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.

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.

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.

Comprehensive transcriptome and methylome analysis delineates the biological basis of hair follicle development and wool-related traits in Merino sheep.

BACKGROUND: Characterization of the molecular mechanisms underlying hair follicle development is of paramount importance in the genetic improvement of wool-related traits in sheep and skin-related traits in humans. The Merino is the most important breed of fine-wooled sheep in the world. In this study, we systematically investigated the complexity of sheep hair follicle development by integrating transcriptome and methylome datasets from Merino sheep skin. RESULTS: We analysed 72 sequence datasets, including DNA methylome and the whole transcriptome of four gene types, i.e. protein-coding genes (PCGs), lncRNAs, circRNAs, and miRNAs, across four embryonic days (E65, E85, E105, and E135) and two postnatal days (P7 and P30) from the skin tissue of 18 Merino sheep. We revealed distinct expression profiles of these four gene types across six hair follicle developmental stages, and demonstrated their complex interactions with DNA methylation. PCGs with stage-specific expression or regulated by stage-specific lncRNAs, circRNAs, and miRNAs were significantly enriched in epithelial differentiation and hair follicle morphogenesis. Regulatory network and gene co-expression analyses identified key transcripts controlling hair follicle development. We further predicted transcriptional factors (e.g. KLF4, LEF1, HOXC13, RBPJ, VDR, RARA, and STAT3) with stage-specific involvement in hair follicle morphogenesis. Through integrating these stage-specific genomic features with results from genome-wide association studies (GWAS) of five wool-related traits in 7135 Merino sheep, we detected developmental stages and genes that were relevant with wool-related traits in sheep. For instance, genes that were specifically upregulated at E105 were significantly associated with most of wool-related traits. A phenome-wide association study (PheWAS) demonstrated that candidate genes of wool-related traits (e.g. SPHK1, GHR, PPP1R27, CSRP2, EEF1A2, and PTPN1) in sheep were also significantly associated with dermatological, metabolic, and immune traits in humans. CONCLUSIONS: Our study provides novel insights into the molecular basis of hair follicle morphogenesis and will serve as a foundation to improve breeding for wool traits in sheep. It also indicates the importance of studying gene expression in the normal development of organs in understanding the genetic architecture of economically important traits in livestock. The datasets generated here are useful resources for functionally annotating the sheep genome, and for elucidating early skin development in mammals, including humans.

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.

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.

Integration of a single-step genome-wide association study with a multi-tissue transcriptome analysis provides novel insights into the genetic basis of wool and weight traits in sheep.

BACKGROUND: Genetic improvement of wool and growth traits is a major goal in the sheep industry, but their underlying genetic architecture remains elusive. To improve our understanding of these mechanisms, we conducted a weighted single-step genome-wide association study (WssGWAS) and then integrated the results with large-scale transcriptome data for five wool traits and one growth trait in Merino sheep: mean fibre diameter (MFD), coefficient of variation of the fibre diameter (CVFD), crimp number (CN), mean staple length (MSL), greasy fleece weight (GFW), and live weight (LW). RESULTS: Our dataset comprised 7135 individuals with phenotype data, among which 1217 had high-density (HD) genotype data (n = 372,534). The genotypes of 707 of these animals were imputed from the Illumina Ovine single nucleotide polymorphism (SNP) 54 BeadChip to the HD Array. The heritability of these traits ranged from 0.05 (CVFD) to 0.36 (MFD), and between-trait genetic correlations ranged from - 0.44 (CN vs. LW) to 0.77 (GFW vs. LW). By integrating the GWAS signals with RNA-seq data from 500 samples (representing 87 tissue types from 16 animals), we detected tissues that were relevant to each of the six traits, e.g. liver, muscle and the gastrointestinal (GI) tract were the most relevant tissues for LW, and leukocytes and macrophages were the most relevant cells for CN. For the six traits, 54 quantitative trait loci (QTL) were identified covering 81 candidate genes on 21 ovine autosomes. Multiple candidate genes showed strong tissue-specific expression, e.g. BNC1 (associated with MFD) and CHRNB1 (LW) were specifically expressed in skin and muscle, respectively. By conducting phenome-wide association studies (PheWAS) in humans, we found that orthologues of several of these candidate genes were significantly (FDR < 0.05) associated with similar traits in humans, e.g. BNC1 was significantly associated with MFD in sheep and with hair colour in humans, and CHRNB1 was significantly associated with LW in sheep and with body mass index in humans. CONCLUSIONS: Our findings provide novel insights into the biological and genetic mechanisms underlying wool and growth traits, and thus will contribute to the genetic improvement and gene mapping of complex traits in sheep.

