Exosomes isolation from bovine serum: qualitative and quantitative comparison between ultracentrifugation, combination ultracentrifugation and size exclusion chromatography, and exoEasy methods.

Exosomes have been extensively studied as disease biomarker in humans, given their role in transporting bioactive molecules. However, despite the great potential of exosomes as noninvasive diagnostic markers and therapeutic nanocarriers for bovine diseases, few studies have been conducted on bovine exosome. Thus, this study aimed to quantitatively and qualitatively compare three isolation methods to identify a suitable method for bovine serum. Exosomes were isolated using ultracentrifugation alone (UC), a combination of ultracentrifugation and size exclusion chromatography (US), or membrane affinity-based exoEasy kit (EE). Isolated particles were evaluated using a range of complementary techniques. Transmission electron microscopy showed that all three isolation methods resulted in particles with a cup-shaped morphology. The particle concentration measured by nanoparticle trafficking analyzer of US was lower compared to those of UC and EE method. As a result of immunoblotting, exosome markers including TSG101, CD81, and HSP70 were detected in US particles, while in UC and EE, only TSG101 expression was confirmed. Particles isolated from UC and EE showed a contamination with the blood protein albumin, whereas particles from US did not show albumin contamination. In addition, to evaluate the possibility of using exosomes as biomarkers, the profiles of the small RNA in the exosomes were compared using the bioanalyzer 2100. As a result, in the EE method, the band of small RNA (25-200 nt) was most prominent, and in the US methods, a distinct band was observed in the small RNA range. Collectively, the purity of exosomes without non-exosomal contamination was highest in the US method. However, for the detection of small RNA, the EE method was found to be the most suitable. Therefore, the results suggest that the optimal isolation method varies depending on the specific purpose of exosome isolation.

Lactiplantibacillus argentoratensis AGMB00912 alleviates salmonellosis and modulates gut microbiota in weaned piglets: a pilot study.

This study aimed to evaluate the efficacy of Lactiplantibacillus argentoratensis AGMB00912 (LA) in reducing Salmonella Typhimurium infection in weaned piglets. The investigation focused on the influence of LA on the gut microbiota composition, growth performance, and Salmonella fecal shedding. The results indicated that LA supplementation significantly improved average daily gain and reduced the prevalence and severity of diarrhea. Fecal analysis revealed reduced Salmonella shedding in the LA-supplemented group. Furthermore, LA notably altered the composition of the gut microbiota, increasing the levels of beneficial Bacillus and decreasing those of harmful Proteobacteria and Spirochaetes. Histopathological examination showed less intestinal damage in LA-treated piglets than in the controls. The study also observed that LA affected metabolic functions related to carbohydrate, amino acid, and fatty acid metabolism, thereby enhancing gut health and resilience against infection. Short-chain fatty acid concentrations in the feces were higher in the LA group, suggesting improved gut microbial activity. LA supplementation enriched the population of beneficial bacteria, including Streptococcus, Clostridium, and Bifidobacterium, while reducing the number of harmful bacteria, such as Escherichia and Campylobacter. These findings indicate the potential of LA as a probiotic alternative for swine nutrition, offering protective effects to the gut microbiota against Salmonella infection.

RNA sequencing provides novel insights into the pathogenesis of naturally occurring myxomatous mitral valve disease stage B1 in beagle dogs.

Myxomatous mitral valve disease (MMVD) is the most common cardiovascular disorder in dogs with a high prevalence, accounting for approximately 75% of all canine heart disease cases. MMVD is a complex disease and shows variable progression from mild valve leakage to severe regurgitation, potentially leading to heart failure. However, the molecular mechanisms and age-related changes that govern disease progression, especially at the early stage (B1) before the development of discernable clinical signs, remain poorly understood. In this prospective study, we aimed to compare gene expression differences between blood samples of aged beagle dogs with stage B1 MMVD and those of healthy controls using RNA sequencing. Clinical evaluation was also conducted, which revealed minimal differences in radiographic and echocardiographic measurements despite distinct biomarker variations between the two groups. Comparative transcriptomics revealed differentially expressed genes associated with extracellular matrix remodeling, prostaglandin metabolism, immune modulation, and interferon-related pathways, which bear functional relevance for MMVD. In particular, the top 10 over- and under-expressed genes represent promising candidates for influencing pathogenic changes in MMVD stage B1. Our research findings, which include identified variations in clinical markers and gene expression, enhance our understanding of MMVD. Furthermore, they underscore the need for further research into early diagnosis and treatment strategies, as, to the best of our knowledge, no prior studies have explored the precise molecular mechanisms of stage B1 in MMVD through total RNA sequencing.

