Cross-Species Comparative DNA Methylation Reveals Novel Insights into Complex Trait Genetics among Cattle, Sheep, and Goats.

The cross-species characterization of evolutionary changes in the functional genome can facilitate the translation of genetic findings across species and the interpretation of the evolutionary basis underlying complex phenotypes. Yet, this has not been fully explored between cattle, sheep, goats, and other mammals. Here, we systematically characterized the evolutionary dynamics of DNA methylation and gene expression in 3 somatic tissues (i.e. brain, liver, and skeletal muscle) and sperm across 7 mammalian species, including 3 ruminant livestock species (cattle, sheep, and goats), humans, pigs, mice, and dogs, by generating and integrating 160 DNA methylation and transcriptomic data sets. We demonstrate dynamic changes of DNA hypomethylated regions and hypermethylated regions in tissue-type manner across cattle, sheep, and goats. Specifically, based on the phylo-epigenetic model of DNA methylome, we identified a total of 25,074 hypomethylated region extension events specific to cattle, which participated in rewiring tissue-specific regulatory network. Furthermore, by integrating genome-wide association studies of 50 cattle traits, we provided novel insights into the genetic and evolutionary basis of complex phenotypes in cattle. Overall, our study provides a valuable resource for exploring the evolutionary dynamics of the functional genome and highlights the importance of cross-species characterization of multiomics data sets for the evolutionary interpretation of complex phenotypes in cattle livestock.

Multiplex fluorescent amplification-refractory mutation system PCR method for the detection of 10 genetic defects in Holstein cattle and its comparison with the KASP genotyping assay.

The common deleterious genetic defects in Holstein cattle include haplotypes 1-6 (HH1-HH6), haplotypes for cholesterol deficiency (HCD), bovine leukocyte adhesion deficiency (BLAD), complex vertebral malformation (CVM) and brachyspina syndrome (BS). Recessive inheritance patterns of these genetic defects permit the carriers to function normally, but homozygous recessive genotypes cause embryo loss or neonatal death. Therefore, rapid detection of the carriers is essential to manage these genetic defects. This study was conducted to develop a single-tube multiplex fluorescent amplification-refractory mutation system (mf-ARMS) PCR method for efficient genotyping of these 10 genetic defects and to compare its efficiency with the kompetitive allele specific PCR (KASP) genotyping assay. The mf-ARMS PCR method introduced 10 sets of tri-primers optimized with additional mismatches in the 3' end of wild and mutant-specific primers, size differentiation between wild and mutant-specific primers, fluorescent labeling of universal primers, adjustment of annealing temperatures and optimization of primer concentrations. The genotyping of 484 Holstein cows resulted in 16.12% carriers with at least one genetic defect, while no homozygous recessive genotype was detected. This study found carrier frequencies ranging from 0.0% (HH6) to 3.72% (HH3) for individual defects. The mf-ARMS PCR method demonstrated improved detection, time and cost efficiency compared with the KASP method for these defects. Therefore, the application of mf-ARMS PCR for genotyping Holstein cattle is anticipated to decrease the frequency of lethal alleles and limit the transmission of these genetic defects.

Investigating the relationship between fluctuations in daily milk yield as resilience indicators and health traits in Holstein cattle.