Genomic analyses and biological validation of candidate genes for rectal temperature as an indicator of heat stress in Holstein cattle.

Heat stress is a major cause of welfare issues and economic losses to the worldwide dairy cattle industry. Genetic selection for heat tolerance has a great potential to positively affect the dairy industry, as the gains are permanent and cumulative over generations. Rectal temperature (RT) is hypothesized to be a good indicator trait of heat tolerance. Therefore, this study investigated the genetic architecture of RT by estimating genetic parameters, performing genome-wide association studies, and biologically validating potential candidate genes identified to be related to RT in Holstein cattle. A total of 33,013 RT records from 7,598 cows were used in this study. In addition, 1,114 cows were genotyped using the Illumina 150K Bovine BeadChip (Illumina, San Diego, CA). Rectal temperature measurements taken in the morning (AMRT) and in the afternoon (PMRT) are moderately heritable traits, with estimates of 0.09 ± 0.02 and 0.04 ± 0.01, respectively. These 2 traits are also highly genetically correlated (r = 0.90 ± 0.08). A total of 10 SNPs (located on BTA3, BTA4, BTA8, BTA13, BTA14, and BTA29) were found to be significantly associated with AMRT and PMRT. Subsequently, gene expression analyses were performed to validate the key functional genes identified (SPAG17, FAM107B, TSNARE1, RALYL, and PHRF1). This was done through in vitro exposure of peripheral blood mononuclear cells (PBMC) to different temperatures (37°C, 39°C, and 42°C). The relative mRNA expression of 2 genes, FAM107B and PHRF1, significantly changed between the control and heat stressed PBMC. In summary, RT is heritable, and enough genetic variability exists to enable genetic improvement of heat tolerance in Holstein cattle. Important genomic regions were identified and biologically validated; FAM107B and PHRF1 are the main candidate genes identified to influence heat stress response in dairy cattle.

Genomic prediction based on a joint reference population for the Xinjiang Brown cattle.

Introduction: Xinjiang Brown cattle constitute the largest breed of cattle in Xinjiang. Therefore, it is crucial to establish a genomic evaluation system, especially for those with low levels of breed improvement. Methods: This study aimed to establish a cross breed joint reference population by analyzing the genetic structure of 485 Xinjiang Brown cattle and 2,633 Chinese Holstein cattle (Illumina GeneSeek GGP bovine 150 K chip). The Bayes method single-step genome-wide best linear unbiased prediction was used to conduct a genomic evaluation of the joint reference population for the milk traits of Xinjiang Brown cattle. The reference population of Chinese Holstein cattle was randomly divided into groups to construct the joint reference population. By comparing the prediction accuracy, estimation bias, and inflation coefficient of the validation population, the optimal number of joint reference populations was determined. Results and Discussion: The results indicated a distinct genetic structure difference between the two breeds of adult cows, and both breeds should be considered when constructing multi-breed joint reference and validation populations. The reliability range of genome prediction of milk traits in the joint reference population was 0.142-0.465. Initially, it was determined that the inclusion of 600 and 900 Chinese Holstein cattle in the joint reference population positively impacted the genomic prediction of Xinjiang Brown cattle to certain extent. It was feasible to incorporate the Chinese Holstein into Xinjiang Brown cattle population to form a joint reference population for multi-breed genomic evaluation. However, for different Xinjiang Brown cattle populations, a fixed number of Chinese Holstein cattle cannot be directly added during multi-breed genomic selection. Pre-evaluation analysis based on the genetic structure, kinship, and other factors of the current population is required to ensure the authenticity and reliability of genomic predictions and improve estimation accuracy.

Vitamin C Alleviates the Negative Effects of Heat Stress on Reproductive Processes by Regulating Amino Acid Metabolism in Granulosa Cells.

