Terpene composition of ewes’ raw milk from nine commercial flocks was analysed from February to July. Ewes’ diet consisted of concentrate and conserved forage in winter (indoor feeding) and part-time grazing from spring (transition and outdoor feeding). Regardless of the feeding, limonene and β-phellandrene were the most abundant monoterpenes and β-caryophyllene showed the highest concentrations among sesquiterpenes. Terpene content increased in the milks of commercial flocks when animals were reared under grazing management. Monoterpenes were detected in the milks of all the commercial flocks throughout the season, whereas sesquiterpenes were only detected in the milks from flocks grazing on non-cultivated community-owned grasslands in which a higher biodiversity of plant species grew. These preliminary results indicated that β-caryophyllene could be a potential pasture-diet marker in the case of milks from animals grazing a higher biodiversity of plant species but in-depth studies including information on terpene composition of plants ingested by the animals are necessary to evaluate the suitability of β-caryophyllene or another terpenoid compound as pasture biomarker.

The current work studied four types of binary antagonist/pathogen bacterial culture system, in order to determine the effect of interaction between two strains of Lactobacillus plantarum and two food-borne pathogens, Listeria monocytogenes and Escherichia coli, in whole UHT milk at 37°C. To determine the type of interaction between the two bacterial populations in co-cultures and to evaluate the antagonistic activity of the lactic acid bacteria (LAB) on the pathogenic bacteria, the growth curves, the kinetic parameters, and the pH profiles of mono- and co-cultures were compared. The Lb. plantarum strains showed different bacteriocin-like inhibitory substance (BLIS) production, auto- and co-inducible. The antibacterial effect of neutralized supernatants of mono and co-cultures harvested at different times of incubation was assessed in order to establish the presence of bacteriocin-like inhibitory-substances (BLIS) and their possible relation to the growth inhibition of the pathogen. The LAB reduced the growth of Esch. coli and of List. monocytogenes by 4 and ∼5 log cycles, respectively and influenced other growth kinetic parameters, such as μmax and lag phase, in the different binary combinations. The growth of the LAB was not relevantly altered by simultaneous growth with the pathogenic strains showing an interaction of amensalism. The pattern of inhibition exerted by the LAB on the pathogens was different; Lb. plantarum LB279 inhibited the growth of List. monocytogenes more effectively than that of Esch. coli. The behaviour of Esch. coli in co-culture with Lb. plantarum WS4174 suggested the presence of metabolic crowding in the mechanism of growth suppression. This exploratory study showed the complexity and specific particularities of the inhibition phenomena between bacterial communities.

The objective of this study was to evaluate the effect of dietary supplementation with selenium (Se) and vitamin E (VitE) on milk yield, composition and renneting parameters in dairy Jonica breed goats. Forty multiparous lactating goats fed the same basal diet were randomly allocated to 1 of 2 dietary treatments: for 135 days, the experimental period. Basal diet was composed by ad libitum mixed vetch-oat hay and a definite amount of a pelleted concentrate. Dietary treatments included: (1) the control diet containing background Se and VitE only, and (2) the experimental diet containing sodium selenite as the Se supplement in concentrate (0·20 mg/head per day of Na-selenite and 20 mg/day per head of VitE). During the trial, no significant differences (P>0·05) were registered in dry matter intake and body weight gain among groups. Goats fed Se-VitE diet showed the highest milk production (P<0·05), and fat (P<0·01) and protein (P<0·05) yields and percentages. Somatic cell count were significantly lower in Se-VitE diet compared with control group (P<0·05). Significant differences (P<0·05) were observed in milk renneting properties. Based on our results, it was concluded that dietary supplementation with SE and VitE led to an increase of milk production and quality in Jonica goat, with positively improvement in milk coagulation aptitude.

An enterotoxigenic Escherichia coli (ETEC) vaccine designed to prevent diarrhoea was inoculated into dairy cows, and the occurrence of clinical mastitis was investigated for 2 years. Half of 480 cows in five farms were subcutaneously inoculated with ETEC vaccine (Imocolibov) twice with a 1-month interval in 2007 and 2008. Fisher's exact test and survival (time to event) analysis with the log-rank test were used to compare vaccinates and controls. In 2007, there was no significant difference in the incidence rate of mastitis between vaccinate (20·3%) and control (17·1%) cows. The rate of death or culling due to mastitis was lower in vaccinated cows (7·4%) than in control cows (29·2%, P=0·07, Fisher's exact test; P=0·02, log-rank test). In 2008, there was no significant difference in both the incidence rate of mastitis and the rate of death or culling due to mastitis. Milk productivity was compared between vaccinates and controls in one farm. Multi-way analysis of variance (ANOVA) was performed for the amount of 4% fat-corrected milk, and there was no significant difference between vaccinates and controls. These results suggest that ETEC vaccine inoculation reduces death or culling due to mastitis, whereas no preventive effect on the development of mastitis was observed.

