Single-nucleotide polymorphisms in genes related to oxidative stress and ion channels in chickens are associated with semen quality and hormonal responses to thermal stress.

Heat stress causes economic loss of livestock industry in tropical areas. Exploring genetic markers for selection of thermotolerance will benefit chicken production. Oxidative stress and fluid-salt balance are tightly associated with thermotolerance and productivity in domestic animals. The study aimed to identify single-nucleotide polymorphisms (SNPs) in genes related to oxidative stress and ion-channel regulation and their associations with semen quality and hormonal responses. In total, 28 SNPs within 11 candidate genes were identified to associate with hormonal changes and semen quality in both broiler- and layer-type Taiwan country chickens (TCCs) after exposure to acute heat stress at 38 °C for 4 h. Acute heat stress significantly affected plasma levels of triiodothyronine and corticosterone and reduced sperm motility, viability, and concentrations in both strains at Day 1 after exposure. In the B-strain TCCs, five SNPs within NDUFA8 and DAB2IP had significant effects on plasma adrenaline and corticosterone levels, and six SNPs within TRPC1, SLC9A9, and TRPC7 markedly affected plasma corticosterone and triiodothyronine levels. In the L2 strain, 15 SNPs within PSMA2, TPK1, MTF1, and CUL1 exerted effects on plasma corticosterone and triiodothyronine levels. Five SNPs within CUL3, TRPC1, and SLC9A9 in the B strain and two SNPs within MTF1 in the L2 strain were associated with sperm concentrations at Day 1 after exposure to heat stress. In conclusion, acute heat stress impaired semen quality and altered plasma corticosterone and triiodothyronine levels in TCCs. Some SNPs involved in oxidative stress and ion-channel regulation were identified to associate with the hormonal and semen alterations. These SNPs in conjunction with differential hormonal responses and semen quality serve as genetic markers for thermotolerance selection in sire lines of chickens.

Genomic regions and pathways associated with thermotolerance in layer-type strain Taiwan indigenous chickens.

This study aimed to investigate genetic markers and candidate genes associated with thermotolerance in a layer-type strain Taiwan indigenous chickens exposed to acute heat stress. One hundred and ninety-two 30-week-old roosters were subjected to acute heat stress. Changes in body temperature (BT, ΔT) were calculated by measuring the difference between the initial BT and the highest BT during heat stress and the results were categorized into dead, susceptible, tolerant, and intermediate groups depending on their survival and ΔT values at the end of the experiment. A genome-wide association study on survival and ΔT values was conducted using the Cochran-Armitage trend test and Fisher's exact test. Association analyses identified 80 significant SNPs being annotated to 23 candidate genes, 440 SNPs to 71 candidate genes, 64 SNPs to 25 candidate genes, and 378 SNPs to 78 candidate genes in the dead versus survivor, tolerant versus susceptible, intermediate versus tolerant, and intermediate versus susceptible groups, respectively. The annotated genes were associated with apoptosis, cellular stress responses, DNA repair, and metabolic oxidative stress. In conclusion, the identified SNPs of candidate genes provide insights into the potential mechanisms underlying physiological responses to acute heat stress in chickens.

Monitoring of genetically close Tsaiya duck populations using novel microsatellite markers with high polymorphism.