Impact of rumen cannulation surgery on rumen microbiota composition in Hanwoo steers.

Rumen cannulation is a surgical technique used to collect rumen contents from ruminants. However, rumen cannulation surgery may potentially impact the composition of the rumen microbiota. This study aimed to examine the longitudinal alterations in the rumen microbiota composition of Hanwoo steers after cannulation surgery. In this study, eight Hanwoo steers were used; four steers underwent rumen cannulation surgery (cannulation group), while the remaining four were left intact (control group). Rumen samples were collected from all eight steers using the stomach tubing method on the day before surgery (day 0) and on postoperative days 1, 4, 7, 10, 14, 17, 21, 24, and 28, resulting in 80 samples (10 timepoints × 8 animals). The microbiota of all 80 samples were analyzed using 16S rRNA gene amplicon sequencing with Quantitative Insights into Microbial Ecology version 2 (QIIME2). There were no significant differences (p > 0.05) in all major phyla and most major genera representing at least 0.5% of total sequences across all 80 samples between the control and cannulation groups on the preoperative and postoperative days. However, while the alpha diversity indices did not differ (p > 0.05) between the two groups on the preoperative day, they significantly differed (p < 0.05) between the two groups on the postoperative days. Further, the overall microbial distribution based on both unweighted and weighted principal coordinate analysis plots significantly differed (p < 0.05) between the two groups on both the preoperative and postoperative days. Orthogonal polynomial contrasts indicated that major genera and microbial diversity in the cannulation group decreased following surgery but returned to their initial states by postoperative day 28. In conclusion, this study demonstrates that rumen cannulation surgery affects some major taxa and microbial diversity, suggesting that the rumen cannulation method can alter the composition of rumen microbiota in Hanwoo steers.

Gut microbiota alteration with growth performance, histopathological lesions, and immune responses in Salmonella Typhimurium-infected weaned piglets.

Salmonella Typhimurium can cause gastroenteritis in weaned piglets, which are particularly vulnerable to dietary changes and dysfunction of their immature organs. The colonization of S. Typhimurium could disrupt the gut microbiota and increase susceptibility to the bacterium. This study aimed to investigate the alterations of gut microbiota in S. Typhimurium-infected weaned piglets. Ten 49-day-old pigs were divided into two groups: S. Typhimurium-inoculated (ST, n = 6) and negative control (NC, n = 4) groups. The body weight and S. Typhimurium fecal shedding were monitored for 14 days after S. Typhimurium inoculation (dpi). The intestinal tissues were collected at 14 dpi; histopathological lesions and cytokine gene expression were evaluated. The gut microbiome composition and short-chain fatty acid concentrations were analyzed in fecal samples collected at 14 dpi. The average daily gain and gut microbiota alpha diversity in ST group tended to be lower than NC group at 14 dpi. Linear discriminant analysis effect size results showed a significant increase in the abundance of two genera and five species, while a significant decrease was observed in the five genera and nine species within the gut microbiota of ST group. Among the significantly less abundant bacteria in the ST group, Lachnospira eligens and Anaerobium acetethylicum produce acetate and butyrate, and may be considered as key S. Typhimurium infection-preventing bacteria. The overall results provide invaluable information about changes in the gut microbiota of S. Typhimurium-infected weaned piglets, which can be used to develop alternative measures to antibiotics and prevent ST bacterial infection.

Genome-wide association study of mammary gland tumors in Maltese dogs.

