Methylome-wide analysis of milk somatic cells upon subclinical mastitis in dairy cattle.

Better understanding of the molecular mechanisms behind bovine mastitis is fundamental for improving the management of this disease, which continues to be of major concern for the dairy industry, especially in its subclinical form. Disease severity and progression depend on numerous aspects, such as livestock genetics, and the interaction between the causative agent, the host, and the environment. In this context, epigenetic mechanisms have proven to have a role in controlling the response of the animal to inflammation. Therefore, in this study we aimed to explore genome-wide DNA methylation of milk somatic cells (SC) in healthy cows (n = 15) and cows affected by naturally occurring subclinical mastitis by Streptococcus agalactiae (n = 12) and Prototheca spp. (n = 11), to better understand the role of SC methylome in the host response to disease. Differentially methylated regions (DMR) were evaluated comparing: (1) Strep. agalactiae-infected versus healthy; (2) Prototheca-infected versus healthy, and (3) mastitis versus healthy and (4) Strep. agalactiae-infected versus Prototheca-infected. The functional analysis was performed at 2 levels. To begin with, we extracted differentially methylated genes (DMG) from promoter DMR, which were analyzed using the Cytoscape ClueGO plug-in. Coupled with this DMG-driven approach, all the genes associated with promoter-methylated regions were fed to the Pathifier algorithm. From the DMR analysis, we identified 1,081 hypermethylated and 361 hypomethylated promoter regions in Strep. agalactiae-infected animals, while 1,514 hypermethylated and 358 hypomethylated promoter regions were identified in Prototheca-infected animals, when compared with the healthy controls. When considering infected animals as a whole group (regardless of the pathogen), we found 1,576 hypermethylated and 460 hypomethylated promoter regions. Both pathogens were associated with methylation differences in genes involved in pathways related to meiosis, reproduction and tissue remodeling. Exploring the whole methylome, in subclinically infected cows we observed a strong deregulation of immune-related pathways, such as nuclear factor kB and toll-like receptors signaling pathways, and of energy-related pathways such as the tricarboxylic acid cycle and unsaturated fatty acid biosynthesis. In conclusion, no evident pathogen-specific SC methylome signature was detected in the present study. Overall, we observed a clear regulation of host immune response driven by DNA methylation upon subclinical mastitis. Further studies on a larger cohort of animals are needed to validate our results and to possibly identify a unique SC methylome that signifies pathogen-specific alterations.

Serum metabolome differences associated with subclinical intramammary infection caused by Streptococcus agalactiae and Prototheca spp. in multiparous dairy cows.

Mastitis is one of the most significant diseases in dairy cows and causes several economic losses. Somatic cell count (SCC) is often used as an indirect diagnostic tool for mastitis, especially for subclinical mastitis (SCM) where no symptoms or signs can be detected. Streptococcus agalactiae is one of the main causes of contagious mastitis, and Prototheca spp. is an alga-inducing environmental mastitis that is not always correlated with increased milk SCC. The aim of this study was to evaluate the changes in the metabolomic profile of blood in relation to subclinical intramammary infection (IMI) in dairy cows. In addition, differences resulting from the etiologic agent causing mastitis were also considered. Forty Holstein-Friesian dairy cows in mid and late lactation were enrolled in this cross-sectional design study. Based on the bacteriological examination of milk, the animals were divided into 3 groups: group CTR (control group; n = 16), group A (affected by SCM with IMI caused by Strep. agalactiae; n = 17), and group P (affected by SCM with IMI caused by Prototheca spp.; n = 7). Blood samples from the jugular vein were collected in tubes containing clot activator; the serum aliquot was stored until metabolomic analysis by 1H-nuclear magnetic resonance spectroscopy. Statistical analysis was conducted by fitting a linear model with the group as the fixed effect and SCC as the covariate. Forty-two metabolites were identified, and among them 10 were significantly different among groups. Groups A and P showed greater levels of His and lactose and lower levels of acetate, Asn, and dimethylamine compared with group CTR. Group A showed high levels of Val, and group P showed high levels of Cit and methylguanidine, as well as lower levels of 3-hydroxybutyrate, acetone, allantoin, carnitine, citrate, and ethanol. These metabolites were related to ruminal fermentations, energy metabolism, urea synthesis and metabolism, immune and inflammatory response, and mammary gland permeability. These results suggest systemic involvement with subclinical IMI and that the metabolic profile of animals with SCM undergoes changes related to the etiological agent of mastitis.