OBJECTIVE: A set of microsatellite markers with high polymorphism from Tsaiya duck were used for the genetic monitoring and genetic structure analysis of Brown and White Tsaiya duck populations in Taiwan. METHODS: The synthetic short tandem repeated probes were used to isolate new microsatellite markers from the genomic DNA of Tsaiya ducks. Eight populations, a total of 566 samples, sourced from Ilan Branch, Livestock Research Institute were genotyped through novel and known markers. The population genetic variables were calculated using optional programs in order to describe and monitor the genetic variability and the genetic structures of these Tsaiya duck populations. RESULTS: In total 24 primer pairs, including 17 novel microsatellite loci from this study and seven previously known loci, were constructed for the detection of genetic variations in duck populations. The average values for the allele number, the effective number of alleles, the observed heterozygosity, the expected heterozygosity, and the polymorphism information content were 11.29, 5.370, 0.591, 0.746, and 0.708, respectively. The results of analysis of molecular variance and principal component analysis indicated a contracting Brown Tsaiya duck cluster and a spreading White Tsaiya duck cluster. The Brown Tsaiya ducks and the White Tsaiya ducks with Pekin ducks were just split to six clusters and three clusters when K was set equal to 6 and 3 in the Bayesian cluster analysis. The individual phylogenetic tree revealed eight taxa, and each individual was assigned to its own population. CONCLUSION: According to our study, the 24 novel microsatellite markers exhibited a high capacity to analyze relationships of inter- and intra-population in those populations with a relatively limited degree of genetic diversity. We suggest that duck farms in Taiwan could use the new (novel) microsatellite set to monitor the genetic characteristics and structures of their Tsaiya duck populations at various intervals in order to ensure quality breeding and conservation strategies.

Genome-wide association study on the body temperature changes of a broiler-type strain Taiwan country chickens under acute heat stress.

Body temperature is the simplest parameter for evaluating the physiological conditions of animals under thermal stress. Genome-wide association studies (GWAS) have identified candidate genes related to economic traits in domestic animals. The present study conducted a GWAS on body temperature changes in a broiler-type strain Taiwan country chickens (TCCs) under acute heat stress. A total of two hundred 30-week-old roosters of a broiler-type strain TCCs were used. The roosters were subjected to acute heat stress at 38 °C for 4 h, and their body temperature was recorded before and during heat stress. The change in body temperature (ΔT) of each rooster was calculated according to the difference between the initial temperature and the highest body temperature during heat stress. The roosters were categorized according to survival and ΔT at the end into dead, susceptible, resistant, and intermediate groups. Collected red blood cells were genotyped using a 600 K chicken single-nucleotide polymorphism (SNP) array. A GWAS for ΔT was conducted using the Cochran-Armitage trend test. Significant SNPs were annotated as candidate genes according to the nearest genes. Results indicated that the ΔT of the heat-resistant group was significantly lower than that of the heat-susceptible group. A total of 17 SNPs belonging to 8 candidate genes, 352 SNPs for 78 candidate genes, and 174 significant SNPs for 63 candidate genes were identified in the association analyses in the dead vs. survival, resistant vs. susceptible, and intermediate vs. susceptible groups, respectively. The annotated candidate genes are associated with apoptosis, cellular response to external stimuli, and signal transduction pathways. In conclusion, the significant SNPs located in and proximal to genes in the GWAS analysis were related to apoptosis or responses to external stimuli which serve as potential candidates underlying physiological adaptation to heat stress or thermotolerance in chickens.

Survey of genetic structure of geese using novel microsatellite markers.

OBJECTIVE: The aim of this study was to create a set of microsatellite markers with high polymorphism for the genetic monitoring and genetic structure analysis of local goose populations. METHODS: Novel microsatellite markers were isolated from the genomic DNA of white Roman geese using short tandem repeated probes. The DNA segments, including short tandem repeats, were tested for their variability among four populations of geese from the Changhua Animal Propagation Station (CAPS). The selected microsatellite markers could then be used to monitor genetic variability and study the genetic structures of geese from local geese farms. RESULTS: 14 novel microsatellite loci were isolated. In addition to seven known loci, two multiplex sets were constructed for the detection of genetic variations in geese populations. The average of allele number, the effective number of alleles, the observed heterozygosity, the expected heterozygosity, and the polymorphism information content were 11.09, 5.145, 0.499, 0.745, and 0.705, respectively. The results of analysis of molecular variance and principal component analysis indicated a contracting white Roman cluster and a spreading Chinese cluster. In white Roman populations, the CAPS populations were depleted to roughly two clusters when K was set equal to 6 in the Bayesian cluster analysis. The founders of private farm populations had a similar genetic structure. Among the Chinese geese populations, the CAPS populations and private populations represented different clads of the phylogenetic tree and individuals from the private populations had uneven genetic characteristics according to various analyses. CONCLUSION: Based on this study's analyses, we suggest that the CAPS should institute a proper breeding strategy for white Roman geese to avoid further clustering. In addition, for preservation and stable quality, the Chinese geese in the CAPS and the aforementioned proper breeding scheme should be introduced to geese breeders.