Disease-related milk losses directly affect dairy herds' profitability and the production efficiency of the dairy industry. Therefore, this study aimed to quantify phenotypic variability in milk fluctuation periods related to diseases and to explore milk fluctuation traits as indicators of disease resilience. By combining high-frequency daily milk yield data with disease records of cows that were treated and recovered from the disease, we estimated milk variability trends within a fixed period around the treatment day of each record for 5 diseases: udder health, reproductive disorders, metabolic disorders, digestive disorders, and hoof health. The average milk yield decreased rapidly from 6 to 8 d before the treatment day for all diseases, with the largest milk reduction observed on the treatment day. Additionally, we assessed the significance of milk fluctuation periods highly related to diseases by defining milk fluctuations as a period of at least 10 consecutive days in which milk yield fell below 90% of the expected milk production values at least once. We defined the development and recovery phases of milk fluctuations using 3,847 milk fluctuation periods related to disease incidences, and estimated genetic parameters of milk fluctuation traits, including milk losses, duration of the fluctuation, variation rate in daily milk yield, and standard deviation of milk deviations for each phase and their genetic correlation with several important traits. In general, the disease-related milk fluctuation periods lasted 21.19 ± 10.36 d with a milk loss of 115.54 ± 92.49 kg per lactation. Compared with the development phase, the recovery phase lasted an average of 3.3 d longer, in which cows produced 11.04 kg less milk and exhibited a slower variation rate in daily milk yield of 0.35 kg/d. There were notable differences in milk fluctuation traits depending on the disease, and greater milk losses were observed when multiple diseases occurred simultaneously. All milk fluctuation traits evaluated were heritable with heritability estimates ranging from 0.01 to 0.10, and moderate to high genetic correlations with milk yield (0.34 to 0.64), milk loss throughout the lactation (0.22 to 0.97), and resilience indicator (0.39 to 0.95). These results indicate that cows with lower milk losses and higher resilience tend to have more stable milk fluctuations, which supports the potential for breeding for more disease-resilient cows based on milk fluctuation traits. Overall, this study confirms the high effect of diseases on milk yield variability and provides insightful information about their relationship with relevant traits in Holstein cattle. Furthermore, this study shows the potential of using high-frequency automatic monitoring of milk yield to assist on breeding practices and health management in dairy cows.

The HSP90AA1 gene is involved in heat stress responses and its functional genetic polymorphisms are associated with heat tolerance in Holstein cows.

As the stress-inducible isoform of the heat-shock protein 90 (HSP90), the HSP90AA1 gene encodes HSP90α and plays an important role in heat stress (HS) response. Therefore, this study aimed to investigate the role of the HSP90AA1 gene in cellular responses during HS and to identify functional SNPs associated with thermotolerance in Holstein cattle. For the in vitro validation experiment of acute HS, cells from the Madin-Darby bovine kidney cell line were exposed to 42°C for 1 h, and various parameters were assessed, including cell apoptosis, cell autophagy, and the cellular functions of HSP90α by using its inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). Furthermore, the polymorphisms identified in the HSP90AA1 gene and their functions related to HS were validated in vitro. Acute HS exposure induced cell apoptosis, cell autophagy, and upregulated expression of the HSP90AA1 gene. Inhibition of HSP90α by 17-AAG treatment had a significant effect on the expression of the HSP90α protein and increased cell apoptosis. However, autophagy decreased in comparison to the control treatment when cells were exposed to 42°C for 1 h. Five SNPs identified in the HSP90AA1 gene were significantly associated with rectal temperature and respiration score in Holstein cows, in which the rs109256957 SNP is located in the 3' untranslated region (3' UTR). Furthermore, we demonstrated that the 3' UTR of HSP90AA1 is a direct target of bta-miR-1224 by cell transfection with exogenous microRNA (miRNA) mimic and inhibitor. The luciferase assays revealed that the SNP rs109256957 affects the regulation of bta-miR-1224 binding activity and alters the expression of the HSP90AA1 gene. Heat stress-induced HSP90AA1 expression maintains cell survival by inhibiting cell apoptosis and increasing cell autophagy. The rs109256957 located in the 3' UTR region is a functional variation and it affects the HSP90AA1 expression by altering its binding activity with bta-miR-1224, thereby associating with the physiological parameters of Holstein cows.

Genetic background of hematological parameters in Holstein cattle based on genome-wide association and RNA sequencing analyses.

