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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 α S1 -CN, α S2 -CN, κ-CN, and β-CN, and 3 whey protein fractions, namely β-lactoglobulin, α-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 × 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 β-CN and α 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, suggesting 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 α 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., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

In recent years, increasing attention has been focused on the genetic evaluation of protein fractions in cow milk with the aim of improving milk quality and technological characteristics. In this context, advances in high-throughput phenotyping by Fourier transform infrared (FTIR) spectroscopy offer the opportunity for large-scale, efficient measurement of novel traits that can be exploited in breeding programs as indicator traits. We took milk samples from 2,558 Holstein cows belonging to 38 herds in northern Italy, operating under different production systems. Fourier transform infrared spectra were collected on the same day as milk sampling and stored for subsequent analysis. Two sets of data (i.e., phenotypes and FTIR spectra) collected in 2 different years (2013 and 2019-2020) were compiled. The following traits were assessed using HPLC: true protein, major casein fractions [α S1 -casein (CN), α S2 -CN, β-CN, κ-CN, and glycosylated-κ-CN], and major whey proteins (β-lactoglobulin and α-lactalbumin), all of which were measured both in grams per liter (g/L) and proportion of total nitrogen (% N). The FTIR predictions were calculated using the gradient boosting machine technique and tested by 3 different cross-validation (CRV) methods. We used the following CRV scenarios: (1) random 10-fold, which randomly split the whole into 10-folds of equal size (9-folds for training and 1-fold for validation); (2) herd/date-out CRV, which assigned 80% of herd/date as the training set with independence of 20% of herd/date assigned as the validation set; (3) forward/backward CRV, which split the data set in training and validation set according with the year of milk sampling (FTIR and gold standard data assessed in 2013 or 2019-2020) using the "old" and "new" databases for training and validation, and vice-versa with independence among them; (4) the CRV for genetic parameters (CRV-gen), where animals without pedigree as assigned as a fixed training population and animals with pedigree information was split in 5-folds, in which 1-fold was assigned to the fixed training population, and 4-folds were assigned to the validation set (independent from the training set). The results (i.e., measures and predictions) of CRV-gen were used to infer the genetic parameters for gold standard laboratory measurements (i.e., proteins assessed with HPLC) and FTIR-based predictions considering the CRV-gen scenario from a bi-trait animal model using single-step genomic BLUP. We found that the prediction accuracies of the gradient boosting machine equations differed according to the way in which the proteins were expressed, achieving higher accuracy when expressed in g/L than when expressed as % N in all CRV scenarios. Concerning the reproducibility of the equations over the different years, the results showed no relevant differences in predictive ability between using "old" data as the training set and "new" data as the validation set and vice-versa. Comparing the additive genetic variance estimates for milk protein fractions between the FTIR predicted and HPLC measures, we found reductions of -19.7% for milk protein fractions expressed in g/L, and -21.19% expressed as % N. Although we found reductions in the heritability estimates, they were small, with values ranging from -1.9 to -7.25% for g/L, and -1.6 to -7.9% for % N. The posterior distributions of the additive genetic correlations (r a ) between the FTIR predictions and the laboratory measurements were generally high (>0.8), even when the milk protein fractions were expressed as % N. Our results show the potential of using FTIR predictions in breeding programs as indicator traits for the selection of animals to enhance milk protein fraction contents. We expect acceptable responses to selection due to the high genetic correlations between HPLC measurements and FTIR predictions., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Udder health in dairy herds is a very important issue given its implications for animal welfare and the production of high-quality milk. Somatic cell count (SCC) is the most widely used means of assessing udder health status. However, differential somatic cell count (DSCC) has recently been proposed as a new and more effective means of evaluating intramammary infection dynamics. Differential SCC represents the combined percentage of