Strategies for the Generation of Gene Modified Avian Models: Advancement in Avian Germline Transmission, Genome Editing, and Applications.

Avian models are valuable for studies of development and reproduction and have important implications for food production. Rapid advances in genome-editing technologies have enabled the establishment of avian species as unique agricultural, industrial, disease-resistant, and pharmaceutical models. The direct introduction of genome-editing tools, such as the clustered regularly interspaced short palindromic repeats (CRISPR) system, into early embryos has been achieved in various animal taxa. However, in birds, the introduction of the CRISPR system into primordial germ cells (PGCs), a germline-competent stem cell, is considered a much more reliable approach for the development of genome-edited models. After genome editing, PGCs are transplanted into the embryo to establish germline chimera, which are crossed to produce genome-edited birds. In addition, various methods, including delivery by liposomal and viral vectors, have been employed for gene editing in vivo. Genome-edited birds have wide applications in bio-pharmaceutical production and as models for disease resistance and biological research. In conclusion, the application of the CRISPR system to avian PGCs is an efficient approach for the production of genome-edited birds and transgenic avian models.

Novel Potassium-Competitive Acid Blocker, Tegoprazan, Protects Against Colitis by Improving Gut Barrier Function.

Inflammatory bowel disease (IBD) is a chronic immune-mediated disorder characterized by prolonged inflammation of the gastrointestinal tract. IBD can result from gut barrier dysfunction, altered gut microbiota, and abnormal intestinal immunity induced by environmental factors in genetically susceptible individuals. Proton pump inhibitors (PPIs) such as rabeprazole are frequently employed for gastric acid inhibition. However, long-term PPI administration can alter the intestinal microbiome composition, possibly worsening IBD severity. The present study revealed that tegoprazan, a potassium-competitive acid blocker, significantly improved colitis in mice and enhanced the intestinal epithelial barrier function. Tegoprazan alleviated gut microbiota dysbiosis and enhanced the growth of Bacteroides vulgatus. In turn, B. vulgatus alleviated intestinal inflammation by inhibiting epithelial adhesion of pathogenic bacteria. Unlike rabeprazole, tegoprazan did not induce gut dysbiosis. Our findings provide novel insights into the potential role of tegoprazan as an intestinal protectant for IBD and as a therapeutic agent for gastric acid-related diseases.

Potassium Channelopathies and Gastrointestinal Ulceration.

Potassium channels and transporters maintain potassium homeostasis and play significant roles in several different biological actions via potassium ion regulation. In previous decades, the key revelations that potassium channels and transporters are involved in the production of gastric acid and the regulation of secretion in the stomach have been recognized. Drugs used to treat peptic ulceration are often potassium transporter inhibitors. It has also been reported that potassium channels are involved in ulcerative colitis. Direct toxicity to the intestines from nonsteroidal anti-inflammatory drugs has been associated with altered potassium channel activities. Several reports have indicated that the long-term use of the antianginal drug Nicorandil, an adenosine triphosphate-sensitive potassium channel opener, increases the chances of ulceration and perforation from the oral to anal regions throughout the gastrointestinal (GI) tract. Several of these drug features provide further insights into the role of potassium channels in the occurrence of ulceration in the GI tract. The purpose of this review is to investigate whether potassium channelopathies are involved in the mechanisms responsible for ulceration that occurs throughout the GI tract.

Direct visualization of deep brain stimulation targets in Parkinson disease with the use of 7-tesla magnetic resonance imaging.

