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.

Characterization of novel lncRNA muscle expression profiles associated with meat quality in beef cattle.

The aim of this study was to identify novel lncRNA differentially expressed (DE) between divergent animals for beef tenderness and marbling traits in Nellore cattle. Longissimus thoracis muscle samples from the 20 most extreme bulls (of 80 bulls set) for tenderness, tender (n = 10) and tough (n = 10) groups, and marbling trait, high (n = 10) and low (n = 10) groups were used to perform transcriptomic analysis using RNA-Sequencing. For tenderness, 29 lncRNA were DE (p-value ≤ 0.01) in tough beef animals in relation to tender beef animals. We observed that genic lncRNAs, for example, lncRNA_595.1, were overlapping exonic part of the PICK gene, while lncRNA_3097.2 and lncRNA_3129.5 overlapped intronic part of the genes GADL1 and PSMD6. The lncRNA associated with PICK1, GADL1, and PMD6 genes were enriched in the pathways associated with the ionotropic glutamate receptor, gamma-aminobutyric acid synthesis, and the ubiquitin-proteasome pathway. For marbling, 50 lncRNA were DE (p-value ≤ 0.01) in high marbling group compared with low marbling animals. The genic lncRNAs, such as lncRNA_3191.1, were overlapped exonic part of the ITGAL gene, and the lncRNA_512.1, lncRNA_3721.1, and lncRNA_41.4 overlapped intronic parts of the KRAS and MASP1 genes. The KRAS and ITGAL genes were enriched in pathways associated with integrin signaling, which is involved in intracellular signals in response to the extracellular matrix, including cell form, mobility, and mediates progression through the cell cycle. In addition, the lincRNAs identified to marbling trait were associated with several genes related to calcium binding, muscle hypertrophy, skeletal muscle, lipase, and oxidative stress response pathways that seem to play a role important in the physiological processes related to meat quality. These findings bring new insights to better understand the biology mechanisms involved in the gene regulation of these traits, which will be valuable for a further investigation of the interactions between lncRNA and mRNAs, and of how these interactions may affect meat quality traits.

Small genetic variation affecting mRNA isoforms associated with marbling and meat color in beef cattle.

The aim of this study was to identify mRNA isoforms and small genetic variants that may be affecting marbling and beef color in Nellore cattle. Longissimus thoracis muscle samples from 20 bulls with different phenotypes (out of 80 bulls set) for marbling (moderate (n = 10) and low (n = 10) groups) and beef color (desirable (n = 10) and undesirable (n = 9) group) traits were used to perform transcriptomic analysis using RNA sequencing. Fourteen and 15 mRNA isoforms were detected as differentially expressed (DE) (P-value ≤ 0.001) between divergent groups for marbling and meat color traits, respectively. Some of those DE mRNA isoforms have shown sites of splicing modified by small structural variants as single nucleotide variant (SNV), insertion, and/or deletion. Enrichment analysis identified metabolic pathways, such as O2/CO2 exchange in erythrocytes, tyrosine biosynthesis, and phenylalanine degradation. The results obtained suggest potential key regulatory genes associated with these economically important traits for the beef industry and for the consumer.

Gene expression profile of the taimen Hucho taimen in response to acute temperature changes.

The endangered cold-water fish species taimen (Hucho taimen) suffer acute temperature changes in culture and wild conditions. Understanding the effects of acute temperature changes on physiological processes of this species is essential for aquaculture practices and conservation. Liver transcriptomic profiles of the taimen (n = 24) exposed to acute temperature decrease (from 20 °C to 10 °C) and acute temperature increase (from 10 °C to 20 °C) was evaluated using high-throughput RNA-Sequencing. Samples were collected at day 0, 1, 7 and 35 in both treatments. Compared to day 0, the total numbers of differentially expressed genes (DEGs) in the taimen after acute temperature decrease were 173, 226 and 42 at day 1, 7 and 35, respectively, and the total numbers of DEGs following acute temperature increase were 260, 253 and 282 at day 1, 7 and 35, respectively. Particularly, 14 key regulatory genes were commonly found between both acute temperature treatments. Functional analysis based on the commonly identified DEGs revealed important metabolic pathways related to metabolism and immune function, suggesting a specific response mechanism of taimen against cold and heat shock. The results may assist in developing management strategies for stress mediation caused by acute temperature changes in the taimen and other cold water fish.