Observational study on the associations between milk yield, composition, and coagulation properties with blood biomarkers of health in Holstein cows.

The considerable increase in the production capacity of individual cows owing to both selective breeding and innovations in the dairy sector has posed challenges to management practices in terms of maintaining the nutritional and metabolic health status of dairy cows. In this observational study, we investigated the associations between milk yield, composition, and technological traits and a set of 21 blood biomarkers related to energy metabolism, liver function or hepatic damage, oxidative stress, and inflammation or innate immunity in a population of 1,369 high-yielding Holstein-Friesian dairy cows. The milk traits investigated in this study included 4 production traits (milk yield, fat yield, protein yield, daily milk energy output), 5 traits related to milk composition (fat, protein, casein, and lactose percentages and urea), 11 milk technological traits (5 milk coagulation properties and 6 curd-firming traits). All milk traits (i.e., production, composition, and technological traits) were analyzed according to a linear mixed model that included the days in milk, the parity order, and the blood metabolites (tested one at a time) as fixed effects and the herd and date of sampling as random effects. Our findings revealed that milk yield and daily milk energy output were positively and linearly associated with total cholesterol, nonesterified fatty acids, urea, aspartate aminotransferase, γ-glutamyl transferase, total bilirubin, albumin, and ferric-reducing antioxidant power, whereas they were negatively associated with glucose, creatinine, alkaline phosphatase, total reactive oxygen metabolites, and proinflammatory proteins (ceruloplasmin, haptoglobin, and myeloperoxidase). Regarding composition traits, the protein percentage was negatively associated with nonesterified fatty acids and ß-hydroxybutyrate (BHB), while the fat percentage was positively associated with BHB, and negatively associated with paraoxonase. Moreover, we found that the lactose percentage increased with increasing cholesterol and albumin and decreased with increasing ceruloplasmin, haptoglobin, and myeloperoxidase. Milk urea increased with an increase in cholesterol, blood urea, nonesterified fatty acids, and BHB, and decreased with an increase in proinflammatory proteins. Finally, no association was found between the blood metabolites and milk coagulation properties and curd-firming traits. In conclusion, this study showed that variations in blood metabolites had strong associations with milk productivity traits, the lactose percentage, and milk urea, but no relationships with technological traits of milk. Specifically, increasing levels of proinflammatory and oxidative stress metabolites, such as ceruloplasmin, haptoglobin, myeloperoxidase, and total reactive oxygen metabolites, were shown to be associated with reductions in milk yield, daily milk energy output, lactose percentage, and milk urea. These results highlight the close connection between the metabolic and innate immunity status and production performance. This connection is not limited to specific clinical diseases or to the transition phase but manifests throughout the entire lactation. These outcomes emphasize the importance of identifying cows with subacute inflammatory and oxidative stress as a means of reducing metabolic impairments and avoiding milk fluctuations.

Effect of intramammary infection and inflammation on milk protein profile assessed at the quarter level in Holstein cows.

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 αS1-CN, αS2-CN, κ-CN, and ß-CN, and 3 whey protein fractions, namely ß-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 ß-CN and αS1-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 αS1-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.

Associations between subclinical intramammary infection and milk fatty acid profile at the quarter level in Holstein cattle.

Mastitis, especially the subclinical form, is the most common economic and health problem in dairy cows. Little is known about changes in milk fatty acid (FA) composition according to infection/inflammation status of the mammary gland. The aim of this study was to investigate the associations between naturally occurring subclinical intramammary infection (IMI) from different pathogens, i.e. Streptococcus agalactiae, Staphylococcus aureus, Streptococcus uberis and Prototheca spp., and the detailed milk FA profile assessed at quarter level in Holstein cows. After an initial bacteriological screening (T0) on 450 Holstein cows reared in three dairy herds, we identified 78 cows positive at the bacteriological examination. These animals were followed up at the quarter level two weeks (T1) and six weeks (T2) after T0. In total, 600 single-quarter samples were obtained at T1 and T2. Individual FAs were determined using the gas chromatography analytical method. Investigated traits were 70 individual FAs, 12 FA groups, and six desaturation indices. The associations between subclinical IMI combined with somatic cell count (SCC) and milk FA profile were investigated using a hierarchical linear mixed model (i.e., observational unit was quarter within cow) with the following fixed effects: days in milk (DIM), parity, herd, SCC, bacteriological status (BACT, positive and negative), 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. The most significant associations were detected at T2. Notably, IMI reduced the proportions of individual short-chain FA, especially 4:0 and 6:0 (-14%), but increased the proportion of the most abundant medium-chain FA (MCFA), 16:0 (+4%). A reduction in the desaturation indices was observed mostly for 14:1 index (-9%), in line with the reduction in 14:1 (-10%). Somatic cell count significantly affected 14 individual FAs. In particular, samples with high SCC (≥200 000) had significantly lower proportions of 8:0, 10:0, 11:0, 12:0, and 13:0 compared with samples with low SCC (<200 000). Increasing SCC in animals positive at the bacteriological examination were associated with a reduction in total MCFA at T2 (while in negative animals, they remained constant across SCC classes), possible evidence that elongation of the FA chain from 11 to 16 carbons is affected by a combination of infection and SCC. This study showed that subclinical IMI and SCC are mainly associated with reductions in the synthesis of FA and the desaturation process in the mammary gland.