OBJECT: A challenge associated with deep brain stimulation (DBS) in treating advanced Parkinson disease (PD) is the direct visualization of brain nuclei, which often involves indirect approximations of stereotactic targets. In the present study, the authors compared T2*-weighted images obtained using 7-T MR imaging with those obtained using 1.5- and 3-T MR imaging to ascertain whether 7-T imaging enables better visualization of targets for DBS in PD. METHODS: The authors compared 1.5-, 3-, and 7-T MR images obtained in 11 healthy volunteers and 1 patient with PD. RESULTS: With 7-T imaging, distinct images of the brain were obtained, including the subthalamic nucleus (STN) and internal globus pallidus (GPi). Compared with the 1.5- and 3-T MR images of the STN and GPi, the 7-T MR images showed marked improvements in spatial resolution, tissue contrast, and signal-to-noise ratio. CONCLUSIONS: Data in this study reveal the superiority of 7-T MR imaging for visualizing structures targeted for DBS in the management of PD. This finding suggests that by enabling the direct visualization of neural structures of interest, 7-T MR imaging could be a valuable aid in neurosurgical procedures.

Altered T2* relaxation time of the hippocampus in major depressive disorder: implications of ultra-high field magnetic resonance imaging.

Previous studies with 1.5 T or 3.0 T magnetic resonance imaging (MRI) have produced mixed results regarding the structural changes of the hippocampus in major depressive disorder (MDD). Subtle region-specific hippocampal tissue changes might be more sensitively detected by measuring the T2* relaxation time (T2*-RT) by ultra-high-field (UHF) MRI, as it provides much higher contrast and sensitivity and consequently greater resolution. We assessed the T2*-RTs of hippocampal sub-regions in 16 MDD patients (9 with recurrent MDD) and 16 control subjects using an UHF 7.0 T MRI system. T2*-RTs of CA1, CA2, CA3, CA4, and subiculum were calculated for both left and right hippocampus. MDD patients had significantly longer T2*-RTs in the right CA1 and subiculum than control subjects. Patients with recurrent MDD had significantly longer T2*-RTs in the right subiculum than those experiencing a first depressive episode, and longer T2*-RTs in the right CA1, CA3, and subiculum than control subjects. Values for T2*-RTs of the right CA3 were significantly correlated with illness duration. In conclusion, we report that T2*-RTs in the right subiculum and CA1 were increased in patients with MDD, especially in cases of recurrent MDD. These findings suggest that region-specific hippocampal damage may be occurring in recurrent depression.

A proposal of new reference system for the standard axial, sagittal, coronal planes of brain based on the serially-sectioned images.

Sectional anatomy of human brain is useful to examine the diseased brain as well as normal brain. However, intracerebral reference points for the axial, sagittal, and coronal planes of brain have not been standardized in anatomical sections or radiological images. We made 2,343 serially-sectioned images of a cadaver head with 0.1 mm intervals, 0.1 mm pixel size, and 48 bit color and obtained axial, sagittal, and coronal images based on the proposed reference system. This reference system consists of one principal reference point and two ancillary reference points. The two ancillary reference points are the anterior commissure and the posterior commissure. And the principal reference point is the midpoint of two ancillary reference points. It resides in the center of whole brain. From the principal reference point, Cartesian coordinate of x, y, z could be made to be the standard axial, sagittal, and coronal planes.

Quantitative analysis of the hippocampus using images obtained from 7.0 T MRI.

In-vivo volumetric measurements of hippocampus have proven to be highly informative for studying various neurological diseases such as Alzheimer's disease. The usefulness of volumetric imaging, however, has been limited due to the poor image resolutions obtained by currently available MRI images. In this study, a new result of volumetric image measurement of the hippocampus using 7.0 T MRI images of high contrast and resolution is described. To verify the usefulness of the proposed method, its reliability and sensitivity were examined and compared with existing imaging techniques such as 1.5 T or 3.0 T MRI imaging. The results of our study with 7.0 T MRI clearly demonstrated superior boundary detection for the hippocampal head, body, and tail compared with low field MRIs. In conclusion, robust and reproducible volumetric measurements as well as 3D images of clear contrast obtained with 7.0 T suggest the usefulness of high field MR imaging and its eventual use for the accurate diagnosis of hippocampal diseases and related research.

Magnetic resonance imaging of the canine brain at 7 T.

