Mice and cattle use distinct pathways for the first cell segregation into inner cell mass (ICM) and trophectoderm (TE) lineages at the blastocyst stage. However, limited knowledge is available regarding the reliable transcriptional networks that orchestrate the complex developmental processes at this stage in nonrodent species. In order to elucidate the site-dominant transcriptomic properties of bovine blastocysts, we separated cell samples into the ICM and TE using both mechanical and chemical methods and performed in silico prescreening for candidate genes that were site-dominantly expressed in bovine blastocysts. We further performed quantitative real-time PCR and in situ hybridization using the site-specific cell samples. As a result, we identified seven ICM-dominant genes and five TE-dominant genes not found in earlier studies. Our findings provide novel insights into the mechanism of cell-fate specification in the pre-implantation bovine embryo.

The aim of the present study was to determine whether the season (hot and cool) and reproductive phase (pregnant and non-pregnant) of the cow affect follicular recruitment and oocyte development. Follicular oocytes were aspirated from Japanese black cows by the ovum pick-up (OPU) method, which was performed 2 to 6 times within 1.5 months in pregnant cows and 2 to 4 times within 2 months in non-pregnant cows, during the hot (July to September) and cool (October to November) seasons. After follicular aspiration, the number and morphology of cumulus-oocyte complexes (COCs) and the developmental competence of oocytes after in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture were evaluated. The quality of aspirated COCs did not differ between the hot and cool seasons, irrespective of the reproductive phase of the donor cows. In the pregnant cows, the season did not affect follicular recruitment, early embryonic development or the quality of embryos. In the non-pregnant cows, however, the mean number of aspirated follicles and collected oocytes decreased during the hot season as compared with the cool season. When the data for the 2 seasons were combined to assess the effects of reproductive phase on oocyte development, the total proportions of cleavage, development into blastocysts and freezable embryos were higher for embryos obtained from pregnant cows (P<0.05) than those obtained from non-pregnant cows. In conclusion, the season did not have any apparent effects on the quality of aspirated COCs and the developmental competence of oocytes after IVM-IVF, but it may affect follicular recruitment in non-pregnant cows. Moreover, the reproductive phase may influence the developmental competence of the recovered oocytes.

ABSTRACT. Bone mineral density (BMD), distribution of its density and bone histomorphometric parameters were evaluated in lumbar vertebra of normally growing miniature pigs. The fourth lumbar vertebra (L4) of the Göttingen miniature pig were used in this cross-sectional study in vitro. The BMD of the miniature pig was similar to that of humans in tendency of gender differences and some growth patterns during puberty. In these regards this animal appears useful as a model for human bone study. However, the trabecular and cortical BMDs of lumbar spine were extremely high value (399.43 ± 26.36 mg/cm 3 in female trabeculae; 973.06 ± 69.55 mg/cm 3 in female cortical bone; 419.04 ± 34.84 mg/cm 3 in male trabeculae; 1038.81 ± 125.72 mg/cm 3 in male cortical bone in pigs 30 months or more). Furthermore, histomorphometric analysis yielded values that were remarkably different from those found in humans. From these results, it was revealed that miniature pig had a higher bone mass and denser trabecular network than human, indicating that its bone is probably stronger. Therefore, care should be taken in choosing the miniature pig as a bone study model. KEY WORDS: bone mineral density, histomorphometric analyses, lumbar spine, miniature pig.J. Vet. Med. Sci. 66(6): 599-609, 2004 Metabolic bone diseases such as osteoporosis are becoming more important because of an aging society in human medicine. These diseases increase the risk of fragility fracture as bone mineral levels decrease, and also reduces their quality of life. Previous studies have reported that bone strength and fracture risk are closely related to bone mineral density (BMD) [6,9,27]. In contrast, several studies have suggested that changes in bone architecture increase fracture risk [29,31,33,34,43]. Therefore, it is necessary to study the changes in both BMD and architecture for more sensitive investigation of the bone disease.Quantitative computed tomography (QCT) has been widely investigated and applied in recent years as a means of non-invasive quantification of BMD. It has been reported that QCT might be most sensitive to changes in bone density caused by rapid bone turnover, such as in menopausal immobilization or hyperthyroidism [9]. QCT can selectively measure both trabecular and cortical bone [9,13,22,38], and the BMD is expressed in milligrams per cubic centimeter (volumetric density) [9]. Ebbesen et al. showed highly correlation between BMD by QCT and compressive strength in the lumbar vertebrae [9].Several studies have reported that bone disorders such as osteoporosis lead to trabecular and cortical bone alterations in humans. These alterations are characterized not only by a reduction of bone mass but also by structural changes in microarchitecture which is measured by histomorphometry of bone [29,31,33]. Therefore, it has been considered that histomorphometric analysis have an important role as contributor to bone strength, in addition to BMD.Recently, miniature pig has been noticed as for experimental animal of bone study. The pig is an excel...