Blood biochemical changes upon subclinical intramammary infection and inflammation in Holstein cattle.

The aim of this study was to investigate the associations between subclinical intramammary infection (IMI) from different pathogens combined with inflammation status and a set of blood biochemical traits including energy-related metabolites, indicators of liver function or hepatic damage, oxidative stress, inflammation, innate immunity, and mineral status in 349 lactating Holstein cows. Data were analyzed with a linear model including the following fixed class effects: days in milk, parity, herd, somatic cell count (SCC), bacteriological status (positive and negative), and the SCC × bacteriological status interaction. Several metabolites had significant associations with subclinical IMI or SCC. Increased SCC was associated with a linear decrease in cholesterol concentrations which ranged from -2% for the class ≥50,000 and <200,000 cells/mL to -11% for the SCC class ≥400,000 cells/mL compared with the SCC class <50,000 cells/mL. A positive bacteriological result was associated with an increase in bilirubin (+24%), paraoxonase (+11%), the ratio paraoxonase/cholesterol (+9%), and advanced oxidation protein product concentration (+23%). Increased SCC were associated with a linear decrease in ferric reducing antioxidant power concentrations ranging from -3% for the class ≥50,000 and <200,000 cells/mL to -9% for the SCC class ≥400,000 cells/mL (respect to the SCC class <50,000 cells/mL). A positive bacteriological result was associated with an increase in haptoglobin concentrations (+19%). Increased SCC were also associated with a linear increase in haptoglobin concentrations, which ranged from +24% for the class ≥50,000 and <200,000 cells/mL (0.31 g/L) to +82% for the SCC class ≥400,000 cells/mL (0.45 g/L), with respect to the SCC class <50,000 cells/mL (0.25 g/L). Increased SCC were associated with a linear increase in ceruloplasmin concentrations (+15% for SCC ≥50,000 cells/mL). The observed changes in blood biochemical markers, mainly acute phase proteins and oxidative stress markers, suggest that cows with subclinical IMI may experience a systemic involvement.

Predicting milk protein fractions using infrared spectroscopy and a gradient boosting machine for breeding purposes in Holstein cattle.

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 [αS1-casein (CN), αS2-CN, ß-CN, κ-CN, and glycosylated-κ-CN], and major whey proteins (ß-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 (ra) 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.

Streptococcus agalactiae and Prototheca spp. induce different mammary gland leukocyte responses in Holstein cows.

