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.

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.

Identification of Novel mRNA Isoforms Associated with Acute Heat Stress Response Using RNA Sequencing Data in Sprague Dawley Rats.

The molecular mechanisms underlying heat stress tolerance in animals to high temperatures remain unclear. This study identified the differentially expressed mRNA isoforms which narrowed down the most reliable DEG markers and molecular pathways that underlie the mechanisms of thermoregulation. This experiment was performed on Sprague Dawley rats housed at 22 °C (control group; CT), and three acute heat-stressed groups housed at 42 °C for 30 min (H30), 60 min (H60), and 120 min (H120). Earlier, we demonstrated that acute heat stress increased the rectal temperature of rats, caused abnormal changes in the blood biochemical parameters, as well as induced dramatic changes in the expression levels of genes through epigenetics and post-transcriptional regulation. Transcriptomic analysis using RNA-Sequencing (RNA-Seq) data obtained previously from blood (CT and H120), liver (CT, H30, H60, and H120), and adrenal glands (CT, H30, H60, and H120) was performed. The differentially expressed mRNA isoforms (DEIs) were identified and annotated by the CLC Genomics Workbench. Biological process and metabolic pathway analyses were performed using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. A total of 225, 5764, and 4988 DEIs in the blood, liver, and adrenal glands were observed. Furthermore, the number of novel differentially expressed transcript lengths with annotated genes and novel differentially expressed transcript with non-annotated genes were 136 and 8 in blood, 3549 and 120 in the liver, as well as 3078 and 220 in adrenal glands, respectively. About 35 genes were involved in the heat stress response, out of which, Dnaja1, LOC680121, Chordc1, AABR07011951.1, Hsp90aa1, Hspa1b, Cdkn1a, Hmox1, Bag3, and Dnaja4 were commonly identified in the liver and adrenal glands, suggesting that these genes may regulate heat stress response through interactions between the liver and adrenal glands. In conclusion, this study would enhance our understanding of the complex underlying mechanisms of acute heat stress, and the identified mRNA isoforms and genes can be used as potential candidates for thermotolerance selection in mammals.

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.

Weighted single-step GWAS and RNA sequencing reveals key candidate genes associated with physiological indicators of heat stress in Holstein cattle.

BACKGROUND: The study of molecular processes regulating heat stress response in dairy cattle is paramount for developing mitigation strategies to improve heat tolerance and animal welfare. Therefore, we aimed to identify quantitative trait loci (QTL) regions associated with three physiological indicators of heat stress response in Holstein cattle, including rectal temperature (RT), respiration rate score (RS), and drooling score (DS). We estimated genetic parameters for all three traits. Subsequently, a weighted single-step genome-wide association study (WssGWAS) was performed based on 3200 genotypes, 151,486 phenotypic records, and 38,101 animals in the pedigree file. The candidate genes located within the identified QTL regions were further investigated through RNA sequencing (RNA-seq) analyses of blood samples for four cows collected in April (non-heat stress group) and four cows collected in July (heat stress group). RESULTS: The heritability estimates for RT, RS, and DS were 0.06, 0.04, and 0.03, respectively. Fourteen, 19, and 20 genomic regions explained 2.94%, 3.74%, and 4.01% of the total additive genetic variance of RT, RS, and DS, respectively. Most of these genomic regions are located in the Bos taurus autosome (BTA) BTA3, BTA6, BTA8, BTA12, BTA14, BTA21, and BTA24. No genomic regions overlapped between the three indicators of heat stress, indicating the polygenic nature of heat tolerance and the complementary mechanisms involved in heat stress response. For the RNA-seq analyses, 2627 genes were significantly upregulated and 369 downregulated in the heat stress group in comparison to the control group. When integrating the WssGWAS, RNA-seq results, and existing literature, the key candidate genes associated with physiological indicators of heat stress in Holstein cattle are: PMAIP1, SBK1, TMEM33, GATB, CHORDC1, RTN4IP1, and BTBD7. CONCLUSIONS: Physiological indicators of heat stress are heritable and can be improved through direct selection. Fifty-three QTL regions associated with heat stress indicators confirm the polygenic nature and complex genetic determinism of heat tolerance in dairy cattle. The identified candidate genes will contribute for optimizing genomic evaluation models by assigning higher weights to genetic markers located in these regions as well as to the design of SNP panels containing polymorphisms located within these candidate genes.

Identification of functional features underlying heat stress response in Sprague-Dawley rats using mixed linear models.

