Identification of novel alternative splicing associated with mastitis disease in Holstein dairy cows using large gap read mapping.

BACKGROUND: Mastitis is a very common disease in the dairy industry that producers encounter daily. Transcriptomics, using RNA-Sequencing (RNA - Seq) technology, can be used to study the functional aspect of mastitis resistance to identify animals that have a better immune response to mastitis. When the cow has mastitis, not only genes but also specific mRNA isoforms generated via alternative splicing (AS) could be differentially expressed (DE), leading to the phenotypic variation observed. Therefore, the objective of this study was to use large gap read mapping to identify mRNA isoforms DE between healthy and mastitic milk somatic cell samples (N = 12). These mRNA isoforms were then categorized based on being 1) annotated mRNA isoforms for gene name and length, 2) annotated mRNA isoforms with different transcript length and 3) novel mRNA isoforms of non - annotated genes. RESULTS: Analysis identified 333 DE transcripts (with at least 2 mRNA isoforms annotated, with at least one being DE) between healthy and mastitic samples corresponding to 303 unique genes. Of these 333 DE transcripts between healthy and mastitic samples, 68 mRNA isoforms are annotated in the bovine genome reference (ARS.UCD.1.2), 249 mRNA isoforms had novel transcript lengths of known genes and 16 were novel transcript lengths of non - annotated genes in the bovine genome reference (ARS.UCD.1.2). Functional analysis including gene ontology, gene network and metabolic pathway analysis was performed on the list of 288 annotated and unique DE mRNA isoforms. In total, 67 significant metabolic pathways were identified including positive regulation of cytokine secretion and immune response. Additionally, numerous DE novel mRNA isoforms showed potential involvement with the immune system or mastitis. Lastly, QTL annotation analysis was performed on coding regions of the DE mRNA isoforms, identifying overlapping QTLs associated with clinical mastitis and somatic cell score. CONCLUSION: This study identified novel mRNA isoforms generated via AS that could lead to differences in the immune response of Holstein dairy cows and be potentially implemented in future breeding programs.

Genetic mechanisms regulating the host response during mastitis.

Mastitis is a very costly and common disease in the dairy industry. The study of the transcriptome from healthy and mastitic milk somatic cell samples using RNA-Sequencing technology can provide measurements of transcript levels associated with the immune response to the infection. The objective of this study was to characterize the Holstein milk somatic cell transcriptome from 6 cows to determine host response to intramammary infections. RNA-Sequencing was performed on 2 samples from each cow from 2 separate quarters, one classified as healthy (n = 6) and one as mastitic (n = 6). In total, 449 genes were differentially expressed between the healthy and mastitic quarters (false discovery rate <0.05, fold change >±2). Among the differentially expressed genes, the most expressed genes based on reads per kilobase per million mapped reads (RPKM) in the healthy group were associated with milk components (CSN2 and CSN3), and in the mastitic group they were associated with immunity (B2M and CD74). In silico functional analysis was performed using the list of 449 differentially expressed genes, which identified 36 significantly enriched metabolic pathways (false discovery rate <0.01), some of which were associated with the immune system, such as cytokine-cytokine interaction and cell adhesion molecules. Seven functional candidate genes were selected, based on the criteria of being highly differentially expressed between healthy and mastitic groups and significantly enriched in metabolic pathways that are relevant to the inflammatory process (GLYCAM1, B2M, CD74, BoLA-DRA, FCER1G, SDS, and NFKBIA). Last, we identified the differentially expressed genes that are located in quantitative trait locus regions previously known to be associated with mastitis, specifically clinical mastitis, somatic cell count, and somatic cell score. It was concluded that multiple genes within quantitative trait locus regions could potentially affect host response to mastitis-causing agents, making some cows more susceptible to intramammary infections. The identification of potential candidate genes with functional, statistical, biological, and positional relevance associated with host defense to infection will contribute to a better understanding of the underlying genetic architecture associated with mastitis. This in turn will improve the sustainability of agricultural practices by facilitating the selection of cows with improved host defense leading to increased resistance to mastitis.

Association of rumination time and health status with milk yield and composition in early-lactation dairy cows.

The objective of this study was to determine the associations of rumination time (RT) and health status with milk yield and milk composition. This study used 339 dairy cows from 4 commercial dairy farms in Ontario, Canada (first lactation, n = 107; second lactation, n = 112; ≥third lactation, n = 120). Rumination time was monitored (24 h/d) using an automated system from 1 to 28 d in milk (DIM). Cows were milked 3×/d on each farm, and 2 farms recorded milk weights at each milking to determine daily milk yield (n = 170). Cows were also monitored for milk composition (fat and protein content) 1×/wk. Last, subclinical ketosis (SCK) was diagnosed 1×/wk; cows with at least one blood sample with ß-hydroxybutyrate ≥1.2 mmol/L postcalving were diagnosed with SCK. Cases of retained placenta, metritis, milk fever, or mastitis during the study period were also recorded. Cows were categorized into 1 of 4 groups: healthy cows that had no SCK or any other health issue (HLT; n = 139); cows that were treated for at least one health issue other than SCK (HLT+; n = 50); SCK cows with no other health problems during transition (HYK; n = 97); or cows that had SCK and one or more other health problems (HYK+; n = 53). All data were summarized by week across cows, and the associations between rumination time and milk yield (n = 170) and milk composition (n = 339) were modeled. Across all lactations, and including all health categories, milk yield increased by week, whereas fat and protein content both decreased by week. A positive association was found between summarized RT and milk yield in first-lactation (+0.006 ± 0.003 kg/min of RT) and second-lactation (+0.015 ± 0.004 kg/min of RT) cows from 4 to 28 DIM, as well as in ≥third-lactation cows; however, the relationship between RT and milk yield differed across weeks in those cows. A negative association between RT and milk fat content was found in ≥third-lactation cows (-0.002 ± 0.00059 percentage points/min of RT). From 4 to 28 DIM, ≥third-lactation HYK and HYK+ cows produced less protein (0.11 ± 0.051 and 0.13 ± 0.056 percentage points, respectively) than HLT cows. Over the 4-wk observation period, first-lactation HYK+ cows tended to deposit 0.11 ± 0.056 percentage points less protein in their milk compared with HLT cows. Second-lactation HYK+ cows produced less milk than HLT cows each week during early lactation. In summary, RT was positively associated with milk yield in early-lactation dairy cows, across all lactations, and negatively associated with milk fat content in ≥third-lactation cows. Further, the results showed that early-lactation cows that experience SCK, particularly with one or more other health problems, might have decreased milk yield and milk protein content.