Genetic parameters and genome-wide association analyses for lifetime productivity in Chinese Holstein cattle.

Lifetime productivity is a trait of great importance to dairy cattle populations as it combines information from production and longevity variables. Therefore, we investigated the genetic background of lifetime productivity in high-producing dairy cattle by integrating genomics and transcriptomics data sets. A total of 3,365,612 test-day milk yield records from 134,029 Chinese Holstein cows were used to define 6 lifetime productivity traits, including lifetime milk yield covering full lifespan and 5 cumulative milk yield traits covering partial lifespan. Genetic parameters were estimated based on univariate and bivariate linear animal models and the Restricted Maximum Likelihood (REML) method. Genome-wide association studies (GWAS) and weighted gene co-expression network analyses (WGCNA) were performed to identify candidate genes associated with lifetime productivity based on genomic data from 3,424 cows and peripheral blood RNA-seq data from 23 cows, respectively. Lifetime milk yield averaged 24,800.8 ± 14,396.6 kg (mean ± SD) across an average of 2.4 parities in Chinese Holstein population. The heritability estimates for lifetime productivity traits ranged from 0.05 (±0.01 for SE) to 0.10 (±0.02 for SE). The estimate of genetic correlation between lifetime milk yield and productive life is 0.88 (±0.3 for SE) while the genetic correlation with 305d milk yield in the first lactation was 0.49 (±0.08 for SE). Absolute values for most genetic correlation estimates between lifetime productivity and type traits were lower than 0.30. Moderate genetic correlations were found between udder related traits and lifetime productivity, such as with udder depth (0.33), rear udder attachment height (0.33), and udder system (0.34). Some single nucleotide polymorphisms and gene co-expression modules significantly associated with lifetime milk yield were identified based on GWAS and WGCNA analyses, respectively. Functional enrichment analyses of the candidate genes identified revealed important pathways related to immune system, longevity, energy utilization and metabolism, and FoxO signaling. The genes NTMT1, FNBP1, and S1PR1 were considered to be the most important candidate genes influencing lifetime productivity in Holstein cows. Overall, our findings indicate that lifetime productivity is heritable in Chinese Holstein cattle and important candidate genes were identified by integrating genomic and transcriptomic data sets.

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.

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.

Accuracy of MRI-Based Radiomics in Diagnosis of Placenta Accreta Spectrum: A PRISMA Systematic Review and Meta-Analysis.

BACKGROUND Placenta accreta syndrome (PAS) can lead to severe obstetric bleeding, and can be life-threatening. This study aimed to assess the precision of radiomics features derived from magnetic resonance imaging (MRI) for diagnosing PAS. MATERIAL AND METHODS A comprehensive search was conducted in the databases PubMed, Embase, Web of Science, and the Cochrane library from inception to October 2023. We included diagnostic accuracy studies utilizing radiomics-MRI in PAS patients, with histopathology serving as the reference standard. The overall diagnostic odds ratio (DOR), sensitivity, specificity, and area under the curve (AUC) were computed to gauge the diagnostic accuracy of MRI-based radiomic features in PAS patients. Quality assessment was performed using the Quality Assessment of Diagnostic Accuracy Studies 2. Statistical analyses were carried out using Stata 14.2, MetaDiSc 1.4, and Review Manager 5.3 software. RESULTS Seven studies involving 672 patients were incorporated. The aggregated DOR, sensitivity, specificity, and AUC for radiomics in detecting PAS were 78% (confidence interval32, 191), 87% (76%, 93%), 92% (89%, 94%), and 0.93 (0.91-0.95), respectively. The meta-analysis revealed notable heterogeneity among the included studies, with no evidence of a threshold effect. Subgroup analysis demonstrated that, in comparison to manual segmentation and validation groups with ≤100 cases and internal validation datasets, automated segmentation, validation groups with >100 cases, and external validation datasets exhibited superior diagnostic performance . CONCLUSIONS Our findings indicate that MRI-based radiomic features perform well in assessing the diagnostic risk of PAS during prenatal diagnosis. This noninvasive and convenient tool may prove valuable in facilitating the identification of PAS.

