Alice Vanzin

Alternative splicing events lead to different mRNA isoforms, potentially translated into proteins with altered structures or impaired functions, or both. This phenomenon may influence the resistance or susceptibility to diseases, such as mastitis. To explore this aspect, this study aims to analyze transcript expression, focusing on cases where multiple mRNA isoforms are present, in milk somatic cells from Holstein cattle affected by subclinical intramammary infection caused by Prototheca spp. (P+, n = 11) or Streptococcus agalactiae (Sa+, n = 11), compared with uninfected animals (Neg, n = 9). The RNA-sequencing data were analyzed using the CLC Genomics Workbench (Qiagen) with a large gap read mapping approach and Bos taurus ARS-UCD1.3 reference genome to identify the differentially expressed transcripts (DET) among the groups. In addition, a functional analysis of the identified DET, combined with the identification of functional variants within the expressed regions, was performed. The comparison P+ versus Neg revealed 27 annotated DET, 11 annotated DET with novel length, and 7 novel DET with no previously annotated associated gene or length. These DET mainly originated from immune-related genes involved in pathways strictly linked to the immune and inflammatory responses (i.e., antigen presentation pathway, MHC class II antigen presentation pathway, macrophage classical activation signaling pathway). In contrast, the Sa+ versus Neg comparison revealed a total of 26 DET, including 17 annotated transcripts, 8 annotated transcripts with a novel length, and 1 novel transcript from a nonannotated gene. In this case, a predominance of enriched pathways related to metabolism and detoxification processes was observed (i.e., FXR/RXR activation, xenobiotic metabolism general signaling pathway, glutathione-mediated detoxification). Functional variants were identified in regions overlapping DET encoded by KRT78, CSN1S1, and MYBPC1, which were downregulated in P+ when compared with Neg group and potentially related to mastitis resistance/susceptibility traits. The transcripts and associated functional variants identified in this study may contribute to a better understanding of bovine mastitis pathogenesis and development, providing useful insights for improving animal health and management strategies.

The definition of dietary models that meet the animal's nutritional needs, improve its physiological state, and preserve the microbiota equilibrium, along with the challenge of reducing environmental effects is essential for producing sustainable, high-quality milk suitable for dairy production. This study aimed to investigate whether a more sustainable diet, with a higher content of alfalfa hay but equal net energy, protein, and NDF levels, has an effect on rumen fluid, feces, and milk microbiota in Sarda sheep. Additionally, it aimed to determine if microbiota changes across different niches correlated with production and traits. Twenty-four animals were divided into 2 groups and fed different diets. Rumen fluid, feces, and milk samples were collected at the beginning (d 1, T0) and at the end of the trial (d 22, T1). Microbial communities were analyzed using Ion GeneStudio S5 for sequencing the V2, V3, V4, V6-V7, V8, and V9 regions of the 16S rRNA gene. Amplicon sequence variant clustering and taxonomic assignment were performed with QIIME2 v2023.9.2. Differential abundant taxa between the 2 experimental groups (H-alfalfa, fed the high-alfalfa content diet, and L-alfalfa, fed the low-alfalfa content diet) were assessed at the 2 time points using the ANCOM-BC plugin of QIIME2, along with the analysis of the diversity indices. Similarly, milk composition, milk coagulation properties and BW, DMI, production, and energy requirements for milk production traits were analyzed for each time point using a linear model with diet as fixed effect. Results confirmed an overall homogeneity of the samples at T0 for milk composition, milk coagulation properties, and energy requirements for milk production, except for DMI. However, no significant difference in DMI was observed at T1 between experimental groups. Regarding the microbiota composition, analysis revealed minor differences between H-alfalfa and L-alfalfa groups in taxa abundances for rumen fluid and feces, both at T0 and 1. In milk, 8 taxa resulted to be enriched at T1 in the treated group. However, all the affected taxa had a low abundance, and no significant correlation was observed with the phenotypic traits. Diets did not affect the microbial structure of rumen fluid and milk, whereas significant differences in beta diversity (Bray-Curtis and unweighted UniFrac) were observed in feces at T1. Five distinct milk taxa resulted positively correlated with 3 taxa in rumen fluid and 2 in feces, supporting the idea that host microbial niches form a network of interconnected communities, even within isolated environments. In addition, a positive correlation was observed between energy requirements for milk production and feces' Oscillospiraceae family, a taxon typical of healthy gut in ruminants. The higher content of alfalfa hay showed no highly significant effects on microbiota composition, milk composition, milk coagulation traits, and energy requirements for milk production. Similarities in chemical composition and energy content between diets likely masked qualitative differences in the ingredients and resulted in minor differences between diet groups. Nevertheless, no adverse effects of the high-alfalfa hay diet were observed, suggesting its higher value as an effective strategy, particularly advantageous from an environmental perspective.