Hematological parameters refer to the assessment of changes in the number and distribution of blood cells, including leukocytes (LES), erythrocytes (ERS), and platelets (PLS), which are essential for the early diagnosis of hematological system disorders and other systemic diseases in livestock. In this context, the primary objectives of this study were to investigate the genomic background of 19 hematological parameters in Holstein cattle, focusing on LES, ERS, and PLS blood components. Genetic and phenotypic (co)variances of hematological parameters were calculated based on the average information restricted maximum likelihood method and 1,610 genotyped individuals and 5,499 hematological parameter records from 4,543 cows. Furthermore, we assessed the genetic relationship between these hematological parameters and other economically important traits in dairy cattle breeding programs. We also carried out genome-wide association studies and candidate gene analyses. Blood samples from 21 primiparous cows were used to identify candidate genes further through RNA sequencing (RNA-seq) analyses. Hematological parameters generally exhibited low-to-moderate heritabilities ranging from 0.01 to 0.29, with genetic correlations between them ranging from -0.88 ± 0.09 (between mononuclear cell ratio and lymphocyte cell ratio) to 0.99 ± 0.01 (between white blood cell count and granulocyte cell count). Furthermore, low-to-moderate approximate genetic correlations between hematological parameters with one longevity, 4 fertility, and 5 health traits were observed. One hundred ninety-nine significant SNP located primarily on the Bos taurus autosomes (BTA) BTA4, BTA6, and BTA8 were associated with 16 hematological parameters. Based on the RNA-seq analyses, 6,687 genes were significantly downregulated and 4,119 genes were upregulated when comparing 2 groups of cows with high and low phenotypic values. By integrating genome-wide association studies (GWAS), RNA-seq, and previously published results, the main candidate genes associated with hematological parameters in Holstein cattle were ACRBP, ADAMTS3, CANT1, CCM2L, CNN3, CPLANE1, GPAT3, GRIP2, PLAGL2, RTL6, SOX4, WDFY3, and ZNF614. Hematological parameters are heritable and moderately to highly genetically correlated among themselves. The large number of candidate genes identified based on GWAS and RNA-seq indicate the polygenic nature and complex genetic determinism of hematological parameters in Holstein cattle.

Three-dimensional force detection and decoupling of a fiber grating sensor for a humanoid prosthetic hand.

A fiber Bragg grating (FBG) based three-dimensional (3D) force sensor for a humanoid prosthetic hand is designed, which can precisely detect 3D force and compensate for ambient temperature. FBG was encapsulated in polydimethylsiloxane (PDMS) for force sensitization and immobilization, and the structural parameters of the sensor were optimized by using finite element simulation, so that its sensitivity to 3D force is enhanced. In the meantime, the calibration experiments for normal force fZ, shear force fX/fY, and temperature were conducted, and the 3D force data were decoupled using the least square (LS) and backpropagation (BP) neural networks decoupling methods, so that an overall decoupling error is 0.038. The results show that the sensor has a simple structure, high sensitivity, high linearity, good creep resistance, and rapid decoupling, providing a successful design for the 3D force detection of a humanoid prosthetic hand.

FBG-based three-dimensional micro-force sensor with axial force sensitivity-enhancing and temperature compensation for micro-forceps.

During retinal microsurgery, excessive interaction force between surgical instruments and intraocular tissue can cause serious accidents such as tissue injury, irreversible retinal damage, and even vision loss. It is essential to accurately sense the micro tool-tissue interaction force, especially for the Ophthalmic Microsurgery Robot. In this study, a fiber Bragg grating (FBG) three-dimensional (3-D) micro-force sensor for micro-forceps is proposed, which is integrated with the drive module as an end-effector and can be conveniently mounted onto the ophthalmic surgical robot. An innovative axial force sensitivity-enhancing structure is proposed based on the principles of flexure-hinge and flexible levers to overcome the low sensitivity of axial force measurement. A dual-grating temperature compensation method is adopted for axial force measurement, which considers the differential temperature sensitivity of the two FBGs. Three FBGs are arranged along the circumference of the guide tube in this study to measure transverse forces and compensate for effects caused by changes in temperature. The experimental results demonstrate that the micro-forceps designed in this study achieved a resolution of 0.13 mN for transverse force and 0.30 mN for axial force. The temperature compensation experiments show that the 3-D micro-force sensor can simultaneously compensate for temperature effects in axial and transverse force measurement.

Estimation of genetic parameters and single-step genome-wide association studies for milk urea nitrogen in Holstein cattle.

