, including samples from different bulls, different freezing protocols, and the biological background of the process. Development of improved protocols for cryopreservation of zona pellucida-intact porcine embryos could greatly impact the swine industry. Our aim was to investigate in vitro development following cryopreservation of embryos from Chinese Meishan (M) and occidental white cross (WC) breeds using a modified protocol described previously (Misumi K et al. 2003 Theriogenology 60, 253-260). First-parity M sows (n = 11) and WC gilts (n = 13) were observed for estrus every 12 h and inseminated at 12 and 24 h after estrous onset within breed using semen from 2 different boars. Females were sacrificed between Days 4.5 and 6 after estrus and embryos were collected using Beltsville embryo culture medium (BECM). Compact morula (CM) or blastocyst stage embryos from each female within breed were randomly allocated either directly into the culture system to serve as controls (68 M and 48 WC embryos) or to undergo cryopreservation. A total of 101 M and 78 WC embryos were cryopreserved using the following protocol: (1) 5 min in BECM + 10% ethylene glycol (EG); (2) 5 min in BECM + 10% EG + 0.27 M sucrose + 1% polyethylene glycol (PEG); and (3) 30 to 45 s in BECM + 40% EG + 0.36 M sucrose + 2% PEG. In the last solution, 5 to 10 embryos in a 5-to 10-µL microdrop attached to a fine glass pipette were exposed to the vapor phase of liquid nitrogen (LN 2 ) for 15 s and then plunged into LN 2 . The pipette tip was broken and the tip and associated frozen microdrop were placed inside an LN 2 -submerged 2-mL cryotube containing a hole in the lid for 1 h. Next, embryos were thawed using a 4-step (5 min each) procedure: (1) BECM + 5% EG + 0.57 M sucrose; (2) BECM + 2...