This study aimed to examine the influence of feed delivery frequency and environmental conditions on daily time budget of lactating dairy cows. The study was carried out in two commercial dairy farms with Holstein herds. Fifty lactating dairy cows milked in automatic milking units (AMS farm) and 96 primiparous lactating dairy cows milked in a conventional milking parlour (conventional farm) were exposed to different frequencies of feed delivery replicated in different periods of the year (warm and mild) that were characterized by different temperature-humidity indices (THI). On each farm, feeding treatments consisted of two different feed delivery frequencies (1× and 2× on the AMS farm; 2× and 3× on the conventional farm). All behaviours of the cows were monitored for the last 8 d of each treatment period using continuous video recording. The two data sets from different farm systems were considered separately for analysis. On both farms, environmental conditions expressed as THI affected time budgets and the pattern of the behavioural indices throughout the day. The variation in the frequency of feed delivery seems to affect the cow's time budget only in a limited way. Standing time of cows on the conventional farm and the time spent by cows in the milking waiting area on the AMS farm both increased in response to increased feeding frequency. Although feed delivery frequency showed limited influence on cow's time budget, the effect on standing time could be carefully considered, especially on farms equipped with AMS where the type of cow traffic system (e.g., milking first) might amplify the negative consequences of more frequent feed delivery. Further investigations are required to evaluate the effect of THI and feed delivery frequency on other aspects of behavioural activity.

The purposes of this study were to investigate the systemic and characteristic metabolites in the serum of dairy goats induced by aflatoxin B1 (AFB1) exposure and to further understand the endogenous metabolic alterations induced by it. A nuclear magnetic resonance (NMR)-based metabonomic approach was used to analyse the metabolic alterations in dairy goats that were induced by low doses of AFB1 (50 µg/kg DM). We found that AFB1 exposure caused significant elevations of glucose, citrate, acetate, acetoacetate, betaine, and glycine yet caused reductions of lactate, ketone bodies (acetate, β-hydroxybutyrate), amino acids (citrulline, leucine/isoleucine, valine, creatine) and cell membrane structures (choline, lipoprotein, N-acetyl glycoproteins) in the serum. These data indicated that AFB1 caused endogenous metabolic changes in various metabolic pathways, including cell membrane-associated metabolism, the tricarboxylic acid cycle, glycolysis, lipids, and amino acid metabolism. These findings provide both a comprehensive insight into the metabolic aspects of AFB1-induced adverse effects on dairy goats and a method for monitoring dairy animals exposed to low doses of AFB1.

The aims of the study described in the Research Communication were to determine the level of influence of some environmental factors on milk yield and quality traits, including lactose, and lactation length in ewes belonging to three different Italian breeds and to estimate the heritability for the same traits. A total of 2138 lactation records obtained from 535 ewes belonging to three different Italian breeds (Comisana, Leccese, and Sarda) were used. Breed significantly affected all of the considered traits. Moreover, year of lambing affected milk yield and lactation length without influence on milk quality traits. Parity affected significantly only the milk yield, whereas type of birth showed its effect on milk yield, fat, protein, and lactose yield. On the whole, the presently reported heritability estimates are within the range of those already obtained in other dairy breeds by other authors, with values for lactation length being very low in all the investigated populations. Considering the heritability estimates for lactose content and yield, to the best of our knowledge, there is a lack of information on these parameters in ovine species and this is the first report on heritability of lactose content and yield in dairy sheep breeds. Our results suggest that genetic variability for milk traits other than lactation length is adequate for selection indicating a good response to selection in these breeds.

The objective of this study was to design and validate a mathematical model to detect post-calving ketosis. The validation was conducted in four commercial dairy farms in Israel, on a total of 706 multiparous Holstein dairy cows: 203 cows clinically diagnosed with ketosis and 503 healthy cows. A logistic binary regression model was developed, where the dependent variable is categorical (healthy/diseased) and a set of explanatory variables were measured with existing commercial sensors: rumination duration, activity and milk yield of each individual cow. In a first validation step (within-farm), the model was calibrated on the database of each farm separately. Two thirds of the sick cows and an equal number of healthy cows were randomly selected for model validation. The remaining one third of the cows, which did not participate in the model validation, were used for model calibration. In order to overcome the random selection effect, this procedure was repeated 100 times. In a second (between-farms) validation step, the model was calibrated on one farm and validated on another farm. Within-farm accuracy, ranging from 74 to 79%, was higher than between-farm accuracy, ranging from 49 to 72%, in all farms. The within-farm sensitivities ranged from 78 to 90%, and specificities ranged from 71 to 74%. The between-farms sensitivities ranged from 65 to 95%. The developed model can be improved in future research, by employing other variables that can be added; or by exploring other models to achieve greater sensitivity and specificity.