The main objectives of this study were to estimate genetic parameters for milk urea nitrogen (MUN) in Holstein cattle and to conduct a single-step (ss)GWAS to identify candidate genes associated with MUN. Phenotypic measurements from 24,435 Holstein cows were collected from March 2013 to July 2019 in 9 dairy farms located in the Beijing area, China. A total of 2,029 cows were genotyped using the Illumina 150K Bovine Bead Chip, containing 121,188 SNP. A single-trait repeatability model was used to evaluate the genetic background of MUN. We found that MUN is a trait with low heritability (0.06 ± 0.004) and repeatability (0.12). Considering similar milk production levels, a lower MUN concentration indicates higher nitrogen digestibility. The genetic correlations between MUN and milk yield, net energy concentration, fat percentage, protein percentage, and lactose percentage were positive and ranged from 0.02 to 0.26. The genetic correlation between MUN and somatic cell score (SCS) was negative (-0.18), indicating that animals with higher MUN levels tend to have lower SCS. Both ssGWAS and pathway enrichment analyses were used to explore the genetic mechanisms underlying MUN. A total of 18 SNP (located on BTA11, BTA12, BTA14, BTA17, and BTA18) were found to be significantly associated with MUN. The genes CFAP77, CAMSAP1, CACNA1B, ADGRB1, FARP1, and INTU are considered to be candidate genes for MUN. These candidate genes are associated with important biological processes such as protein and lipid metabolism and binding to specific proteins. This set of candidate genes, metabolic pathways, and their functions provide a better understanding of the genomic architecture and physiological mechanisms underlying MUN in Holstein cattle.

Epigenomics of rats' liver and its cross-species functional annotation reveals key regulatory genes underlying short term heat-stress response.

Molecular responses to heat stress are multifaceted and under a complex cellular post-transcriptional control. This study explores the epigenetic and transcriptional alterations induced by heat stress (42 °C for 120 min) in the liver of rats, by integrating ATAC-seq, RNA-Seq, and WGBS information. Out of 2586 differential ATAC-seq peaks induced by heat stress, 36 up-regulated and 22 down-regulated transcript factors (TFs) are predicted, such as Cebpα, Foxa2, Foxo4, Nfya and Sp3. Furthermore, 150,189 differentially methylated regions represent 2571 differentially expressed genes (DEGs). By integrating all data, 45 DEGs are concluded as potential heat stress response markers in rats. To comprehensively annotate and narrow down predicted markers, they are integrated with GWAS results of heat stress parameters in cows, and PheWAS data in humans. Besides better understanding of heat stress responses in mammals, INSR, MAPK8, RHPN2 and BTBD7 are proposed as candidate markers for heat stress in mammals.

Genomic prediction of service sire effect on female reproductive performance in Holstein cattle: A comparison between different methods, validation population and marker densities.

Reproductive traits of dairy cattle are bound to the actual efficiency of farm operation, which therefore show great economic importance. Among them, some traits were deemed to be simultaneously affected by service sire and mating cow. Service sires are proved to play an important role in reproduction process of cows. However, limited study explored the genetic effect of service sire (GESS), let alone the genomic prediction of this effect. In the present study, 2244 genotyped bulls together with phenotypic records were used to predict the GESS on conception rate, 56-day non-return rate, calving ease, stillbirth and gestation length. The feasibilities of multi-step genomic best linear unbiased predictor (msGBLUP) and single-step genomic best linear unbiased predictor (ssGBLUP) were investigated under different scenarios, that is, different marker densities and validation population. The predictive accuracies and unbiasedness for GESS ranged from 0.159 to 0.647 and from 0.202 to 2.018, respectively, when validated on young bulls, while the accuracies and unbiasedness ranged from 0.409 to 0.802 and 0.333 to 1.146 when validated on random split data sets. It is feasible to predict GESS on reproductive traits by using a linear mixed model and genomic data, and high-density marker panel had limited contribution to the prediction. This research investigated the potential factors that influence the genomic prediction of GESS on reproductive traits and indicated the possibility of genomic selection on GESS, both in ideal and practical circumstances.

Effects of DNA Methylation on Gene Expression and Phenotypic Traits in Cattle: A Review.

Gene expression in cells is determined by the epigenetic state of chromatin. Therefore, the study of epigenetic changes is very important to understand the regulatory mechanism of genes at the molecular, cellular, tissue and organ levels. DNA methylation is one of the most studied epigenetic modifications, which plays an important role in maintaining genome stability and ensuring normal growth and development. Studies have shown that methylation levels in bovine primordial germ cells, the rearrangement of methylation during embryonic development and abnormal methylation during placental development are all closely related to their reproductive processes. In addition, the application of bovine male sterility and assisted reproductive technology is also related to DNA methylation. This review introduces the principle, development of detection methods and application conditions of DNA methylation, with emphasis on the relationship between DNA methylation dynamics and bovine spermatogenesis, embryonic development, disease resistance and muscle and fat development, in order to provide theoretical basis for the application of DNA methylation in cattle breeding in the future.