The purpose of this study was to describe relevant canine brain structures as seen on T2-weighted images following magnetic resonance (MR) imaging at 7 T and to compare the results with imaging at 1.5 T. Imaging was performed on five healthy laboratory beagle dogs using 1.5 and 7 T clinical scanners. At 1.5 T, spin echo images were acquired, while gradient echo images were acquired at 3 T. Image quality and conspicuity of anatomic structures were evaluated qualitatively by direct comparison of the images obtained from the two different magnetic fields. The signal-to-nose ratio (SNR) and contrast-to-noise ratio (CNR) were calculated and compared between 1.5 and 7 T. The T2-weighted images at 7 T provided good spatial and contrast resolution for the identification of clinically relevant brain anatomy; these images provided better delineation and conspicuity of the brain stem and cerebellar structures, which were difficult to unequivocally identify at 1.5 T. However, frontal and parietal lobe and the trigeminal nerve were difficult to identify at 7 T due to susceptibility artifact. The SNR and CNR of the images at 7 T were significantly increased up to 318% and 715% compared with the 1.5 T images. If some disadvantages of 7 T imaging, such as susceptibility artifacts, technical difficulties, and high cost, can be improved, 7 T clinical MR imaging could provide a good experimental and diagnostic tool for the evaluation of canine brain disorders.

A proinflammatory cytokine interleukin-32beta promotes the production of an anti-inflammatory cytokine interleukin-10.

A new proinflammatory cytokine interleukin-32 (IL-32) has six isoforms. Although IL-32 can be detected in sera from patients suffering from Crohn's disease and rheumatoid arthritis, it is unclear which isoforms are involved. To this end, we investigated the functions of the most abundant IL-32beta by generating K562-IL-32beta stable cell lines. This report confirms, using IL-32 small interfering RNA, that IL-32beta induces an anti-inflammatory cytokine IL-10 in K562-IL-32beta cells and U937 promonocytic cells, which express endogenous IL-32beta upon phorbol 12-myristate 13-acetate (PMA) treatment, and monocyte-derived dendritic cells (DC) upon lipopolysaccharide (LPS) treatment. Interleukin-32beta was induced in monocyte-derived macrophages by LPS and in monocyte-derived DC by LPS, poly(I:C), or anti-CD40 antibody, but was not induced by PMA. We showed that IL-32beta expression was increased in a time-dependent manner in monocyte-derived DC upon LPS treatment and peaked at 24 hr. Production of IL-10 was exactly coincident with IL-32beta expression, but IL-1beta and tumour necrosis factor-alpha production peaked at 6 hr after LPS treatment, then steeply declined. Interleukin-12 p40 was induced at 9 hr and gradually increased until 48 hr, at which time IL-32beta and IL-10 were no longer increased. Knock-down of IL-32beta by IL-32 small interfering RNA led to the decrease of IL-10, but the increase of IL-12 in monocyte-derived DC, which means that IL-32beta promotes IL-10 production, but limits IL-12 production. We also showed that IL-10 neutralization increases IL-12, IL-1beta and tumour necrosis factor-alpha production, which implies that IL-10 suppresses such proinflammatory cytokines. Taken together, our results suggest that IL-32beta upregulates the production of an anti-inflammatory cytokine IL-10, and then IL-10 suppresses proinflammatory cytokines.

Imaging and analysis of lenticulostriate arteries using 7.0-Tesla magnetic resonance angiography.

