Effects of Cyclic High Ambient Temperature on Muscle Imidazole Dipeptide Content in Broiler Chickens.

Imidazole dipeptides possess important bioregulatory properties in animals. This study aimed to evaluate the effect of high ambient temperature on muscle imidazole dipeptides (carnosine, anserine, and balenine) in broiler chickens. Sixteen 14-day-old male broiler chickens were divided into two groups, which were reared under thermoneutral (25 ± 1 °C) or cyclic high ambient temperature (35 ± 1 °C for 8 h/day) for 4 weeks. Chickens exposed to cyclic high ambient temperatures displayed lower skeletal muscle anserine and carnosine content than control chickens. Balenine could not be detected in the pectoral muscle of either group. The pectoral muscles of broiler chickens kept under cyclic high-temperature exhibited significantly lower mRNA expression of carnosine synthase 1, which synthesizes carnosine and anserine; but a significantly higher mRNA expression of carnosinase 2, which degrades carnosine and anserine. Our results suggest that heat exposure decreases pectoral imidazole dipeptide content in broiler chickens. This may be attributed to a lower expression of imidazole dipeptide-synthesizing genes, but higher levels of genes involved in their degradation.

Effects of Delaying Post-hatch Feeding on the Plasma Metabolites of Broiler Chickens Revealed by Targeted and Untargeted Metabolomics.

Exogenous nutrients are essential for body and skeletal muscle growth in newly hatched chicks, and delaying post-hatch feeding negatively affects body growth, meat yield, and meat quality. The aim of this study was to investigate the effects of delayed post-hatch feeding on the metabolic profiles of broiler chickens using a combination of targeted and untargeted metabolomics. Newly hatched chicks had either immediate free access to feed (freely fed chicks) or no access to feed from 0 to 2 days of age (delayed-fed chicks); both groups were subsequently provided feed ad libitum until 13 days of age. Untargeted metabolomic analysis was performed using gas chromatography-mass spectrometry, whereas targeted metabolomic analysis of amino acids was performed using high-performance liquid chromatography with ortho-phthalaldehyde derivatization. Delayed feeding increased the plasma levels of sucrose, maltose, serotonin, lactitol, gentiobiose, xylitol, threonic acid, and asparagine, and decreased the plasma levels of creatinine, aspartic acid, and glutamic acid. In addition, the digestibility of the nitrogen-free extract (starch and sugar) and the cecal butyric acid concentration increased in chicks subjected to delayed feeding. In contrast, delayed feeding did not affect muscle protein degradation or digestibility in chicks. Taken together, our results indicate that delaying feeding until 48 h post-hatch alters multiple metabolic pathways, which are accompanied by changes in intestinal carbohydrate digestion and cecal butyric acid content in broiler chickens.

Population structure of Vietnamese pigs using mitochondrial DNA.

The D-loop region on mitochondrial DNA (mtDNA) is frequently used for analyses of maternal lineages within domestic animal species. There are many native pig breeds in Vietnam, but their origins remain unclear. This study investigated maternal lineages using the D-loop region on mtDNA of 260 samples collected from native pigs in 20 provinces across Vietnam. The D-loop region of all samples was amplified and sequenced. We obtained 713 bp sequences of the D-loop region for each sample excluding the repeat region, and variants on this region were used to construct a phylogenetic tree. We detected 50 haplotypes from Vietnamese native pigs, with 27 novel haplotypes. Phylogenetic tree analysis showed two haplotype groups: one for the MTSEA group, frequently found in domestic pigs in the mountainous areas of Cambodia and Laos; and the D2 group, found in pigs originating from Chinese pigs. No European haplotype was found. Haplotypes in northeast Vietnam comprised only haplotypes of the D2 group, whereas in areas from the northwest mountains to the south, we found haplotypes belonging to both the D2 and MTSEA groups. This study suggested that both origins contributed to maternal lineages of current populations of Vietnamese native pigs.