Since global temperature is expected to rise by 2 °C in 2050 heat stress may become the most severe environmental factor. In the study, we illustrate the application of mixed linear models for the analysis of whole transcriptome expression in livers and adrenal tissues of Sprague-Dawley rats obtained by a heat stress experiment. By applying those models, we considered four sources of variation in transcript expression, comprising transcripts (1), genes (2), Gene Ontology terms (3), and Reactome pathways (4) and focussed on accounting for the similarity within each source, which was expressed as a covariance matrix. Models based on transcripts or genes levels explained a larger proportion of log2 fold change than models fitting the functional components of Gene Ontology terms or Reactome pathways. In the liver, among the most significant genes were PNKD and TRIP12. In the adrenal tissue, one transcript of the SUCO gene was expressed more strongly in the control group than in the heat-stress group. PLEC had two transcripts, which were significantly overexpressed in the heat-stress group. PER3 was significant only on gene level. Moving to the functional scale, five Gene Ontologies and one Reactome pathway were significant in the liver. They can be grouped into ontologies related to DNA repair, histone ubiquitination, the regulation of embryonic development and cytoplasmic translation. Linear mixed models are valuable tools for the analysis of high-throughput biological data. Their main advantages are the possibility to incorporate information on covariance between observations and circumventing the problem of multiple testing.

Analysis of Genomic Alternative Splicing Patterns in Rat under Heat Stress Based on RNA-Seq Data.

Heat stress is one of the most severe challenges faced in livestock production in summer. Alternative splicing as an important post-transcriptional regulation is rarely studied in heat-stressed animals. Here, we performed and analyzed RNA-sequencing assays on the liver of Sprague-Dawley rats in control (22 °C, n = 5) and heat stress (4 °C for 120 min, H120; n = 5) groups, resulting in the identification of 636 differentially expressed genes. Identification analysis of the alternative splicing events revealed that heat stress-induced alternative splicing events increased by 20.18%. Compared with other types of alternative splicing events, the alternative start increased the most (43.40%) after heat stress. Twenty-eight genes were differentially alternatively spliced (DAS) between the control and H120 groups, among which Acly, Hnrnpd and mir3064 were also differentially expressed. For DAS genes, Srebf1, Shc1, Srsf5 and Ensa were associated with insulin, while Cast, Srebf1, Tmem33, Tor1aip2, Slc39a7 and Sqstm1 were enriched in the composition of the endoplasmic reticulum. In summary, our study conducts a comprehensive profile of alternative splicing in heat-stressed rats, indicating that alternative splicing is one of the molecular mechanisms of heat stress response in mammals and providing reference data for research on heat tolerance in mammalian livestock.

SOD1 Gene Silencing Promotes Apoptosis and Suppresses Proliferation of Heat-Stressed Bovine Granulosa Cells via Induction of Oxidative Stress.

Heat stress (HS) compromises dairy cattle reproduction by altering the follicular dynamics, oocyte maturation, and normal physiological function of ovarian granulosa cells (GCs), eventually resulting in oxidative damage and cell apoptosis. To protect the cells from oxidative damage, the Superoxide dismutase-1 (SOD1) degraded the hydrogen peroxide (H2O2) to oxygen (O2) and water. The objective of the current study was to investigate the impact of SOD1 silencing on intracellular ROS accumulation, cell viability, MMP, hormone synthesis (P4, E2), cell proliferation, and apoptosis in GCs under HS. The mechanistic role of SOD1 regulation in the heat-stressed GCs was explored. SOD1 gene was successfully silenced in GCs and confirmed at both transcriptional and translational levels. We found that silencing of SOD1 using siRNA under HS aggravated intracellular accumulation of reactive oxygen species, apoptosis, disrupted the mitochondrial membrane potential (MMP), altered transition of the cell cycle, and impaired synthesis of progesterone (P4) and estrogen (E2) in GCs. The associative apoptotic, steroidogenic, and cell cycle genes (BAX, Caspase-3, STAR, Cyp11A1, HSP70, PCNA, and CyclinB1) were used to confirm the results. These results identify a novel role of SOD1 in the modulation of bovine ovarian GC apoptosis, which provides a target for improving the fertility of heat-stressed dairy cows in summer.

Comprehensive RNA-Seq Profiling Reveals Temporal and Tissue-Specific Changes in Gene Expression in Sprague-Dawley Rats as Response to Heat Stress Challenges.

