During routine microbiological examination of milk samples from dairy cows without clinical signs of mastitis, quarter milk samples of 231 dairy cows from 12 herds were investigated for the presence of coagulase-negative staphylococci (CNS). The isolates were identified on the basis of colony morphology, Gram staining, catalase and coagulase test and the commercial kit, API Staph. CNS was detected in 29% (67/231) of the cows. A total of seven CNS species were identified with the most prevalent being Staphylococcus (Staph.) chromogenes (30%) and Staph. haemolyticus (28·8%), followed by Staph. simulans (11·2%), Staph. xylosus (11·2%), Staph. epidermidis (7·5%), Staph. hyicus (6·3%) and Staph. sciuri (5%). The predominant species, Staph. chromogenes and Staph. haemolyticus, were further characterized by antibiotic susceptibility testing using the agar disc diffusion method (Kirby-Bauer) and by pulsed-field gel electrophoresis (PFGE). Considerable resistance to ampicillin and penicillin was observed in both species. Isolates with identical or highly similar PFGE profiles were detected at the herd level despite a marked heterogeneity seen for both species. On the basis of somatic cell count, absence of clinical signs of inflammation and heterogeneity of genotypes, we assume that CNS isolated in this study could not be considered as important causative agents of the bovine mammary gland inflammation.

In this prospective observational study rectal and vaginal temperature of 82 (26 primiparous, 56 multiparous) early post-partum healthy dairy cows that calved without intervention within 3 months and did not show clinical signs of infectious and metabolic diseases were continuously measured and evaluated for associations with plausible factors during the first 10 days in milk (DIM). During May, June and July mean (±sd) temperature humidity index (THI) was 60·1±5; 66·8±5·6 and 74·2±4·3, respectively. Environmental conditions had a negligible effect on body temperature (BT) during May (P<0·05). During June and July, however, the ambient temperature and THI influenced BT (P<0·05). Furthermore, plausible factors like parity, DIM, months and time of day had an effect on BT (P<0·05). Overall, primiparous cows demonstrated 0·2°C greater BT during the first 10 DIM than multiparous cows. The effect of parity, however, on BT varied between DIM according to month (P<0·001). During this 3-month study period all cows demonstrated BT rhythms; however, the amplitude of BT increased from May to July (0·3 to 0·7°C). A greater proportion of the vaginal temperature measurements exceeded a threshold tested (⩾39·5°C) during July (46·8%) than in June (33·9%) and May (19·3%). Overall the percentage of BT values above a threshold of ⩾39·5°C was lower during the period 6.00–10.00 compared with the remaining 20 h (P<0·05). Therefore this study concluded that the BT of healthy post-partum dairy cows during the period 1–10 DIM post partum is greater compared with the reference range of 38·6 to 39·5°C reported by others and is influenced by parity, DIM, time of day and THI. When the association between BT and THI increased the reliability of threshold levels of BT (⩾39·5°C) decreased.

Intake of dietary phyto-oestrogens has received a great deal of attention owing to their potential influence on hormone-sensitive cancers such as breast and prostate cancer. Cows’ milk contains phyto-oestrogens and the content varies according to the composition of the feed and the type and amount of legumes used. In this study we evaluated the proliferative effect of milk (whey) with different phyto-oestrogen content in human breast (MCF-7) and prostate cancer cells (PC-3). Milk was obtained from cows fed either a birdsfoot trefoil-timothy silage based ration (B1) or two different red clover silage based diets (R1 and R2) resulting in total phyto-oestrogen contents of 403, 1659 and 1434 ng/ml for the B1, R1 and R2 diets, respectively. Whey was produced from the milk and added to cell culture medium in concentrations up to 10% for MCF-7 cells and 5% for PC-3 cells. Cell proliferation was measured fluorometrically after 7 d for MCF-7 cells and 5 d for PC-3 cells. There was no significant difference in the proliferative effect of whey from the different dietary treatments at any of the whey concentrations tested. An anti-proliferative effect (P<0·01) of 5 and 10% whey was seen when tested in the presence of 10 pm oestradiol in the medium. This effect was independent of dietary treatment of cows. Whey induced a significant (P<0·01) proliferative response in PC-3 cells independent of dietary treatment. Purified equol in concentrations similar to equol concentrations in milk decreased PC-3 cell proliferation, and therefore the stimulatory effect of whey in PC-3 cells is believed to be mediated by other bioactives than equol. In conclusion, our results suggest that using whey in these proliferation assays, it was not possible to discriminate between milk with high or low levels of phyto-oestrogens.