This Technical Research Communication describes new analytical methods for free, unbound glutamic acid and glutamine in protein-free blood plasma and milk and introduces the use of quantitation of free amino groups in the same matrices for descriptive and analytical purposes. The present enzymatic-fluorometric methods are easily performed within one working day, allowing for ‘high throughput’ assays of animal trials. These assays could support and enable further studies in lactation physiology with the objective of improved metabolic health.

High concentrations of non-esterified fatty acid (NEFA) and β-hydroxybutyrate (BHBA) in cows’ blood caused by ketosis are associated with inflammatory states. We hypothesised that ketosis in postparturient dairy cows would result in altered levels on inflammation-related proteins not only in plasma but also in the milk fat globule membranes (MFGM). Thirty cows were selected from a dairy farm in Heilongjiang, China. Inflammatory milk fat globule membrane proteins were detected using ELISA kits, and a fully automatic biochemical analyser was used to measure the concentrations of BHBA, NEFA, glucose (GLU) and triglyceride (TG) in plasma. MFGM protein from milk of ketotic cows contained significantly different concentrations of acute-phase response proteins (complement C3 (C3), prothrombin (F2), alpha-1-acid glycoprotein (ORM1), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), alpha-2-HS-glycoprotein (AHSG), complement C9 (C9), complement regulatory protein variant 4 (CD46)) in comparison with milk from non-ketotic cows. Blood concentrations of C3, complement C9 (C9), tumour necrosis factor α (TNFα), MFGM C3, monocyte differentiation antigen CD14 (CD14) and ORM1 levels were correlated with energy balance. ITIH4 and CD46 increased, and AHSG and ORM1 decreased before the onset of ketosis. These biomarkers offer potential as predictors and monitors of ketosis in at-risk cows.

The aim of this work was to fit and compare three non-linear models (Wood, Milkbot and diphasic) to model lactation curves from two approaches: with and without cow random effect. Knowing the behaviour of lactation curves is critical for decision-making in a dairy farm. Knowledge of the model of milk production progress along each lactation is necessary not only at the mean population level (dairy farm), but also at individual level (cow-lactation). The fits were made in a group of high production and reproduction dairy farms; in first and third lactations in cool seasons. A total of 2167 complete lactations were involved, of which 984 were first-lactations and the remaining ones, third lactations (19 382 milk yield tests). PROC NLMIXED in SAS was used to make the fits and estimate the model parameters. The diphasic model resulted to be computationally complex and barely practical. Regarding the classical Wood and MilkBot models, although the information criteria suggest the selection of MilkBot, the differences in the estimation of production indicators did not show a significant improvement. The Wood model was found to be a good option for fitting the expected value of lactation curves. Furthermore, the three models fitted better when the subject (cow) random effect was considered, which is related to magnitude of production. The random effect improved the predictive potential of the models, but it did not have a significant effect on the production indicators derived from the lactation curves, such as milk yield and days in milk to peak.

This Research Communication describes, for the first time, the detection of HSP70 in saliva of dairy cows. Thermal stress is a major environmental stress that limits animal growth, metabolism, and productivity. The cellular response to heat stress involves the synthesis of heat shock proteins (HSPs), presumably to protect the functional stability of cells at increasing temperatures. HSP70 has been found to be present in cattle blood serum and may also be present in other secretory fluids, such as saliva, as already observed in humans. The aim of this study was to detect heat shock protein HSP70 in bovine saliva. Saliva samples were taken from higher- (n = 5) and lower milk producing (n = 5) Holstein-Friesian cows in summer and in winter for the detection of HSP70. HSP70 concentrations were assayed using the ELISA technique. Salivary HSP70 concentrations ranged from 0·524 to 12·174 ng/ml in cows. Higher salivary HSP70 concentrations were significantly associated with higher milk production and higher environmental temperature, but not with rectal temperature.

