The Detection of Bovine Estrus by Lactoferrin Monoclonal Antibody.

To improve reproductive performance in cattle, the accurate detection of estrus and optimization of insemination relative to ovulation are necessary. However, poor heat detection by farm staff leads to a decreased conception rate, thus inflicting economic damage to the beef and dairy industries. This study aimed to develop monoclonal antibodies (mAb) that can specifically bind to the bovine lactoferrin (bLF) protein, which we have previously demonstrated to be overexpressed in bovine cervical mucus during estrus. Female rats were intraperitoneally immunized with bLF protein as the antigen. Anti-bLF mAbs were then purified by affinity chromatography, and their binding affinity for the bLF antigen was examined using ELISA. We found a high binding affinity between mAbs and bLF. Finally, we developed a rapid bovine heat detection kit using the anti-bLF mAbs that we generated and tested on cervical mucus from 12 cows (estrous synchronization, n = 2; natural cycling, n = 10). We found that the kits accurately detected estrus. Overall, our fabricated heat detection kit based on rat anti-bLF mAbs could pave the way for the development of potent tools for heat detection devices for dairy cattle, thereby preventing economic loss.

Author Correction: Comprehensive genome and transcriptome analyses reveal genetic relationship, selection signature, and transcriptome landscape of small-sized Korean native Jeju horse.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genome-Wide Transcriptome and Metabolome Analyses Provide Novel Insights and Suggest a Sex-Specific Response to Heat Stress in Pigs.

Heat stress (HS) negatively impacts pig production and swine health. Therefore, to understand the genetic and metabolic responses of pigs to HS, we used RNA-Seq and high resolution magic angle spinning (HR-MAS) NMR analyses to compare the transcriptomes and metabolomes of Duroc pigs (n = 6, 3 barrows and 3 gilts) exposed to heat stress (33 °C and 60% RH) with a control group (25 °C and 60% RH). HS resulted in the differential expression of 552 (236 up, 316 down) and 879 (540 up, 339 down) genes and significant enrichment of 30 and 31 plasma metabolites in female and male pigs, respectively. Apoptosis, response to heat, Toll-like receptor signaling and oxidative stress were enriched among the up-regulated genes, while negative regulation of the immune response, ATP synthesis and the ribosomal pathway were enriched among down-regulated genes. Twelve and ten metabolic pathways were found to be enriched (among them, four metabolic pathways, including arginine and proline metabolism, and three metabolic pathways, including pantothenate and CoA biosynthesis), overlapping between the transcriptome and metabolome analyses in the female and male group respectively. The limited overlap between pathways enriched with differentially expressed genes and enriched plasma metabolites between the sexes suggests a sex-specific response to HS in pigs.

A Gene-Set Enrichment and Protein-Protein Interaction Network-Based GWAS with Regulatory SNPs Identifies Candidate Genes and Pathways Associated with Carcass Traits in Hanwoo Cattle.

Non-synonymous SNPs and protein coding SNPs within the promoter region of genes (regulatory SNPs) might have a significant effect on carcass traits. Imputed sequence level data of 10,215 Hanwoo bulls, annotated and filtered to include only regulatory SNPs (450,062 SNPs), were used in a genome-wide association study (GWAS) to identify loci associated with backfat thickness (BFT), carcass weight (CWT), eye muscle area (EMA), and marbling score (MS). A total of 15, 176, and 1 SNPs were found to be significantly associated (p < 1.11 × 10-7) with BFT, CWT, and EMA, respectively. The significant loci were BTA4 (CWT), BTA6 (CWT), BTA14 (CWT and EMA), and BTA19 (BFT). BayesR estimated that 1.1%~1.9% of the SNPs contributed to more than 0.01% of the phenotypic variance. So, the GWAS was complemented by a gene-set enrichment (GSEA) and protein-protein interaction network (PPIN) analysis in identifying the pathways affecting carcass traits. At p < 0.005 (~2,261 SNPs), 25 GO and 18 KEGG categories, including calcium signaling, cell proliferation, and folate biosynthesis, were found to be enriched through GSEA. The PPIN analysis showed enrichment for 81 candidate genes involved in various pathways, including the PI3K-AKT, calcium, and FoxO signaling pathways. Our finding provides insight into the effects of regulatory SNPs on carcass traits.