The aim of this study was to evaluate possible associations between three SNPs at the oxytocin locus (AM234538: g.28C>T; g.204A>G and g.1627G>T) and two productive traits, milk yield and milkability, in Italian Mediterranean river buffaloes. Effects of parity, calving season and month of production were also evaluated. A total of 41 980 test-day records belonging to 219 lactations of 163 buffalo cows were investigated. The allele call rate was 98·8% and the major allele frequency for all the investigated loci was 0·76. The OXT genotype was significantly associated with milk yield (P=0·029). The TT genotype showed an average daily milk yield approximately 1·7 kg higher than GT buffaloes. Such a difference represents about 23% more milk/d. A large dominance effect (−1·17±0·43 kg) was estimated, whereas the contribution of OXT genotype (r2OXT) to the total phenotypic variance in milk yield was equal to 0·06. The TT genotype showed higher values also for the milk flow, even though the estimated difference did not reach a level of statistical significance (P=0·07). Such an association, among the first reported for the oxytocin locus in ruminants, should be tested on a population scale and possible effects on milk composition traits should be evaluated in order to supply useful indications for the application of marker-assisted selection programmes in river buffaloes.

The objective of this study was to provide insight into the biological mechanisms underlying mammary development and the role of the ovaries in prepubertal caprine mammogenesis using a serial ovariectomy approach. Young Alpine goats were ovariectomized (Ovx) or sham-operated (Int) at three periods before puberty (G1=1 month, G2=2 month and G3=3 months of age) and one after puberty (G7=7 months of age). The goats were slaughtered at 9 months of age and mammary glands were removed. Ovariectomy performed at 1, 2 and 3 months of age caused a 50% reduction in DNA concentration, in mammary tissue taken from the parenchyma-stroma border region. Morphological analysis of mammary tissue sections indicated that the parenchymal structures of Ovx goats were negatively affected by ovariectomy. Goats ovariectomized before 2 months of age (Ovx-1 and Ovx-2) showed a significant decrease in the percent of cells proliferating in mammary glands of 9-month old goats (proliferating cell nuclear antigen expression and antigen Ki67-positive cell number). Also, goats ovariectomized at 1 and 2 months of age had reduced matrix metalloprotease 2 activity at 9 months of age. E-cadherin was strongly decreased in goats ovariectomized before 2 months of age (80 and 85% in Ovx-1 and Ovx-2 goats, respectively). Quantitative PCR analysis of transcripts encoding for oestrogen (ERα) and progesterone receptors (PR) and immunodetection of ERα showed that ovariectomy at 1 and 2 months of age strongly inhibited the transcription of ERα and PR in the mammary gland. We conclude that ovariectomy before 3 months of age markedly impaired parenchymal development. These findings suggest that prepubertal mammogenesis in goats depends on the ovaries to initiate mammary epithelial cell proliferation and mammary gland remodelling.

Fresh cheeses from pasteurised (80°C for 15 s), homogenised–pasteurised (15+3 MPa at 60°C; 80°C for 15 s) or ultra-high pressure homogenised milks (300 MPa and inlet temperature of 30°C) were produced in order to evaluate different technological aspects during cheese-making and to study their microbial shelf life. Although the coagulation properties of milk were enhanced by ultra-high pressure homogenisation (UHPH), the cheese-making properties were somewhat altered; both conventional homogenisation and UHPH of milk provoked some difficulties at cutting the curd due to crumbling and improper curd matting due to poor cohesion of the grains. Cheese-milk obtained by UHPH showed a higher microbiological quality than milk obtained by conventional treatments. Starter-free fresh cheeses made from UHPH-treated milk showed less syneresis during storage and longer microbiological shelf-life than those from conventionally treated milk samples.