The objective of the studies reported in this research communication was to investigate differences in composition and enzymatic activities in bulk milk samples provided from Swedish dairy farms with different management systems, i.e. automated (AMS) and conventional milking systems (CMS). A bulk milk sample was collected from each of 104 dairy farms, 51 using AMS and 53 using CMS, located in the same geographical region. Sampling took place within two consecutive days during the indoor period (October). Milk samples were analysed for contents of total fat and protein, free fatty acids (FFA), caseins and whey proteins, somatic cell count (SCC), pH, plasmin and plasminogen derived activities, and total proteolysis. Our results showed a lower protein content and higher SCC in bulk milk from AMS herds compared with milk from CMS herds. Plasmin, plasminogen and total plasmin/ plasminogen derived activities were lower in milk from AMS herds but despite this, total casein and the ß-casein fraction as % of total protein were lower in milk from AMS herds than in milk from herds using CMS. Total proteolysis was higher in milk from AMS herds, suggesting that other proteases than plasmin, e.g. cellular and bacterial proteases, contributed to the degradation of casein. This was supported by a positive correlation between SCC and total proteolysis (P < 0·01), as well as a negative correlation between total proteolysis and ß-casein fraction (P < 0·05). In conclusion, comparing the quality of bulk milk from commercial dairy herds using AMS and CMS, respectively, several differences were observed, suggesting a significant effect from management system.

This research communication describes the profile of gene expression related to the synthesis of yak milk as determined via quantitative reverse transcription polymerase chain reaction (RT-qPCR). Significant up-regulation during lactation were observed in genes related to fatty acid (FA) uptake from blood (LPL, CD36), intracellular FA transport (FABP3), intracellular FA activation of long- and short-chain FAs (ACSS1, ACSS2, ACSL1), de novo synthesis (ACACA), desaturation (SCD), triacyglycerol (TAG) synthesis (AGPAT6, GPAM, LPIN1), lipid droplet formation (PLIN2, BTN1A1, XDH), ketone body utilisation (BDH1, OXCT1), and transcription regulation (THRSP, PPARGC1A). In particular, intracellular de novo FA synthesis (ACSS2, ACACA, and FABP3) and TAG synthesis (GPAM, AGPAT6, and LPIN1), whose regulation might be orchestrated as part of the gene network under the control of SERBF1 in the milk fat synthesis process, were more activated compared to levels in dairy cows. However, the genes involved in lipid droplet formation (PLIN2, XDH, and BTN1A1) were expressed at lower levels compared to those in dairy cows, where these genes are mainly controlled by the PPARG regulator.

The aim of the study reported in this Research Communication was to analyse the variations of milk cortisol concentrations in response to the relocation of dairy cows between production groups. Milk cortisol measured during 3 consecutive days did not vary significantly in cows without environmental perturbation. However, relocation of cows caused a significant increase of cortisol in milk starting from the first milking after the group change. This suggests that cortisol in milk can be a suitable biomarker to assess the HPA response of dairy cows to a short/medium-term environmental challenge.

The work reported in this Research Communication describes the modification in epithelial cell populations during the first and the last month of milking in Holstein Friesian cows that have undergone different management during the dry period, and we report the differential expression of CD49f+ and cytokeratin18+ cell subpopulations. Twenty six cows were randomly divided into 2 balanced groups that were housed at stocking density of either 11 m2 (CTR) or 5 m2 from 21 ± 3 d before the expected calving until calving. Cells collected from milk samples taken in early lactation and late lactation were directly analysed for CD45, CD49f, cytokeratin 14, cytokeratin 18 and cell viability. We observed a differential expression with a significant reduction in CD49f+ (P < 0·01) and cytokeratin 18+ (P < 0·05) cells in early lactation. Differences were still evident in late lactation but were not significant. These observations suggest that mammary epithelial cell immunophenotypes could be associated with different animal management in the dry period and we hypothesise they may have a role as biomarkers for mammary gland function in dairy cows.

Myeloperoxidase (MPO) is an important enzyme, which is one of the components of the antibacterial system in neutrophils and monocytes. MPO participates in the inflammatory response in multiple locations in the body, including the mammary glands. As a result of the activity of MPO, many oxidising compounds as well as reactive oxygen species are generated. It seems that myeloperoxidase may be a marker linking inflammation processes and oxidative stress. So far, there are no literature data on the association between the MPO gene polymorphism and the antioxidant properties of milk. The aim of the study was to analyse the effect of g.9476869G > A polymorphism of myeloperoxidase (MPO) gene and age of cows on the antioxidant activity of milk and other milk traits in Polish Holstein-Friesian cows. Polymorphism of MPO gene was identified by the PCR-RFLP method using the HphI endonuclease. The total antioxidant capacity of milk samples was measured by the Trolox Equivalent Antioxidant Capacity (TEAC) method. It was found that the GG genotype was the most frequent (0·606). The genotype at the tested MPO locus and the age of the animals affected the antioxidant activity of milk. Milk from cows with the GA genotype was characterised by a significantly higher antioxidant activity than milk from cows with the GG genotype (P < 0·0001). The analysis of interaction showed that cows with the GA genotype and older than 6·5 years produced milk with a significantly higher antioxidant activity compared with younger cows with the same genotype (P < 0·0001), as well as cows with the GG genotype of all ages (P < 0·0001).