Heat stress-induced biochemical alterations in ovarian follicles compromise the function of granulosa cells (GCs) and the developmental competence of oocytes. Summer heat stress can have a far-reaching negative impact on overall fertility and reproductive success. Together with the heat stress, the rise of assisted reproductive technologies (ART), potential confounding hazards of in vitro handling and the absence of systemic body support in ART makes it imperative to study the heat stress ameliorative effects of vitamin C under in vitro conditions. Using in vitro heat stress treatment of 43 °C for two hours in bovine GCs, we studied the effects of vitamin C on cell growth, oxidative stress, apoptosis and cell cycle progression together with a comprehensive metabolomics profiling. This study investigates the molecular milieu underlying the vitamin C (VC)-led alleviation of heat-related disruptions to metabolic processes in bovine GCs. The supplementation of VC ameliorated the detrimental effects of heat stress by reducing oxidative stress and apoptosis while restoring cell proliferation. Normal cell function restoration in treated GCs was demonstrated through the finding of significantly high levels of progesterone. We observed a shift in the metabolome from biosynthesis to catabolism, mostly dominated by the metabolism of amino acids (decreased tryptophan, methionine and tyrosine) and the active TCA cycle through increased Succinic acid. The Glutathione and tryptophan metabolism were important in ameliorating the inflammation and metabolism nexus under heat stress. Two significant enzymes were identified, namely tryptophan 2,3-dioxygenase (TDO2) and mitochondrial phenylalanyl-tRNA synthetase (FARS2). Furthermore, our findings provide insight into the significance of B-complex vitamins in the context of heat stress during VC supplementation. This study underscores the importance of VC supplementation in heat stress and designates multiple metabolic intervention faucets in the context of ameliorating heat stress and enhancing reproductive efficiency.

Identification of Key Genes Associated with Heat Stress in Rats by Weighted Gene Co-Expression Network Analysis.

Heat stress has been a big challenge for animal survival and health due to global warming. However, the molecular processes driving heat stress response were unclear. In this study, we exposed the control group rats (n = 5) at 22 °C and the other three heat stress groups (five rats in each group) at 42 °C lasting 30, 60, and 120 min, separately. We performed RNA sequencing in the adrenal glands and liver and detected the levels of hormones related to heat stress in the adrenal gland, liver, and blood tissues. Weighted gene co-expression network analysis (WGCNA) was also performed. Results showed that rectal temperature and adrenal corticosterone levels were significantly negatively related to genes in the black module, which was significantly enriched in thermogenesis and RNA metabolism. The genes in the green-yellow module were strongly positively associated with rectal temperature and dopamine, norepinephrine, epinephrine, and corticosterone levels in the adrenal glands and were enriched in transcriptional regulatory activities under stress. Finally, 17 and 13 key genes in the black and green-yellow modules were identified, respectively, and shared common patterns of changes. Methyltransferase 3 (Mettl3), poly(ADP-ribose) polymerase 2 (Parp2), and zinc finger protein 36-like 1 (Zfp36l1) occupied pivotal positions in the protein-protein interaction network and were involved in a number of heat stress-related processes. Therefore, Parp2, Mettl3, and Zfp36l1 could be considered candidate genes for heat stress regulation. Our findings shed new light on the molecular processes underpinning heat stress.

RNA-Seq Analysis of Peripheral Whole Blood from Dairy Bulls with High and Low Antibody-Mediated Immune Responses-A Preliminary Study.

Enhancing the immune response through breeding is regarded as an effective strategy for improving animal health, as dairy cattle identified as high immune responders are reported to have a decreased prevalence of economically significant diseases. The identification of differentially expressed genes (DEGs) associated with immune responses might be an effective tool for breeding healthy dairy cattle. In this study, antibody-mediated immune responses (AMIRs) were induced by the immunization of hen egg white lysozyme (HEWL) in six Chinese Holstein dairy bulls divided into high- and low-AMIR groups based on their HEWL antibody level. Then, RNA-seq was applied to explore the transcriptome of peripheral whole blood between the two comparison groups. As a result, several major upregulated and downregulated genes were identified and attributed to the regulation of locomotion, tissue development, immune response, and detoxification. In addition, the result of the KEGG pathway analysis revealed that most DEGs were enriched in pathways related to disease, inflammation, and immune response, including antigen processing and presentation, Staphylococcus aureus infection, intestinal immune network for IgA production, cytokine-cytokine receptor interaction, and complement and coagulation cascades. Moreover, six genes (BOLA-DQA5, C5, CXCL2, HBA, LTF, and COL1A1) were validated using RT-qPCR, which may provide information for genomic selection in breeding programs. These results broaden the knowledge of the immune response mechanism in dairy bulls, which has strong implications for breeding cattle with an enhanced AMIR.