Milk for cheese production in Ireland is predominantly produced by pasture-fed spring-calving herds. Consequently, there are marked seasonal changes in milk composition, which arise from the interactive lactational, dietary and environmental factors. In this study, Cheddar cheese was manufactured on a laboratory scale from milk taken from a spring calving herd, over a 9-month lactation cycle between early April and early December. Plasmin activity of 6-months-old Cheddar cheese samples generally decreased over ripening time. One-dimensional urea-polyacrylamide gel electrophoresis (PAGE) of cheese samples taken after 6 months of ripening showed an extensive hydrolysis of caseins, with the fastest hydrolysis of αs1-caseins in cheeses made in August. A proteomic comparison between cheeses produced from milk taken in April, August and December showed a reduction in levels of β-casein and appearance of additional products, corresponding to low molecular weight hydrolysis products of the caseins. This study has demonstrated that a seasonal milk supply causes compositional differences in Cheddar cheese, and that proteomic tools are helpful in understanding the impact of those differences.

Proteomic analysis of bovine, caprine, buffalo, equine and camel milk highlighted significant interspecies differences. Camel milk was found to be devoid of β-lactoglobulin, whereas β-lactoglobulin was the major whey protein in bovine, buffalo, caprine, and equine milk. Five different isoforms of κ-casein were found in camel milk, analogous to the micro-heterogeneity observed for bovine κ-casein. Several spots observed in 2D-electrophoretograms of milk of all species could tentatively be identified as polypeptides arising from the enzymatic hydrolysis of caseins. The understanding gained from the proteomic comparison of these milks may be of relevance both in terms of identifying sources of hypoallergenic alternatives to bovine milk and detection of adulteration of milk samples and products.

Since sheep's milk is mainly used for cheese making and milk chemical composition and fatty acids (FA) profile affect cheese yield and quality, the objective of this study was to determine the effects of different feeding levels on milk chemical composition and FA profile, as well as on plasma FA profile, and on insulin and leptin concentrations. Twenty-four sheep were assigned to three homogeneous sub-groups. Throughout the experimental period each group was fed the same diet but in quantities which met 70% (under-feeding), 100% (control) and 130% (over-feeding) of their respective energy and crude protein requirements. The results showed that the underfed sheep had higher milk fat content compared with overfed. In blood plasma the concentrations of C18:0 and C18:1 in the underfed sheep were significantly higher compared with control and overfed sheep. The concentrations of leptin and insulin were significantly higher in overfed compared with underfed sheep. Underfeeding reduced the concentrations of short chain FA (SCFA) and medium chain FA (MCFA) and increased that of C18:0 and mono unsaturated FA (MUFA) in sheep milk fat compared with controls and overfed. The concentrations of C18:0, long chain FA (LCFA) and monounsaturated FA (MUFA) in milk were significantly higher and those of SCFA, MCFA and saturated FA (SFA) significantly lower in the underfed compared with the overfed sheep. In conclusion, long term under- and over-feeding affected the sheep milk chemical composition and FA profile which consequently has an impact on milk products yield (cheese and yogurt) and quality (human health).

The high nutritional value of whey makes it an interesting substrate for the development of fermented foods. The aim of this work was to evaluate the growth and proteolytic activity of sixty-four strains of lactic acid bacteria in whey to further formulate a starter culture for the development of fermented whey-based beverages. Fermentations were performed at 37°C for 24 h in 10 and 16% (w/v) reconstituted whey powder. Cultivable populations, pH, and proteolytic activity (o-phthaldialdehyde test) were determined at 6 and 24 h incubation. Hydrolysis of whey proteins was analysed by Tricine SDS-PAGE. A principal component analysis (PCA) was applied to evaluate the behaviour of strains. Forty-six percent of the strains grew between 1 and 2 Δlog CFU/ml while 19% grew less than 0·9 Δlog CFU/ml in both reconstituted whey solutions. Regarding the proteolytic activity, most of the lactobacilli released amino acids and small peptides during the first 6 h incubation while streptococci consumed the amino acids initially present in whey to sustain growth. Whey proteins were degraded by the studied strains although to different extents. Special attention was paid to the main allergenic whey protein, β-lactoglobulin, which was degraded the most by Lactobacillus acidophilus CRL 636 and Lb. delbrueckii subsp. bulgaricus CRL 656. The strain variability observed and the PCA applied in this study allowed selecting appropriate strains able to improve the nutritional characteristics (through amino group release and protein degradation) and storage (decrease in pH) of whey.