This paper aimed at evaluating the effect of summer transhumance to mountain pastures of dairy cows of different breeds on cheese-making ability of milk. Data were from 649 dairy cows of specialized (Holstein Friesian and Brown Swiss) dual purpose (Simmental) and local (mostly Rendena and Alpine Grey) breeds. The Fourier-Transform Infra-Red Spectra (FTIRS) of their milk samples were collected before and after transhumance in 109 permanent dairy farms, and during transhumance in 14 summer farms (with multi-breeds herds) of the Trento Province, north-eastern Italy. A variety of 18 traits describing milk coagulation, curd firming, cheese yield and nutrients recovery in curd/loss in whey were predicted on the basis of FTIRS collected at the individual cow level. Moving the cows to summer farms improved curd firming traits but reduced cheese yields because of an increase of water and fat lost in the whey. During summer grazing, most of cheese-making traits improved, often non-linearly. The milk from summer farms supplementing cows with more concentrates showed better curd firming and cheese yield, because of lower fat lost in the whey. The breed of cows affected almost all the traits with a worst cheese-making ability for milk samples of Holsteins through all the trial, and interacted with concentrate supplementation because increasing compound feed tended to improve cheese-making traits for all breed, with the exception of local breeds for coagulation time and of Brown Swiss for curd firming time. In general, summer transhumance caused a favourable effect on cheese-making aptitude of milk, even though with some difference according to parity, initial days in milk, breed and concentrate supplementation of cows.

The aim of this Regional Research Communication was to validate a panel of 30 microsatellite markers recommended by FAO/ISAG for studies of biodiversity in cattle to improve the characterisation of Cuban buffalo populations. The water buffalo (Bubalus bubalis) is an economically important livestock species. Therefore, research focused on the study of the genetic relationships among water buffalo populations is useful to support conservation decisions and to design breeding schemes. Twenty-eight of the 30 tested regions were amplified, one of which (ETH10) turned out to be monomorphic. A total of 143 alleles were observed in the Cuban water buffalo population. The average number of alleles per locus was 5·04. The number of alleles per polymorphic locus ranged from two (INRA 63 and MM12) to nine (ETH185). The observed and expected heterozygosity ranged from 0·108 (HAUT24) to 0·851 (CSSM66) and 0·104 (MM12) to 0·829(INRA32), respectively. The polymorphic information content (PIC) ranged from 0·097 (MM12) to 0·806 (INRA32), and the overall value for these markers was 0·482. Within the population, inbreeding estimates (FIS) was positive in 14 of the 30 loci analysed. This study thus highlights the usefulness of heterologous bovine microsatellite markers to assess the genetic variability in Cuban water buffalo breeds. Furthermore, the results can be utilised for future breeding strategies and conservation.

Bovine mastitis, an inflammation of the udder, results in reduced milk production and poor milk quality. Mastitis is usually, but not always, a response to pathogen infection. High milk yield can produce oxidative stress in the mammary tissue. High milk yield is also known to be associated with bovine mastitis. Thus, in the current study, we hypothesised that oxidative stress increases inflammatory responses in bovine mammary cells. To examine the hypothesis, we produced cellular oxidative stress and investigated resulting inflammatory responses in bovine mammary alveolar cells (MAC-T). To produce oxidative stress, cells were treated with the reactive oxygen species (ROS; e.g., superoxide anion)-producing agent, menadione (MD; 0–10 µm; 6 h). To ensure the ROS-induced responses, cells were pretreated with an antioxidant NAC (0–10 mm; 1 h). Results showed that MD elevated intracellular ROS levels and protein expression of cyclooxygenase-2 (COX-2), a biomarker of inflammation. Pretreatment of cells with NAC attenuated MD-induced COX-2 expression by scavenging intracellular ROS and enhancing intracellular glutathione levels. MD-induced COX-2 expression was mediated by activation of extracellular signal receptor-activated kinase 1/2 (ERK1/2), Akt, and nuclear factor-kappa B (NF-κB). NAC attenuated activation of these intracellular signalling molecules. Treatment of cells with pharmacological inhibitors for ERK1/2, Akt, and NF-κB confirmed the association of these signalling pathways in MD-induced COX-2 expression. These results support our hypothesis that oxidative stress, which is found in high-yielding dairy cows, can produce cellular inflammation in bovine mammary alveolar cells and prevention of oxidative stress can attenuate such pathological responses. This may be relevant for cases of clinical mastitis for which no pathogen can be isolated.

