mRNA-Seq of testis and liver tissues reveals a testis-specific gene and alternative splicing associated with hybrid male sterility in dzo.

Alternative splicing (AS) plays an important role in the co-transcription and post-transcriptional regulation of gene expression during mammalian spermatogenesis. The dzo is the male F1 offspring of an interspecific hybrid between a domestic bull (Bos taurus ♂) and a yak (Bos grunniens ♀) which exhibits male sterility. This study aimed to identify the testis-specific genes and AS associated with hybrid male sterility in dzo. The iDEP90 program and rMATS software were used to identify the differentially expressed genes (DEG) and differential alternative splicing genes (DSG) based on RNA-seq data from the liver (n = 9) and testis (n = 6) tissues of domestic cattle, yak, and dzo. Splicing factors (SF) were obtained from the AmiGO2 and the NCBI databases, and Pearson correlation analysis was performed on the differentially expressed SFs and DSGs. We focused on the testis-specific DEGs and DSGs between dzo and cattle and yak. Among the top 3,000 genes with the most significant variations between these 15 samples, a large number of genes showed testis-specific expression involved with spermatogenesis. Cluster analysis showed that the expression levels of these testis-specific genes were dysregulated during mitosis with a burst downregulation during the pachynema spermatocyte stage. The occurrence of AS events in the testis was about 2.5 fold greater than in the liver, with exon skipping being the major AS event (81.89% to 82.73%). A total of 74 DSGs were specifically expressed in the testis and were significantly enriched during meiosis I, synapsis, and in the piRNA biosynthesis pathways. Notably, STAG3 and DDX4 were of the exon skipping type, and DMC1 was a mutually exclusive exon. A total of 36 SFs were significantly different in dzo testis, compared with cattle and yak. DDX4, SUGP1, and EFTUD2 were potential SFs leading to abnormal AS of testis-specific genes in dzo. These results show that AS of testis-specific genes can affect synapsis and the piRNA biosynthetic processes in dzo, which may be important factors associated with hybrid male sterility in dzo.

The interspecific hybrid offspring of a domestic bull (Bos taurus) and a yak (Bos grunniens) display heterosis in meat and milk production. The hybrid offspring are particularly adaptable to the harsh environments of the Qinghai-Tibet Plateau. However, the male F1 to F3 offspring of this interspecies hybrid are infertile, and spermatogenesis is arrested at meiosis preventing the prolonged utilization of the benefits of heterosis. This study aimed to identify the testis-specific genes and alternative splicing (AS) associated with hybrid male sterility using RNA-Seq data from the liver and testis tissues of domestic cattle, yaks, and their F1 offspring (dzo). The expression of the testis-specific genes became disordered during mitosis and meiosis in dzo. Their testis-specific genes with AS events were enriched during synapsis and in the piRNA biosynthetic processes. In addition, we identified the potential splicing factors associated with abnormal testis-specific AS gene expression in dzo. These results reveal the important role of AS in the meiotic arrest of dzo.

When Livestock Genomes Meet Third-Generation Sequencing Technology: From Opportunities to Applications.

Third-generation sequencing technology has found widespread application in the genomic, transcriptomic, and epigenetic research of both human and livestock genetics. This technology offers significant advantages in the sequencing of complex genomic regions, the identification of intricate structural variations, and the production of high-quality genomes. Its attributes, including long sequencing reads, obviation of PCR amplification, and direct determination of DNA/RNA, contribute to its efficacy. This review presents a comprehensive overview of third-generation sequencing technologies, exemplified by single-molecule real-time sequencing (SMRT) and Oxford Nanopore Technology (ONT). Emphasizing the research advancements in livestock genomics, the review delves into genome assembly, structural variation detection, transcriptome sequencing, and epigenetic investigations enabled by third-generation sequencing. A comprehensive analysis is conducted on the application and potential challenges of third-generation sequencing technology for genome detection in livestock. Beyond providing valuable insights into genome structure analysis and the identification of rare genes in livestock, the review ventures into an exploration of the genetic mechanisms underpinning exemplary traits. This review not only contributes to our understanding of the genomic landscape in livestock but also provides fresh perspectives for the advancement of research in this domain.

Evaluation of MYBL1 as the master regulator for pachytene spermatocyte genes dysregulated in interspecific hybrid dzo.