Background: A genome-wide association study (GWAS) is a valuable tool for investigating genetic and phenotypic variation in many diseases. Objective: The objective of this study was to identify variations in the genomes of Maltese dogs that are associated with the mammary gland tumor (MGT) phenotype and to assess the association between each biological condition and MGT phenotype in Maltese dogs. Methods: DNA was extracted from 22 tumor samples and 11 whole blood samples from dogs with MGTs. Genome-wide single-nucleotide polymorphism (SNP) genotyping was performed, and the top 20 SNPs associated with various conditions and genetic variations were mapped to their corresponding gene locations. Results: The genotyping process successfully identified 173,662 loci, with an overall genotype completion rate of 99.92%. Through the quality control analysis, 46,912 of these SNPs were excluded. Allelic tests were conducted to generate Manhattan plots, which showed several significant SNPs associated with MGT phenotype in intergenic region. The most prominent SNP, located within a region associated with transcription and linked to the malignancy grade of MGT, was identified on chromosome 5 (p = 0.00001) though there may be lack of statistical significance. Other SNPs were also found in several genes associated with oncogenesis, including TNFSF18, WDR3, ASIC5, STAR, and IL1RAP. Conclusion: To our knowledge, this is the first GWAS to analyze the genetic predisposition to MGT in Maltese dogs. Despite the limited number of cases, these analyzed data could provide the basis for further research on the genetic predisposition to MGTs in Maltese dogs.

Raw potato starch diet supplement in weaned pigs could reduce Salmonella Typhimurium infection by altering microbiome composition and improving immune status.

Backgorund: Salmonella enterica serovar Typhimurium (ST) is one of the causative agents of gastroenteritis in pigs. Pigs fed a diet supplemented with raw potato starch (RPS) have improved gut health by the alteration of the microbiota composition and production of short-chain fatty acids (SCFAs). This study aimed to evaluate the effects of RPS supplementation in reducing infection severity and fecal shedding in ST-infected pigs. Methods: The weaned experimental pigs were divided into two groups: CON (n = 6) fed a corn/soybean-based diet and TRT (n = 6) supplemented with 5% RPS. After 21 d, the pigs were inoculated with ST, and their body weight, clinical signs, and fecal shedding of ST were monitored for 14 d. At 14 d post-inoculation (dpi), the jejunum, cecum, ileum, and colon tissues were collected from euthanized pigs, and histopathological lesions and cytokine gene expression were compared. Additionally, blood samples at 2 dpi were analyzed for gene ontology enrichment. Moreover, the gutmicrobiome was analyzed using 16S rRNA metagenomic sequencing, and the SCFA concentration was measured using gas chromatography. Results: The average daily weight gain was significantly higher in TRT than in CON during the ST infection period; however, histopathological lesion scores were significantly lower in TRT than in CON. The relative abundance of nine genera of butyrate- and acetate-producing bacteria significantly increased in TRT compared with that of only two acetate-producing bacteria in CON. Among the genes involved in the immune response, IL-18 expression level was significantly lower in the jejunum and colon in TRT than in CON. Furthermore, Reg3γ expression was significantly different in the cecum and colon of both groups. Conclusion: The diet supplemented with RPS in weaned pigs could result in predominance of butyrate- and acetate-producing bacteria, reducing the severity of ST infection by improving the immune status.

Pathobiological analysis of african swine fever virus contact-exposed pigs and estimation of the basic reproduction number of the virus in Vietnam.

BACKGROUND: African swine fever (ASF), caused by African swine fever virus (ASFV), is a fatal disease affecting wild and domestic pigs. Since China reported the first ASF outbreak in August 2018, ASFV has swept over the neighbouring Asian countries. However, studies involving experimental pig-to-pig ASFV transmission in Vietnam are lacking. The main objective of this experimental study was to demonstrate the pathobiological characteristics of ASFV contact-exposed pigs and estimate their basic reproduction number (R0) in Vietnam. Fifteen pigs were randomly divided into two groups: experimental (n = 10) and negative control (n = 5) groups. One pig in the experimental group was intramuscularly inoculated with ASFV strain from Vietnam in 2020 and housed with the uninoculated pigs during the study period (28 days). RESULTS: The inoculated pig died 6 days post-inoculation, and the final survival rate was 90.0%. We started observing viremia and excretion of ASFV 10 days post-exposure in contact-exposed pigs. Unlike the surviving and negative control pigs, all necropsied pigs showed severe congestive splenomegaly and moderate-to-severe haemorrhagic lesions in the lymph nodes. The surviving pig presented with mild haemorrhagic lesions in the spleen and kidneys. We used Susceptible-Infectious-Removed models for estimating R0. The R0 values for exponential growth (EG) and maximum likelihood (ML) were calculated to be 2.916 and 4.015, respectively. In addition, the transmission rates (ß) were estimated to be 0.729 (95% confidence interval [CI]: 0.379-1.765) for EG and 1.004 (95% CI: 0.283-2.450) for ML. CONCLUSIONS: This study revealed pathobiological and epidemiological information in about pig-to-pig ASFV transmission. Our findings suggested that culling infected herds within a brief period of time may mitigate the spread of ASF outbreaks.