Analysis of CircRNA Expression in Peripheral Blood of Holstein Cows in Response to Heat Stress.

The present study aimed to identify key circRNAs and pathways associated with heat stress in blood samples of Holstein cows, which will provide new insights into the molecular mechanisms driving heat stress in cows. Hence, we evaluated changes in milk yield, rectal temperature, and respiratory rate of experimental cows between heat stress (summer) and non-heat stress (spring) conditions with two comparisons, including Sum1 vs. Spr1 (same lactation stage, different individuals, 15 cows per group) and Sum1 vs. Spr2 (same individual, different lactation stages, 15 cows per group). Compared to both Spr1 and Spr2, cows in the Sum1 group had a significantly lower milk yield, while rectal temperature and respiratory rate were significantly higher (p < 0.05), indicating that cows in the Sum1 group were experiencing heat stress. In each group, five animals were chosen randomly to undergo RNA-seq. The results reveal that 140 and 205 differentially expressed (DE) circRNAs were screened in the first and second comparisons, respectively. According to the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, these DE circRNAs were mainly enriched in five signaling pathways, including choline metabolism, the PI3K/AKT signaling pathway, the HIF-1 signaling pathway, the longevity-regulating pathway, and autophagy. Then, we obtained the top 10 hub source genes of circRNAs according to the protein-protein interaction networks. Among them, ciRNA1282 (HIF1A), circRNA4205 (NR3C1), and circRNA12923 (ROCK1) were enriched in multiple pathways and identified as binding multiple miRNAs. These key circRNAs may play an important role in the heat stress responses of dairy cows. These results provide valuable information on the involvement of key circRNAs and their expression pattern in the heat stress response of cows.

Fecal microbiota and their association with heat stress in Bos taurus.

BACKGROUND: Humans have been influencing climate changes by burning fossil fuels, farming livestock, and cutting down rainforests, which has led to global temperature rise. This problem of global warming affects animals by causing heat stress, which negatively affects their health, biological functions, and reproduction. On the molecular level, it has been proved that heat stress changes the expression level of genes and therefore causes changes in proteome and metabolome. The importance of a microbiome in many studies showed that it is considered as individuals' "second genome". Physiological changes caused by heat stress may impact the microbiome composition. RESULTS: In this study, we identified fecal microbiota associated with heat stress that was quantified by three metrics - rectal temperature, drooling, and respiratory scores represented by their Estimated Breeding Values. We analyzed the microbiota from 136 fecal samples of Chinese Holstein cows through a 16S rRNA gene sequencing approach. Statistical modeling was performed using a negative binomial regression. The analysis revealed the total number of 24 genera and 12 phyla associated with heat stress metrics. Rhizobium and Pseudobutyrivibrio turned out to be the most significant genera, while Acidobacteria and Gemmatimonadetes were the most significant phyla. Phylogenetic analysis revealed that three heat stress indicators quantify different metabolic ways of animals' reaction to heat stress. Other studies already identified that those genera had significantly increased abundance in mice exposed to stressor-induced changes. CONCLUSIONS: This study provides insights into the analysis of microbiome composition in cattle using heat stress measured as a continuous variable. The bacteria highly associated with heat stress were highlighted and can be used as biomarkers in further microbiological studies.

Genetic parameters for dairy calf and replacement heifer wellness traits and their association with cow longevity and health indicators in Holstein cattle.