The effect of copper(II) ions (Cu+2) on the structure of β-lactoglobulin (β-lg) was investigated spectroscopically using UV-visible, fluorescence and circular dichroism (CD) and calorimetrically using isothermal titration calorimetry (ITC), at different temperatures. Results of the UV-visible studies showed that adding Cu+2 to β-lg solution caused increasing turbidity, indicative of protein aggregation. It was noticeable that the rate of increasing turbidity was directly proportional to increasing temperature. The far-UV CD studies displayed that the Cu+2 cannot induce any significant changes in the secondary structures of β-lg at different temperatures. Also, the ITC data indicated that the binding process of Cu+2 to β-lg is mainly entropically driven. The results highlight that copper ions cause the tertiary structure of β-lg to change and induce a slightly open structure leading to the formation of supramolecular aggregates in β-lg which may result in the reduced allergenicity of β-lg and its increased use in industrial applications.

Efficient milking systems, in terms of labour demand, capital investment and cow udder health are critical to successful dairy herd expansion. The objective of this study was to establish the effect of two primary influencing factors on efficient milking performance, i.e. parlour size (number of milking units) and pre-milking routine (full and nil) of spring-calved cows, in a single-operator side-by-side, swing-over milking parlour. Efficiency parameters investigated in a 5×2 factorial design included milk-flow and yield, row time, over-milking duration and operator idle time. Five combinations of parlour size (14, 18, 22, 26 and 30 milking units) each with two different pre-milking routines (Full: spray, strip, wipe, attach clusters, and Nil: attach clusters) were examined with one milking operator. The trial was carried out over 40 milking sessions and cows (up to 120) were randomly assigned to groups (n=14, 18, 22, 26 or 30) before each milking session. Row within a milking session was the experimental unit. The experiment was carried out at both peak and late lactation. The data were analysed with a mixed model using GenStat 13.2. The full pre-milking routine reduced time to milk let-down and milking time, increased average flow rate but did not affect milk yield. As milking unit number increased, the duration of over-milking (defined as time at milk flow rate <0·2 kg/min) increased more with a full compared with nil routine. Thus, the use of pre-milking preparation decreased milking time per cow but as parlour size increased, milking row times, as well as the proportion of cows that were over-milked, also increased, thereby reducing overall efficiency. These results have implications for milking management in single-operator swing-over, tandem and rotary parlours with seasonally calved herds.

Cows' and donkey milks (raw and thermally processed) and respective whey were analysed for quantification of major proteins. Two different chromatographic approaches, size exclusion (SE-HPLC) and reversed-phase high performance liquid chromatography (RP-HPLC) both coupled to UV detection were used. Usefulness of these methods for routine control of the effect of thermal processing was evaluated. The external standard method was used to calibrate the SE-HPLC and RP-HPLC systems. Concerning quantification of β-lactoglobulin (β-lg), α-lactalbumin (α-la), lysozyme (lys), and total casein (cn), no significant differences between results obtained by SE-HPLC and by RP-HPLC (t-test, P>0·05) were observed for raw milks and whey. Heating of cows' milk promoted aggregation of denatured proteins as observed by SE-HPLC, whereas α-la and β-lg from donkey milk were stable to thermal processing at 100°C (5 min). Lys was quantified in donkey raw milk and whey however, in thermally processed donkey milk lys was denatured and could not be quantified by HPLC.