Understanding heat stress physiology and identifying reliable biomarkers are paramount for developing effective management and mitigation strategies. However, little is known about the molecular mechanisms underlying thermal tolerance in animals. In an experimental model of Sprague-Dawley rats subjected to temperatures of 22 ± 1°C (control group; CT) and 42°C for 30 min (H30), 60 min (H60), and 120 min (H120), RNA-sequencing (RNA-Seq) assays were performed for blood (CT and H120), liver (CT, H30, H60, and H120), and adrenal glands (CT, H30, H60, and H120). A total of 53, 1,310, and 1,501 differentially expressed genes (DEGs) were significantly identified in the blood (P < 0.05 and |fold change (FC)| >2), liver (P < 0.01, false discovery rate (FDR)-adjusted P = 0.05 and |FC| >2) and adrenal glands (P < 0.01, FDR-adjusted P = 0.05 and |FC| >2), respectively. Of these, four DEGs, namely Junb, P4ha1, Chordc1, and RT1-Bb, were shared among the three tissues in CT vs. H120 comparison. Functional enrichment analyses of the DEGs identified in the blood (CT vs. H120) revealed 12 biological processes (BPs) and 25 metabolic pathways significantly enriched (FDR = 0.05). In the liver, 133 BPs and three metabolic pathways were significantly detected by comparing CT vs. H30, H60, and H120. Furthermore, 237 BPs were significantly (FDR = 0.05) enriched in the adrenal glands, and no shared metabolic pathways were detected among the different heat-stressed groups of rats. Five and four expression patterns (P < 0.05) were uncovered by 73 and 91 shared DEGs in the liver and adrenal glands, respectively, over the different comparisons. Among these, 69 and 73 genes, respectively, were proposed as candidates for regulating heat stress response in rats. Finally, together with genome-wide association study (GWAS) results in cattle and phenome-wide association studies (PheWAS) analysis in humans, five genes (Slco1b2, Clu, Arntl, Fads1, and Npas2) were considered as being associated with heat stress response across mammal species. The datasets and findings of this study will contribute to a better understanding of heat stress response in mammals and to the development of effective approaches to mitigate heat stress response in livestock through breeding.

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.

Identification of novel molecular markers of mastitis caused by Staphylococcus aureus using gene expression profiling in two consecutive generations of Chinese Holstein dairy cattle.

BACKGROUND: Mastitis in dairy cows caused by Staphylococcus aureus is a major problem hindering economic growth in dairy farms worldwide. It is difficult to prevent or eliminate due to its asymptomatic nature and long persistence of infection. Although transcriptomic responses of bovine mammary gland cells to pathogens that cause mastitis have been studied, the common responses of peripheral blood leukocytes to S. aureus infection across two consecutive generations of dairy cattle have not been investigated. METHODS: In the current study, RNA-Seq was used to profile the transcriptomes of peripheral blood leukocytes sampled from S. aureus-infected mothers and their S. aureus-infected daughters, and also healthy non-infected mothers and their healthy daughters. Differential gene expression was evaluated as follows: 1) S. aureus-infected cows versus healthy non-infected cows (S vs. H, which include all the mothers and daughters), 2) S. aureus-infected mothers versus healthy non-infected mothers (SM vs. HM), and 3) S. aureus-infected daughters versus healthy non-infected daughters (SMD vs. HMD). RESULTS: Analysis of all identified expressed genes in the four groups (SM, SMD, HM, and HMD) showed that EPOR, IL9, IFNL3, CCL26, IL26 were exclusively expressed in both the HM and HMD groups, and that they were significantly (P <  0.05) enriched for the cytokine-cytokine receptor interaction pathway. A total of 17, 13 and 10 differentially expressed genes (DEGs) (FDR P adj. < 0.1 and |FC| > 1.2) were detected in the three comparisons, respectively. DEGs with P <  0.05 and |FC| > 2 were used for functional enrichment analyses. For the S vs. H comparison, DEGs detected included CCL20, IL13 and MMP3, which are associated with the IL-17 signaling pathway. In the SM vs. HM and SMD vs. HMD comparisons, five (BLA-DQB, C1R, C2, FCGR1A, and KRT10) and six (BLA-DQB, C3AR1, CFI, FCAR, FCGR3A, and LOC10498484) genes, respectively, were involved in the S. aureus infection pathway. CONCLUSIONS: Our study provides insights into the transcriptomic responses of bovine peripheral blood leukocytes across two generations of cattle naturally infected with S. aureus. The genes highlighted in this study could serve as expression biomarkers for mastitis and may also contain sequence variation that can be used for genetic improvement of dairy cattle for resilience to mastitis.