Misregulation of spermatogenesis transcription factors (TF) in hybrids can lead to misexpression, which is a mechanism for hybrid male sterility (HMS). We used dzo (male offspring of Bos taurus ♂ × Bos grunniens ♀) in bovines to investigate the relationship of the key TF with HMS via RNA sequencing and assay for transposase-accessible chromatin with high-throughput sequencing analyses. RNA sequencing showed that the widespread misexpression in dzo was associated with spermatogenesis-related genes and somatic or progenitor genes. The transition from leptotene or zygotene spermatocytes to pachytene spermatocytes may be the key stage for meiosis arrest in dzo. The analysis of TF-binding motif enrichment revealed that the male meiosis-specific master TF MYB proto-oncogene like 1 (MYBL1, known as A-MYB) motif was enriched on the promoters of downregulated pachytene spermatocyte genes in dzo. Assay for transposase-accessible chromatin with high-throughput sequencing revealed that TF-binding sites for MYBL1, nuclear transcription factor Y, and regulatory factor X were enriched in the low-chromatin accessibility region of dzo. The target genes of the MYBL1-binding motif were associated with meiosis-specific genes and significantly downregulated in dzo testis. The transcription factor MYBL1 may be the candidate master regulator for pachytene spermatocyte genes dysregulated in interspecific HMS dzo. This study reported that a few upstream TF regulation changes might exert a cascading effect downstream in a regulatory network as a mechanism for HMS.

N6-methyladenosine RNA demethylase ALKBH5 is testis-specifically downregulated in hybrid male sterile dzo and is a target gene of bta-miR-200a.

N6-methyladenosine (m6A) is the most common RNA methylation modification in mammals, which is controlled in the male germline to ensure coordinated gene expression in the entire process of spermatogenesis. Dzo is the male offspring of a cross between the domestic cattle (Bos taurus) and yak (Bos grunniens), and is sterile. This study aimed to investigate whether m6A-associated genes are linked with dzo male sterility. The mRNA expression pattern of m6A-associated genes and spermatogenesis-related genes modified by m6A was characterized in cattle, yak, and dzo. Compared with fertile cattle and yak, m6A erasing (ALKBH5 and FTO), writing (METTL14, WTAP, and ZC3H13), and reading (YTHDC2, YTHDF1, and YTHDF2) were testis-specifically downregulated in infertile dzo. The expression of m6A target genes in spermatogonial self-renewal and proliferation (BCL6B, FOXO1, TAF4B, and FGFR1) and differentiation genes (DNMT3B and SOHLH2) were dereguleted in dzo testis. Immunofluorescent staining showed that intense ALKBH5 immunoreactivity was present in spermatogonia, primary spermatocytes, and round spermatids of cattle and yak testis. However, the number of ALKBH5 immunoreactive-positive cells were significantly reduced in dzo testis, especially in primary spermatocytes and round spermatids. Whole genome bisulfite-seq data showed that the promoter regions of FTO and YTHDC2 genes were hypermethylated in dzo testis. Moreover, bta-miR-200a was significantly downregulated in dzo testis, and it targeted the m6A-associated genes such as ALKBH5, FTO, WTAP, and YTHDF2. In conclusion, mRNA of ALKBH5 was testis-specifically downregulated in dzo, which may be because fewer specific spermatogenic cells express this gene. The role of m6A-associated genes in dzo male sterility and the interaction of DNA methylation and miRNA with m6A-associated protein expression need to be further explored.

Inheritance patterns of leukocyte gene expression under heat stress in F1 hybrid cattle and their parents.

Crossbreeding capitalizes on heterosis effects and results in increased performance of crossbred animals. Dominance hypothesis and overdominance hypothesis are 2 common models proposed to explain heterosis. Differential gene expression between parents and hybrids is hypothesized to be responsible for heterosis. This study aimed to investigate the heat tolerance and inheritance patterns of leukocyte transcriptomics in F1 hybrid cattle (Angus males × Droughtmaster females) and their parents Red Angus (AN) and Droughtmaster (DR) under heat stress. According to the respiratory rate and heat tolerance coefficient index, DR was better adapted to heat stress than AN. The physiological responses to heat stress of F1 hybrids were similar to AN. We identified 802 differentially expressed genes in leukocytes between AN and DR under heat stress using mRNA sequencing. Compared with AN, upregulated genes in DR were enriched in biological processes of response to stress, external and chemical stimulus, and cytokine, cell surface receptor signaling pathway, and cardiovascular system development. In contrast, upregulated genes in AN were enriched in B cell activation and regulation of B cell activation. Gene expression levels can be inherited additively or nonadditively and are classified into additive (35%), dominance (44%), and overdominance and underdominance (18%) modes in F1 hybrids and their parents. Inheritance patterns of gene expression showed that 97% (249/255) of the dominant genes were classified as paternal AN dominant in hybrids. The paternal imprinted PEG10 gene and its regulatory transcription factor MYC showed an AN dominant expression pattern. The MYC interacted with most AN dominant genes. These transcriptomic analyses revealed that DR and AN had specific cellular and humoral immunity and cardiovascular systems development function under heat stress. Inheritance pattern analyses from gene expression partly explained phenotypic differences between parents and F1 hybrids. The paternal imprinted PEG10 gene interaction with transcription factor MYC may contribute to explaining paternal dominant gene expression in hybrids.