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.

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.

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.

Extraction and Elevation of Cell-Free DNA under Mastitis and Heat Stress in Dairy Cattle.

In this study, four methods (phenol-chloroform protocol, sodium iodide kit, QIAamp DNA Blood Mini Kit, and TIANamp Micro DNA Kit) were used to extract cell-free DNA (cfDNA) from cattle blood, and the yield and purity of cfDNA varied in four different methods from 0.36 to 0.84 ng/mL for yield and 0.67 to 1.80 (A260/A280) for purity. Compared with other methods, the TIANamp Micro DNA kit performed better in both cfDNA amount and purity (p < 0.05); furthermore, blood cfDNA levels were significantly increased in Holstein dairy cows under the influence of heat stress (p < 0.01) and mastitis (p < 0.0001), which showed a potential power to discriminate mastitis (AUC = 0.99, 95% CI = 0.97 to 1.00) or heat stress (AUC = 0.86, 95% CI = 0.73 to 0.98) in cows. In brief, we established a complete experimental system for the extraction of cfDNA from cattle blood based on the high-yielding method of the TIANamp Micro DNA Kit and showed the effect of mastitis and heat stress on cfDNA levels in cattle blood for the first time. Our findings suggested that cfDNA in cattle blood may be a useful marker to measure mastitis and heat stress in dairy cattle.

Mesothelial cyst of uterus in a nullipara patient: A case report.

INTRODUCTION: Uterine mesothelial cysts represent a diagnostic challenge because of their low incidence, with very few cases reported in the English literature. PATIENT CONCERNS: We report the case of a 27-year-old nullipara woman complaining of self-discovery of a mass in the abdomen for 1 week. Supersonic examination revealed a pelvic cystic lesion measuring 8.9 × 8.2 cm. The patient underwent exploratory single-port laparoscopic surgery and had a large uterine cystic mass located within the posterior wall of the uterus. DIAGNOSIS: After excision of the uterine cyst, the final histopathological diagnosis was uterine mesothelial cyst. INTERVENTIONS: We treated her with a single-port laparoscopic uterine cystectomy. OUTCOMES: Close follow-up of the case for 2 years showed that the patient was free of any symptoms, and no recurrence was noted. LESSONS: Uterine mesothelial cysts are extremely rare. They are often misdiagnosed by clinicians as extrauterine masses or cystic degeneration of leiomyomas. This report aims to share a rare case of uterine mesothelial cyst and improve gynecologists' academic vision of the disease.

Genomic and transcriptomic analyses enable the identification of important genes associated with subcutaneous fat deposition in Holstein cows.

Subcutaneous fat deposition has many important roles in dairy cattle, including immunological defense and mechanical protection. The main objectives of this study are to identify key candidate genes regulating subcutaneous fat deposition in high-producing dairy cows by integrating genomic and transcriptomic datasets. A total of 1654 genotyped Holstein cows are used to perform a genome-wide association study (GWAS) aiming to identify genes associated with subcutaneous fat deposition. Subsequently, weighted gene co-expression network analyses (WGCNA) are conducted based on RNA-sequencing data of 34 cows and cow yield deviations of subcutaneous fat deposition. Lastly, differentially expressed (DE) mRNA, lncRNA, and differentially alternative splicing genes are obtained for 12 Holstein cows with extreme and divergent phenotypes for subcutaneous fat deposition. Forty-six protein-coding genes are identified as candidate genes regulating subcutaneous fat deposition in Holstein cattle based on GWAS. Eleven overlapping genes are identified based on the analyses of DE genes and WGCNA. Furthermore, the candidate genes identified based on GWAS, WGCNA, and analyses of DE genes are significantly enriched for pathways involved in metabolism, oxidative phosphorylation, thermogenesis, fatty acid degradation, and glycolysis/gluconeogenesis pathways. Integrating all findings, the NID2, STARD3, UFC1, DEDD, PPP1R1B, and USP21 genes are considered to be the most important candidate genes influencing subcutaneous fat deposition traits in Holstein cows. This study provides novel insights into the regulation mechanism underlying fat deposition in high-producing dairy cows, which will be useful when designing management and breeding strategies.