Overview of Folic Acid Supplementation Alone or in Combination with Vitamin B12 in Dairy Cattle during Periparturient Period.

The periparturient period is the period from three weeks before calving to three weeks post-calving. This period is important in terms of health, productivity and profitability, and is fundamental to successful lactation. During this period, the animal experiences stress because of hormonal changes due to pregnancy and the significant rise in milk production. In addition, a negative energy balance usually occurs, because the demand for nutrients to sustain milk production increases by more than the nutrient supply during the periparturient period. The immunity of dairy cattle is suppressed around parturition, which increases their susceptibility to infections. Special care regarding nutrition can reduce the risks of metabolism and immunity depression, which dairy cattle face during the periparturient span. Folic acid is relevant in this regard because of its critical role in the metabolism to maintain lactational performance and to improve health. Being a donor of one-carbon units, folic acid has a vital role in DNA and RNA biosynthesis. Generally, the folic acid requirements of dairy cattle can be met by the microbial synthesis in the rumen; however, in special cases, such as during the periparturient period, the requirement for this vitamin strictly increases. Vitamin B12 also has a critical role in the metabolism as a coenzyme of the enzyme methionine synthase for the transfer of a methyl group from folic acid to homocysteine for the regeneration of methionine. In the current review, we highlight the issues facing periparturient dairy cattle, and relevant knowledge and practices, and point out future research directions for utilization of the associated vitamins in ruminants, especially during the periparturient period.

RNAi-Mediated Silencing of Catalase Gene Promotes Apoptosis and Impairs Proliferation of Bovine Granulosa Cells under Heat Stress.

Heat stress in dairy cattle is recognized to compromise fertility by altering the functions of ovarian follicle-enclosed cells, e.g., oocyte and granulosa cells (GCs). Catalase is an antioxidant enzyme that plays a significant role in cellular protection against oxidative damage by the degradation of hydrogen peroxide to oxygen and water. In this study, the role and mechanism of CAT on the heat stress (HS)-induced apoptosis and altered proliferation of bovine GCs were studied. The catalase gene was knocked-down successfully in bovine GCs at both the transcriptional and translational levels. After a successful knockdown using siRNA, GCs were divided into HS (40 °C + NC and 40 °C + CAT siRNA) and 38 °C + NC (NC) groups. The GCs were then examined for ROS, viability, mitochondrial membrane potential (MMP), cell cycle, and biosynthesis of progesterone (P4) and estrogen (E2) hormones. The results indicated that CAT silencing promoted ROS production and apoptosis by up-regulating the Bcl-2-associated X protein (BAX) and Caspase-3 genes both at the transcriptional and translational levels. Furthermore, the knockdown of CAT markedly disrupted the MMP, impaired the production of P4 and E2, altered the progression of the G1 phase of the cell cycle, and decreased the number of cells in the S phase. This was further verified by the down-regulation of proliferating cell nuclear antigen (PCNA), CyclinB1, steroidogenic acute regulatory protein (STAR), and cytochrome P450 family 11 subfamily A member 1 (Cyp11A1) genes. Our study presented a novel strategy to characterize how CAT can regulate cell proliferation and apoptosis in GCs under HS. We concluded that CAT is a broad regulatory marker in GCs by regulating apoptosis, cellular progression, and simultaneously by vital fluctuations in hormonal signaling. Our findings infer a crucial evidence of how to boost the fertility of heat-stressed cows.

Heat Stress Impairs the Physiological Responses and Regulates Genes Coding for Extracellular Exosomal Proteins in Rat.

Heat stress (HS) is challenging in humans and animals as it is a complicated regulatory mechanism. This prompted us to characterize the physiological and molecular responses of a HS-animal model. In this study, a rat model system was developed by using three temperature treatments (40 ℃, 42 ℃, and 43 ℃) and sixteen biochemical indicators in blood at 42 ℃ for 30 min (H30), 60 min (H60), and 120 min (H120). In addition, transcriptomic profiling was carried out in H120-rats' blood, liver, and adrenal gland samples for detection of the genes of interest. Our findings demonstrated that the adrenocorticotropic hormone, catalase, prolactin, growth hormone, and lactic acid have significant spatiotemporal variation in the H120-rats as compared with the control. Furthermore, through transcriptomic screening, we documented a high ratio of differentially expressed genes (DEGs) in adrenal glands, liver, and blood, respectively. Among them, Nup153, Plxnb2, Stx7, Hspa9, Chordc1, Pde4d, Gm2α, and Rnf125 were associated with the regulation of HS and immune response processes. Notably, 36 and 314 of DEGs in blood and adrenal glands were detected in the composition of the extracellular exosome, respectively. Furthermore, the correlation analysis between gene transcripts and biochemical indicator levels identified the Lgals3, S1006, Fn1,F2, and Kng1l1 as key candidate genes for HS encoding extracellular exosomal proteins. On the basis of our results, it was concluded that the current rat model provides a molecular basis for future research in HS resistance in humans and livestock.