Identification of novel mRNA isoforms associated with meat tenderness using RNA sequencing data in beef cattle.

The Warner-Bratzler shear force (WBSF) and myofibrillar fragmentation index (MFI) are complementary methodologies used to measure beef tenderness. Longissimus thoracis samples from the 20 most extreme bulls (out of 80 bulls set) for WBSF (tender (n = 10) and tough (n = 10)) and MFI (high (n = 10) and low (n = 10)) traits were collected to perform transcriptomic analysis using RNA-Sequencing. All analysis were performed through CLC Genomics Workbench. A total of 39 and 27 transcripts for WBSF and MFI phenotypes were DE, respectively. The possible DE novel mRNA isoforms, for WBSF and MFI traits, are myosin encoders (e.g. MYL1 and MYL6). In addition, we identified potential mRNA isoforms related to genes affecting the speed fibers degradation during the meat aging process. The DE novel transcripts are transcripted by genes with biological functions related to oxidative process, energy production and striated muscle contraction. The results suggest that the identified mRNA isoforms could be used as potential candidate to select animals in order to improve meat tenderness.

Use of gene expression profile to identify potentially relevant transcripts to myofibrillar fragmentation index trait.

The myofibrillar fragmentation index (MFI) is an indicative trait for meat tenderness. Longissimus thoracis muscle samples from the 20 most extreme bulls (out of 80 bulls set) for MFI (high (n = 10) and low (n = 10) groups) trait were used to perform transcriptomic analysis, using RNA Sequencing (RNA-Seq). An average of 24.616 genes was expressed in the Nellore muscle transcriptome analysis. A total of 96 genes were differentially expressed (p value ≤ 0.001) between the two groups of divergent bulls for MFI. The HEBP2 and BDH1 genes were overexpressed in animals with high MFI. The MYBPH and MYL6, myosin encoders, were identified. The differentially expressed genes were related to increase mitochondria efficiency, especially in cells under oxidative stress conditions, and these also were related to zinc and calcium binding, membrane transport, and muscle constituent proteins, such as actin and myosin. Most of those genes were involved in metabolic pathways of oxidation-reduction, transport of lactate in the plasma membrane, and muscle contraction. This is the first study applying MFI phenotypes in transcriptomic studies to identify and understand differentially expressed genes for beef tenderness. These results suggest that differences detected in gene expression between high and low MFI animals are related to reactive mechanisms and structural components of oxidative fibers under the condition of cellular stress. Some genes may be selected as positional candidate genes to beef tenderness, MYL6, MYBPH, TRIM63, TRIM55, TRIOBP, and CHRNG genes. The use of MFI phenotypes could enhance results of meat tenderness studies.

Transcriptome profiling of muscle in Nelore cattle phenotypically divergent for the ribeye muscle area.

This study aimed to use RNA-Seq to identify differentially expressed genes (DEGs) in muscle of uncastrated Nelore males phenotypically divergent for ribeye muscle area (REA). A total of 80 animals were phenotyped for REA, and 15 animals each with the highest REA and the lowest REA were selected for analyses. DEGs found (N = 288) belonging to families related to muscle cell growth, development, motility and proteolysis, such as actin, myosin, collagen, integrin, solute carrier, ubiquitin and kelch-like. Functional analysis showed that many of the significantly enriched gene ontology terms were closely associated with muscle development, growth, and degradation. Through co-expression network analysis, we predicted three hub genes (PPP3R1, FAM129B and UBE2G1), these genes are involved in muscle growth, proteolysis and immune system. The genes expression levels and its biological process found this study may result in differences in muscle deposition, and therefore, Nelore animals with different REA proportions.