The objective of this Research Communication was to use polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) analysis to investigate a region of the bovine TLR4 gene (TLR4) in pasture-fed New Zealand (NZ) Holstein-Friesian × Jersey (HF × J) cross dairy cows and to determine whether gene variation was associated with milk production traits. Genetic variation was observed, with two variants (A and B) containing a single nucleotide polymorphism (SNP) (c.2021C/T) that was non-synonymous and putatively results in a p.Thr674Ile substitution in the transmembrane/cytoplasmic domain of TLR4. Variant A was associated with higher milk yields, but lower milk fat percentages, whereas B was associated with lower milk yields, but higher fat and protein percentages. Cows of genotype AA produced more milk than AB or BB cows, but the milk produced by AA cows contained less fat than AB or BB cows.

The purpose of the study reported in this Research Communication was to evaluate alterations in concentration of total antioxidant capacity (TAC) of plasma, plasma total thiols as markers of oxidative protein damage and malondialdehyde (as a final product of lipid peroxidation) in samples obtained at different stages of the lactation cycle and dry period of dairy cows. We found that TAC was significantly lower in the primiparous cows compared to multiparous cows. This study clearly demonstrates a need for monitoring primiparous cows during the production cycle, especially when they are faced with severe metabolic conditions. Furthermore, TAC may be a sensitive, reliable and useful indicator for measurement of cumulative effects of antioxidants as an addition to metabolic profile tests, which are currently used to analyse dairy cattle health.

The purpose of the study described in this Research Communication was to report the full characterisation of the goat and sheep oxytocin-neurophysin I gene (OXT), their promoters and amino acid sequences. Using the genomic DNA as template, we sequenced and compared the whole OXT gene (3 exons), plus 958/960 nucleotides at the 5′ flanking region and 478/477 nucleotides at the 3′ flanking region, in 46 sheep and 24 goats belonging to different breeds/genetic types reared in Italy, Greece and Germany. The comparison of the obtained sequences showed a high degree of genetic variability at these loci. In particular, we focused on the SNP g.438T > C as possible example of trans-specific polymorphism. This SNP alters a putative binding site of the transcription factor Oct-1. The set-up of a luciferase assay confirmed that the C variant of this SNP negatively affects the promoter activity of the sheep OXT gene. The results of this study suggest that the SNP g.438T > C might be useful to promote association studies with traits/physiological processes controlled by this hormone.

Recently published work as described by the authors highlighted the extent of Complement activity in bovine milk. Localised mastitis infection occurring in the mammary glands of dairy cows is readily detectable by the levels of somatic cells in milk. Thus, it is opportune to monitor Complement activity in milks in association with the animal's innate immune response to mammary infection. Preliminary screening of milk samples taken randomly showed that milk with a high somatic cell count (SCC) reduced growth of the Complement-sensitive strain E. coli O111 to a greater extent (P < 0·05) than when the marker microorganism was grown in milk heated for the purpose of inactivating Complement. A follow-up study set out to determine the effect on Complement activity when a sub-clinical mastitis infection was induced in the mammary gland of four lactating dairy cows. The effect of Str. dysgalactiae spp. dysgalactiae inoculation into selected individual udder quarters of the mammary glands of each animal was followed by monitoring of SCC levels in the milks from the segregated udder samples during subsequent milking. At 72 and 96 h post inoculation (PI), the SCCs for the challenged quarter were increased compared to normal values. At the same time, the bactericidal sequestration assay identified increased E. coli O111 inhibition that can be directly linked to greater Complement activity in those quarter milks affected by induced inflammation. Thus, it can be identified that the high SCC milks were more effective in limiting E. coli O111 growth. Milks from the unchallenged quarters in all four cows were significantly less effective at reducing growth of the assay strain (P < 0·05).

An ELISA assay targeting specific activation components of the Complement pathways confirmed that greater bacterial inhibition observed during the bactericidal sequestration assay was attributable to higher Complement activity in the milk samples from the affected quarters, i.e., with higher SCC. The induced infection was confirmed as self-limiting in three of the affected animals and their SCC returned to normal levels within 14 d PI, while the fourth cow required brief antibiotic intervention.