Multi-organ metastases of primary chondrosarcoma of the mammary gland of a spayed Toy Poodle.

We report the clinical and histopathological findings associated with multi-organ metastases of primary mammary chondrosarcoma in a 12-year-old spayed female Toy Poodle. At post-mortem examination, multifocal, sharply demarcated, grey-white to bright brown, round nodules of variable size were randomly distributed in the lungs, myocardium, liver, pancreas, spleen, intestinal tract and kidneys. Histologically, immature cartilage structures and primitive mesenchymal cells were seen in these organs. Neoplastic cells located in the cartilaginous basophilic extracellular matrix had cytoplasmic vacuolation and round vesicular nuclei and were stained with Safranin O and Alcian blue. To the best of our knowledge, this is the first report of a multi-organ metastatic chondrosarcoma that originated in the mammary gland of a dog.

Dihydroquercetin Supplementation Improved Hepatic Lipid Dysmetabolism Mediated by Gut Microbiota in High-Fat Diet (HFD)-Fed Mice.

Dihydroquercetin (DHQ) is a natural flavonoid with multiple bioactivities, including hepatoprotective effects. This study aimed to investigate whether DHQ improved lipid dysmetabolism in the body, especially in the liver, and whether there is a relationship between hepatic metabolism and altered gut flora in high-fat diet (HFD)-induced mice. HFD-induced mice were given 50 mg/kg body weight DHQ intragastrically for 10 weeks. The data showed that DHQ reduced body weight, the weight of the liver and white adipose tissue as well as serum leptin, LPS, triglyceride and cholesterol levels. RNA-seq results indicated that DHQ down-regulated lipogenesis-related genes and up-regulated fatty acid oxidation-related genes, including MOGAT1 and CPT1A. Furthermore, DHQ had a tendency to decrease hepatic cholesterol contents by reducing the mRNA levels of cholesterol synthesis genes such as FDPS and HMGCS1. 16S rRNA sequencing analysis indicated that DHQ significantly decreased the richness of Lactococcus, Lachnoclostridium, and Eubacterium_xylanophilum_group. Correlation analysis further demonstrated that these bacteria, Lactococcus and Eubacterium_xylanophilum_group in particular, had significantly positive correlation with lipid and cholesterol synthesis genes, and negative correlation with fatty acid oxidation genes. In conclusion, DHQ could improve hepatic lipid dysmetabolism potentially by improved gut microbial community, which may be used as an intervention strategy in hepatic metabolism diseases.

Erratum to: Evaluation of adenosine triphosphate testing for on-farm cleanliness monitoring compared to microbiological testing in an empty pig farrowing unit.

[This corrects the article DOI: 10.5187/jast.2020.62.5.682.].

Evaluation of adenosine triphosphate testing for on-farm cleanliness monitoring compared to microbiological testing in an empty pig farrowing unit.

Careful cleaning and disinfection of pigpens is essential to prevent disease spread and avoid the resultant economic losses. Hygiene in pigpens is generally evaluated by visual monitoring supplemented with bacteriological monitoring, which includes counting the total aerobic bacteria (TAB) and/or fecal indicator bacteria (FIB). However, these methods present drawbacks such as time and labor requirements. As adenosine triphosphate (ATP) is ubiquitous in all living organisms including microorganisms, this study aimed to directly compare the results of microbial assessment and ATP quantification, and to suggest possible detailed application methods of the ATP test for hygiene evaluation in pigpens of a farrowing unit. Before and after standard cleaning procedures, samples were collected from the floor corner, floor center, and feeding trough of four pigpens at different time points. No FIB were detected and both the TAB and ATP levels were significantly decreased in the floor center area after cleaning. FIB were continuously detected after cleaning and disinfection of the floor corners, and there was no significant ATP level reduction. The feeding trough did not show any significant difference in these values before and after cleaning, indicating insufficient cleaning of this area. The levels of TAB and ATP after cleaning were significantly correlated and the average ATP value was significantly lower in the absence of FIB than in their presence. In the absence of standard references, a more thorough hygiene management could be achieved evenly by supplementing cleaning or disinfection based on the lowest ATP results obtained at the cleanest test site, which in the present study was the floor center. Overall, these results indicate that the on-farm ATP test can be used to determine the cleanliness status, in addition to visual inspection, as an alternative to laboratory culture-based testing for the presence of microorganisms.