Quantification of Nτ -Methylhistidine and Nπ-Methylhistidine in Chicken Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

The concentration of Nτ-methylhistidine in plasma provides an index of skeletal muscle protein breakdown. This study aimed to establish a quantitative method for measuring the concentrations of Nτ-methylhistidine and its isomer Nπ-methylhistidine in chicken plasma, using liquid chromatography-tandem mass spectrometry with stable isotope dilution analysis. The acceptable linear ranges of detection were 1.56-50.00 µmol/L for Nτ-methylhistidine and 0.78-25.00 µmol/L for Nπ-methylhistidine. The proposed method detected changes in the plasma levels of Nτ-methylhistidine and Nπ-methylhistidine in response to fasting and re-feeding. These results suggest that the method developed in this study can be used for the simultaneous measurement of Nτ-methylhistidine and Nπ-methylhistidine in chicken plasma.

Detection of long terminal repeat loci derived from endogenous retrovirus in junglefowl using whole-genome sequencing.

Endogenous retroviruses (ERVs) are genetic elements present in the genome that retain traces of past viral infections. Characterization of ERVs can provide crucial insights into avian evolution. This study aimed to identify novel long terminal repeat (LTR) loci derived from ERVs (ERV-LTRs) absent in the reference genome using whole-genome sequencing data of red junglefowl, gray junglefowl, Ceylon junglefowl, and green junglefowl. In total, 835 ERV-LTR loci were identified across the four Gallus species. The numbers of ERV-LTRs loci detected in red junglefowl and its subspecies gray junglefowl, Ceylon junglefowl, and green junglefowl were 362, 216, 193, and 128, respectively. The phylogenetic tree was congruent with previously reported trees, suggesting the potential for inferring relationships among past junglefowl populations from the identified ERV-LTR loci. Of the detected loci, 306 ERV-LTRs were identified near or within the genes, and some were associated with cell adhesion. The detected ERV-LTR sequences were classified as endogenous avian retrovirus family, avian leukosis virus subgroup E, Ovex-1, and murine leukemia virus-related ERVs. In addition, the sequence of the EAV family was divided into four patterns by combining the U3, R, and U5 regions. These findings contribute to a more comprehensive understanding of the characteristics of junglefowl ERVs.

Detection of non-reference porcine endogenous retrovirus loci in the Vietnamese native pig genome.

The Vietnamese native pig (VnP)-a porcine breed with a small body-has proven suitable as a biomedical animal model. Here, we demonstrate that, compared to other breeds, VnPs have fewer copies of porcine endogenous retroviruses (PERVs), which pose a risk for xenotransplantation of pig organs to humans. More specifically, we sought to characterize non-reference PERVs (nrPERVs) that were previously unidentified in the reference genome. To this end, we used whole-genome sequencing data to identify nrPERV loci with long terminal repeat (LTR) sequences in VnPs. RetroSeq was used to estimate nrPERV loci based on the most current porcine reference genome (Sscrofa11.1). LTRs were detected using de novo sequencing read assembly near the loci containing the target site duplication sequences in the inferred regions. A total of 21 non-reference LTR loci were identified and separated into two subtypes based on phylogenetic analysis. Moreover, PERVs within the detected LTR loci were identified, the presence of which was confirmed using conventional PCR and Sanger sequencing. These novel loci represent previously unknown PERVs as they have not been identified in the porcine reference genome. Thus, our RetroSeq method accurately detects novel PERV loci, and can be applied for development of a useful biomedical model.

The phenotypic characteristics and relational database for Vietnamese native pig populations.

