The objectives of this research were to confirm the effect of compounds derived from milk fermented by Lactobacillus helveticus (LH-2) on the nonspecific host defence system, and isolate and characterize the active peptides that mediate the immune response. The cell-free supernatant obtained from the fermented milk and its fractions were tested in vitro for immuno-modulating activity using murine macrophages (RAW 264·7 cell line). Cytokine production (Interleukin-6 (IL-6), Tumor Necrosis Factor-α (TNF-α), and Interleukin-1β (IL1-β)), nitric oxide (NO) production and phagocytosis were used as biomarkers. Macrophages stimulated with cell-free supernatant of fermented milk showed higher production of cytokines and NO compared with macrophages stimulated with LPS (Lipopolysaccharide) and a commercial immunomodulator derived from β-casein (f54-59). Phagocytosis was observed by macrophages stimulated with the supernatant. Two of nine fractions collected from the supernatant using size exclusion chromatography produced the highest response when used to stimulate macrophages. The results of the dose-response study of the effect of the fraction with the highest stimulation effect on the production of TNF-α showed a direct correlation between protein concentration and TNF-α release. The fraction contained four novel peptides, three derived from the hydrolysis of β-casein and one from the hydrolysis of α-lactalbumin. These results confirm that fermentation of milk by Lactobacillus helveticus (LH-2) results in the production of specific peptides capable of modulating macrophage activity.

Three models for the estimation of milk, fat and protein daily yield (DY) based on a.m. (AM) or p.m. (PM) milkings were compared. A total of 518 766 test-day records from 5078 dairy cattle farms obtained between March 2004 and April 2008 were analysed. The DY model was a linear model with DY as a dependent variable. In the PYR model and the DYR model, partial yield ratios (AM:DY and PM:DY) and daily yield ratios (DY:AM and DY:PM), respectively, were used as a dependent variable in the first step. In the second step, DY was estimated as a partial yield divided (PYR model) or multiplied (DYR model) by the estimated yield ratio from the first step. Models included the effect of partial yield (only in the DY model), milking interval, stage (month) of lactation and parity. Analysis of variance indicated that partial yield was the most important source of variation for the DY model whereas milking interval had the biggest effect in the PYR model and the DYR model. Differences in accuracy (correlation between the true and the estimated DY) between the models were negligible. On the other hand, models differed in the amount of bias (average error). The DYR model on average overestimated DY by 0·13 kg, 0·01 kg and 0·01 kg for milk, fat and protein, respectively. For the other two models the overall bias was almost zero. However, the DY model overestimated low and underestimated high DY owing to the well known regression property. The DYR model progressively overestimated high DY. These problems were not observed with the PYR model which seemed to be the best model. In this paper a relatively old topic was analysed and discussed from a new point of view, where the estimation of DY is based on modelling biologically more stable partial yield ratios rather than yield values from a.m. or p.m. milking.

The effectiveness of three natural compounds in slowing down the deterioration of microbial quality of Fior di latte cheese is addressed. In particular, the control of the growth of spoilage microorganisms and the determination of the Microbiological Stability Limit (MAL) were the main goals. A Central Composite Design (CCD) strategy was adopted to highlight possible combined effects between three essential oils: sage and two types of lemon. Results showed an increase in the MAL value for all cheese samples packaged with the antimicrobial compounds, compared with the control cheese. Moreover, the combinations of the three essential oils indicated that the substances can act in a synergistic or antagonistic way, depending on their concentrations. In particular, a synergistic effect was evident when the three compounds were used at about 3000 ppm.