A sampling and estimation method for monitoring poultry red mite (Dermanyssus gallinae) infestation on caged-layer poultry farms.

BACKGROUND: The poultry red mite, Dermanyssus gallinae, is a serious problem in the laying hen industry worldwide. Currently, the foremost control method for D. gallinae is the implementation of integrated pest management, the effective application of which necessitates a precise monitoring method. OBJECTIVES: The aim of the study was to propose an accurate monitoring method with a reliable protocol for caged-layer poultry farms, and to suggest an objective classification for assessing D. gallinae infestation on caged-layer poultry farms according to the number of mites collected using the developed monitoring method. METHODS: We compared the numbers of mites collected from corrugated cardboard traps, regarding with length of sampling periods, sampling sites on cage, and sampling positions in farm buildings. The study also compared the mean numbers of mites collected by the developed method with the infestation levels using by the conventional monitoring methods in 37 caged-layer farm buildings. RESULTS: The statistical validation provided the suitable monitoring method that the traps were installed for 2 days on feed boxes at 27 sampling points which included three vertical levels across nine equally divided zones of farms. Using this monitoring method, the D. gallinae infestation level can be assessed objectively on caged-layer poultry farms. Moreover, the method is more sensitive than the conventional method in detecting very small populations of mites. CONCLUSIONS: This method can be used to identify the initial stages of D. gallinae infestation in the caged-layer poultry farms, and therefore, will contribute to establishment of effective control strategies for this mite.

Prevalence of poultry red mite (Dermanyssus gallinae) in Korean layer farms and the presence of avian pathogens in the mite.

The poultry red mite, Dermanyssus gallinae, is a blood-feeding parasite of layer hens and a potential vector of several avian infectious agents. High infestation with D. gallinae in layer farm buildings could result in economic losses, and the mites may act as a reservoir of avian pathogens within farms. This study aimed to assess the prevalence of D. gallinae in layer farm buildings in Korea and to investigate avian pathogens in the collected mites. The mite samples were collected from 36 Korean layer farm buildings on 21 farms nationwide. Information obtained from each farm building included the flock size, flock age, methods for controlling D. gallinae, and cleaning status. Association between these variables and the population density of D. gallinae was analyzed. Additionally, the presence of 10 avian pathogens was assessed using DNA samples from mites collected in 16 farm buildings. The prevalence of D. gallinae was 75% at the farm building level (90.5% at the farm level). Repetitive cleaning procedures for each building were significantly related with the mite infestation level, and the most influential factor for determining the mite population in the layer farm buildings. In the 16 DNA samples, we detected avian pathogenic Escherichia coli (n = 6), wild-type fowlpox virus (n = 3), wild-type Marek's disease virus (n = 2), chicken anemia virus (n = 1), and fowl adenovirus (n = 1). These findings suggest that repetitive cleaning procedures for the layer farm buildings could decrease the numbers of D. gallinae which may transmit avian pathogens within the farm.

Effects of graded concentrations of supplemental selenium on selenium concentrations in tissues and prediction equations for estimating dietary selenium intake in pigs.

The experiment was conducted to determine the effects of graded dietary selenium (Se) on organ weight and Se concentrations in tissues and to develop equations for estimating dietary Se intake in pigs. Sixteen barrows (initial body weight = 30.0 ± 2.6) were allotted to four dietary treatments including graded Se supplementations with 0, 1, 5, and 50 mg/kg of diet. The experimental diets fed to the pigs for 30 d, and then the pigs were euthanized, and the organs, muscle, and urine samples were collected. The hair and blood samples of pigs were collected on d 15 and 30. Equations were developed for predicting daily Se intake using the Se concentration in plasma, hair, liver, kidneys, muscle, or urine. For graded dietary Se concentrations, linear and quadratic effects on the final body weight, weight and relative weight of liver and kidneys were not observed. The Se concentration in plasma, hair, liver, kidneys, muscle, and urine were linearly and quadratically increased as dietary Se concentration increased (P < 0.001). The dietary Se concentration was positively correlated with the Se concentrations in the plasma, organs, muscle, and urine (r > 0.81, P < 0.001). The equations for estimating dietary Se intake using the Se concentration in the plasma, hair, or organ as an independent variable were significant (P < 0.05). In conclusion, the dietary Se concentration was well reflected in the Se concentration in the plasma, hair, liver, kidneys, and urine. The Se concentration in the plasma, hair, liver, and kidneys can be used as an independent variable for estimating the Se intake.