Emergence of differentially regulated pathways associated with the development of regional specificity in chicken skin.

BACKGROUND: Regional specificity allows different skin regions to exhibit different characteristics, enabling complementary functions to make effective use of the integumentary surface. Chickens exhibit a high degree of regional specificity in the skin and can serve as a good model for when and how these regional differences begin to emerge. RESULTS: We used developing feather and scale regions in embryonic chickens as a model to gauge the differences in their molecular pathways. We employed cosine similarity analysis to identify the differentially regulated and co-regulated genes. We applied low cell techniques for expression validation and chromatin immunoprecipitation (ChIP)-based enhancer identification to overcome limited cell availabilities from embryonic chicken skin. We identified a specific set of genes demonstrating a high correlation as being differentially expressed during feather and scale development and maturation. Some members of the WNT, TGF-beta/BMP, and Notch family known to be involved in feathering skin differentiation were found to be differentially regulated. Interestingly, we also found genes along calcium channel pathways that are differentially regulated. From the analysis of differentially regulated pathways, we used calcium signaling pathways as an example for further verification. Some voltage-gated calcium channel subunits, particularly CACNA1D, are expressed spatio-temporally in the skin epithelium. These calcium signaling pathway members may be involved in developmental decisions, morphogenesis, or epithelial maturation. We further characterized enhancers associated with histone modifications, including H3K4me1, H3K27ac, and H3K27me3, near calcium channel-related genes and identified signature intensive hotspots that may be correlated with certain voltage-gated calcium channel genes. CONCLUSION: We demonstrated the applicability of cosine similarity analysis for identifying novel regulatory pathways that are differentially regulated during development. Our study concerning the effects of signaling pathways and histone signatures on enhancers suggests that voltage-gated calcium signaling may be involved in early skin development. This work lays the foundation for studying the roles of these gene pathways and their genomic regulation during the establishment of skin regional specificity.

The association of genetic variations in the promoter region of myostatin gene with growth traits in Duroc pigs.

Average daily gain (ADG) and feed efficiency (FE) are important factors for assessing productivity in farm animals. Myostatin (MSTN), previously called GDF8, is a member of transforming growth factor ß (TGFß) superfamily. It is a negative regulator for both embryonic development and adult homeostasis of skeletal muscle. In this study, the genotypes of MSTN g.435G > A and g.447A > G SNPs in Duroc pigs were determined. The 435GG/447AA individually had significantly higher ADG (P < 0.01), body weight at 70 d (P < 0.05) and 150 d (P < 0.01), and a lower age at 110 kg (P < 0.01) than 435AA/447GG individuals. Dose dependent genetic additive effects were found for the negative effects of the 435A/447 G allele for ADG and body weight on 70 d and 150 d. The 435A/447 G allele also increased the age at 110 kg about 1.47 and 4.53% for 1 and 2 copies, respectively. The MSTN 435 G/447A allele increased the age at 110 kg about 1.41 and 4.47% for 1 and 2 copies, respectively. Overall, the two mutated MSTN 435A/447G allele had negative effects on ADG (P < 0.01), body weight at 70 d (P < 0.05), and 150 d (P < 0.001) and increased the age at 110 kg (P < 0.001). The present study provided evidence that MSTN g.435G > A and g.447A > G affected growth in Duroc pigs. The effects of the mutated alleles were additive with the maximal effects resulting from two copies of the wild-type allele. Selection for the 435 G/447A allele is expected to increase ADG, body weight and decrease the age at 110 kg in Duroc pigs and might be used in porcine breeding programs.

Pedigree-based analyses of changes in genetic variability in three major swine breeds in Taiwan after a disease outbreak.