The aim of the study was to test the effect of rumen-inert fat supplements of different chemical forms or containing different unsaturated/saturated (U/S) fatty acid contents on milk production, milk composition and liver and blood metabolic variables of high-yielding dairy cows in the peripartal period. Thirty Holstein-Friesian dairy cows were divided into three equal groups and fed a corn silage-based diet, without fat supplementation (control) or supplemented with 11·75 MJ NEl per day of calcium soaps of palm oil fatty acids (CAS; U/S=61/39) or with 11·75 MJ NEl per day of hydrogenated palm oil triglyceride (HTG; U/S=6/94). Each diet was fed from 25±2 d prior to the expected calving to 100±5 d post partum. Compared with the control, both CAS and HTG supplementation resulted in an increase of the average milk yield. Milk fat content and fat-corrected milk yield were higher in the HTG group but lower in the CAS group than in the control group. In all groups liver triglyceride concentrations (TGL) increased from 15 d prepartum to 5 d post partum, and then decreased thereafter. At 5 d TGL was lower in the HTG group than control or CAS cows. No significant differences were detected in TGL among dietary treatments at 15 d prepartum and 25 d post partum. Higher plasma glucose and insulin and lower non-esterified fattay acids and β-hydroxybutyrate concentrations and aspartate aminotransferase activity were measured in the HTG group than in the control or CAS groups at 5 d or 25 d post partum. Our results show that HTG may provide a better energy supply for high-yielding dairy cows in negative energy balance than CAS around calving.

Staphylococcus aureus is the most common aetiologic agent of contagious bovine mastitis. It is characterized by a wide array of virulence factors. The differences among strains jeopardize the development of effective vaccines against Staph. aureus mastitis. We tested the immunogenicity of a peptide subunit vaccine coding for three different adhesion factors, fibrinogen-binding protein (Efb), fibronectin-binding protein A (FnbpA) and clumping factor A (ClfA). Then we evaluated the influence of some virulence factors on the ability of specific anti-adhesin antibodies to react with sixteen Staph. aureus strains isolated from bovine subclinical mastitis. Immunization with the recombinant adhesins stimulated a strong humoural (IgG and IgA) and mucosal IgA immune response in all animals tested. Hyperimmune serum recognized with diverse efficiency the sixteen Staph. aureus strains and this circumstance correlated well with the level of expression of adhesins. Among the different virulence factors considered to classify strains, spa gene polymorphisms showed the strongest influence on isolate reactions to hyperimmune serum. Our results indicate the importance of a disease- and environment-specific analysis of isolates. Thus, as opposed to other pathogens to obtain an effective vaccine we should characterize multiple strains and identify the prevalent virulence factors expressed.

Two types of artificial neural networks, multilayer perceptron (MLP) and self-organizing feature map (SOM) were used to detect mastitis by automatic milking systems (AMS) using a new mastitis indicator that combined two previously reported indicators based on higher electrical conductivity (EC) and lower quarter yield (QY). Four MLPs with four combinations of inputs were developed to detect infected quarters. One input combination involved principal components (PC) adopted for addressing multi-collinearity in the data. The PC-based MLP model was superior to other non-PC-based models in terms of less complexity and higher predictive accuracy. The overall correct classification rate (CCR), sensitivity and specificity of this model were 90·74%, 86·90% and 91·36%, respectively. The SOM detected the stage of progression of mastitis in a quarter within the mastitis spectrum and revealed that quarters form three clusters: healthy, moderately ill and severely ill. The clusters were validated using k-means clustering, ANOVA and least significant difference. Clusters reflected the characteristics of healthy and subclinical and clinical mastitis, respectively. We conclude that the PC based model based on EC and QY can be used in AMS to detect mastitis with high accuracy and that the SOM model can be used to monitor the health status of the herd for early intervention and possible treatment.

The aim of this study was to investigate the effects of unhydrolysed/intact casein and eight different sodium casein hydrolysates (a–h) on the viability and growth of human cancer cell lines. Both human Jurkat T cells and Caco-2 cells were incubated with increasing concentrations of the test compounds (0·5–10% v/v) for 24 h. Cell viability was assessed using the MTT, lactate dehydrogenase (LDH) release and Trypan Blue assays. Cell growth was monitored using the MTT, Trypan Blue and Bromodeoxyuridine (BrdU) proliferation assays. Casein hydrolysates b, c and f had an inhibitory effect on the viability and growth of both cell lines. The casein hydrolysates did not negatively affect the membrane integrity of both Jurkat and Caco-2 cells. In Jurkat cells hydrolysates a and h had an inhibitory effect on DNA synthesis after 24 h, while in Caco-2 cells DNA synthesis was not affected. In conclusion, we found that the different casein hydrolysates had cell-specific effects which target particular functions within the cell. Overall, casein hydrolysates had no effect on membrane integrity while they had varied effects on mitochondrial activity and DNA synthesis in the different cell lines.