In this study, we investigated the association between natural subclinical intramammary infection (IMI) caused by Streptococcus agalactiae and Prototheca spp. and milk differential cell counts assessed by cytofluorimetric analysis in Holstein cows. After an initial bacteriological screening on 188 animals and a second assessment carried out 2 wk later aimed at confirming the bacteriological status, we collected milk samples from 47 animals and performed (1) milk composition analyses; (2) somatic cell counts and differential somatic cell counts (DSCC); and (3) cytofluorimetric analyses. Before statistical analyses, animals with co-infections were filtered out. Bacteriological status (negative, positive for Strep. agalactiae, or positive for Prototheca spp.) significantly affected the investigated traits. Compared with culture-negative samples, those that were positive for Strep. agalactiae and Prototheca spp. had higher SCS (+61% and +49%, respectively), DSCC (+4% and +19%, respectively), log polymorphonuclear neutrophil (PMN)-lymphocyte (LYM) counts (+59% and +71%, respectively), and log macrophage (MAC) counts (+63% and +72%, respectively). The individual leukocyte populations determined by cytofluorimetric analysis confirmed that mastitis infection increased the proportion of PMN in the milk samples compared with culture-negative samples, particularly when caused by Strep. agalactiae (+51%). In the case of MAC, the 2 pathogens behaved in opposite ways: Strep. agalactiae increased MAC by 41%, whereas Prototheca decreased MAC by 25%. Prototheca infection strongly increased the proportion of total T lymphocytes (TL; +87%) and T-helper lymphocytes (+83%). Accordingly, the (PMN+MAC):TL ratio increased with Strep. agalactiae infection (+95%) and decreased with Prototheca infection (-43%) compared with culture-negative samples. These results suggest the prevalence of an adaptive immune response and chronic inflammation in Prototheca infection, and an innate immune response to Strep. agalactiae. This knowledge might provide an important contribution to the development of novel and effective diagnostics and therapeutics.

Impact of somatic cell count combined with differential somatic cell count on milk protein fractions in Holstein cattle.

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 αS1-CN, αS2-CN, κ-CN, and ß-CN, and 3 whey protein fractions, namely ß-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 αS1-CN, ß-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 ß-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.

Associations between ultrasound hepatic measurements, body measures, and milk production traits in Holstein cows.

Ultrasound (US) imaging has been proposed as a noninvasive tool for monitoring liver dysfunction in dairy cows. This study, carried out on 306 clinically healthy Holstein cows in the first 120 d of lactation kept in 2 herds in northern Italy, aimed at investigating the association between US imaging-derived traits, namely predicted liver triacylglycerol content (pTAG, mg/g), liver depth (LD, mm), portal vein depth (PVD, mm) and area (PVA, mm2), and body size measurements, body condition score (BCS), and milk productivity indicators. Transcutaneous US examination, milk sampling, body size measurements (withers height and heart girth), and BCS were collected once from all cows in 10 sampling batches. The body weights (BW) of a subsample of 73 cows were recorded and used together with an existing data set of BW and measures of Holstein Friesian cows (n = 399) to develop a regression equation to predict BW, which was then used to compute productivity indicators by scaling the milk production traits to predicted BW. Body size measures, BCS, milk traits, and productivity indicators were classified (low, medium, and high) in 0.75 units of standard deviation of the residuals generated from a linear model that included the effects of parity, days in milk, and sampling batch. Liver pTAG, PVA, PVD, and LD were analyzed with a sequence of linear mixed models that included the fixed effects of days in milk and parity and the random effect of sampling batch as common terms, whereas the classes of body and milk traits and the productivity indicators were included one by one. The US-related traits were found to be associated with body size measurements and BCS. Specifically, pTAG was inversely related to BCS, whereas PVD and LD increased with increasing heart girth, BCS, and predicted BW. Generally, no relevant associations were observed between the US parameters and milk production traits, including when expressed in terms of productivity. In conclusion, this study suggests that US measures of liver dimensions of clinically healthy cows are related to their size, whereas pTAG concentrations reflect body condition status, with no particular implications for milk production and productivity. Moreover, healthy cows seemed able to counteract the metabolic stress of the first 120 d of the lactation period without straining liver functionality. Finally, US imaging proved to be a promising technique to assess liver metabolic conditions. However, further studies are needed to confirm its potential as a noninvasive tool for monitoring liver conditions in healthy cows.

The ß-casein (CSN2) A2 allelic variant alters milk protein profile and slightly worsens coagulation properties in Holstein cows.