In this study we wanted to investigate the associations between naturally occurring subclinical intramammary infection (IMI) caused by different etiological agents (i.e., Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, and Prototheca spp.), in combination with somatic cell count (SCC), on the detailed milk protein profile measured at the individual mammary gland quarter. An initial bacteriological screening (time 0; T0) conducted on individual composite milk from 450 Holstein cows reared in 3 herds, was performed to identify cows with subclinical IMI. We identified 78 infected animals which were followed up at the quarter level at 2 different sampling times: T1 and T2, 2 and 6 wk after T0, respectively. A total of 529 quarter samples belonging to the previously selected animals were collected at the 2 sampling points and analyzed with a reversed phase HPLC (RP-HPLC) validated method. Specifically, we identified and quantified 4 caseins (CN), namely alpha S1-CN, alpha S2-CN, kappa-CN, and beta-CN, and 3 whey protein fractions, namely beta-lactoglobulin, alpha-lactalbumin, and lactoferrin (LF), which were later expressed both quantitatively (g/L) and qualitatively (as a percentage of the total milk nitrogen content, % N). Data were analyzed with a hierarchical linear mixed model with the following fixed effects: days in milk (DIM), parity, herd, SCC, bacteriological status (BACT), and the SCC x BACT interaction. The random effect of individual cow, nested within herd, DIM and parity was used as the error term for the latter effects. Both IMI (i.e., BACT) and SCC significantly reduced the proportion of beta-CN and alpha S1-CN, ascribed to the increased activity of both milk endogenous and microbial proteases. Less evident alterations were found for whey proteins, except for LF, which being a glycoprotein with direct and undirect antimicrobial activity, increased both with IMI and SCC, suggest ing its involvement in the modulation of both the innate and adaptive immune response. Finally, increasing SCC in the positive samples was associated with a more marked reduction of total caseins at T1, and alpha S1-CN at T2, suggesting a synergic effect of infection and inflammation, more evident at high SCC. In conclusion, our work helps clarify the behavior of protein fractions at quarter level in animals having subclinical IMI. The inflammation status driven by the increase in SCC, rather the infection, was associated with the most significant changes, suggesting that the activity of endogenous proteolytic enzymes related to the onset of inflammation might have a pivotal role in directing the alteration of the milk protein profile.