Dioxins are lipophilic compounds with a small molecular weight and are highly persistent, bioaccumulative and toxic. Dioxin detoxification is associated with an increased production of reactive oxygen species (ROS). In physiological conditions the body is protected against ROS and their toxic products by a wide range of antioxidant systems. We hypothesize that the imbalance between ROS production, associated with dioxin exposure, and the antioxidant defence capacity, may lead to oxidative stress, with consequent increased consumption of antioxidants and accumulation of toxic compounds in blood and tissues. The objective of this study was to evaluate the effect of exposure to dioxins on the plasma redox status of lactating buffalo cows. To this aim, the major liposoluble (retinol and α-tocopherol) and water-soluble (ascorbate) antioxidants, the superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity, the total antioxidant capacity (TAC), as well as specific protein oxidation markers (protein bound carbonyls and nitro-tyrosine) and lipid oxidation markers (hydroperoxides), were chosen as indices of blood redox status. The concentration of antioxidants, protein-bound carbonyls (PC), nitro-tyrosine (N-Tyr), and hydroperoxides (LPO), the SOD and GPx activity, and the TAC were measured in plasma samples obtained from buffalo cows exposed to environmental levels of dioxins higher (n=21, group A) or lower (n=29; group B) than those permitted. Plasma titres of antioxidants, as measured by HPLC, and the total antioxidant capacity, as measured by trolox equivalents capacity, were higher in group B than in A. Similarly, SOD and GPx activities were higher in group B than in A. Conversely, plasma levels of PC, N-Tyr and LPO, as measured by ELISA, were higher in group A than in B. Our results suggest that exposure to dioxins impairs the plasma antioxidant defence system of lactating buffalo cows, and that metabolic processes associated with dioxin detoxification might induce or enhance oxidation of protein and lipids. This adverse effect on blood redox status might have negative implications for animal health and reproduction, and might compromise animal welfare.

The present study investigated the effect of age at first lambing (AFL) on the performance of Lacaune sheep under intensive management conditions. Records from 3088 maiden sheep from one farm, for the period 2005–2010, were classified into four experimental groups: group E (early) ewes with AFL ⩽390 d; group M (middle) with AFL of 391–450 d; group L (late) with AFL of 451–510 d; and group A (aged) with AFL ⩾511 d. The higher the number of lactations, the lower were the yield/lactation and yield/Day in Milk. Ewes from group M up to 450 days old lambed 0·2 times more often and had 0·25 more lactations than the ewes from group L; in addition, the former group lambed 0·5 times more often and had 0·49 more lactations than ewes from group A. Group A had the lowest yield per lifetime; the yield followed the sequence: group E (1032 l) >group M (1051 l) >group L (989 l) > group A (859 l) (P<0·0001). Yield/lactation was affected by AFL during the first three lactations (P<0·05). Group E produced significantly less milk in the first lactation than ewes from groups M, L and A. AFL correlated negatively with the number of lactations in life (r= −0·26; P<0·0001) and with total milk yield (r= −209; P<0·0001). The productive performance between the ewes that reached puberty early (n=404) or late (n=2684) in life were different (P<0·0001) with the early sheep having a lower total milk yield (591 v. 1073 l, P<0·0001). In conclusion, the current study indicates that the optimal AFL for the Lacaune breed under an intensive dairy system is between 390 and 450 d. Ewes with AFL earlier than 390 d or later than 450 d are likely to have a shorter productive life and a lower lifetime milk production.