The combination of ultrasound and heat (thermoultrasound) is an emerging food preservation technique that retains higher quantities of bioactive components compared with current thermal pasteurization practice, but has not yet been assessed for pasteurizing human milk. Artificially contaminated human milk samples were treated with ultrasound (20 kHz, 150 watts) with and without heating. The retention of four human milk proteins was quantified by biochemical assay and laser scattering particle sizing was used to determine the extent of homogenization. While ultrasonic treatment was effective at inactivating Escherichia coli (D4 °C=5·94 min), Staphylococcus epidermidis exhibited resistance (D4 °C=16·01 min). Thermoultrasonic treatment was considerably more effective (Esch. coli D45 °C=1·74 min, D50 °C=0·89 min; Staph. epidermidis D45 °C=2·08 min, D50 °C=0·94 minutes) with a predicted retention (2·8 min treatment, 50°C) of secretory IgA lysozyme, lactoferrin and bile salt stimulated lipase of 91, 80, 77, and 45%, respectively. Homogenization of the milk samples occurred after 5 min and 2 min of ultrasonic and thermoultrasonic treatment, respectively. Thermoultrasonic treatment is an effective method for pasteurizing donor human milk and retaining a greater proportion of bioactive components compared with current practices. However, further studies are required to assess the practicality of applying this technique routinely to donor human milk.

Hormone-sensitive lipase (LIPE) plays a fundamental role in the regulation of energy balance by releasing free fatty acids from adipose triacylglycerol stores. These fatty acids can be subsequently transferred to other body compartments to be oxidized or employed in other biochemical reactions. This enzymic function is particularly important in lactating animals because the synthesis of milk components involves the mobilization of lipid depots to satisfy the large energy demands of the mammary gland. In the current study, we partially sequenced the goat LIPE gene in several individuals. In doing so, we identified two synonymous polymorphisms at exons 2 (c.327C>A>T, triallelic polymorphism) and 3 (c.558C>T). Moreover, we found a mis-sense polymorphism at exon 6 (c.1162G>T) that involves an alanine to serine substitution at position 388. Analysis with Polyphen and Panther softwares revealed that this amino acid replacement is expected to be neutral. Performance of an association analysis with a variety of milk traits revealed that goat LIPE genotype has highly suggestive effects on milk yield (P=0·0032) as well as on C18:3 n-6g (P=0·0051), trans-10 cis-12 CLA (P=0·007) and C12:0 (P=0·0084) milk contents. These associations are concordant with the preference of LIPE to selectively mobilize medium-chain and unsaturated fatty acids.

The MAC-T cell line has been used extensively to investigate bovine mammary epithelial cell function. A lactogenic phenotype is generally induced in this cell line by a combination of dexamethasone, insulin and prolactin and has typically been assessed by milk protein production. Few studies have focused on identifying other factors that may affect milk protein synthesis in the MAC-T cell line, and none have considered the lipid class distribution of MAC-T cells as a component of the lactogenic phenotype. Growth hormone (GH) has been shown to increase milk protein synthesis both in vivo and in mammary cell models, and has been shown to alter the lipogenic profile of mammary explant models. We tested the hypothesis that MAC-T cells would respond directly to GH and that the response would include alterations to the lipid class distribution as well as to milk protein gene expression, leading to a more appropriate model for mammary cell function than treatment with dexamethasone, insulin and prolactin alone. Differentiated cells expressed GH receptor mRNA, and addition of GH to the differentiation medium significantly induced production of α-s1 casein and α-lactalbumin mRNA. GH also significantly affected the proportion of triacylglycerol and sphingomyelin. These results indicate that GH is an important factor in inducing a lactogenic phenotype in the MAC-T cell line, and support the possibility of a direct effect of GH on milk synthesis in vivo.