Pedigree analysis was performed in three major Taiwanese swine breeds to evaluate the genetic variability in the current population and determine the main reason for genetic diversity (GD) loss after the occurrence of foot-and-mouth disease (FMD) in Taiwan. The pedigree files of the Duroc, Landrace, and Yorkshire breeds, containing 60,237, 87,177, and 34,373 records, respectively, were analyzed. We divided the population into two subpopulations (pre-1998 and post-1998) to determine the role of FMD in GD loss. Pedigree completeness and related indicators were analyzed to evaluate the pedigree quality, and several parameters were used to measure the levels of GD and further used to determine the major cause of GD loss. The pedigree completeness indexes for the different breeds were higher than 0.60, and the trend was enhanced after the FMD outbreak. The estimated proportion of random genetic drift in GD loss increased in all breeds over time (from 62.64% to 78.44% in Duroc; from 26.26% to 57.99% in Landrace; and from 47.97% to 55.00% in Yorkshire, respectively). The effective population size of Duroc and Landrace were increased by the time (Duroc: from 61.73 to 84.75; Landrace: from 108.70 to 113.64); however, it shows opposite trend in Yorkshire population (decline from 86.21 to 50.00). In summary, the occurrence of FMD led to the major loss of GD loss by random genetic drift. Therefore, for the recovery of GD, breeders in Taiwan should increase the effective population size with newly imported genetic materials and adjust the breeding strategy to reduce the inbreeding rate.

Molecular characterization of hypoxia and hypoxia-inducible factor 1 alpha (HIF-1α) from Taiwan voles (Microtus kikuchii).

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that senses and adapts cells to hypoxic environmental conditions. HIF-1 is composed of an oxygen-regulated α subunit (HIF-1α) and a constitutively expressed ß subunit (HIF-1ß). Taiwan voles (Microtus kikuchii) are an endemic species in Taiwan, found only in mountainous areas greater than 2000m above sea level. In this study, the full-length HIF-1α cDNA was cloned and sequenced from liver tissues of Taiwan voles. We found that HIF-1α of Taiwan voles had high sequence similarity to HIF-1α of other species. Sequence alignment of HIF-1α functional domains indicated basic helix-loop-helix (bHLH), PER-ARNT-SIM (PAS) and C-terminal transactivation (TAD-C) domains were conserved among species, but sequence variations were found between the oxygen-dependent degradation domains (ODDD). To measure Taiwan vole HIF-1α responses to hypoxia, animals were challenged with cobalt chloride, and HIF-1α mRNA and protein expression in brain, lung, heart, liver, kidney, and muscle was assessed by quantitative RT-PCR and Western blot analysis. Upon induction of hypoxic stress with cobalt chloride, an increase in HIF-1α mRNA levels was detected in lung, heart, kidney, and muscle tissue. In contrast, protein expression levels showed greater variation between individual animals. These results suggest that the regulation of HIF-1α may be important to the Taiwan vole under cobalt chloride treatments. But more details regarding the evolutionary effect of environmental pressure on HIF-1α primary sequence, HIF-1α function and regulation in Taiwan voles remain to be identified.

Whole-genome de novo sequencing reveals unique genes that contributed to the adaptive evolution of the Mikado pheasant.

Background: The Mikado pheasant (Syrmaticus mikado) is a nearly endangered species indigenous to high-altitude regions of Taiwan. This pheasant provides an opportunity to investigate evolutionary processes following geographic isolation. Currently, the genetic background and adaptive evolution of the Mikado pheasant remain unclear. Results: We present the draft genome of the Mikado pheasant, which consists of 1.04 Gb of DNA and 15,972 annotated protein-coding genes. The Mikado pheasant displays expansion and positive selection of genes related to features that contribute to its adaptive evolution, such as energy metabolism, oxygen transport, hemoglobin binding, radiation response, immune response, and DNA repair. To investigate the molecular evolution of the major histocompatibility complex (MHC) across several avian species, 39 putative genes spanning 227 kb on a contiguous region were annotated and manually curated. The MHC loci of the pheasant revealed a high level of synteny, several rapidly evolving genes, and inverse regions compared to the same loci in the chicken. The complete mitochondrial genome was also sequenced, assembled, and compared against four long-tailed pheasants. The results from molecular clock analysis suggest that ancestors of the Mikado pheasant migrated from the north to Taiwan about 3.47 million years ago. Conclusions: This study provides a valuable genomic resource for the Mikado pheasant, insights into its adaptation to high altitude, and the evolutionary history of the genus Syrmaticus, which could potentially be useful for future studies that investigate molecular evolution, genomics, ecology, and immunogenetics.