Prediction of hub genes associated with intramuscular fat content in Nelore cattle.

BACKGROUND: The aim of this study was to use transcriptome RNA-Seq data from longissimus thoracis muscle of uncastrated Nelore males to identify hub genes based on co-expression network obtained from differentially expressed genes (DEGs) associated with intramuscular fat content. RESULTS: A total of 30 transcriptomics datasets (RNA-Seq) obtained from longissimus thoracis muscle were selected based on the phenotypic value of divergent intramuscular fat content: 15 with the highest intramuscular fat content (HIF) and 15 with the lowest intramuscular fat content (LIF). The transcriptomics datasets were aligned with a reference genome and 65 differentially expressed genes (DEGs) were identified, including 21 upregulated and 44 downregulated genes in HIF animals. The normalized count data from DEGs was then used for co-expression network construction. From the co-expression network, four modules were identified. The topological properties of the network were analyzed; those genes engaging in the most interactions (maximal clique centrality method) with other DEGs were predicted to be hub genes (PDE4D, KLHL30 and IL1RAP), which consequently may play a role in cellular and/or systemic lipid biology in Nelore cattle. Top modules screened from the gene co-expression network were identify. The two candidate modules had clear associated biological pathways related to fat development, cell adhesion, and muscle differentiation, immune system, among others. The hub genes belonged in top modules and were downregulated in HIF animals. PDE4D and IL1RAP have known effects on lipid metabolism and the immune system through the regulation of cAMP signaling. Given that cAMP is known to play a role in lipid systems, PDE4D and IL1RAP downregulation may contribute to increased levels of intracellular cAMP and thus may have effects on IF content differences in Nelore cattle. KLHL30 may have effects on muscle metabolism. Klhl protein families play a role in protein degradation. However, the downregulation of this gene and its role in lipid metabolism has not yet been clarified. CONCLUSIONS: The results reported in this study indicate candidate genes and molecular mechanisms involved in IF content difference in Nelore cattle.

Prediction of meat quality traits in Nelore cattle by near-infrared reflectance spectroscopy.

The main definition for meat quality should include factors that affect consumer appreciation of the product. Physical laboratory analyses are necessary to identify factors that affect meat quality and specific equipment is used for this purpose, which is expensive and destructive, and the analyses are usually time consuming. An alternative method to performing several beef analyses is near-infrared reflectance spectroscopy (NIRS), which permits to reduce costs and to obtain faster, simpler, and nondestructive measurements. The objective of this study was to evaluate the feasibility of NIRS to predict shear force [Warner-Bratzler shear force (WBSF)], marbling, and color (*a = redness; b* = yellowness; and L* = lightness) in meat samples of uncastrated male Nelore cattle, that were approximately 2-yr-old. Samples of longissimus thoracis (n = 644) were collected and spectra were obtained prior to meat quality analysis. Multivariate calibration was performed by partial least squares regression. Several preprocessing techniques were evaluated alone and in combination: raw data, reduction of spectral range, multiplicative scatter correction, and 1st derivative. Accuracies of the calibration models were evaluated using the root mean square error of calibration (RMSEC), root mean square error of prediction (RMSEP), coefficient of determination in the calibration (R2C), and prediction (R2P) groups. Among the different preprocessing techniques, the reduction of spectral range provided the best prediction accuracy for all traits. The NIRS showed a better performance to predict WBSF (RMSEP = 1.42 kg, R2P = 0.40) and b* color (RMSEP = 1.21, R2P = 0.44), while its ability to accurately predict L* (RMSEP = 1.98, R2P = 0.16) and a* (RMSEP = 1.42, R2P = 0.17) was limited. NIRS was unsuitable to predict subjective meat quality traits such as marbling in Nelore cattle.