High mortality and involuntary culling rates cause great economic losses to the worldwide dairy cattle industry. However, there is low emphasis on wellness traits in replacement animals (dairy calves and replacement heifers) during their development stages in modern dairy cattle breeding programs. Therefore, the main objectives of this study were to estimate genetic parameters of wellness traits in replacement cattle (replacement wellness traits) and obtain their genetic correlations with 12 cow health and longevity traits in the Chinese Holstein population. Seven replacement wellness traits were analyzed, including birth weight, survival from 3 to 60 d (Sur1), survival from 61 to 365 d (Sur2), survival from 366 d to the first calving (Sur3), calf diarrhea, calf pneumonia, and calf serum total protein (STP). Single and bivariate animal models were employed to estimate (co)variance components using the data from 189,980 Holstein cattle. The genetic correlations between replacement wellness traits and cow longevity, health traits were calculated by employing bivariate models, including 6 longevity traits and 6 health traits (clinical mastitis, metritis, ketosis, displaced abomasum, milk fever, and hoof health or hoof disease). The estimated heritabilities (± SE) were 0.335 (± 0.008), 0.088 (± 0.005), 0.166 (± 0.006), 0.102 (±0 .006), 0.048 (± 0.003), 0.063 (± 0.004), and 0.170 (± 0.019) for birth weight, Sur1, Sur2, Sur3, pneumonia, diarrhea, and STP, respectively. The majority of the genetic correlations among the 7 replacement wellness traits were negligible. The genetic correlations among Sur1, Sur2, and Sur3 ranged from 0.112 (Sur1 and Sur3) to 0.445 (Sur1 and Sur2) when fitting a linear model (estimates in the observed scale), and from 0.560 (Sur1 and Sur3) to 0.773 (Sur1 and Sur2) when fitting a threshold model (estimates in the liability scale). The genetic correlations between replacement wellness and cow longevity were low (absolute value lower than 0.30), but some of them were significantly different from zero. Compared with other replacement wellness traits, Sur3 and STP had relatively high genetic correlations with cow longevity. Replacement wellness traits are heritable and can be improved through direct genetic and genomic selection. The results from the current study will contribute for better balancing dairy cattle breeding goals to genetically improve dairy cattle wellness in the period from birth to first calving.

Metabolome profiling of plasma reveals different metabolic responses to acute cold challenge between Inner-Mongolia Sanhe and Holstein cattle.

Low-temperature conditions influence cattle productivity and survivability. Understanding the metabolic regulations of specific cattle breeds and identifying potential biomarkers related to cold challenges are important for cattle management and optimization of genetic improvement programs. In this study, 28 Inner-Mongolia Sanhe and 22 Holstein heifers were exposed to -25°C for 1 h to evaluate the differences in metabolic mechanisms of thermoregulation. In response to this acute cold challenge, altered rectal temperature was only observed in Holstein cattle. Further metabolome analyses showed a greater baseline of glycolytic activity and mobilization of AA in Sanhe cattle during normal conditions. Both breeds responded to the acute cold challenge by altering their metabolism of volatile fatty acids and AA for gluconeogenesis, which resulted in increased glucose levels. Furthermore, Sanhe cattle mobilized the citric acid cycle activity, and creatine and creatine phosphate metabolism to supply energy, whereas Holstein cattle used greater AA metabolism for this purpose. Altogether, we found that propionate and methanol are potential biomarkers of acute cold challenge response in cattle. Our findings provide novel insights into the biological mechanisms of acute cold response and climatic resilience, and will be used as the basis when developing breeding tools for genetically selecting for improved cold adaptation in cattle.

Transcriptome Analyses Reveal Essential Roles of Alternative Splicing Regulation in Heat-Stressed Holstein Cows.

Heat stress (HS) severely impacts the productivity and welfare of dairy cows. Investigating the biological mechanisms underlying HS response is crucial for developing effective mitigation and breeding strategies. Therefore, we evaluated the changes in milk yield, physiological indicators, blood biochemical parameters, and alternative splicing (AS) patterns of lactating Holstein cows during thermoneutral (TN, N = 19) and heat-stress (HS, N = 17) conditions. There was a significant (p < 0.05) decline in milk yield as physiological indicators increased after exposure to natural HS conditions. The levels of eight out of 13 biochemical parameters of HS were also significantly altered in the presence of HS (p < 0.05). These results demonstrate that HS negatively influences various biological processes of Holstein cows. Furthermore, we investigated AS events based on the RNA-seq data from blood samples. With HS, five common types of AS events were generally increased by 6.7−38.9%. A total of 3470 AS events corresponding to 3143 unique genes were differentially alternatively spliced (DSGs) (p-adjusted < 0.05) between TN and HS groups. The functional annotation results show that the majority of DSGs are involved in mRNA splicing and spliceosomal complex, followed by enrichment in immune and metabolic processes. Eighty-seven out of 645 differentially expressed genes (DEGs) (fold change ≥ 1.5 and false discovery rate < 0.05) overlapped with DSGs. Further analyses showed that 20 of these genes were significantly enriched for the RNA splicing, RNA binding, and RNA transport. Among them, two genes (RBM25 and LUC7L3) had strong interrelation and co-expression pattern with other genes and were identified as candidate genes potentially associated with HS responses in dairy cows. In summary, AS plays a crucial role in changing the transcriptome diversity of heat-stress-related genes in multiple biological pathways and provides a different regulation mechanism from DEGs.