Differentially expressed transcripts in shell glands from low and high egg production strains of chickens using cDNA microarrays.

We have constructed a tissue-specific in-house cDNA microarray to identify differentially expressed transcripts in shell glands from low (B) and high (L2) egg production strains of Taiwanese country chickens during their egg-laying period. The shell gland cDNA library was constructed from the high egg production strain. cDNA clones (7680) were randomly selected and their 5'-end sequences characterized. After excluding overlapping sequences, an in-house cDNA microarray, representing 2743 non-redundant transcripts, was generated for functional genomic studies. Using our microarray, we have successfully identified 85 differentially expressed transcripts from the two different strains of chicken shell glands. In this study, 34 of these transcripts were associated with signal transduction, protein biosynthesis, cell adhesion, cellular metabolism, skeletal development, cell organization and biogenesis. We selected a number of the differentially expressed transcripts for further validation using semi-quantitative RT-PCR. These included elongation factor 2 (EEF2), ovocalyxin-32 (OCX-32) and annexin A2 (ANXA2) which were expressed at high levels in the chicken shell glands of the B strain and, in contrast, the coactosin-like protein (COTL1), transcription factor SOX18 and MX protein were more highly expressed in the L2 strain. Our results suggest that these differentially expressed transcripts may be suitable to use as molecular markers for high rates of egg production, and now need to be investigated further to assess whether they can be applied for use in breeding selection programs in Taiwanese country chickens.

Efficient SNP Discovery by Combining Microarray and Lab-on-a-Chip Data for Animal Breeding and Selection.

The genetic markers associated with economic traits have been widely explored for animal breeding. Among these markers, single-nucleotide polymorphism (SNPs) are gradually becoming a prevalent and effective evaluation tool. Since SNPs only focus on the genetic sequences of interest, it thereby reduces the evaluation time and cost. Compared to traditional approaches, SNP genotyping techniques incorporate informative genetic background, improve the breeding prediction accuracy and acquiesce breeding quality on the farm. This article therefore reviews the typical procedures of animal breeding using SNPs and the current status of related techniques. The associated SNP information and genotyping techniques, including microarray and Lab-on-a-Chip based platforms, along with their potential are highlighted. Examples in pig and poultry with different SNP loci linked to high economic trait values are given. The recommendations for utilizing SNP genotyping in nimal breeding are summarized.

Melting analysis on microbeads in rapid temperature-gradient inside microchannels for single nucleotide polymorphisms detection.

A continuous-flow microchip with a temperature gradient in microchannels was utilized to demonstrate spatial melting analysis on microbeads for clinical Single Nucleotide Polymorphisms (SNPs) genotyping on animal genomic DNA. The chip had embedded heaters and thermometers, which created a rapid and yet stable temperature gradient between 60 °C and 85 °C in a short distance as the detection region. The microbeads, which served as mobile supports carrying the target DNA and fluorescent dye, were transported across the temperature gradient. As the surrounding temperature increased, the fluorescence signals of the microbeads decayed with this relationship being acquired as the melting curve. Fast DNA denaturation, as a result of the improved heat transfer and thermal stability due to scaling, was also confirmed. Further, each individual microbead could potentially bear different sequences and pass through the detection region, one by one, for a series of melting analysis, with multiplex, high-throughput capability being possible. A prototype was tested with target DNA samples in different genotypes (i.e., wild and mutant types) with a SNP location from Landrace sows. The melting temperatures were obtained and compared to the ones using a traditional tube-based approach. The results showed similar levels of SNP discrimination, validating our proposed technique for scanning homozygotes and heterozygotes to distinguish single base changes for disease research, drug development, medical diagnostics, agriculture, and animal production.