The objective of the study was to evaluate the relationship of veterinary clinical assessments of lameness to probability estimates of lameness predicted from vertical kinetic measures. We hypothesized that algorithm-derived probability estimates of lameness would accurately reflect vertical measures in lame limbs even though vertical changes may not inevitably occur in all lameness. Kinetic data were collected from sound (n=179) and unilaterally lame (n=167) dairy cattle with a 1-dimensional, parallel force plate system that registered vertical ground reaction force signatures of all four limbs as cows freely exited the milking parlour. Locomotion was scored for each hind limb using a 1–5 locomotion score system (1=sound, 5=severely lame). Pain response in the interdigital space was quantified with an algometer and pain response in the claw was quantified with a hoof tester fitted with a pressure gage. Lesions were assigned severity scores (1=minimal pathology to 5=severe pathology). Lameness diminished the magnitude of peak ground reaction forces, average ground reaction forces, Fourier transformed ground reaction forces, stance times and vertical impulses in the lame limbs of unilaterally lame cows. The only effect of lameness on the opposite sound limb was increased magnitude of stance times and vertical impulses in unilaterally lame cows. Symmetry measures of the peak ground reaction forces, average ground reaction forces, Fourier transformed ground reaction forces, stance times and vertical impulses between the left and right hind limbs were also affected in unilateral lameness. Paradoxically, limbs with clinically similar lesion and locomotion scores and pain responses were associated with a broad range of load-transfer off the limb. Substantial unloading and changes in the vertical limb variables occurred in some lameness while minimal unloading and changes in vertical limb variables occurred in other lameness. Corresponding probability estimates of lameness accurately reflected changes in the vertical parameters of limbs and generated low probability estimates of lameness when minimal unloading occurred. Failure to transfer load off limbs with pain reactions, locomotion abnormalities and lesions explained much of the limited sensitivity in lameness detection with vertical limb variables.

The objectives of this study were to analyse buffalo milk fat composition, to verify the activity of Delta(9)-desaturase enzyme in the mammary gland, as well as to estimate additive genetic variances for milk, fat and protein yield, and milk cis-9,trans-11 conjugated linoleic acid percentage (cis-9,trans-11 CLA%). A total of 3929 lactation milk yields (MY) records from 2130 buffaloes and 1598 lactation fat (FY) and protein (PY) yield records from 914 buffaloes were analysed. For cis-9,trans-11 CLA%percentage, a total of 661 milk samples from 225 buffaloes, daughters of 8 sires, belonging to 4 herds and calving in 2003 and 2004, were used. The genetic parameters and variance components were estimated by Restricted Maximum Likelihood applying an animal model. The fixed effects considered in the model were: contemporary group (herd, year, calving season) and age at calving (linear and quadratic effects) and lactation length (linear and quadratic effects) as covariables. Additive genetic and permanent environment effects were considered as random. The MY, FY, PY and CLA% means were 1482±355 kg, 90·1±24·6 kg, 56·9±15·2 kg and 0·69±0·16%, respectively. Heritability estimates for MY, FY, PY and CLA% were 0·28±0·05, 0·26±0·11, 0·25±0·11 and 0·35±0·14, respectively. There is enough additive genetic variation for buffalo milk, protein and fat yield to improve these traits through selection. The cis-9,trans-11 CLA% can be enhanced by selection in buffaloes and will contribute to improving human health. The activity and efficiency of Delta(9)-desaturase in the mammary was measured and confirmed.

Sensitive methods that are currently used to monitor proteolysis by plasmin in milk are limited due to their high cost and lack of standardisation for quality assurance in the various dairy laboratories. In this study, four methods, trinitrobenzene sulphonic acid (TNBS), reverse phase high pressure liquid chromatography (RP-HPLC), gel electrophoresis and fluorescamine, were selected to assess their suitability for the detection of proteolysis in milk by plasmin. Commercial UHT milk was incubated with plasmin at 37°C for one week. Clarification was achieved by isoelectric precipitation (pH 4·6 soluble extracts) or 6% (final concentration) trichloroacetic acid (TCA). The pH 4·6 and 6% TCA soluble extracts of milk showed high correlations (R2 > 0·93) by the TNBS, fluorescamine and RP-HPLC methods, confirming increased proteolysis during storage. For gel electrophoresis, extensive proteolysis was confirmed by the disappearance of α- and β-casein bands on the seventh day, which was more evident in the highest plasmin concentration. This was accompanied by the appearance of α- and β-casein proteolysis products with higher intensities than on previous days, implying that more products had been formed as a result of casein breakdown. The fluorescamine method had a lower detection limit compared with the other methods, whereas gel electrophoresis was the best qualitative method for monitoring β-casein proteolysis products. Although HPLC was the most sensitive, the TNBS method is recommended for use in routine laboratory analysis on the basis of its accuracy, reliability and simplicity.

Standard method for the determination of the activity of milk coagulants is the rotating bottle method, where clotting time is defined as the time when visually observable flocculation starts. Aim of this study was to verify whether it is possible to determine milk clotting time by rotational viscometry. Using three different coagulants and reconstituted milk of different pH and temperature, flocculation time and viscosity in steady shear was determined, and milk gelation was monitored by small amplitude oscillating shear rheometry. The results show that, independent of pH and temperature, milk clotting time is related to an apparent viscosity of 7·24±0·45 mPa.s, indicating that rotational viscometry can be used for the screening of flocculation time with an accuracy of approximately 6%.