Differential Responses of Physiological Parameters, Production Traits, and Blood Metabolic Profiling between First- and Second-Parity Holstein Cows in the Comparison of Spring versus Summer Seasons.

Heat stress (HS) negatively influences cows' welfare and productivity. Therefore, a better understanding of the physiological and molecular mechanisms of HS responses from multiple parities is paramount for the development of effective management and breeding strategies. In comparison with first-parity cows in the spring (Spring-1), first-parity cows in the summer (Summer-1) had a significantly higher rectal temperature (RT), respiration rate (RR), drooling score (DS), and daily activity (DA), while lower (P < 0.05) daily rumination (DR), seven-day average milk yield (7AMY), milk yield on sampling day (MY_S), milk yield on test day (MY_T), and lactose percentage (LP) were observed. When comparing the spring (Spring-2) and summer (Summer-2) of the second-parity cows, significant differences were also found in RT, RR, DS, DA, and DR (P < 0.05), corresponding to similar trends with the first parity while having smaller changes. Moreover, significantly negative impacts on performance traits were only observed on fat percentage (FP) and LP. These results showed that there were different biological responses between first- and second-parity Holstein cows. Further, 18 and 17 metabolites were involved in the seasonal response of first- and second-parity cows, respectively. Nine differential metabolites were shared between the two parities, and pathway analyses suggested that cows had an inhibited tricarboxylic acid cycle, increased utilization of lipolysis, and a dysregulated gut microbiome during the summer. The metabolites identified exclusively for each parity highlighted the differences in microbial response and host amino acid metabolism between two parities in response to HS. Moreover, glucose, ethanol, and citrate were identified as potential biomarkers for distinguishing individuals between Spring-1 and Summer-1. Ethanol and acetone were better predictors for distinguishing individuals between Spring-2 and Summer-2. Taken together, the present study demonstrated the impact of naturally induced HS on physiological parameters, production traits, and the blood metabolome of Holstein cows. There are different biological responses and regulation mechanisms between first- and second-parity Holstein cows.

Transcriptome Reveals Granulosa Cells Coping through Redox, Inflammatory and Metabolic Mechanisms under Acute Heat Stress.

Heat stress affects granulosa cells (GCs) and the ovarian follicular microenvironment, causing poor oocyte developmental competence and fertility. This study aimed to investigate the physical responses and global transcriptomic changes in bovine GCs to acute heat stress (43 °C for 2 h) in vitro. Heat-stressed GCs exhibited transient proliferation senescence and resumed proliferation at 48 h post-stress, while post-stress immediate culture-media change had a relatively positive effect on proliferation resumption. Increased accumulation of reactive oxygen species and apoptosis was observed in the heat-stress group. In spite of the upregulation of inflammatory (CYCS, TLR2, TLR4, IL6, etc.), pro-apoptotic (BAD, BAX, TNFSF9, MAP3K7, TNFRSF6B, FADD, TRADD, RIPK3, etc.) and caspase executioner genes (CASP3, CASP8, CASP9), antioxidants and anti-apoptotic genes (HMOX1, NOS2, CAT, SOD, BCL2L1, GPX4, etc.) were also upregulated in heat-stressed GCs. Progesterone and estrogen hormones, along with steroidogenic gene expression, declined significantly, in spite of the upregulation of genes involved in cholesterol synthesis. Out of 12,385 differentially expressed genes (DEGs), 330 significant DEGs (75 upregulated, 225 downregulated) were subjected to KEGG functional pathway annotation, gene ontology enrichment, STRING network analyses and manual querying of DEGs for meaningful molecular mechanisms. High inflammatory response was found to be responsible for oxidative-stress-mediated apoptosis of GCs and nodes towards the involvement of the NF-κB pathway and repression of the Nrf2 pathway. Downregulation of MDM4, TP53, PIDD1, PARP3, MAPK14 and MYC, and upregulation of STK26, STK33, TGFB2, CDKN1A and CDKN2A, at the interface of the MAPK and p53 signaling pathway, can be attributed to transient cellular senescence and apoptosis in GCs. The background working of the AMPK pathway through upregulation of AKT1, AMPK, SIRT1, PYGM, SLC2A4 and SERBP1 genes, and downregulation of PPARGCIA, IGF2, PPARA, SLC27A3, SLC16A3, TSC1/2, KCNJ2, KCNJ16, etc., evidence the repression of cellular transcriptional activity and energetic homeostasis modifications in response to heat stress. This study presents detailed responses of acute-heat-stressed GCs at physical, transcriptional and pathway levels and presents interesting insights into future studies regarding GC adaptation and their interaction with oocytes and the reproductive system at the ovarian level.