polymorphonuclear neutrophils and lymphocytes (PMN-LYM) in the total SCC, with macrophages (MAC) accounting for the remaining proportion. The aim of this study was to evaluate the association between SCC and DSCC and the detailed milk protein profile in a population of 1,482 Holstein cows. A validated reversed-phase HPLC method was used to quantify 4 caseins (CN), namely α S1 -CN, α S2 -CN, κ-CN, and β-CN, and 3 whey protein fractions, namely β-lactoglobulin, α-lactalbumin, and lactoferrin, which were expressed both quantitatively (g/L) and qualitatively (as a percentage of the total milk nitrogen content, %N). A linear mixed model was fitted to explore the associations between somatic cell score (SCS) combined with DSCC and the protein fractions expressed quantitatively and qualitatively. We ran an additional model that included DSCC expressed as PMN-LYM and MAC counts, obtained by multiplying the percentages of PMN-LYM and MAC by SCC for each cow in the data set. When the protein fractions were expressed as grams per liter, SCS was significantly negatively associated with almost all the casein fractions and positively associated with the whey protein α-lactalbumin, while DSCC was significantly associated with α S1 -CN, β-CN, and α-lactalbumin, but in the opposite direction to SCS. We observed the same pattern with the qualitative data (i.e., %N), confirming opposite effects of SCS and DSCC on milk protein fractions. The PMN-LYM count was only slightly associated with the traits of concern, although the pattern observed was the same as when both SCS and DSCC were included in the model. The MAC count, however, generally had a greater impact on many casein fractions, in particular decreasing both β-CN content (g/L) and proportion (%N), and exhibited the opposite pattern to the PMN-LYM count. Our results show that information obtained from both SCS and DSCC may be useful in assessing milk quality and protein fractions. They also demonstrate the potential of MAC count as a novel udder health trait., (© 2022, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The quantities and proportions of protein fractions have notable effects on the nutritional and technological value of milk. Although much is known about the effects of genetic variants on milk proteins, the complex relationships among the set of genes and pathways regulating the different protein fractions synthesis and secretion into milk in dairy cows are still not completely understood. We conducted genome-wide association studies (GWAS) for milk nitrogen fractions in a cohort of 1,011 Brown Swiss cows, which uncovered 170 significant single nucleotide polymorphism (SNPs), mostly located on BTA6 and BTA11. Gene-set analysis and the network-based Associated Weight Matrix approach revealed that the milk proteins associated genes were involved in several biological functions, particularly ion and cation transmembrane transporter activity and neuronal and hormone signalling, according to the structure and function of casein micelles. Deeper analysis of the transcription factors and their predicted target genes within the network revealed that GFI1B, ZNF407 and NR5A1 might act as master regulators of milk protein synthesis and secretion. The information acquired provides novel insight into the regulatory mechanisms controlling milk protein synthesis and secretion in bovine mammary gland and may be useful in breeding programmes aimed at improving milk nutritional and/or technological properties.

The fatty acid (FA) profile has a considerable impact on the nutritional and technological quality of milk and dairy products. The molecular mechanism underlying the regulation of fat metabolism in bovine mammary gland have been not completely elucidated. We conducted genome-wide association studies (GWAS) across 65 milk FAs and fat percentage in 1,152 Brown Swiss cows. In total, we identified 175 significant single nucleotide polymorphism (SNPs) spanning all chromosomes. Pathway analyses revealed that 12:0 was associated with the greatest number of overrepresented categories/pathways (e.g. mitogen-activated protein kinase (MAPK) activity and protein phosphorylation), suggesting that it might play an important biological role in controlling milk fat composition. An Associated Weight Matrix approach based on SNP co-associations predicted a network of 791 genes related to the milk FA profile, which were involved in several connected molecular pathways (e.g., MAPK, lipid metabolism and hormone signalling) and undetectable through standard GWAS. Analysis of transcription factors and their putative target genes within the network identified BACH2, E2F3 and KDM5A as key regulators of milk FA metabolism. These findings contribute to increasing knowledge of FA metabolism and mammary gland functionality in dairy cows and may be useful in developing targeted breeding practices to improve milk quality.