The purpose of this study was to analyze human lenticulostriate arteries (LSAs) obtained non-invasively by 7.0-T MRI. A three-dimensional time-of-flight (3D TOF) magnetic resonance angiography (MRA) technique was used with an investigational 7.0-T MRI scanner with a radio-frequency coil that was optimized and designed for angiographic purposes. We obtained images from 16 healthy volunteers (8 males and 8 females, mean age 21 +/- 2.7 years). For direct comparison of LSA images with digital subtraction angiography (DSA), we also obtained 7.0-T MRA and DSA images from one patient, a 27-year-old woman with a posterior fossa arteriovenous malformation (AVM). We then analyzed the characteristics of LSAs using a custom data analysis method with MatLab for quantitative analysis. Analysis of LSA images included shape and number of branches and origins, findings that are essential and useful for quantification of LSA abnormalities in both healthy controls and patients. Ultra-high-field MRA provided clear anatomic delineation of the LSAs, thereby suggesting that 7.0-T MRA may be a promising technique for microvascular imaging of the LSAs.

Profiling of transcripts and proteins modulated by the E7 oncogene in the lung tissue of E7-Tg mice by the omics approach.

The E6 and E7 oncoproteins of human papilloma virus (HPV) type 16 have been known to cooperatively induce the immortalization and transformation of primary keratinocytes. We established an E7 transgenic mouse model to screen HPV-related biomakers using the omics approach. The methods used to identify HPV-modulated factors were genomics analysis by microarray using the Affymetrix 430 2.0 array to screen E7-modulated genes, and proteomics analysis using nano-LC-ESI-MS/MS to screen E7-modulated proteins with the lung tissue of E7 transgenic mice. According to omics data, cyclin B1, cyclin E2, topoisomerase IIα, calnexin, activated leukocyte cell adhesion molecule CD166, actinin α1, diaphorase 1, gelsolin, platelet glycoprotein, and annexin A2 and A4 were up-regulated in the E7-Tg mice, while proteoglycan 4, sarcolipin, titin, vimentin, drep 1, troponin and cofilin-1 were down-regulated. We further confirmed the significance of differences between the expression levels of the selected factors in E7-Tg and non-Tg mice by real-time PCR. Genes related to cancer cell adhesion, cell cycle and migration, proliferation and apoptosis, as well as to the intermediate filament network and to endoplasmic reticulum proteins, were selected. Taken together, the results suggest that the E7 oncogene modulates the expression levels of cell cycle-related (cyclin B1, cyclin E2) and cell adhesion- and migration-related (actinin α1, CD166) factors, which may play important roles in cellular transformation in cancer. In addition, the solubilization of the rigid intermediate filament network by specific proteolysis mediated via up-regulating gelsolin and down-regulating cofilin-1, as well as increased levels of endoplasmic reticulum protein calnexin with chaperone functions, might also be involved in E7-lung epithelial cells.

Evaluation of MR angiography at 7.0 Tesla MRI using birdcage radio frequency coils with end caps.

The purpose of this study was to evaluate the feasibility of MR angiography (MRA) at 7.0 Tesla (T) using optimized birdcage (BC) coils with simple end cap configurations. Shielded 16-rung high-pass BC coils were built with identical geometry and compared with different sizes and locations of end caps. To determine whether the end cap configuration was effective, the signal intensity profiles along the superior-inferior (S-I) direction were analyzed in phantom and in vivo human experiments. The effects were also investigated in two-dimensional (2D) and three-dimensional (3D) time-of-flight (TOF) MRA experiments. The signal intensity profiles showed that B1 homogeneity at the service end, that is, the end cap side, was improved as the diameter of the end caps increased and the end cap became closer to the coil end ring. The results of 2D and 3D TOF experiments showed the best improvement of vessel visibility at the BC coil with an 80% end cap, when compared with BC coils with other end cap sizes or without an end cap. In conclusion, the BC coil with an end cap was effective for improving S-I directional homogeneity and suitable for MRA applications, especially at ultrahigh field MRI, such as 7.0T.

Functional MR angiography with 7.0 T Is direct observation of arterial response during neural activity possible?

We propose a new type of functional imaging in magnetic resonance imaging (MRI), a functional MR angiography (fMRA). As it is known, arterial responses during neural activities have been studied in animals, but little is known about the human brain in-vivo. Proposed fMRA at ultra-high field strength, 7.0 Tesla (T), has a potential for a direct visualization of vascular responses of those blood vessels related to the neural activity during the tasks, such as the hand movement or checker board stimulation, that is, fMRA. The results of this paper clearly indicate that there is the possibility that one can directly observe the vascular changes in individual blood vessels related to the tasks in human brain in-vivo, similar to fMRI.