Although there are a number of Vietnamese native pig (VnP) populations, some are on the verge of extinction, and therefore adequate management and conservation are necessary. In this study, we conducted a field survey of VnP populations and analyzed interrelationships among their characteristics. We also established a relational database for management of field data on these populations. For data collection, we conducted interviews with farmers and visual inspection of 32 VnP populations in 22 provinces of Vietnam, as well as taking photographs of individual animals. Data on the characteristics of VnP populations were subjected to multiple correspondence analysis (MCA). For establishment of the database, normalization and table partitioning were performed to eliminate redundancy and ensure consistency of the collected data items. Passport data, characteristics data, and image data were collected from a total of 1,918 VnPs and entered as a normalized table. Upon MCA, most of the populations were not separated from each other, but the Mong Cai, O Lam, and Chu Prong populations were separated from the other populations. Thus, we have constructed a relational database from comprehensive information on the characteristics of VnP populations.

Characteristic features of porcine endogenous retroviruses in Vietnamese native pigs.

We aimed to clarify the genomic characteristics of porcine endogenous retroviruses (PERVs) in Vietnamese native pig (VnP) breeds. First, we investigated genetic polymorphisms in ß- and γ-like PERVs, and we then measured the copy numbers of infectious γ-like PERVs (PERV-A, B, and C). We purified genomic DNA from 15 VnP breeds from 12 regions all over the country and three Western pig breeds as controls, and investigated genetic polymorphisms in all known PERVs, including the beta (ß)1-4 and gamma (γ)1-5 groups. PERVs of ß1, ß2, ß3, and γ4 were highly polymorphic with VnP-specific haplotypes. We did not identify genetic polymorphisms in ß4, γ1, or γ2 PERVs. We then applied a real-time polymerase chain reaction-based method to estimate copy numbers of the gag, pol, and env genes of γ1 PERVs (defined as A, B, and C). VnP breeds showed significantly lower copy number of the PERV genes compared with the Western pig breeds (on average, 16.2 and 35.7 copies, respectively, p < .05). Two VnP breeds showed significantly higher copy number compared with the other VnPs (p < .05). Our results elucidated that VnPs have specific haplotypes and a low copy number of PERV genes.

Evaluation of genetic richness among Vietnamese native pig breeds using microsatellite markers.

We have elucidated genetic relationships of Vietnamese native pigs (VNP) using preliminarily collected samples by a single-nucleotide polymorphism (SNP) array. In order to confirm our previous results and compare with the results of a previous study using microsatellite (MS) markers, we aimed to characterize genetic diversity and population structure in wider varieties (24 breeds from 21 Provinces) of VNP across the country using 20 polymorphic MS markers recommended by ISAG/FAO (International Society for Animal Genetics/Food and Agriculture Organization) for diversity study. In this study, we collected 1,136 DNA samples of the VNPs and three exotic breeds. Our results revealed that the average number of alleles and allelic richness across the loci in VNPs were 10.0 and 7.6, which were higher than those of exotic breeds. Genomic components among VNPs were subjected to the sampling locations. Interestingly, Co Binh Thuan showed remarkable genetic feature compared to the other VNPs, because the habitation of Co Binh Thuan was relatively far from the other breeds. The results of this study provided useful information for exploitation, conservation, and development trends of the VNP breeds. More recently, African swine fever caused significant damage to most of the VNP populations. Therefore, our findings will help a reconstruction scheme of the VNP genetic resources.

Improvement of the developmental competence of porcine oocytes collected from early antral follicles by cytoplast fusion.