Milk proteins genetic variants have long attracted interest as they are associated with important issues relating to milk composition and technological properties. An important debate has recently opened at an international level on the role of ß-casein (ß-CN) A1 and A2 polymorphisms, toward human health. For this reason, a lot of efforts has been put into the promotion of A2 milk by companies producing and selling A1-free milk, leading the farmers and breeders to switch toward A2 milk production without paying attention on the potential effect of the processability of milk into cheese. The aim of the present work was to evaluate the effects of ß-CN, specifically the A1 and A2 allelic variants, on the detailed milk protein profile and cheese-making traits in individual milk samples of 1,133 Holstein Friesian cows. The protein fractions were measured with reversed-phase (RP)-HPLC (expressed in g/L and % N), and the cheese-making traits, namely milk coagulation properties, cheese yield, and curd nutrient recoveries assessed at the individual level, with a nano-scale cheese-making procedure. The ß-CN (CSN2), κ-CN (CSN3), and ß-lactoglobulin (LGB) genetic variants were first identified through RP-HPLC and then confirmed through genotyping. Estimates of the effects of protein genotypes were obtained using a mixed inheritance model that considered, besides the standard nuisance variables (i.e., days in milk, parity, and herd-date), the milk protein genes located on chromosome 6 (CSN2, CSN3) and on chromosome 11 (LGB), and the polygenic background of the animals. Milk protein genes (CSN2, CSN3, and LGB) explained an important part of the additive genetic variance in the traits evaluated. The ß-CN A1A1 was associated with a significantly lower production of whey proteins, particularly of ß-lactoglobulin (-8.2 and -6.8% for g/L and % N, respectively) and α-lactalbumin (-4.7 and -4.4% for g/L and % N, respectively), and a higher production of ß-CN (6.8 and 6.1% for g/L and % N, respectively) with respect to the A2A2 genotype. Regarding milk cheese-making ability, the A2A2 genotype showed the worst performance compared with the other genotypes, particularly with respect to the BA1, with a higher rennet coagulation time (7.1 and 28.6% compared with A1A1 and BA1, respectively) and a lower curd firmness at 30 min. Changes in milk protein composition through an increase in the frequency of the A2 allele in the production process could lead to a worsening of the coagulation and curd firming traits.

Quarter-level analyses of the associations among subclinical intramammary infection and milk quality, udder health, and cheesemaking traits in Holstein cows.

In this study, we investigated associations among subclinical intra-mammary infection (IMI) and quarter-level milk composition, udder health indicators, and cheesemaking traits. The dataset included records from 450 Holstein cows belonging to three dairy herds. After an initial screening (T0) to identify animals infected by Streptococcus agalactiae, Streptococcus uberis, Staphylococcus aureus, and Prototheca spp., 613 quarter milk samples for 2 different sampling times (T1 and T2, 1 mo after T1) were used for analysis. Milk traits were analyzed using a hierarchical linear mixed model including the effects of days in milk, parity and herd, and bacteriological and inflammatory category [culture negative with somatic cell count (SCC) <200,000 cells/mL; culture negative with SCC ≥200,000 cells/mL; or culture positive]. All udder health indicators were associated with increased SCC and IMI at both sampling times. The largest effects were detected at T2 for milk lactose (-7% and -5%) and milk conductivity (+9% and +8%). In contrast, the increase in differential SCC (DSCC) in samples with elevated SCC was larger at T1 (+17%). Culture-negative samples with SCC ≥200,000 cells/mL had the highest SCC and greatest numbers of polymorphonuclear-neutrophils-lymphocytes and macrophages at both T1 and T2. Regarding milk cheesemaking ability, samples with elevated SCC showed the worst pattern of curd firmness at T1 and T2. At T2, increased SCC and IMI induced large decreases in recoveries of nutrients into the curd, in particular recovered protein (-14% and -16%) and recovered fat (-12% and -14%). Different behaviors were observed between Strep. agalactiae and Prototheca spp., especially at T2. In particular, samples that were positive for Strep. agalactiae had higher proportions of DSCC (+19%) compared with negative samples with low SCC, whereas samples that were positive for Prototheca spp. had lower DSCC (-11%). Intramammary infection with Prototheca spp. increased milk pH compared with culture-negative samples (+3%) and negative samples that had increased SCC (+2%). The greatest impairment in curd firmness at 30 min from rennet addition was observed for samples that were positive for Prototheca spp. (-99% compared with negative samples, and -98% compared with negative samples with high SCC). These results suggest that IMI caused by Prototheca spp. have detrimental effects on milk technological traits that deserve further investigation of the mechanisms underlying animals' responses to infection.

Genetic parameters of differential somatic cell count, milk composition, and cheese-making traits measured and predicted using spectral data in Holstein cows.

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 (k20), 0.45 with rennet coagulation time estimated using the curd firming over time equation (RCTeq), -0.39 with asymptotic potential curd firmness, -0.26 with maximum curd firmness (CFmax), 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 CFmax (-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), RCTeq (0.53 and 0.41), tmax (0.38 and 0.48). Log MAC count was correlated with k20 (+0.34), and log PMN-LYM count was correlated with CFmax (-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.