Evaluation of heat stress effects on cellular and transcriptional adaptation of bovine granulosa cells.

BACKGROUND: Heat stress is known to affect follicular dynamics, oocyte maturation, and fertilization by impairing steroidogenic ability and viability of bovine granulosa cell (bGCs). The present study explored the physiological and molecular response of bGCs to different heat stress intensities in-vitro. We exposed the primary bGCs to heat stress (HS) at 39 °C, 40 °C and 41 °C along with control samples (38 °C) for 2 h. To evaluate the impact of heat stress on bGCs, several in vitro cellular parameters including cell apoptosis, intracellular reactive oxygen species (ROS) accumulation and HSP70 kinetics were assessed by flow cytometry, florescence microscopy and western blot, respectively. Furthermore, the ELISA was performed to confirm the 17ß-estradiol (E2) and progesterone (P4) levels. In addition, the RNA sequencing (RNA-Seq) method was used to get the molecular based response of bGCs to different heat treatments. RESULTS: Our findings revealed that the HS significantly decreased the cell viability, E2 and P4 levels in bGCs, whereas, increased the cellular apoptosis and ROS. Moreover, the RNA-Seq experiments showed that all the treatments (39 °C, 40 °C and 41 °C) significantly regulated many differentially expressed genes (DEGs) i.e. BCL2L1, STAR, CYP11A1, CASP3, SOD2, HSPA13, and MAPK8IP1 and pathways associated with heat stress, apoptosis, steroidogenesis, and oxidative stress. Conclusively, our data demonstrated that the impact of 40 °C treatment was comparatively detrimental for cell viability, apoptosis and ROS accumulation. Notably, a similar trend of gene expression was reported by RT-qPCR for RNA-seq data. CONCLUSIONS: Our study presented a worthy strategy for the first time to characterize the cellular and transcriptomic adaptation of bGCs to heat stress (39, 40 and 41 °C) in-vitro. The results infer that these genes and pathways reported in present study could be useful candidates/indicators for heat stress research in dairy cattle. Moreover, the established model of bGCs to heat stress in the current study provides an appropriate platform to understand the mechanism of how heat-stressed bGCs can affect the quality of oocytes and developing embryo.

Corticosterone tissue-specific response in Sprague Dawley rats under acute heat stress.

Our study evaluated the physiological responses to acute heat stress in rats via body temperature and tissue corticosterone levels, and investigated the relative tissue response to heat stress based on corticosterone. Body temperature of rats under 22 °C (control) and 42 °C for 30 (H30), 60 (H60) and 120 min (H120) was measured. Correspondingly, corticosterone was analyzed in 11 tissues (adrenal, brain, heart, kidney, liver, lung, leg muscle, blood, stomach, spleen and small intestine). Analysis of variance and correlations were conducted on body temperature and corticosterone levels. The receiver operating characteristic (ROC) analyzed the thermo-sensitivity via corticosterone. Body temperature of rats in H30, H60 and H120 groups were higher (P < 0.05) than the control. Compared to the control, corticosterone levels of heart, stomach and small intestine at H30, corticosterone levels in adrenal, leg muscle and stomach at H60, and corticosterone levels in adrenal, heart, lung, stomach and small intestine at H120 differed (P < 0.05). The corticosterone in lung tissue was an excellent indicator of acute heat stress, with an area under the curve (AUC) of 1.00 at H60 and H120. In order to improve the prediction of acute heat stress, models combining corticosterone levels of multiple tissues reached an AUC of 1.00 for H30, and the sensitivity increased to 100% for H60 and H120. In conclusion, changes in the patterns and thermosensitivity of corticosterone levels associated with the duration of heat stress across body tissues were evidenced. The single and multi-organizational corticosterone models serve as indicators for evaluating heat stress across different time periods.