This work studied the behaviour of caseinomacropeptide (CMP) in a whey protein fractionation process based on the selective precipitation of α-lactalbumin (α-la) in an acid medium. Three different acids (hydrochloric, citric and lactic) and different operating conditions (protein concentration, temperature and pH) were considered to perform the precipitation step. Under the optimised precipitation conditions obtained for α-la (pH 4, 55°C, initial α-la concentration around 12 g/l) CMP presents quite similar behaviour to that observed for β-lactoglobulin (β-lg), namely remaining in the supernatant fraction. However, at a lower pH value (3·5) the amount of precipitated CMP increases up to 72% when citric acid is added. This behaviour could be due to the fact that CMP is close to its isoelectric point, which allows a supernatant fraction enriched in β-lg that is almost free from the rest of proteins in sweet whey.

Proteinase activity of Lactobacillus fermentum IFO3956 cells was higher when they were grown on milk-based media than on 10% reconstituted skim milk. The lowest protease activity was observed when cells were grown on milk-free media. The extraction of milk-induced cell-bound proteases from Lb. fermentum IFO3956 was most efficient using 1% Tween 80 while the use of 1% SDS inhibited all proteolytic activity. Two bands of ∼35 and >100 kDa were observed by zymogram, indicating that proteolytic activity corresponded to the presence of at least two types of enzymes or two molecular forms of one enzyme. Mass spectrometry analyses of αS1-casein hydrolysates detected 24 peptides with sizes ranging from 5 to 36 amino acids, including 9 phosphorylated peptides, resulting from the fermentation of Lb. fermentum IFO3956 of αS1-casein. Most of the identified peptides originated from the N-terminal portion of αS1-casein. The studied bacterial strain could hydrolyze αS1-casein in many sites including the epitopes triggering the allergic reactions against αS1-casein e.g. at the positions 23, 30, 41, 71, 91, 98, 126, 179. After hydrolysis of αS1-casein with Lb. fermentum IFO3956 the recognition and the binding of this casein to IgE from the pooled sera of 18 patients with cow's milk allergy was significantly reduced.

The aim of the study was to describe the characteristics of cleaning procedures for milking equipment applied in intensive dairy farms in Lombardy (Italy) and to study their relationships with bacterial count of bulk milk and hygienic condition of milking machine components. A group of 22 dairy farms was visited twice (winter and summer) in order to collect bulk tank milk and post-rinse water samples and swabs from liners and milk receiver. Samples were analysed to determine: standard plate count (SPC), laboratory pasteurization count (LPC), psychrotrophic bacteria count (PBC), coliform count (CC) and Escherichia coli. Cleaning procedures were monitored using electronic milk flow meters with specific software for the measurement of the duration of each cleaning phase, circulating solution temperature and electrical conductivity, turbulence and water filling percentage of pipelines. The results showed that farms classified as high and low milk total bacteria count significantly differed both in terms of liners and receiver bacterial contamination and in terms of water temperature reached during the detergent phase of cleaning milking equipment. Significant positive correlations were found among total bacteria count in milk and bacterial contamination of the liners. Maximum water temperature reached during the cleaning cycle of milking equipment was very low (34·4±8·9°C on average); most of the observations (88·6%) corresponded to water temperatures <45°C. Cleaning temperature was related to psychrotrophic bacteria count of milk and post-rinse water and coliform count in liners. Routine check and regulation of water temperature during the washing phase of the milking machine can be a simple and effective way to control one of the main risk factors for bacteriological quality of bulk tank milk.

This work aimed to test if milk preserved with bronopol can be reliably used for fatty acid determination. Dairy production and milk quality are often monitored regularly to assess performance and contribute to selection indices. With evidence that fat composition can be influenced by selective breeding, there might be an interest in using samples collected in routine testing to evaluate individual cow fatty acid profiles, contributing to breeding indices. However, most recording services use a preservative such as bronopol and there is no published record if this influences subsequent fatty acid analysis. This study used milk from an oil seed supplementation trial, generating a wide range of milk fatty acid profiles, to test if the concentration of 31 individual fatty acids determined by GC were influenced by bronopol. Provided preserved samples are subsequently frozen, milk treated with bronopol can reliably be used to evaluate fatty acid composition in most cases; however bronopol might influence a few long-chain fatty acids present in relatively low concentrations. This is one small step towards simplifying milk compositional analysis but it could ultimately streamline the inclusion of milk fat quality into breeding indices, either with a view to ‘healthier’ milk or potentially reducing methane output and the environmental impact of dairy production.