Investigation of Metabolome Underlying the Biological Mechanisms of Acute Heat Stressed Granulosa Cells.

Heat stress affects granulosa cells and the ovarian follicular microenvironment, ultimately resulting in poor oocyte developmental competence. This study aims to investigate the metabo-lomics response of bovine granulosa cells (bGCs) to in vitro acute heat stress of 43 °C. Heat stress triggers oxidative stress-mediated apoptosis in cultured bGCs. Heat-stressed bGCs exhibited a time-dependent recovery of proliferation potential by 48 h. A total of 119 metabolites were identified through LC-MS/MS-based metabolomics of the spent culture media, out of which, 37 metabolites were determined as differentially involved in metabolic pathways related to bioenergetics support mechanisms and the physical adaptations of bGCs. Multiple analyses of metabolome data identified choline, citric acid, 3-hydroxy-3-methylglutaric acid, glutamine, and glycocyamine as being upregulated, while galactosamine, AICAR, ciliatine, 16-hydroxyhexadecanoic acid, lysine, succinic acid, uridine, xanthine, and uraconic acid were the important downregulated metabolites in acute heat stress. These differential metabolites were implicated in various important metabolic pathways directed towards bioenergetics support mechanisms including glycerophospholipid metabolism, the citrate cycle (TCA cycle), glyoxylate and dicarboxylate metabolism, and serine, threonine, and tyrosine metabolism. Our study presents important metabolites and metabolic pathways involved in the adaptation of bGCs to acute heat stress in vitro.

Genome-wide identification and functional prediction of long non-coding RNAs in Sprague-Dawley rats during heat stress.

BACKGROUND: Heat stress (HS) is a major stress event in the life of an animal, with detrimental upshots in production and health. Long-non-coding RNAs (lncRNAs) play an important role in many biological processes by transcriptional regulation. However, no research has been reported on the characterization and functionality of lncRNAs in heat-stressed rats. RESULTS: We studied expression levels of lncRNAs in rats during HS, using strand-specific RNA sequencing. Six rats, three in each of Control (22 ± 1 °C) and H120 (42 °C for 120 min) experimental groups, were used to screen for lncRNAs in their liver and adrenal glands. Totally, 4498 and 7627 putative lncRNAs were identified in liver and adrenal glands of the Control and H120 groups, respectively. The majority of lncRNAs were relatively shorter and contained fewer exons than protein-coding transcripts. In total, 482 (174 up-regulated and 308 down-regulated) and 271 (126 up-regulated and 145 down-regulated) differentially-expressed lncRNAs (DElncRNAs, P < 0.05) were identified in the liver and adrenal glands of the Control and H120 groups, respectively. Furthermore, 1274, 121, and 73 target differentially-expressed genes (DEGs) in the liver were predicted to interact with DElncRNAs based on trans-/cis- and sequence similarity regulatory modes. Functional annotation analyses indicated that these DEGs were mostly significantly enriched in insulin signalling, myeloid leukaemia, and glucagon signalling pathways. Similarly, 437, 73 and 41 target DEGs in the adrenal glands were mostly significantly enriched in the cell cycle (trans-prediction) and lysosome pathways (cis-prediction). The DElncRNAs interacting with DEGs that encode heat shock proteins (HSPs) may play an important role in HS response, which include Hsf4, Dnaja1, Dnajb4, Hsph1 and Hspb1 in the liver, and Dnajb13 and Hspb8 in the adrenal glands. The strand-specific RNA sequencing findings were also further verified through RT-qPCR. CONCLUSIONS: This study is the first to provide a detailed characterization and functional analysis of expression levels of lncRNAs in liver and adrenal glands of heat-stressed rats, which provides basis for further studies on the biological functions of lncRNAs under heat stress in rats and other mammalian species.