Efficacy of chromoendoscopy with indigocarmine for the detection of ascending colon and cecum lesions.

OBJECTIVE: Previous data suggest that routine chromoendoscopy may increase detection rates of diminutive or flat lesions. The aim of this study was to evaluate the usefulness of chromoendoscopy in the ascending colon and cecum, where the incidence of diminutive or flat lesions is high. MATERIAL AND METHODS: Between June 2006 and September 2006, a total of 316 consecutive patients were prospectively enrolled in this study. The patients were randomly divided into two groups (control group: 158 patients, chromoendoscopy group: 158 patients). If the quality of bowel preparation was poor or cecal intubation was not achieved, the patient was excluded from the study. In the control group, the ascending colon and cecum were observed twice without chromoendoscopy. In the chromoendoscopy group, the cecum and ascending colon were reinspected following staining with indigocarmine solution after conventional examination of the cecum and ascending colon. Finally, a total of 151 and 149 patients were enrolled in the control and chromoendoscopy groups, respectively. RESULTS: The chromoendoscopy group differed significantly from the control group in the number of additionally detected polyps (control: 14 versus chromoendoscopy: 62, p<0.001) and in the number of patients with additionally detected polyps (control: 12 versus chromoendoscopy: 50, p<0.001). Multivariate analysis revealed that detection of polyps after indigocarmine spraying was independently associated with a high body mass index and older age (p = 0.045 and p = 0.006, respectively). CONCLUSIONS: With chromoendoscopy using indigocarmine, more polyps can be detected in the ascending colon and cecum as compared with using conventional colonoscopy.

A fusion PET-MRI system with a high-resolution research tomograph-PET and ultra-high field 7.0 T-MRI for the molecular-genetic imaging of the brain.

We have developed a positron emission tomography (PET) and magnetic resonance imaging (MRI) fusion system for the molecular-genetic imaging (MGI) of the in vivo human brain using two high-end imaging devices: the HRRT-PET, a high-resolution research tomograph dedicated to brain imaging on the molecular level, and the 7.0 T-MRI, an ultra-high field version used for morphological imaging. HRRT-PET delivers high-resolution molecular imaging with a resolution down to 2.5 mm full width at half maximum (FWHM), which allows us to observe the brain's molecular changes using the specific reporter genes and probes. On the other front, the 7.0 T-MRI, with submillimeter resolution images of the cortical areas down to 250 mum, allows us to visualize the fine details of the brainstem areas as well as the many cortical and subcortical areas. The new PET-MRI fusion imaging system will provide many answers to the questions on neurological diseases as well as cognitive neurosciences. Some examples of the answers are the quantitative visualization of neuronal functions by clear molecular and genetic bases, as well as diagnoses of many neurological diseases such as Parkinson's and Alzheimer's. The salient point of molecular-genetic imaging and diagnosis is the fact that they precede the morphological manifestations, and hence, the early and specific diagnosis of certain diseases, such as cancers.

Observation of the lenticulostriate arteries in the human brain in vivo using 7.0T MR angiography.

BACKGROUND AND PURPOSE: We sought to examine the feasibility of observing the lenticulostriate arteries (LSAs) noninvasively by ultrahigh-field MRI with 7.0T. METHODS: We used 3-dimensional time-of-flight MR angiography with a radiofrequency coil optimized for 7.0T MRI. We examined the LSAs of 6 healthy subjects and compared 7.0T MR angiography images with 1.5T ones to examine the potentials of ultrahigh-field MRI for angiography. RESULTS: The results show clear details of LSAs and their distribution in the normal healthy subjects with large variations in the shapes, the number of branches and the sites of origin. We also observed substantial differences between the left and right sides within each subject. Although we studied only 6 subjects, we found no age- or gender-related differences in the LSAs. CONCLUSIONS: The visualization of microvasculature of the brain, such as LSAs, using 7.0T MR angiography, is possible in in vivo human studies noninvasively. We, therefore, believe that it could play a major role in the study of small vascular abnormalities, such as the early stages of cerebral strokes.