The Utility of the Ultrasound "Superimposed-Line" Sign at the Junction of the Vomer and Maxilla in First-Trimester Screening for Fetal Cleft Palate: A Case-Control Study.

INTRODUCTION: The current retrospective case-control study evaluates the diagnostic value of screening for a fetal cleft palate by using the ultra-sound-based observation of the "superimposed-line" sign appearing at the junction of the vomer and maxilla in the first trimester of pregnancy. METHODS: Retrospective analyses were performed of ultrasonographic images of nuchal translucency obtained during the first trimester of pregnancy (11-13+6 weeks) from 45 fetuses with a cleft palate later confirmed following parturition or induced labor (cases) and 4,500 normal fetuses confirmed after parturition (controls). Ultrasonographic features of the "superimposed-line" sign were observed and recorded, and between-group comparisons were performed. RESULTS: The "superimposed-line" sign was absent in 39 cases (86.67%), including 4 (8.89%) with simple secondary hard palate cleft and 35 (77.78%) with secondary hard palate cleft complicated by a primary cleft palate. The "superimposed-line" sign was shown in 6 cases (13.33%), including 2 (4.44%) with a simple secondary soft palate cleft, 1 (2.22%) with a simple secondary bifid uvula, and 3 (6.67%) with a simple primary cleft palate. Among the 4,500 controls, 31 fetuses showed an absence of the "superimposed-line" sign (0.69%) and 4,469 showed the "superimposed-line" sign (99.31%). The between-group difference was significant (p < 0.05). The sensitivity, specificity, positive predictive value, and negative predictive values of the "superimposed-line" sign in the first trimester of pregnancy for predicting fetal cleft palate were 86.67% (39/45), 99.31% (4,469/4,500), 55.71% (39/70), and 99.86% (4,469/4,475), respectively. CONCLUSION: The "superimposed-line" sign did not appear in fetuses with secondary hard palate cleft and primary cleft palate only when a secondary hard palate cleft is present. The sign appeared in normal fetuses and those with a simple primary cleft palate, simple secondary soft palate cleft, or a simple secondary bifid uvula. Based on these results, we propose that the "superimposed-line" sign in the mid-sagittal plane of the fetal face in the first trimester of pregnancy (11-13+6 weeks) is an important tool in screening for fetal cleft palate, especially secondary hard palate cleft.

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.

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.

Improvement in L-ornithine production from mannitol via transcriptome-guided genetic engineering in Corynebacterium glutamicum.

BACKGROUND: L-Ornithine is an important medicinal intermediate that is mainly produced by microbial fermentation using glucose as the substrate. To avoid competition with human food resources, there is an urgent need to explore alternative carbon sources for L-ornithine production. In a previous study, we constructed an engineered strain, Corynebacterium glutamicum MTL13, which produces 54.56 g/L of L-ornithine from mannitol. However, compared with the titers produced using glucose as a substrate, the results are insufficient, and further improvement is required. RESULTS: In this study, comparative transcriptome profiling of MTL01 cultivated with glucose or mannitol was performed to identify novel targets for engineering L-ornithine-producing strains. Guided by the transcriptome profiling results, we modulated the expression of qsuR (encoding a LysR-type regulator QsuR), prpC (encoding 2-methylcitrate synthase PrpC), pdxR (encoding a MocR-type regulator PdxR), acnR (encoding a TetR-type transcriptional regulator AcnR), CGS9114_RS08985 (encoding a hypothetical protein), and CGS9114_RS09730 (encoding a TetR/AcrR family transcriptional regulator), thereby generating the engineered strain MTL25 that can produce L-ornithine at a titer of 93.6 g/L, representing a 71.6% increase as compared with the parent strain MTL13 and the highest L-ornithine titer reported so far for C. glutamicum. CONCLUSIONS: This study provides novel indirect genetic targets for enhancing L-ornithine accumulation on mannitol and lays a solid foundation for the biosynthesis of L-ornithine from marine macroalgae, which is farmed globally as a promising alternative feedstock.