Genotype-by-environment interaction in Holstein heifer fertility traits using single-step genomic reaction norm models.

BACKGROUND: The effect of heat stress on livestock production is a worldwide issue. Animal performance is influenced by exposure to harsh environmental conditions potentially causing genotype-by-environment interactions (G × E), especially in highproducing animals. In this context, the main objectives of this study were to (1) detect the time periods in which heifer fertility traits are more sensitive to the exposure to high environmental temperature and/or humidity, (2) investigate G × E due to heat stress in heifer fertility traits, and, (3) identify genomic regions associated with heifer fertility and heat tolerance in Holstein cattle. RESULTS: Phenotypic records for three heifer fertility traits (i.e., age at first calving, interval from first to last service, and conception rate at the first service) were collected, from 2005 to 2018, for 56,998 Holstein heifers raised in 15 herds in the Beijing area (China). By integrating environmental data, including hourly air temperature and relative humidity, the critical periods in which the heifers are more sensitive to heat stress were located in more than 30 days before the first service for age at first calving and interval from first to last service, or 10 days before and less than 60 days after the first service for conception rate. Using reaction norm models, significant G × E was detected for all three traits regarding both environmental gradients, proportion of days exceeding heat threshold, and minimum temperature-humidity index. Through single-step genome-wide association studies, PLAG1, AMHR2, SP1, KRT8, KRT18, MLH1, and EOMES were suggested as candidate genes for heifer fertility. The genes HCRTR1, AGRP, PC, and GUCY1B1 are strong candidates for association with heat tolerance. CONCLUSIONS: The critical periods in which the reproductive performance of heifers is more sensitive to heat stress are trait-dependent. Thus, detailed analysis should be conducted to determine this particular period for other fertility traits. The considerable magnitude of G × E and sire re-ranking indicates the necessity to consider G × E in dairy cattle breeding schemes. This will enable selection of more heat-tolerant animals with high reproductive efficiency under harsh climatic conditions. Lastly, the candidate genes identified to be linked with response to heat stress provide a better understanding of the underlying biological mechanisms of heat tolerance in dairy cattle.

Cell-Free Virus-Host Chimera DNA From Hepatitis B Virus Integration Sites as a Circulating Biomarker of Hepatocellular Cancer.

BACKGROUND AND AIMS: Early recurrence of hepatocellular carcinoma (HCC) after surgical resection compromises patient survival. Timely detection of HCC recurrence and its clonality is required to implement salvage therapies appropriately. This study examined the feasibility of virus-host chimera DNA (vh-DNA), generated from junctions of hepatitis B virus (HBV) integration in the HCC chromosome, as a circulating biomarker for this clinical setting. APPROACH AND RESULTS: HBV integration in 50 patients with HBV-related HCC was determined by the Hybridization capture-based next-generation sequencing (NGS) platform. For individual HCC, the vh-DNA was quantified by specific droplet digital PCR (ddPCR) assay in plasma samples collected before and 2 months after surgery. HBV integrations were identified in 44 out of 50 patients with HBV-related HCC. Tumor-specific ddPCR was developed to measure the corresponding vh-DNA copy number in baseline plasma from each patient immediately before surgery. vh-DNA was detected in 43 patients (97.7%), and the levels correlated with the tumor sizes (detection limit at 1.5 cm). Among the plasma collected at 2 months after surgery, 10 cases (23.3%) still contained the same signature vh-DNA detected at baseline, indicating the presence of residual tumor cells. Nine of them (90%) experienced HCC recurrence within 1 year, supporting vh-DNA as an independent risk factor in predicting early recurrence. Analysis of circulating vh-DNA at recurrence further helped identify the clonal origin. A total of 81.8% of recurrences came from original HCC clones sharing the same plasma vh-DNA, whereas 18.2% were from de novo HCC. CONCLUSIONS: vh-DNA was shown to be a circulating biomarker for detecting the tumor load in majority of patients with HBV-related HCC and aided in monitoring residual tumor and recurrence clonality after tumor resection.