The traditional utilization of donkeys (Equus asinus) as dairy animals has recently attracted substantial scientific interest with regard to human nutrition. Donkey milk is well tolerated by infants with cows’ milk allergy, useful in the treatment of human immune-related diseases, in the prevention of atherosclerosis, and in-vitro studies showed an anti-proliferative effect. Active 3-3′-5-triiodothyronine (T3) in colostrum and milk could play different physiological roles, systemic and paracrine, for both the mother and the suckling offspring. The aim was to evaluate whether thyroid hormones (TH) concentrations in milk and blood of lactating donkeys change with the advancing lactation and whether they can be affected by dietary supplementation with several trace elements, some of them directly involved with TH synthesis (I), metabolism (Se) and action (Zn). Sixteen lactating jennies were divided into two groups (CTL and TE). Mixed feed for TE was added with Zn, Fe, Cu, Mn, I, Se and Co. Every 2 weeks milk and blood samples were collected at 11·00. Total concentrations of T3 in milk (T3M) and T3 and T4 in plasma (T3P and T4P) were assayed using ELISA kits, validated for the donkey species. T3M was not correlated with TH concentrations in blood, did not change with the stage of lactation, and was significantly higher in TE (4·09±0·07 ng/ml, mean±se) than in CTL group (3·89±0·08 ng/ml). T4P (81·8±5·2 ng/ml) and T3P (15·2±1 ng/ml) significantly changed with time, but were not significantly affected by dietary treatment. T3P/T4P ratio was significantly lower in TE group. This study indicates that in donkey milk the concentration of T3, a human-like bioactive compound, can be affected by trace elements intake.

Precipitation of calcium phosphates occurs in dairy products and depending on pH and ionic environment, several salts with different crystallinity can form. The present study aimed to investigate the effects of NaCl and citrate on the characteristics of precipitates obtained from model solutions of calcium phosphate at pH 6·70 maintained constant or left to drift. The ion speciation calculations showed that all the starting solutions were supersaturated with respect to dicalcium phosphate dihydrate (DCPD), octacalcium phosphate (OCP) and hydroxyapatite (HAP) in the order HAP>OCP>DCPD. X-ray diffraction (XRD) and Fourier transform infrared (FTIR) analyses of the precipitates showed that DCPD was formed at drifting pH (acidic final pH) whereas poor crystallised calcium deficient apatite was mainly formed at constant pH (6·70). Laser light scattering measurements and electron microscopy observations showed that citrate had a pronounced inhibitory effect on the crystallisation of calcium phosphates both at drifting and constant pH. This resulted in the decrease of the particle sizes and the modification of the morphology and the microstructure of the precipitates. The inhibitory effect of citrate mainly acted by the adsorption of the citrate molecules onto the surfaces of newly formed nuclei of calcium phosphate, thereby changing the morphology of the growing particles. These findings are relevant for the understanding of calcium phosphate precipitation from dairy byproducts that contain large amounts of NaCl and citrate.

Staphylococcus aureus is one of the most important pathogens associated with bovine mastitis. Recent studies have shown that Staph. aureus strains may differ in virulence, and in their ability to disseminate across commercial dairy herds. The goal of this study was to determine whether Staph. aureus isolates differed in their ability to colonize mammary tissue, and whether such differences could be related to molecular characteristics. Quarter milk and mammary tissues of 22 cows from two dairy herds, were collected at slaughter and bacteriological analysis was performed. All Staph. aureus isolates were characterized by Pulsed Field Gel Electrophoresis (PFGE) and microarray. Overall 45 mammary quarters were infected and 20 Staph. aureus isolates were identified. The bacteria were mostly recovered from both milk and tissue of the same quarter in significantly higher numbers from herd A cows compared with herd B. Molecular characterization of the isolates showed distinct PFGE profiles for isolates from each herd. Differences in virulence factors between herds A and B isolates were evidenced The genes for enterotoxin D, J and R were present in herd A, those for G, I, N, M, O and U were shown in herd B, whilst both components of the leukocidin lukD/E genes were only carried by herd A isolates. Furthermore, all herd A isolates showed β-haemolysin activity, which was absent in all but one isolate from herd B. Therefore our data indicate that Staph. aureus isolates showing differences in their ability to disseminate and colonize across quarters, also have significantly different virulence characteristics.