Genetic correlations between fertility traits and milk composition and fatty acids in Holstein-Friesian, Brown Swiss, and Simmental cattle using recursive models.

This study aimed to investigate the genetic and putative causal relationships between fertility traits [i.e., days open (DO) and calving rate (CR)] and milk quality, composition, and fatty acid contents (milk composition traits) in Holstein-Friesian, Brown Swiss, and Simmental cattle, using recursive models within a Bayesian framework. Trivariate animal models were run, each including one fertility trait, one milk composition trait, and, in all models, milk yield. The DO and CR data were merged with the test days closest to the insemination date for milk composition traits. After editing, 16,468 to 23,424 records for Holstein-Friesian, 23,424 to 46,660 for Brown Swiss, and 26,105 to 35,574 for Simmental were available for the analyses. Recursive animal models were applied to investigate the possible causal influences of milk composition traits on fertility and the genetic relationships among these traits. The results suggested a potential cause-and-effect relationship between milk composition traits and fertility traits, with the first trait influencing the latter. We also found greater recursive effects of milk composition traits on DO than on CR, the latter with some putative differences among breeds in terms of sensitivity. For instance, the putative causal effects of somatic cell score on CR (on the observed scale, %) varied from -0.96 to -1.39%, depending on the breed. Concerning fatty acids, we found relevant putative effects of C18:0 on CR, with estimates varying from -7.8 to -9.9%. Protein and casein percentages, and short-chain fatty acid showed larger recursive effects on CR, whereas fat, protein, and casein percentages, C16:0, C18:0, and long-chain fatty acid had larger effects on DO. The results obtained suggested that these milk traits could be considered as effective indicators of the effects of animal metabolic and physiological status on fertility.

Associations between differential somatic cell count and milk yield, quality, and technological characteristics in Holstein cows.

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.

Genetic parameters for fertility traits assessed in herds divergent in milk energy output in Holstein-Friesian, Brown Swiss, and Simmental cattle.

In this study, we aimed to investigate differences in the genetics of fertility traits (heritability of traits and correlations between traits in divergent environments) in dairy cows of different production levels defined on the basis of the herd-average daily milk energy output (herd-dMEO). Data were obtained from Holstein-Friesian (n = 37,359 for fertility traits, 381,334 for dMEO), Brown Swiss (n = 79,638 for fertility traits, 665,697 for dMEO), and Simmental cows (n = 63,048 for fertility traits, 448,445 for dMEO) reared in northeastern Italy. Fertility traits under study were interval from calving to first service, interval from first service to conception, days open, calving interval, calving rate, and nonreturn rate at d 56. We classified herds into low and high productivity based on the herd-average dMEO (inferred using mixed effects models). We estimated genetic parameters using Bayesian bivariate animal models, where expressions of a phenotype in the low and high dMEO herds were taken as being different-albeit correlated-traits. Fertility traits were more favorable in Simmental than in Holstein-Friesian cows, whereas for all traits, Holstein-Friesian had the highest estimates of intraherd heritability [ranging from 0.021 (0.006-0.038) to 0.126 (0.10-0.15)] and Simmental the lowest [ranging from 0.008 (0.001-0.017) to 0.101 (0.08-0.12)]. The genetic correlations between fertility traits and dMEO were moderate and unfavorable, ranging, in absolute values, from 0.527 (0.37-0.68) to 0.619 (0.50-0.73) in Holstein-Friesian; from 0.339 (0.20-0.47) to 0.556 (0.45-0.66) in Brown Swiss; and from 0.340 (0.10-0.60) to 0.475 (0.33-0.61) in Simmental cattle. The only exception was the nonreturn rate at d 56, which had weak genetic correlations with dMEO in all 3 breeds. The herd correlations between fertility and dMEO tended to be modest and favorable and the residual correlations modest and variable. The heritability of fertility traits tended to be greater in the low dMEO than in the high dMEO herds in the case of the Holstein-Friesians, but not in the case of the Brown Swiss or Simmentals. The additive genetic correlations between fertility traits in the low and high dMEO herds were always lower than 1 [0.329 (-0.17 to 0.85) to 0.934 (0.86 to 0.99)] for all traits considered in all breeds. The correlation was particularly low for the threshold characters and the interval from first service to conception in Holstein-Friesian, suggesting that the relative performances of genotypes vary significantly between herds of different dMEO levels. Although there was large variability in the estimates, results might support making separate genetic evaluations of fertility in the different herd production groups. Our results also indicate that Simmental, a dual-purpose breed, has higher fertility and lower environmental sensitivity than Holstein-Friesian, with Brown Swiss being intermediate.