In the present study, we propose an alternative technique called cytoplast fusion to improve the maturation rate and developmental competence of growing oocytes collected from early antral follicles in pigs. We examined whether the fusion of a growing oocyte with the cytoplast from a fully-grown oocyte (CFR group) could better promote maturation and developmental competence of the growing oocyte compared to germinal vesicle (GV) transfer (GVTR group). After 44 h of in vitro maturation (IVM), most growing oocytes (GR group) were still arrested at the GV stage (64.0 ± 5.1%); this number was significantly higher (P < 0.01) than that of the other groups. No matured oocyte was observed in the GR group. The maturation rate of GVTR oocytes was significantly improved (18.8 ± 3.5%) compared with that of growing oocytes. The proportion of oocytes that reached the metaphase-II (M-II) stage in the CFR group (37.8 ± 2.0%) was significantly higher (P < 0.05) than that in the GVTR group, although still lower than that in the control group (75.2 ± 4.4%). No blastocyst was derived from growing oocytes. Among in vitro fertilized GVTR oocytes, 3.0 ± 1.9% developed into blastocysts; however, this percentage showed an insignificant increase compared with the GR group. On the other hand, the percentage of CFR embryos that developed into blastocysts (12.0 ± 4.3%) was significantly higher than that of GR embryos (0.0%), although still lower than that of control embryos (27.0 ± 5.5%). Total cell number in blastocysts in the GVTR group (23.3 ± 6.9) was significantly lower (P < 0.05) than that in the control group (50.4 ± 5.0). Meanwhile, the total cell number in blastocysts derived from CFR oocytes (36.3 ± 4.8) was comparable to that of the control group. In summary, cytoplast fusion significantly improves maturation rate and developmental competence of growing oocytes compared with GV transfer.

Identification of tamaraw (Bubalus mindorensis) from natural habitat-derived fecal samples by PCR-RFLP analysis of cytochrome b gene.

Fecal DNA analysis is a useful tool for the investigation of endangered species. Tamaraw (Bubalus mindorensis) is endemic to the Philippine island of Mindoro but knowledge of its genetic and ecological information is limited. In this study, we developed a species identification method for tamaraw by fecal DNA analysis. Eighteen feces presumed to be from tamaraw were collected in Mount Iglit-Baco National Park and species-known feces from domestic buffaloes and cattle were obtained from a farm. Additionally, one species-unknown fecal sample was obtained in Mount Aruyan Preserve, where the sighting of tamaraw has not been reported in recent years. Based on DNA sequence data previously reported, the genus Bubalus- and tamaraw-specific primers for PCR of cytochrome b gene were newly designed. The Bubalus-specific primer yielded a 976 bp fragment of cytochrome b for all fecal samples from tamaraw and domestic buffaloes, but not for cattle, whereas the tamaraw-specific primer yielded a 582 bp fragment for all tamaraw fecal samples and for one of the four domestic buffalo samples. PCR-RFLP (restriction fragment length polymorphism) analysis of the 976 bp PCR fragment with AvrII or BsaXI provided distinct differences between tamaraw and domestic buffalo. PCR-RFLP analysis also showed that the species-unknown sample obtained in Mount Aruyan Preserve, originates from tamaraw.

[Time-dependence evaluation of the primary visual cortex by 1H MR-spectroscopy using a stimulation control system].

UNLABELLED: Proton magnetic resonance spectroscopy ((1)H MRS) is used to evaluate tumors and their remaining function under the assumption that event-related inspection and a method of data collection dependent on time are difficult. Therefore, this study examined the possibility that time depended on in MRS with a visual cortex. We conducted experiments using the following stimulations: REST: eyes are in a closed state, CONTROL: eyes are in an open state, and Continuous: consecutive stimulation and time dependence in the visual cortex. We enabled dependence in time by collecting data with a system that controlled stimulation and experimented on stimulation for a short time using this system. The results showed that no significant difference in metabolites was seen in the REST, CONTROL, and Consecutive stimulations. However, a significant difference was seen in Cr and NAA with time-dependent stimulation. Therefore, we considered functional magnetic resonance spectroscopy (fMRS) useful because time-dependent stimulation showed a meaningful difference with REST.

[Response by visual stimulation with the BOLD effect and motion artifact: identification of the activating signal and motion artifact].