Immune gene expression profiles in swine inguinal lymph nodes with different viral loads of porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) infection has been suggested as an acquired immunodeficiency disorder. However, the immunopathogenesis of PCV2 infection is still not fully clarified. In the present study, 35 inguinal lymph nodes (LNs) with different levels of PCV2 load obtained from postwaening multisystemic wasting syndrome (PMWS)-affected pigs and 7 from healthy subclinically PCV2-infected pigs were selected. The LNs were subsequently ranked by their PCV2 loads to mimic the progression of PCV2 infection-associated lesion development. The expressions of 96 selected immune genes in these LNs were assessed by the integration of several reverse transcription quantitative real-time polymerase chain reaction experiments. Hierarchical cluster analysis of the gene expression profiles resulted in 5 major clusters (A, B, C, D, and E). Different clusters of immune gene expression profiles were compatible with the divergent functions of various immune cell subpopulations. 61 out of 96 selected genes belonged to cluster C and were mainly involved in the activation of dendritic cells and B and T lymphocytes. The expression levels of these genes were generally up-regulated in the LNs obtained from PMWS-affected pigs with relatively lower PCV2 loads. However, the up-regulated level tended to reduce or turned into down-regulation as the PCV2 load increased. Genes belonging to cluster B, involved in T cell receptor signaling, became silenced as the PCV2 load increased. The expression profiles of macrophage-associated genes were either independent from or positively correlated with the PCV2 load, such as those in clusters A and E and in cluster D, respectively. In addition, the principle component analysis of the expression of the 96 selected genes in the 42 inguinal LNs revealed that 53.10% and 72.29% of the total data variants could be explained by the top-3 and top-7 principle components, respectively, suggesting that the disease development of PCV2 infection may be associated with a few major and some minor factors. In conclusion, assessment of immune gene expression profiles in LNs supports a close interaction between immune activation and suppression during the progression of PMWS development.

Differential expression of porcine testis proteins during postnatal development.

The development of the testes includes changes in cell morphology and endocrine levels that are essential for the maturation of males. A large number of novel proteins are expressed throughout testis development and play important roles in spermatogenesis. Differences in protein expressions during the development of porcine testes have not been systematically studied. The purpose of this study was to investigate differential protein expression in porcine testes during postnatal development. Testes from four pigs each at 1wk, 3mo, and 1yr of age were used for a proteomic analysis. Expression levels of 264 protein spots were quantified using the Melanie 3 software. In total, 108 protein spots showed more than 2-fold differences (P<0.05) among developmental stages, and 90 of them were successfully identified by mass spectrometry. The proteins were sorted based on whether the expression levels increased with age (36.1%), decreased with age (38.0%), or fluctuated among different developmental stages (25.9%). In total, 69 unique gene products were further classified according to their gene ontology annotations. A majority of the proteins are organelle proteins (41%) with the nucleus and mitochondria being the main organelles. About 45% of the proteins have a protein binding domain and are likely involved in protein-protein interactions. Finally, a large proportion of these differentially expressed proteins are involved in cellular (25%) and metabolic (22%) processes. Identifying these differentially expressed proteins should be valuable for exploring developmental biology and the pathology of male reproduction.

A reference map and identification of porcine testis proteins using 2-DE and MS.

The development of the testis is essential for maturation of male mammals. A complete understanding of proteins expressed in the testis will provide biological information on many reproductive dysfunctions in males. The purposes of this study were to apply a proteomic approach to investigating protein composition and to establish a 2-D PAGE reference map for porcine testis proteins. MALDI-TOF MS was performed for protein identification. When 1 mg of total proteins was assayed by 2-D PAGE and stained with colloidal CBB, more than 400 proteins with a pI of pH 3-10 and M(r) of 10-200 kDa could be detected. Protein expression varied among individuals, with CV between 4.7 and 131.5%. A total of 447 protein spots were excised for identification, among which 337 spots were identified by searching the mass spectra against the NCBInr database. Identification of the remaining 110 spots was unsuccessful. A 2-D PAGE-based porcine testis protein database has been constructed on the basis of the results and will be published on the WWW. This database should be valuable for investigating the developmental biology and pathology of porcine testis.