Genome-wide association and pathway analysis of carcass and meat quality traits in Piemontese young bulls.

A key concern in beef production is how to improve carcass and meat quality traits. Identifying the genomic regions and biological pathways that contribute to explaining variability in these traits is of great importance for selection purposes. In this study, genome wide-association (GWAS) and pathway-based analyses of carcass traits (age at slaughter (AS), carcass weight (CW), carcass daily gain (CDG), conformation score and rib-eye muscle area) and meat quality traits (pH, Warner-Bratzler shear force, purge loss, cooking loss and colour parameters (lightness, redness, yellowness, chroma, hue)) were conducted using genotype data from the 'GeneSeek Genomic Profiler Bovine LD' array in a cohort of 1166 double-muscled Piemontese beef cattle. The genome wide-association analysis was based on the GRAMMAR-GC approach and identified 37 significant single nucleotide polymorphisms (SNPs), which were associated with 12 traits (P<5 × 10-5). In particular, 14 SNPs associated with CW, CDG and AS were located at 38.57 to 38.94 Mb on Bos taurus autosome 6 and mapped within four genes, that is, Leucine Aminopeptidase 3, Family with Sequence Similarity 184 Member B, Non-SMC Condensin I Complex Subunit G and Ligand-Dependent Nuclear Receptor Corepressor-Like. Strong pairwise linkage disequilibrium was found in this region. For meat quality traits, most associations were 1 SNP per trait, except for a signal on BTA25 (at ~11.96 Mb), which was significant for four of the five meat colour parameters assessed. Gene-set enrichment analyses yielded significant results for six traits (right-sided hypergeometric test, false discovery rate <0.05). In particular, several pathways related to transmembrane transport (i.e., oxygen, calcium, ion and cation) were overrepresented for meat colour parameters. The results obtained provide useful information for genomic selection for beef production and quality in the Piemontese breed.

Genetic and genomic analyses of latent variables related to the milk fatty acid profile, milk composition, and udder health in dairy cattle.

The aim of this study was to perform genetic, genome-wide association (GWAS), and gene-set enrichment analyses with latent variables related to milk fatty acid profile (i.e., fatty acids factor scores; FAF), milk composition, and udder health in a cohort of 1,158 Italian Brown Swiss cows. The phenotypes under study were 12 FAF previously identified through factor analysis and classified as follows: de novo FA (F1), branched-chain FA-milk yield (F2), biohydrogenation (F3), long-chain fatty acids (F4), desaturation (F5), short-chain fatty acids (F6), milk protein and fat contents (F7), odd fatty acids (F8), conjugated linoleic acids (F9), linoleic acid (F10), udder health (F11) and vaccelenic acid (F12). (Co)variance components were estimated for factor scores using a Bayesian linear animal model via Gibbs sampling. The animals were genotyped with the Illumina BovineSNP50 BeadChip v.2 (Illumina Inc., San Diego, CA). A single marker regression model was fitted for GWAS analysis. The gene-set enrichment analysis was run on the GWAS results using the Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway databases to identify the ontologies and pathways associated with the FAF. Marginal posterior means of the heritabilities of the aforementioned FAF ranged from 0.048 for F12 to 0.310 for F5. Factors F1 and F6 had the highest number of relevant genetic correlations with the other traits. The genomic analysis detected a total of 39 significant SNP located on 17 Bos taurus autosomes. All latent variables produced signals except for F2 and F10. The traits with the highest number of significant associations were F11 (17) and F12 (7). Gene-set enrichment analyses identified significant pathways (false discovery rate 5%) for F3 and F7. In particular, systemic lupus erythematosus was enriched for F3, whereas the MAPK (mitogen-activated protein kinase) signaling pathway was overrepresented for F7. The results support the existence of important and exploitable genetic and genomic variation in these latent explanatory phenotypes. Information acquired might be exploited in selection programs and when designing further studies on the role of the putative candidate genes identified in the regulation of milk composition and udder health.

Milk protein composition in purebred Holsteins and in first/second-generation crossbred cows from Swedish Red, Montbeliarde and Brown Swiss bulls.