This study was designed to characterize the effect of medetomidine (Med) on canine electroencephalography (EEG), to evaluate the use of quantitative EEG for assessing sedation levels and to explore the correlation between the serum concentration of Med and the quantitative EEG. Four groups of dogs were given Med at doses of 20, 40, 80 and 160 microg/kg (Med-20, Med-40, Med-80 and Med-160 groups). Following Med administration, there was synchrony between each unipolar EEG lead. On EEG power spectrum analysis of the bipolar leads, all groups showed a significant depression of the 14-30 Hz components. The power of the 1-3 Hz component in the Med-80 and Med-160 groups was significantly increased, although there were few changes in the other groups. Similar results were obtained from raw data analysis. As a result of quantitative EEG analysis, spectrum edge frequency 90 analysis (SEP90) showed that the frequency was significantly reduced in all groups after Med administration. A dose-response effect was observed in all groups except for the Med-160 group. Both of these EEG analyses were significantly correlated with the serum concentration of Med. However, the result of the SPF90 analysis sugested a stronger correlation than that for median edge frequency analysis. In conclusion, care must be taken in veterinary clinical diagnoses when Med is used during EEG recording, as Med may cause increased activity in the low frequency band and a decrease in high frequency band activity. In addition, quantitative EEG analysis may be useful in assessing the depth of sedation and in further studies on Med administration.

The purpose of this study was to evaluate the effects of the administration of an alpha2-adrenoceptor agonist alone and in combination with other derivatives on brain wave activity. In addition, the diagnostic values of the electroencephalogram (EEG) for judging the depth of the balanced anaesthesia with an alpha2-adrenoceptor agonist was evaluated. The treatments comprised 20 microg/kg medetomidine (Me-20), 80 microg/kg medetomidine (Me-80), 20 microg/kg medetomidine and 0.5 mg/kg midazolam (Me-Mi) administered intramuscularly, and 20 microg/kg medetomidine with 0.5 mg/kg midazolam and 0.1 mg/kg butorphanol (Me-Mi-Bu). The EEG was recorded continuously at pre-administration, and at 7, 10, 20, 30, 45 and 60 min after administration. The recorded data were analysed by separating the power spectrum into 1-3, 4-7, 8-13 and 14-30 Hz bands. Spectral-edge analysis was used to calculate the spectral edge frequency 90 (SEF90) and the median edge frequency (MEF). Time-related changes in power spectrum analysis showed a significant increase in the Me-80 group in the 1-3 Hz band. The power for 1-3 Hz in the Me-80 group was significantly higher than in all the other groups. In the 14-30 Hz band, there was a significant reduction of power in all groups following administration of the agents. The SEF90 frequencies were significantly reduced in all groups except for the Me-20 group after administration of the agents. The SEF90 frequencies in the Me-20, Me-Mi and Me-Mi-Bu were all significantly higher than those in the Me-80 group. However, there was no significant difference between the Me-20, Me-Mi and Me-Mi-Bu groups in any analyses. Our results demonstrated that the changes in quantitative EEG made by the Me-Mi-Bu and Me-Mi groups were similar to those made by Me-20 groups. Present results suggest that the EEG should be interpreted with caution in assessing the anaesthetic level in balanced anaesthesia in dogs.

The objective was to determine if immature bovine oocytes with cumulus cells at the germinal vesicle (GV) stage could be vitrified by aluminum sheets (AS; pieces of sheet-like aluminum foil). Cleavage rates in fertilized oocytes previously vitrified by the AS procedure were higher than those vitrified by a nylon-mesh holder (NM) procedure (89.3 ± 2.1% vs. 65.0 ± 3.7%). Cleaved embryos derived from the AS but not from the NM procedures developed to blastocysts. Furthermore, to investigate the effects of vitrifying GV oocytes on cytoplasmic structure and on the ability to undergo cytoplasmic changes, the intracellular phospholipid membrane (IM) was stained with the lipophilic fluorescent dye, 3,3'-dioctadecyloxa-carbocyanine perchlorate. After vitrification by AS, the IM remained intact relative to that of oocytes vitrified by NM. During in vitro maturation, reorganization of the IM was also undamaged in oocytes vitrified by AS before oocyte maturation, and the IM within oocytes vitrified by the NM procedure was evidently impaired. Finally, vitrification (AS) was used for GV oocytes collected using the ovum pick-up method. A bull calf was born after in vitro production and subsequent embryo transfer. The vitrification techniques described herein should facilitate generation of viable in vitro production bovine blastocysts using oocytes recovered using the ovum pick-up method.

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We investigated the effects of twice-weekly follicular punctures of ovaries with or without corpus luteum (CL) on follicular and luteal dynamics. A cross-over design was used, with each cow (seven Japanese Black beef cows) being assigned to one of the three groups at 2-month intervals. Follicular punctures were performed twice weekly for three consecutive weeks until day 20 (oestrus = day 0). All visible follicles (diameter >3 mm) in the ovaries bearing CL (ipsilateral group) or those in the contralateral ovaries (contralateral group) were aspirated. As a control, all visible follicles in both ovaries were aspirated (bilateral group). Follicular development, CL formation and progesterone concentrations in each cow were monitored from days 0 to 30. Follicular growth profiles in the punctured ovaries during/after puncture treatment were similar, irrespective of the presence of follicles in the unpunctured ovary and the CL in the punctured or unpunctured ovaries. After puncture, two cows (28.6%) each in the ipsilateral and bilateral groups did not exhibit behavioural oestrus until day 30, whereas all cows in the contralateral group exhibited oestrus. CL growth and increase in progesterone concentrations after the last follicular puncture in the bilateral group were delayed when compared with those in the ipsilateral group. Our results indicate that the presence of follicles in the unpunctured ovary and the CL in the punctured or unpunctured ovaries does not significantly influence follicular growth in punctured ovaries during/after puncture treatment. However, follicular puncture in ovaries bearing CL may disturb or delay oestrus occurrence after treatment.