Mastitis is one of the most prevalent diseases in dairy cattle and is the cause of considerable economic losses. Alongside somatic cell count (SCC), differential somatic cell count (DSCC) has been recently introduced as a new indicator of intramammary infection. The DSCC is expressed as a count or a proportion (%) of polymorphonuclear neutrophils plus lymphocytes (PMN-LYM) in milk somatic cells. These numbers are complemented to total somatic cell count or to 100 by macrophages (MAC). The aim of this study was to investigate the genetic variation and heritability of DSCC, and its correlation with milk composition, udder health indicators, milk composition, and technological traits in Holstein cattle. Data used in the analysis consisted in single test-day records from 2,488 Holstein cows reared in 36 herds located in northern Italy. Fourier-transform infrared (FTIR) spectroscopy was used to predict missing information for some milk coagulation and cheese-making traits, to increase sample size and improve estimation of the genetic parameters. Bayesian animal models were implemented via Gibbs sampling. Marginal posterior means of the heritability estimates were 0.13 for somatic cell score (SCS); 0.11 for DSCC, MAC proportion, and MAC count; and 0.10 for PMN-LYM count. Posterior means of additive genetic correlations between SCS and milk composition and udder health were low to moderate and unfavorable. All the relevant genetic correlations between the SCC traits considered and the milk traits (composition, coagulation, cheese yield and nutrients recovery) were unfavorable. The SCS showed genetic correlations of −0.30 with the milk protein proportion, −0.56 with the lactose proportion and −0.52 with the casein index. In the case of milk technological traits, SCS showed genetic correlations of 0.38 with curd firming rate (k 20), 0.45 with rennet coagulation time estimated using the curd firming over time equation (RCT eq), −0.39 with asymptotic potential curd firmness, −0.26 with maximum curd firmness (CF max), and of −0.31 with protein recovery in the curd. Differential somatic cell count expressed as proportion was correlated with SCS (0.60) but had only 2 moderate genetic correlations with milk traits: with lactose (−0.32) and CF max (−0.33). The SCS was highly correlated with the log PMN-LYM count (0.79) and with the log MAC count (0.69). The 2 latter traits were correlated with several milk traits: fat (−0.38 and −0.43 with PMN-LYM and MAC counts, respectively), lactose percentage (−0.40 and −0.46), RCT eq (0.53 and 0.41), t max (0.38 and 0.48). Log MAC count was correlated with k 20 (+0.34), and log PMN-LYM count was correlated with CF max (−0.26) and weight of water curd as percentage of weight of milk processed (−0.26). The results obtained offer new insights into the relationships between the indicators of udder health and the milk technological traits in Holstein cows. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the associations between differential somatic cell count (DSCC) and milk quality and udder health traits, and for the first time, between DSCC and milk coagulation properties and cheesemaking traits in a population of 1,264 Holstein cows reared in northern Italy. Differential somatic cell count represents the combined proportions of polymorphonuclear neutrophils plus lymphocytes (PMN-LYM) in the total somatic cell count (SCC), with macrophages (MAC) making up the remaining proportion. The milk traits investigated in this study were milk yield (MY), 8 traits related to milk composition and quality (fat, protein, casein, casein index, lactose, urea, pH, and milk conductivity), 9 milk coagulation traits [3 milk coagulation properties (MCP) and 6 curd firming (CF) traits], 7 cheesemaking traits, 3 cheese yield (CY) traits, and 4 milk nutrient recovery in the curd (REC) traits. A linear mixed model was fitted to explore the associations between SCS combined with DSCC and the aforementioned milk traits. An additional model was run, which included DSCC expressed as the PMN-LYM and MAC counts, obtained by multiplying the percentage of PMN-LYM and MAC by SCC in the milk for each cow in the data set. The unfavorable association between SCS and milk quality and technological traits was confirmed. Increased DSCC was instead associated with a linear increase in MY, casein index, and lactose proportion and a linear decrease in milk fat and milk conductivity. Accordingly, DSCC was favorably associated with all MCP and CF traits (with the exception of the time needed to achieve maximum, CF), particularly with rennet coagulation time, and it always displayed linear relationships. Differential somatic cell count was also positively associated with the recovery of milk nutrients in the curd (protein, fat, and energy), which increased linearly with increasing DSCC. The PMN-LYM count was rarely associated with milk traits, even though the pattern observed confirmed the results obtained when both SCS and DSCC were included in the model. The MAC count, however, showed the opposite pattern: MY, casein index, and lactose percentage decreased and milk conductivity increased with an increasing MAC count. No significant association was found between PMN-LYM count and MCP, CF, CY, and REC traits, whereas MAC count was unfavorably associated with MCP, CF traits, some CY traits, and all REC traits. Our results showed that the combined information derived from SCS and DSCC might be useful to monitor milk quality and cheesemaking-related traits. [ABSTRACT FROM AUTHOR]