This study used functional magnetic resonance imaging (fMRI) to evaluate brain function. fMRI data were collected by a block paradigm, and brain function was evaluated. However, irrelevant artifacts caused by the BOLD effect frequently occur in analytical processing. Therefore, there is a limit to obtaining an adequate activating reaction in the analytical system, which is normally equipped with MRI. However, there is a limit to obtaining a sufficient activating reaction in the analytical system, which is normally equipped with MRI. It has become standard practice to use an analytical system such as statistical parametric mapping (SPM). The response of movement of cerebrospinal fluid (CSF) artifact is high. Therefore, disappearance of the CSF artifact was done by statistical analysis. Then, the response of the signal from the beginning of stimulation was examined. As a result, the activating signal and the artifact signal could be identified.

Influence of moving sight by turning over frequency.

In late years, f-MRI (functional magnetic resonance imaging) are used for analysis of a visual function. In case of the study for a visual function, the checker flag pattern consists of white square and black square is usually used for sight stimulation. In sight stimulation using this pattern, a color of square (black and white) changes alternately at constant frequency. It is usually called pattern reversal stimulation. When this pattern is used for stimulation, the subject sometimes feels movement for the shown stimulation pattern. We think that this sensation of movement relates to spatial frequency (that means the size of a square of a stimulation pattern) of a stimulation image and a turning over frequency of a stimulation pattern strongly. Our objectives of this study are the following. 1) Clarify a part of the brain that is a cause of this moving sensation. 2) Investigate the relationship among the magnitude of this sensation, spatial frequency and turning over frequency. Three normal adults were used for the subject. This time, turning over frequency was changed to 8 Hz by 1 Hz step under fixed spatial frequency. Under this condition, we examined the state of activation of a V1 area. In addition, we examined whether BOLD effect varied with a change of a stimulation color. In this experiment, we used blue and yellow color instead of black and white for stimulation, and the reaction was examined.

[Indoor air pollution due to 2-ethyl-1-hexanol airborne concentrations, emission sources and subjective symptoms in classroom users].

OBJECTIVE: 2-Ethyl-1-hexanol (2E1H) is a volatile organic compound (VOC) which seldom attracts attention in Japan. This study aimed at clarifying changes in its concentration over time, emission sources, and students' symptoms in classrooms of a university building where indoor air was found to be markedly polluted with 2E1H. METHODS: From March 2001 through September 2002, we measured VOC concentrations in Building A, constructed in 1998, as well as Building B (Sept. 2002), constructed over 30 years ago and considered as a control. Airborne concentrations of 13 carbonyl compounds were quantified with diffusive samplers and high-performance liquid chromatography, and those of 41 other VOCs with an active sampling method using charcoal tubes and a gas chromatograph with a mass spectrometer (GC-MS). In August 2002, we also measured VOC emissions from the floors using double-cylinder chambers and the airborne concentrations of phthalate esters by filtration sampling, both by GC-MS. Subjective symptoms in 315 student classroom users in Building A and 275 in Building B were surveyed in July 2002 with anonymous self-administered questionnaires. RESULTS: 2E1H concentrations in Building A, which exceeded the Japanese recommended threshold of total VOCs (400 microg/m3) in some measurements, tended to be lower in winter and higher in summer, and did not show any tendency for decrease over time. No association was found between indoor concentrations of phthalate esters and those of 2E1H. The concentrations clearly differed between rooms, related to emission rates from the floors. Carpeting materials had been placed directly on the concrete floors in rooms with higher emission levels, whereas the carpeting materials and the concrete floor did not make contact in the room where emission was lower. The odds ratio for subjective symptoms with students in classrooms in Building A was not higher than in Building B where the 2E1H concentrations were low. However, a few students limited to Building A did complain of problems with the nasal passages, throat and lower airways. CONCLUSION: Compounds containing 2-ethyl-1-hexyl moiety are presumably hydrolyzed to emit 2E1H when the backing of carpeting material is in contact with concrete floors. Although no significant difference was observed in symptoms between the student groups in the two buildings, this was possibly due to the small sample size. Measures to prevent 2E1H emission and dose-response relationships in sensitive individuals should be studied further.