Development and application of arachnomelia syndrome genetic detection in four Chinese dual-purpose cattle populations.

Arachnomelia syndrome (AS) is an autosomal recessive hereditary disorder in cattle, and affected calves are usually stillborn and characterized by complex anomalies. Therefore, identification of the carrier animals based on genetic tests is important for the control and elimination of this defect. The aim of this study was to build an effective workflow to routinely screen the AS mutations in bovine MOSC1 and SUOX genes and determine individuals carrying the AS mutations in four Chinese cattle populations. By combining the fluorescence-labeled PCR and capillary electrophoresis, we established a convenient and cost-effective workflow to detect two AS casual mutations simultaneously. Sanger sequencing was further used as a validation criterion and showed that 100% of the tests (37/37) had consistent results with genotype calls determined by our established workflow. Then, 582 bulls and 1-926 cows from Chinese dual-purpose cattle populations of Simmental, Sanhe, Shuxuan, and Xinjiang Brown were subjected to AS detection. The results showed that four bulls and 11 cows in the Simmental population, and six bulls and six cows in the Sanhe population were identified as AS carriers with the MOCS1 mutation c.1224_1225delCA. However, no animal was found to carry the c.363_364insG mutation in the SUOX gene. The frequencies of AS carriers were 1.08% and 1.65% in the Simmental and Sanhe populations, respectively, with a frequency of 1.076% in four populations. The pedigree analysis found that all carriers could be traced back to a common ancestor, the German Simmental sire ROMEL. Those findings suggested that this genetic defect spread into China mainly through the wide use of ROMEL. In conclusion, the occurrence of AS has not had a wide impact on the Chinese cattle industry; however, a screening system and mating strategy should be employed to gradually eliminate this recessive gene from the Chinese dual-purpose cattle population.

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.

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.

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.

JAC4 Protects from X-ray Radiation-Induced Intestinal Injury by JWA-Mediated Anti-Oxidation/Inflammation Signaling.

Radiation-induced intestinal injury is one of the major side effects in patients receiving radiation therapy. There is no specific treatment for radiation-induced enteritis in the clinic. We synthesized a compound, named JAC4, which is an agonist and can increase JWA protein expression. JWA has been shown to reduce oxidative stress, DNA damage, anti-apoptosis, and anti-inflammatory; in addition, the small intestine epithelium showed dysplasia in JWA knockout mice. We hypothesized that JAC4 might exert a protective effect against radiation-induced intestinal damage. Herein, X-ray radiation models were built both in mice and in intestinal crypt epithelial cells (IEC-6). C57BL/6J mice were treated with JAC4 by gavage before abdominal irradiation (ABI); the data showed that JAC4 significantly reduced radiation-induced intestinal mucosal damage and increased the survival rate. In addition, radiation-induced oxidative stress damage and systemic inflammatory response were also mitigated by JAC4 treatment. Moreover, JAC4 treatment alleviated DNA damage, decreased cell apoptosis, and maintained intestinal epithelial cell proliferation in mice. In vitro data showed that JAC4 treatment significantly inhibited ROS formation and cell apoptosis. Importantly, all the above protective effects of JAC4 on X-ray radiation-triggered intestinal injury were no longer determined in the intestinal epithelium of JWA knockout mice. Therefore, our results provide the first evidence that JAC4 protects the intestine from radiation-induced enteritis through JWA-mediated anti-oxidation/inflammation signaling.

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.