Udder health has a crucial role in sustainable milk production, and various reports have pointed out that changes in udder condition seem to affect milk mineral content. The somatic cell count (SCC) is the most recognized indicator for the determination of udder health status. Recently, a new parameter, the differential somatic cell count (DSCC), has been proposed for a more detailed evaluation of intra-mammary infection patterns. Specifically, the DSCC is the combined proportions of polymorphonuclear neutrophils and lymphocytes (PMN-LYM) on the total SCC, with macrophages (MAC) representing the remainder proportion. In this study, we evaluated the association between DSCC in combination with SCC on a detailed milk mineral profile in 1,013 Holstein Friesian cows reared in 5 herds. An Inductively Coupled Plasma- Optical Emission Spectrometry (ICP-OES) was used to quantify 32 milk mineral elements. Two different linear mixed models were fitted to explore the associations between the milk mineral elements and first, the DSCC combined with SCC, and second, DSCC expressed as the PMN-LYM and MAC counts, obtained by multiplying the proportion of PMN-LYM and MAC by SCC. We observed a significant positive association between SCC and milk Na, S and Fe levels. Differential somatic cell count showed an opposite behavior to the one displayed by SCC, with a negative association with Na and positive association with K milk concentrations. When considering DSCC as count, Na and K showed contrasting behavior when associated to PMN-LYM or MAC counts, with decreasing of Na content and increasing K when associated to increasing PMN-LYM counts, and increasing Na and decreasing K when associated to increasing MAC count. These findings confirmed that an increase in SCC is associated with altered milk Na and K amounts. Moreover, MAC count seemed to mirror SCC patterns, with the worsening of inflammation. Differently, PMN-LYM count exhibited patterns of associations with milk Na and K contents attributable more to LYM than PMN, given the non-pathological condition of the vast majority of the investigated population. An interesting association was observed for milk S content, which increased with increasing of inflammatory conditions (i.e., increased SCC and MAC count) probably attributable to its relationship with milk proteins, especially whey proteins. Moreover, milk Fe content showed positive associations with the PMN-LYM population, highlighting its role in immune regulation during inflammation. Further studies including individuals with clinical condition are needed to have a comprehensive view of milk mineral behavior during udder health impairment.

Early detection of bovine subclinical mastitis may improve treatment strategies and reduce the use of antibiotics. Herein, individual milk samples from Holstein cows affected by subclinical mastitis induced by S. agalactiae and Prototheca spp. were analyzed by untargeted and targeted mass spectrometry approaches to assess changes in their peptidome profiles and identify new potential biomarkers of the pathological condition. Results showed a higher amount of peptides in milk positive on the bacteriological examination when compared with the negative control. However, the different pathogens seemed not to trigger specific effects on the milk peptidome. The peptides that best distinguish positive from negative samples are mainly derived from the most abundant milk proteins, especially from β- and α_s1-casein, but also include the antimicrobial peptide casecidin 17. These results provide new insights into the physiopathology of mastitis. Upon further validation, the panel of potential discriminant peptides could help the development of new diagnostic and therapeutic tools.

The causes of variation in the milk mineral profile of dairy cattle during the first phase of lactation were studied under the hypothesis that the milk mineral profile partially reflects the animals' metabolic status. Correlations between the minerals and the main milk constituents (i.e., protein, fat, and lactose percentages), and their associations with the cows' metabolic status indicators were explored. The metabolic status indicators (MET) that we used were blood energy-protein metabolites [nonesterified fatty acids, β-hydroxybutyrate (BHB), glucose, cholesterol, creatinine, and urea], and liver ultrasound measurements (predicted triacylglycerol liver content, portal vein area, portal vein diameter and liver depth). Milk and blood samples, and ultrasound measurements were taken from 295 Holstein cows belonging to 2 herds and in the first 120 d in milk (DIM). Milk mineral contents were determined by ICP-OES; these were considered the response variable and analyzed through a mixed model which included DIM, parity, milk yield, and MET as fixed effects, and the herd/date as a random effect. The MET traits were divided in tertiles. The results showed that milk protein was positively associated with body condition score (BCS) and glucose, and negatively associated with BHB blood content; milk fat was positively associated with BHB content; milk lactose was positively associated with BCS; and Ca, P, K and S were the minerals with the greatest number of associations with the cows' energy indicators, particularly BCS, predicted triacylglycerol liver content, glucose, BHB and urea. We conclude that the protein, fat, lactose, and mineral contents of milk partially reflect the metabolic adaptation of cows during lactation and within 120 DIM. Variations in the milk mineral profile were consistent with changes in the major milk constituents and the metabolic status of cows.