Comprehensive genome and transcriptome analyses reveal genetic relationship, selection signature, and transcriptome landscape of small-sized Korean native Jeju horse.

The Jeju horse, indigenous to the Jeju Island in Korea may have originated from Mongolian horses. Adaptations to the local harsh environment have conferred Jeju horse with unique traits such as small-sized body, stocky head, and shorter limbs. These characteristics have not been studied previously at the genomic level. Therefore, we sequenced and compared the genome of 41 horses belonging to 6 breeds. We identified numerous breed-specific non-synonymous SNPs and loss-of-function mutants. Demographic and admixture analyses showed that, though Jeju horse is genetically the closest to the Mongolian breeds, its genetic ancestry is independent of that of the Mongolian breeds. Genome wide selection signature analysis revealed that genes such as LCORL, MSTN, HMGA2, ZFAT, LASP1, PDK4, and ACTN2, were positively selected in the Jeju horse. RNAseq analysis showed that several of these genes were also differentially expressed in Jeju horse compared to Thoroughbred horse. Comparative muscle fiber analysis showed that, the type I muscle fibre content was substantially higher in Jeju horse compared to Thoroughbred horse. Our results provide insights about the selection of complex phenotypic traits in the small-sized Jeju horse and the novel SNPs identified will aid in designing high-density SNP chip for studying other native horse breeds.

Transcriptome analysis and identification of significantly differentially expressed genes in Holstein calves subjected to severe thermal stress.

RNA-Seq analysis was used to characterize transcriptome response of Holstein calves to thermal stress. A total of eight animals aged between 2 and 3 months were randomly selected and subjected to thermal stress corresponding to a temperature humidity index of 95 in an environmentally controlled house for 12 h consecutively for 3 days. A set of 15,787 unigenes were found to be expressed and after a threshold of threefold change, and a Q value <0.05; 502, 394, and 376 genes were found to be differentially expressed on days 1, 2, and 3 out of which 343, 261 and 256 genes were upregulated and 159, 133, and 120 genes were downregulated. Only 356 genes out of these were expressed on all 3 days, and only they were considered as significantly differentially expressed. KEGG pathway analysis revealed that ten pathways were significantly enriched; the top two among them were protein processing in endoplasmic reticulum and MAPK signaling pathways. These results suggest that thermal stress triggered a complex response in Holstein calves and the animals adjusted their physiological and metabolic processes to survive. Many of the genes identified in this study have not been previously reported to be involved in thermal stress response. The results of this study extend our understanding of the animal's response to thermal stress and some of the identified genes may prove useful in the efforts to breed Holstein cattle with superior thermotolerance, which might help in minimizing production loss due to thermal stress.

Genomic structure, expression and association study of the porcine FSD2.

The fibronectin type III and SPRY domain containing 2 (FSD2) on porcine chromosome 7 is considered a candidate gene for pork quality, since its two domains, which were present in fibronectin and ryanodine receptor. The fibronectin type III and SPRY domains were first identified in fibronectin and ryanodine receptor, respectively, which are candidate genes for meat quality. The aim of this study was to elucidate the genomic structure of FSD2 and functions of single nucleotide polymorphisms (SNPs) within FSD2 that are related to meat quality in pigs. Using a bacterial artificial chromosome clone sequence, we revealed that porcine FSD2 consisted of 13 exons encoding 750 amino acids. In addition, FSD2 was expressed in heart, longissimus dorsi muscle, psoas muscle, and tendon among 23 kinds of porcine tissues tested. A total of ten SNPs, including four missense mutations, were identified in the exonic region of FSD2, and two major haplotypes were obtained based on the SNP genotypes of 633 Berkshire pigs. Both haplotypes were associated significantly with intramuscular fat content (IMF, P < 0.020) and moisture percentage (MP, P < 0.002). Moreover, haplotype 2 was associated with meat color, affecting yellowness (P = 0.002). These haplotype effects were further supported by the alteration of putative protein structures with amino acid substitutions. Taken together, our results suggest that FSD2 haplotypes are involved in regulating meat quality including IMF, MP, and meat color in pigs, and may be used as meaningful molecular makers to identify pigs with preferable pork quality.