Non-Coding Transcriptome Maps across Twenty Tissues of the Korean Black Chicken, Yeonsan Ogye.

Yeonsan Ogye is a rare Korean domestic chicken breed whose entire body, including feathers and skin, has a unique black coloring. Although some protein-coding genes related to this unique feature have been examined, non-coding elements have not been widely investigated. Thus, we evaluated coding and non-coding transcriptome expression and identified long non-coding RNAs functionally linked to protein-coding genes in Ogye. High-throughput RNA sequencing and DNA methylation sequencing were performed to profile the expression of 14,264 Ogye protein-coding and 6900 long non-coding RNA (lncRNA) genes and detect DNA methylation in 20 different tissues of an individual Ogye. Approximately 75% of Ogye lncRNAs and 45% of protein-coding genes showed tissue-specific expression. For some genes, tissue-specific expression levels were inversely correlated with DNA methylation levels in their promoters. Approximately 39% of tissue-specific lncRNAs displayed functional associations with proximal or distal protein-coding genes. Heat shock transcription factor 2-associated lncRNAs appeared to be functionally linked to protein-coding genes specifically expressed in black skin tissues, more syntenically conserved in mammals, and differentially expressed in black relative to in white tissues. Pending experimental validation, our findings increase the understanding of how the non-coding genome regulates unique phenotypes and can be used for future genomic breeding of chickens.

Major medical causes by breed and life stage for dogs presented at veterinary clinics in the Republic of Korea: a survey of electronic medical records.

BACKGROUND: Age and breed are considered the greatest risk factors for disease prevalence and mortality in companion dogs. Understanding the prevalence of diseases, in relation to age and breed, would support appropriate guidance for future health care strategies and provide useful information for the early diagnosis of diseases. The purpose of this study was to investigate the major medical causes for dogs visiting primary-care veterinary clinics in the Republic of Korea, stratified by age and breed. METHODS: A total of 15,531 medical records of canine patients were analyzed from 11 veterinary clinics who shared data from January 1, 2016 to December 31, 2016. An electronic medical record (EMR) system was used for data collection, which included the animal identification number, age, breed, gender, neuter status, clinical information, and diagnosis. EMR data were classified using the International Classification of Disease system from the World Health Organization; presenting signs or diagnoses were identified according to breed and life stage. RESULTS: Within the age groups, preventive medicine (16.7% confidence intervals (CI) [15.9-17.5]) was the most common cause for clinic visits for the <1 year and 1-3 year groups. Additionally, neutering surgery (6.6% CI [6.0-7.1]) and patella luxation (1.4% CI [1.8-2.7]) were frequently performed in these age groups. In the 4-6 year group, otitis externa (8.8% CI [7.8-10.0]) and dermatitis or eczema (8.5% CI [7.5-9.6]) were common medical problems. In older dogs (>10 year), the prevalences of heart disease, kidney disease, Cushing's disease, and mammary tumors were higher than in the other age groups. Small and toy breed dogs comprised 67.7% of all dogs in this analysis. For all breeds, otitis externa, dermatitis or eczema, vomiting, and diarrhea were common medical problems. DISCUSSION: This study identified the most common medical disorders and differences in prevalences of diseases, according to age and breeds. The information from EMRs for dogs visiting primary-care veterinary clinics can provide background knowledge that is required to enable a better understanding of disease patterns and occurrence by age and breeds. The information from this study could enable the creation of strategies for preventing diseases and enable the identification of health problems for more effective disease management in companion dogs.

Whole genome and transcriptome maps of the entirely black native Korean chicken breed Yeonsan Ogye.