Promoter hypermethylation of PIWI/piRNA pathway genes associated with diminished pachytene piRNA production in bovine hybrid male sterility.

Hybrid male sterility (HMS) is a postzygotic reproductive isolation mechanism that enforces speciation. A bovine example of HMS is the yattle (also called dzo), an interspecies hybrid of taurine cattle (Bos taurus) and yak (Bos grunniens). The molecular mechanisms underlying HMS of yattle are not well understood. Epigenetic modifications of DNA methylation and P-element induced wimpy testis (PIWI)-interacting RNA (piRNAs) are important regulators in spermatogenesis. In this study, we investigated DNA methylation patterns and piRNA expression in adult testes in hybrid infertile yattle bulls and fertile cattle and yak bulls using whole genome bisulphite-seq and small RNA-seq. Promoter hypermethylation in yattle were associated with DNA methylation involved in gamete generation, piRNA metabolic processes, spermatogenesis, and spermatid development (P < 2.6 × 10-5). Male infertility in yattle was associated with the promoter hypermethylation-associated silencing of PIWI/piRNA pathway genes including PIWIL1, DDX4, PLD6, MAEL, FKBP6, TDRD1 and TDRD5. The downstream effects of silencing these genes were diminished production of 29- to 31- nucleotide pachytene piRNAs in yattle testes. Hypermethylation events at transposable element loci (LINEs, SINEs, and LTRs) were found in yattle. LINE-derived prepachytene piRNAs increased and SINE-derived prepachytene piRNAs were reduced in yattle testes. Our data suggests that DNA methylation affects the PIWI/piRNA pathway and is involved in gene expression and pachytene piRNA production during spermatogenesis in bovine HMS. DNA hypermethylation and disruption of piRNA production contributed to unsuccessful germ cell development that may drive bovine HMS.

Comparison of Y-chromosome-linked TSPY, TSPY2, and PRAMEY genes in Taurus cattle, yaks, and interspecific hybrid bulls.

Domestic yaks (Bos grunniens) and domestic Taurus cattle (Bos taurus) are closely related. An interesting phenomenon in interspecific crossings is male sterility in the F1 hybrid (yattle) and F2 backcross, with no late meiotic cells or spermatids in the seminiferous tubules. The mammalian Y chromosome is crucial for spermatogenesis and male fertility. This study investigated the copy number variations and mRNA of Y-transitional region genes TSPY2 (testis specific protein, Y-linked 2 and testis-specific Y-encoded protein 3-like) and PRAMEY (preferentially expressed antigen in melanoma, Y-linked), and Y-ampliconic region genes TSPY (testis-specific Y-encoded protein 1-like), ZNF280BY (zinc finger protein 280B, Y-linked) and HSFY (heat-shock transcription factor, Y-linked) in mature testes from Taurus cattle, yaks, and yattle. Phylogenetic trees divided 33 copies of TSPY into major 2 types (TSPY-T1 and TSPY-T2), 19 copies of TSPY2 into 2 types (TSPY2-T1 and T2), and 8 copies of PRAMEY into 4 types (PRAMEY-T1 to T4). Searching by the Basic Local Alignment Search Tool of the TSPY2 coding sequences in GenBank revealed that TSPY2 was conserved in Bovidae. The TSPY2-T2 sequences were absent, whereas PRAMEY-T2 and PRAMEY-T4 were amplified on the yak Y chromosome. The average copy numbers of TSPY-T2 and ZNF280BY were significantly different between cattle and yaks. The TSPY-T2, TSPY2, PRAMEY, ZNF280BY, and HSFY genes were uniquely or predominantly expressed in testes. Reverse-transcription quantitative PCR showed that the TSPY-T2, PRAMEY-T2, HSFY, ZNF280BY, protamine 1 (PRM1), and protamine 2 (PRM2) genes were almost not expressed in yattle. The PRM1 and PRM2 genes are used as positive markers for spermatozoa. Thus, our results showed that the genomic structure of the Y-transitional and Y-ampliconic region differed between Taurus cattle and yaks. Dysregulated expression of Y-ampliconic region genes TSPY-T2, HSPY, ZNF280BY, and Y-transitional region gene PRAMEY-T2 may be associated with hybrid male sterility in yattle.

MiR-1246 is upregulated and regulates lung cell apoptosis during heat stress in feedlot cattle.