Renovation of a drop embryo cultures system by using refined mineral oil and the effect of glucose and/or hemoglobin added to a serum-free medium.

This study was conducted to evaluate whether refining mineral oil and the addition of hemoglobin and/or glucose to a serum-free medium could improve in vitro-development of embryos cultured in a chemically semi-defined microdroplet culture system. Block strain, outbred (ICR) mouse 1- or 2-cell embryos were cultured in 5 microl droplets of Chatot, Ziomek and Bavister medium overlaid with mineral oil of different types, and preimplantation development to the blastocyst stage was subsequently monitored. In the experiment 1, either Sigma (M-8410) or BDH (GPR) mineral oil with or without washing was used for embryo culture and, distilled water (DW) or culture medium was used as a washing agent. As results, better (P<0.0001) development of 1-cell embryos was found in the Sigma than in the BDH; more blastocysts developed in Sigma oil washed with culture medium than in the others (37% vs. 0%). Subsequently, 1- (experiment 2) or 2-cell (experiment 3) embryos were cultured in the droplets overlaid with medium-washed Sigma oil, to which 0.001 mg/ml hemoglobin and/or 5.6 mM glucose were supplemented at the 1-cell and the 4-cell stages, respectively. Regardless of embryo stages, blastocyst formation was significantly improved by the addition of hemoglobin (54 to 48% vs. 42 to 31% in 1-cell and 83 to 78% vs. 65 to 68% in 2-cell embryos) and this effect was independent of glucose addition. In conclusion, the selection and washing of mineral oil, and the addition of hemoglobin is beneficial for improving the efficacy of a drop embryo culture system using a serum-free medium.

Pain dynamics observed by functional magnetic resonance imaging: differential regression analysis technique.

PURPOSE: To observe the dynamic responses of the cortical areas related to the pain processing by using the differential regression analysis (DRA) technique in functional magnetic resonance imaging (fMRI) and investigation of pain mechanisms. MATERIALS AND METHODS: For pain studies, thermal stimulation was applied by immersing the index finger into a hot bath of water with a temperature of 50-52 degrees C. Motor (finger tapping) and visual (flickering light) stimulation experiments were conducted to elucidate the physiological differences between the simple sensory tasks and pain tasks. To obtain dynamic responses, T values (regression analysis) were sequentially estimated by using a series of shifted differential window functions (narrow width). RESULTS: By using the DRA technique, well-defined prompt responses were observed for both motor and visual stimuli. On the other hand, in the pain experiment, a set of sequentially varying responses was observed for the thalamus (Thal), the dorsal anterior cingulate cortex (dACC), the caudal ACC (cACC), and the rostral ACC (rACC). This time-dependent response suggests the dynamics of pain signal processing in cortical areas. CONCLUSION: The results support the hypothesis that the activated areas are similar to the previously reported pain processing areas; however, new sequential responses were observed, suggesting that the technique may reveal dynamics of pain perception and their pathway, important elements in understanding the mechanism of pain. The DRA technique can provide a new opportunity for many spatiotemporal analyses, for example, the physiologically complex and little-studied physiological phenomena, such as pain dynamics.

Production of nuclear transfer-derived piglets using porcine fetal fibroblasts transfected with the enhanced green fluorescent protein.