The aim of this study was to analyze milk protein composition in purebred and crossbred dairy cattle and estimate the effects of individual sources of variation on the investigated traits. Milk samples were collected from 505 cows from three commercial farms located in Northern Italy, some of which had originated from crossbreeding programs, although most were purebred Holsteins (HO). The basic crossbreeding scheme was a three-breed rotational system using Swedish Red (SR) semen on HO cows (SR×HO), Montbeliarde (MO) semen on SR×HO cows (MO×(SR×HO)) and HO semen again on MO×(SR×HO) cows. A smaller number of purebred HO from each of the herds were mated inverting the breed order (MO×HO and SR×(MO×HO)) or using Brown Swiss (BS) bulls (BS×HO) then MO bulls (MO×(BS×HO)). Milk samples were analyzed by reverse-phase HPLC to obtain protein fraction amounts (g/l) and proportions (% of total true protein). Traits were analyzed using a linear model, which included the fixed effects of herd-test-day (HTD), parity, days in milk and breed combination. Results showed that milk protein fractions were influenced by HTD, stage of lactation, parity and breed combination. The increase in protein concentration during lactation was due in particular to ß-casein (ß-CN), α S1-CN and ß-lactoglobulin (ß-LG). The higher protein content of primiparous milk was mainly due to higher concentrations of all casein fractions. The milk from crossbred cows had higher contents and proportions of κ-CN and α-lactalbumin (α-LA), lower proportions of ß-LG and greater proportion of caseins/smaller in whey proteins on milk true protein than purebred HO. The three-way crossbreds differed from two-way crossbreds only in having greater proportions of α-LA in their milk. Of the three-way crossbreds, the SR sired cows yielded milk with a smaller content and proportion of ß-LG than the MO sired cows, and, consequently, a higher proportion of caseins than whey proteins. Results from this study support the feasibility of using crossbreeding programs to alter milk protein profiles with the aim of improving milk quality and cheese-making properties.

Factors affecting variations in the detailed fatty acid profile of Mediterranean buffalo milk determined by 2-dimensional gas chromatography.

Buffalo milk is the world's second most widely produced milk, and increasing attention is being paid to its composition, particularly the fatty acid profile. The objectives of the present study were (1) to characterize the fatty acid composition of Mediterranean buffalo milk, and (2) to investigate potential sources of variation in the buffalo milk fatty acid profile. We determined the profile of 69 fatty acid traits in 272 individual samples of Mediterranean buffalo milk using gas chromatography. In total, 51 individual fatty acids were identified: 24 saturated fatty acids, 13 monounsaturated fatty acids, and 14 polyunsaturated fatty acids. The major individual fatty acids in buffalo milk were in the order 16:0, 18:1 cis-9, 14:0, and 18:0. Saturated fatty acids were the predominant fraction in buffalo milk fat (70.49%); monounsaturated and polyunsaturated fatty acids were at 25.95 and 3.54%, respectively. Adopting a classification based on carbon-chain length, we found that medium-chain fatty acids (11-16 carbons) represented the greater part (53.7%) of the fatty acid fraction of buffalo milk, whereas long-chain fatty acids (17-24 carbons) and short-chain fatty acids (4-10 carbons) accounted for 32.73 and 9.72%, respectively. The n-3 and n-6 fatty acids were 0.46 and 1.77%, respectively. The main conjugated linoleic acid, rumenic acid, represented 0.45% of total milk fatty acids. Herd/test date and stage of lactation were confirmed as important sources of variation in the fatty acid profile of buffalo milk. The percentages of short-chain and medium-chain fatty acids in buffalo milk increased in early lactation (+0.6 and +3.5%, respectively), whereas long-chain fatty acids decreased (-4.2%). The only exception to this pattern was butyric acid, which linearly decreased from the beginning of lactation, confirmation that its synthesis is independent of malonyl-CoA. These results seem to suggest that in early lactation the mobilization of energy reserves may have less influence on the fatty acid profile of buffalo milk than that of cow milk, probably due to a shorter and less severe period of negative energy balance. Parity affected the profiles of a few traits and had the most significant effects on branched-chain fatty acids. This work provided a detailed overview of the fatty acid profile in buffalo milk including also those fatty acids present in small concentrations, which may have beneficial effects for human health. Our results contributed also to increase the knowledge about the effects of some of the major factors affecting buffalo production traits and fatty acid concentrations in milk, and consequently its technological and nutritional properties.