Globally, heat stress seriously threatens productivity of cattle. The objective of this study was to identify novel miRNAs that regulated heat stress in feedlot cattle. Experiment was conducted under heat stress and normal conditions. With profiling miRNAs of each feedlot cattle, our results showed the level of miR-1246 was significantly increased in these heat-stressed cattle (P < 0.05). Furthermore, by using bioinformatics analysis and luciferase reporter assays combined with qPCR and western blot, we found miR-1246 negatively regulated poly (C) binding protein 2 (PCBP2) and cAMP response element binding protein-like 2 (CREBL2) mRNA and protein levels through binding to the 3'-UTR region (P < 0.05); further, it inhibited heat-induced apoptosis in lung cells. Finally, our results suggested that miR-1246 plays an important role in heat stress and it has the potential to be a novel modulation factor for heat stress in feedlot cattle.

New Insights on Water Buffalo Genomic Diversity and Post-Domestication Migration Routes From Medium Density SNP Chip Data.

The domestic water buffalo is native to the Asian continent but through historical migrations and recent importations, nowadays has a worldwide distribution. The two types of water buffalo, i.e., river and swamp, display distinct morphological and behavioral traits, different karyotypes and also have different purposes and geographical distributions. River buffaloes from Pakistan, Iran, Turkey, Egypt, Romania, Bulgaria, Italy, Mozambique, Brazil and Colombia, and swamp buffaloes from China, Thailand, Philippines, Indonesia and Brazil were genotyped with a species-specific medium-density 90K SNP panel. We estimated the levels of molecular diversity and described population structure, which revealed historical relationships between populations and migration events. Three distinct gene pools were identified in pure river as well as in pure swamp buffalo populations. Genomic admixture was seen in the Philippines and in Brazil, resulting from importations of animals for breed improvement. Our results were largely consistent with previous archeological, historical and molecular-based evidence for two independent domestication events for river- and swamp-type buffaloes, which occurred in the Indo-Pakistani region and close to the China/Indochina border, respectively. Based on a geographical analysis of the distribution of diversity, our evidence also indicated that the water buffalo spread out of the domestication centers followed two major divergent migration directions: river buffaloes migrated west from the Indian sub-continent while swamp buffaloes migrated from northern Indochina via an east-south-eastern route. These data suggest that the current distribution of water buffalo diversity has been shaped by the combined effects of multiple migration events occurred at different stages of the post-domestication history of the species.

Effects of traditional Chinese medicine formula on ruminal fermentation, enzyme activities and nutrient digestibility of beef cattle.

This study was conducted to evaluate effects of traditional Chinese medicine formula (TCMF) combined with several herbs on ruminal fermentation, enzyme activities and nutrient digestibility. Twenty finishing bulls were assigned to control or different TCMFs (Yufeisan-1, -2, -3; 2.5% dry matter (DM) in concentrate). Results showed that DM intake was higher (P < 0.05) in the Yufeisan-3 group than others. Compared to control, apparent digestibility of crude protein and neutral detergent fiber were increased (P < 0.05) by Yufeisan-3. No changes were observed in ruminal pH, concentrations of ammonia-N, microbial crude protein and total volatile fatty acid, whereas ratio of acetate to propionate was lower (P < 0.05) and propionate proportion tended to be higher (P < 0.1) in three TCMFs than control. Ruminal xylanase (P = 0.061) and carboxymethylcellulase (P < 0.05) activities were higher in Yufeisan-3 than control. No changes were observed in abundance of total bacteria, fungi and protozoa, whereas Fibrobacter succinogenes (P = 0.062) and Ruminococcus flavefaciens (P < 0.05) were increased and total methanogens was reduced (P = 0.069) by Yufeisan-3 compared to control. Yufeisan-3 improved nutrient digestibility and ruminal enzyme activity, and modified fermentation and microbial community, maybe due to the presence of Herba agastaches, Cortex phellodendri and Gypsum fibrosum.

Transcriptomic analysis of Rongchang pig brains and livers.

Recent developments in high-throughput RNA sequencing (RNA-seq) technology have led to a dramatic impact on our understanding of the structure and expression profiles of the mammalian transcriptome. To gain insights into the usefulness of swine production and biomedical model, the transcriptome profiling of Rongchang pig brains and livers was characterized using RNA-seq technology to uncover functional candidate molecules. In the study, total RNAs from brains and livers of Rongchang pig were sequenced and 8.6Gb sequencing data was obtained. This analysis revealed tissue specificity through the identification of 5575 and 4600 differentially expressed genes (DEGs) in brains and livers, respectively and the functional analysis of DEGs. Furthermore, 83 neuropeptide gene transcripts, 69 neuropeptide receptor gene transcripts, 10 pro-neuropeptide convertase gene transcripts and many other neuropeptide related protein gene transcripts were identified. Totally, the major characteristics of the transcriptional profiles of Rongchang pig brains and livers were present.