Host transcriptome and microbiome interactions in Holstein cattle under heat stress condition.

Climate change affects animal physiology. In particular, rising ambient temperatures reduce animal vitality due to heat stress and this can be observed at various levels which included genome, transcriptome, and microbiome. In a previous study, microbiota highly associated with changes in cattle physiology, which included rectal temperature, drooling score and respiratory score, were identified under heat stress conditions. In the present study, genes differentially expressed between individuals were selected representing different additive genetic effects toward the heat stress response in cattle in their production condition. Moreover, a correlation network analysis was performed to identify interactions between the transcriptome and microbiome for 71 Chinese Holstein cows sequenced for mRNA from blood samples and for 16S rRNA genes from fecal samples. Bioinformatics analysis was performed comprising: i) clustering and classification of 16S rRNA sequence reads, ii) mapping cows' transcripts to the reference genome and their expression quantification, and iii) statistical analysis of both data types-including differential gene expression analysis and gene set enrichment analysis. A weighted co-expression network analysis was carried out to assess changes in the association between gene expression and microbiota abundance as well as to find hub genes/microbiota responsible for the regulation of gene expression under heat stress. Results showed 1,851 differentially expressed genes were found that were shared by three heat stress phenotypes. These genes were predominantly associated with the cytokine-cytokine receptor interaction pathway. The interaction analysis revealed three modules of genes and microbiota associated with rectal temperature with which two hubs of those modules were bacterial species, demonstrating the importance of the microbiome in the regulation of gene expression during heat stress. Genes and microbiota from the significant modules can be used as biomarkers of heat stress in cattle.

Joint Transcriptome and Metabolome Analysis Prevails the Biological Mechanisms Underlying the Pro-Survival Fight in In Vitro Heat-Stressed Granulosa Cells.

Previous studies reported the physical, transcriptome, and metabolome changes in in vitro acute heat-stressed (38 °C versus 43 °C for 2 h) bovine granulosa cells. Granulosa cells exhibited transient proliferation senescence, oxidative stress, an increased rate of apoptosis, and a decline in steroidogenic activity. In this study, we performed a joint integration and network analysis of metabolomic and transcriptomic data to further narrow down and elucidate the role of differentially expressed genes, important metabolites, and relevant cellular and metabolic pathways in acute heat-stressed granulosa cells. Among the significant (raw p-value < 0.05) metabolic pathways where metabolites and genes converged, this study found vitamin B6 metabolism, glycine, serine and threonine metabolism, phenylalanine metabolism, arginine biosynthesis, tryptophan metabolism, arginine and proline metabolism, histidine metabolism, and glyoxylate and dicarboxylate metabolism. Important significant convergent biological pathways included ABC transporters and protein digestion and absorption, while functional signaling pathways included cAMP, mTOR, and AMPK signaling pathways together with the ovarian steroidogenesis pathway. Among the cancer pathways, the most important pathway was the central carbon metabolism in cancer. Through multiple analysis queries, progesterone, serotonin, citric acid, pyridoxal, L-lysine, succinic acid, L-glutamine, L-leucine, L-threonine, L-tyrosine, vitamin B6, choline, and CYP1B1, MAOB, VEGFA, WNT11, AOX1, ADCY2, ICAM1, PYGM, SLC2A4, SLC16A3, HSD11B2, and NOS2 appeared to be important enriched metabolites and genes, respectively. These genes, metabolites, and metabolic, cellular, and cell signaling pathways comprehensively elucidate the mechanisms underlying the intricate fight between death and survival in acute heat-stressed bovine granulosa cells and essentially help further our understanding (and will help the future quest) of research in this direction.