Background: Yeonsan Ogye (YO), an indigenous Korean chicken breed (Gallus gallus domesticus), has entirely black external features and internal organs. In this study, the draft genome of YO was assembled using a hybrid de novo assembly method that takes advantage of high-depth Illumina short reads (376.6X) and low-depth Pacific Biosciences (PacBio) long reads (9.7X). Findings: The contig and scaffold NG50s of the hybrid de novo assembly were 362.3 Kbp and 16.8 Mbp, respectively. The completeness (97.6%) of the draft genome (Ogye_1.1) was evaluated with single-copy orthologous genes using Benchmarking Universal Single-Copy Orthologs and found to be comparable to the current chicken reference genome (galGal5; 97.4%; contigs were assembled with high-depth PacBio long reads (50X) and scaffolded with short reads) and superior to other avian genomes (92%-93%; assembled with short read-only or hybrid methods). Compared to galGal4 and galGal5, the draft genome included 551 structural variations including the fibromelanosis (FM) locus duplication, related to hyperpigmentation. To comprehensively reconstruct transcriptome maps, RNA sequencing and reduced representation bisulfite sequencing data were analyzed from 20 tissues, including 4 black tissues (skin, shank, comb, and fascia). The maps included 15,766 protein-coding and 6,900 long noncoding RNA genes, many of which were tissue-specifically expressed and displayed tissue-specific DNA methylation patterns in the promoter regions. Conclusions: We expect that the resulting genome sequence and transcriptome maps will be valuable resources for studying domestic chicken breeds, including black-skinned chickens, as well as for understanding genomic differences between breeds and the evolution of hyperpigmented chickens and functional elements related to hyperpigmentation.

Aberrant gene expression patterns in extraembryonic tissue from cloned porcine embryos.

The abnormal development of embryos reconstructed by somatic cell nuclear transfer (SCNT) is considered to be associated with consequent changes in gene expression following errors in epigenetic reprogramming. In this study, we carried out SCNT using donor fibroblast cells derived from 3-way hybrids (Landrace×Duroc×Yorkshire). A total of 655 SCNT embryos were transferred, and 6.97±2.3 cloned fetuses were successfully recovered from three surrogates at gestational day 30. An analysis of the 6.97±2.3 cloned embryos revealed that most had severe extraembryonic defects. The extraembryonic tissue from the SCNT embryos was abnormally small compared with that of the control. To investigate the differentially expressed genes between the SCNT and control extraembryonic tissues, we compared the gene expression profiles of the extraembryonic tissues from gestational day 30 cloned pig embryos with those from the control using an annealing control primer-based GeneFishing polymerase chain reaction. As a result, we found that a total of 50 genes were differentially expressed by utilizing 120 ACPs, 38 genes of which were known. Among them, 26 genes were up-regulated, whereas 12 genes were down-regulated. Real-time RT-PCR showed that apoptosis-related genes were expressed significantly higher in SCNT extraembryonic tissue than in the control, whereas metabolism-related genes were expressed at significantly lower levels in the SCNT extraembryonic tissue. These observations strongly indicate that early gestational death of SCNT embryo is caused, at least in part, by the disruption of developing extraembryonic tissues as a result of aberrant gene expression, which results in abnormal apoptosis and metabolism.

Spent mushroom substrate influences elk (cervus elaphus canadensis) hematological and serum biochemical parameters.

The objective of this study was to evaluate the effect of spent mushroom substrate (SMS) derived from Pleurotus eryngii on the hematological and biochemical blood properties of elk. A total of 18, two and three-year-old elk were fed three different levels of SMS (0, 15 and 20%) in a corn-wheat bran diet for 80 days. The results indicated significantly high levels of blood monocytes, hemoglobin (Hb), and hematocrit (HCT) in elk fed 15% or 20% SMS (p<0.05) compared to control animals. Serum blood urea nitrogen (BUN) and glucose concentrations were also significantly elevated in elk fed both 15% and 20% SMS. The inclusion of SMS in the elk diet did not affect serum total cholesterol, triglyceride, or low density lipoprotein (LDL)-cholesterol concentrations; however, high density lipoprotein (HDL)-cholesterol concentration was significantly increased in SMS-fed groups. In addition, 20% SMS in the diet increased serum iron and testosterone concentrations in elk. These results indicate that adding SMS to the diet of elk can increase their Hgb, serum BUN, glucose, and HDL-cholesterol concentration; therefore, diets containing SMS may enhance the physiologic condition of elk during growth.