A system for somatic cell nuclear transfer (SCNT) was developed and led to the successful production of GFP-transfected piglets. In experiment 1, two groups of SCNT couplets reconstructed with porcine fetal fibroblasts (PFF) and enucleated sow (S) or gilt oocytes (G): 1). received a simultaneous electrical fusion/activation (S-EFA or G-EFA groups), or 2). were electrically fused followed by activation with ionomycin (S-EFIA or G-EFIA groups), or 3). were subjected to electrical fusion and subsequent activation by ionomycin, followed by 6-dimethylaminopurine treatment (S-EFIAD or G-EFIAD groups). The frequency of blastocyst formation was significantly higher in S-EFA (26%) compared with that observed in the other experimental groups (P < 0.05), but not with S-EFIA (23%). Sow oocytes yielded significantly higher cleavage frequencies (68%-69%) and total cell numbers of blastocysts when compared with gilt oocytes, regardless of fusion/activation methods (P < 0.05). However, the ratio of inner cell mass (ICM)/total cells in G-EFA and S-EFA was significantly lower than in the other groups (P < 0.05). In experiment 2, SCNT couplets reconstructed with PFF cultured in the presence or absence of serum and enucleated sow oocytes were subjected to EFA. There were no effects of serum starvation on cell-cycle synchronization, developmental competence, total cell numbers, and ratio of ICM/total cells. In experiment 3, SCNT couplets reconstructed with PFF transfected with an enhanced green fluorescence protein (EGFP) gene using FuGENE-6 and enucleated sow oocytes were subjected to EFA and cultured for 7 days. Expression frequencies of GFP gene during development were 100%, 78%, 72%, 71%, and 70% in fused, two-cell, four to eight cells, morulae, and blastocysts, respectively. In experiment 4, SCNT embryos derived from different recipient cytoplasts (sows or gilts) and donor karyoplasts (PFF or GFP-transfected) were subjected to EFA and transferred to the oviducts of surrogates. The pregnancy rates in SCNT embryos derived from sow oocytes (66%-69%) were higher than those with gilt oocytes (23%-27%) regardless of donor cell types. One live offspring from GFP-SCNT embryos and two from PFF-SCNT embryos were delivered. Microsatellite analysis confirmed that the clones were genetically identical to the donor cells and polymerase chain reaction (PCR) from genomic DNA of cloned piglets and subsequent southern blot analysis confirmed the integration of EGFP gene into chromosomes.

Improved development of ICR mouse 2-cell embryos by the addition of amino acids to a serum-, phosphate-and glucose-free medium.

This study was conducted to evaluate how exogenous amino acids could affect preimplantation development of ICR mouse embryos. Two-cell embryos collected from naturally mated mice were cultured in amino acid-, glucose- and phosphate-free preimplantation (P)-1 medium. In Experiments 1, 19 amino acids (aa; 1% and 0.5% of MEM essential and nonessential amino acid solutions, respectively) were added to P-1 medium supplemented with either fatty acid-free bovine serum albumin (BSA; 3 mg/mL) or human follicular fluid (hFF; 10%). Regardless of BSA or hFF addition, embryo development to the morula (84 to 86% vs. 97 to 100%) and the blastocyst (54% vs. 93 to 94%) stages was significantly (P<0.05) enhanced by the addition of aa compared with no addition. In Experiment 2, the cell number of blastomeres and inner cell mass (ICM) cells in blastocysts and the ratio of ICM cell to trophectodermal cell (TE) were evaluated after aa addition. In both BSA- and hFF-containing P-1 medium, a significant increase in total blastomere number were found after aa addition (47 to 52 vs. 62 to 63 cells) compared with no addition. However, the ICM/TE ratio was not significantly affected by aa supplementation in both media, while ICM cell number was greatly increased after aa addition in hFF-containing medium (12 vs. 17 cells). When blastocysts were further cultured up to 162 hr post-hCG injection, development to the hatched blastocyst stage was significantly promoted by aa addition (0% vs. 11 to 20%) in both BSA- and hFF-containing media. In conclusion, aa significantly promote the preimplantation development to the hatched blastocyst stage and such effect mainly exerted on supporting blastomere proliferation.