Aqueous solutions of reconstituted whey protein- concentrate (WPC) & isolate (WPI) powders were sonicated at 20 kHz in a batch process for 1–60 min. Sonication at 20 kHz increased the clarity of WPC solutions largely due to the reduction in the size of the suspended insoluble aggregates. The gel strength of these solutions when heated at 80°C for 20 min also increased with sonication, while gelation time and gel syneresis were reduced. These improvements in gel strength were observed across a range of initial pH values, suggesting that the mechanism for gel promotion is different from the well known effects of pH. Examining the microstructure of the whey protein gels indicated a compact network of densely packed whey protein aggregates arising from ultrasound treatment. Comparable changes were not observed with whey protein isolate solutions, which may reflect the absence of larger aggregates in the initial solution or differences in composition.

In this work, a method based on Raman spectroscopy in combination with Principal Component Analysis (PCA) and Partial Least Square-Discriminant Analysis (PLS-DA) has been developed for the rapid differentiation of heterofermentative related lactobacilli. In a first approach, Lactobacillus kefir strains were discriminated from other species of heterofermentative lactobacilli: Lb. parakefir and Lb. brevis. After this first approach, PCA allowed for a clear differentiation between Lb. parakefir and Lb.brevis. For the first level of discrimination, PCA was performed on the whole spectra and also on delimited regions, defined taking into consideration the loading values. The best regions allowing a clear differentiation between Lb. kefir and non-Lb. kefir strains were found to be: the 1700–1500 cm−1, 1500–1185 cm−1 and 1800–400 (whole spectrum) cm−1 Raman ranges. In order to develop a classification rule, PLS-DA was carried out on the mentioned regions. This method permitted the discrimination and classification of the strains under study in two groups: Lb. kefir and non-Lb. kefir. The model was further validated using lactobacilli strains from different culture collections or strains isolated from kefir grains previously identified using molecular methods. The second approach based on PCA was also performed on the whole spectra and on delimited regions, being the regions 1700–1500 cm−1, 1500–1185 cm−1 and 1185–1020 cm−1, i.e., those allowing the clearest discrimination between Lb. parakefir and Lb. brevis. The results obtained in this work, allowed a clear discrimination within heterofermentative lactobacilli strains, proteins being the biological structures most determinant for this discrimination.

CD4+ T cells play a key role in the immune response of pathogen-induced mastitis in dairy cattle. Mammary gland factor STAT5b is involved in the regulation of CD4+T cell differentiation during inflammatory response and milk production. Little is known about the genetic variation effects of bovine CD4 and STAT5b genes on somatic cell score (SCS) and milk production traits in dairy cattle. The aim of the study was to investigate the single nucleotide polymorphisms (SNPs) of bovine CD4 and STAT5b in Chinese Holsteins and to analyse their association with estimated breeding values (EBVs) for SCS and milk production traits. In the present study, SNPs of CD4 (NC_007303 g.13598C>T) and STAT5b (NC_007317 g.31562 T>C) were identified and genotyped in Chinese Holstein population. The results showed that both SNPs were significantly associated with the EBVs for milk yield and protein yield in Chinese Holstein cows, and the SNP in CD4 was associated with the EBV for SCS (P<0·01). The additive effect of CD4 SNP on protein yield was significant (P<0·05), and the dominant effect of STAT5b SNP was significant on milk yield and protein yield (P<0·01). Cows with combination genotype C7 (CCTT: CD4 g.13598C>T and STAT5b g.31562 T>C) had the highest SCS EBV but lower milk yield, while cows with C2 (TTTC) produced more milk, fat and protein than the other eight combination genotypes. These results suggested that the SNPs in CD4 and STAT5b may be potential genetic markers for SCS and milk/protein yields selecting and warrant further functional research.

Dry matter, protein, fat, pH, mineral (Na, K, Ca and Mg) and trace elements (Fe, Cu, Zn and Se) concentrations were determined in samples of goats’ milk and in fresh, semi-hard and hard cheeses to study the effect of the goats’ diet and the type of rennet used for the cheese processing of the Palmero Protected Designation of Origin cheeses. Two groups of 20 Palmero goats were fed 2 different diets: a Palmero diet (PD supplied by native forages adapted to subhumid areas, which had a high ratio of long fibre to concentrates (65:35), and an actual diet (AD), the most commonly used by goat farmers, with a low ratio of long fibre to concentrates (35:65). In general, the cheese samples from goats fed with PD had higher mean Ca, Zn, Cu and Se concentrations than the samples obtained from AD fed goats. The diet exhibited a greater influence on the chemical composition of the cheeses than the rennet used in their production. Applying a stepwise linear discriminant analysis a complete percentage of correct classifications of the three types of cheeses according to the diet of the goats was observed.