Whole-genome resequencing of Hanwoo (Korean cattle) and insight into regions of homozygosity.

BACKGROUND: Hanwoo (Korean cattle), which originated from natural crossbreeding between taurine and zebu cattle, migrated to the Korean peninsula through North China. Hanwoo were raised as draft animals until the 1970s without the introduction of foreign germplasm. Since 1979, Hanwoo has been bred as beef cattle. Genetic variation was analyzed by whole-genome deep resequencing of a Hanwoo bull. The Hanwoo genome was compared to that of two other breeds, Black Angus and Holstein, and genes within regions of homozygosity were investigated to elucidate the genetic and genomic characteristics of Hanwoo. RESULTS: The Hanwoo bull genome was sequenced to 45.6-fold coverage using the ABI SOLiD system. In total, 4.7 million single-nucleotide polymorphisms and 0.4 million small indels were identified by comparison with the Btau4.0 reference assembly. Of the total number of SNPs and indels, 58% and 87%, respectively, were novel. The overall genotype concordance between the SNPs and BovineSNP50 BeadChip data was 96.4%. Of 1.6 million genetic differences in Hanwoo, approximately 25,000 non-synonymous SNPs, splice-site variants, and coding indels (NS/SS/Is) were detected in 8,360 genes. Among 1,045 genes containing reliable specific NS/SS/Is in Hanwoo, 109 genes contained more than one novel damaging NS/SS/I. Of the genes containing NS/SS/Is, 610 genes were assigned as trait-associated genes. Moreover, 16, 78, and 51 regions of homozygosity (ROHs) were detected in Hanwoo, Black Angus, and Holstein, respectively. 'Regulation of actin filament length' was revealed as a significant gene ontology term and 25 trait-associated genes for meat quality and disease resistance were found in 753 genes that resided in the ROHs of Hanwoo. In Hanwoo, 43 genes were located in common ROHs between whole-genome resequencing and SNP chips in BTA2, 10, and 13 coincided with quantitative trait loci for meat fat traits. In addition, the common ROHs in BTA2 and 16 were in agreement between Hanwoo and Black Angus. CONCLUSIONS: We identified 4.7 million SNPs and 0.4 million small indels by whole-genome resequencing of a Hanwoo bull. Approximately 25,000 non-synonymous SNPs, splice-site variants, and coding indels (NS/SS/Is) were detected in 8,360 genes. Additionally, we found 25 trait-associated genes for meat quality and disease resistance among 753 genes that resided in the ROHs of Hanwoo. These findings will provide useful genomic information for identifying genes or casual mutations associated with economically important traits in cattle.

Neuronal genes for subcutaneous fat thickness in human and pig are identified by local genomic sequencing and combined SNP association study.

Obesity represents a major global public health problem that increases the risk for cardiovascular or metabolic disease. The pigs represent an exceptional biomedical model related to energy metabolism and obesity in humans. To pinpoint causal genetic factors for a common form of obesity, we conducted local genomic de novo sequencing, 18.2 Mb, of a porcine QTL region affecting fatness traits, and carried out SNP association studies for backfat thickness and intramuscular fat content in pigs. In order to relate the association studies in pigs to human obesity, we performed a targeted genome wide association study for subcutaneous fat thickness in a cohort population of 8,842 Korean individuals. These combined association studies in human and pig revealed a significant SNP located in a gene family with sequence similarity 73, member A (FAM73A) associated with subscapular skin-fold thickness in humans (rs4121165, GC-corrected p-value  = 0.0000175) and with backfat thickness in pigs (ASGA0029495, p-value  = 0.000031). Our combined association studies also suggest that eight neuronal genes are responsible for subcutaneous fat thickness: NEGR1, SLC44A5, PDE4B, LPHN2, ELTD1, ST6GALNAC3, ST6GALNAC5, and TTLL7. These results provide strong support for a major involvement of the CNS in the genetic predisposition to a common form of obesity.