The objectives of this preliminary investigation were to evaluate the feasibility of transrectal colour Doppler sonography (TCDS) for determining blood flow of the pudendoepigastric trunk in cows with experimentally induced Escherichia coli Mastitis. Five primiparous Holstein dairy cows, 4–6 months after calving, were examined in two trials. All monitored udder quarters were initially clinically healthy, somatic cell count (SCC) was <50 000 cells/ml and bacteriologically negative. The blood flow of the left and the right pudendoepigastric trunk was described by the blood flow volume (BFV). In the methodological part of the study, the intra-observer precision of the method was evaluated. The coefficients of variation of the BFV were 7·1% for the left and 9·4% for the right pudendoepigastric trunk. The intraclass correlation coefficients of the BFV were 0·99 (P<0·001) for the left and 0·75 (P=0·004) for the right vessel. BFV did not differ significantly between the left and the right side nor between pre- and post-milking nor between oestrus and dioestrus. In the experimental part of the study, significant differences of increasing BFV between 0 and 12 h p.i. (post infectionem) (P=0·043) and decreasing BFV between 12 and 24 h p.i. (P=0·043) were discovered for the pudendoepigastric trunk of the infected right side. In the left-right (control-infection) comparison a significant increase of the right BFV was observed at 12 h p.i. (P=0·043). The difference of an increasing SCC correlated positively with the difference of an increasing BFV between 0 and 12 h p.i. (Spearman's ρ=1·00; P=0·043) for the right infected side. It was shown that TCDS is a reproducible technique for investigating pathological mammary blood flow changes at an early stage of acute mastitis.

Leptin is an autocrine and paracrine factor which affects the development and function of mammary gland. The purpose of this study was to investigate the presence and regulatory effect of leptin in Chinese Guan Zhong dairy goat mammary gland from the virgin state to involution. The protein expression and localization of leptin and its long form receptor (OB-Rb) were detected by western blot and a confocal laser scanning microscope. Explants were cultured to detect the impacts of leptin on mammary gland, western blot was used to research leptin signal transduction pathway in pregnancy, lactation and involution. Leptin and amounts of OB-Rb protein were high throughout the virgin stage and at the beginning of pregnancy, lower at mid-pregnancy and throughout lactation, then higher at involution. Immunofluorescence performed with the anti-leptin and anti-leptin receptor antibody showed labelling located in adipose, epithelial cells, or extracellular matrix at different stages. The localization of leptin and OB-Rb revealed that leptin induced the expression of OB-Rb specifically and controlled the development and physiological function of the mammary gland by binding to OB-Rb. Leptin stimulated the proliferation and differentiation of ductal epithelial cells in pregnancy by JAK-MAPK signal pathway, enhanced the amount of β-casein in cultured lactating mammary gland by JAK-STAT5 signal pathway, made the mammary duct disappear and induced apoptosis of mammary epithelial cells and mammary gland restitution by JAK-STAT3 signal pathway in involution. Overall, this study demonstrated the importance and complexity of leptin and OB-Rb during mammary gland development and provides a valuable resource for future research in this area.

Bovine colostrum and milk antibodies of calving and lactating cows immunized with a multivalent vaccine consisting of whole cells of three different species of pathogenic bacteria including four strains of enterotoxigenic Escherischia coli, five strains of enteropathogenic Esch. coli, three strains of enteroinvasive Esch. coli, two strains of Samonella typhi, and one strain each of Shigellia dysenteriae, Sh. sonnei and Sh. flexneri were generated, respectively. A significantly elevated activity and titre of specific IgG from bovine immune colostrum were seen for only 5 days after calving of immunized cows, however, the levels of specific IgG could be obtained continuously from the milk of immunized lactating cows until the 11th week of the entire experiment period. Subsequently, we observed that the high specific IgG activity in immune milk was relatively stable under pH 5·0–7·0 at 37°C. Of importance, we identified that the specific IgG preserved its biological function for high antigen-binding activity at pH 5·5–6·5 for 30 min of heat treatment at 70°C and for 350 s at 72°C. Our findings suggest that the specific IgG from milk antibodies of immunized lactating cows may be used as an abundant source of hyper-immune products for prevention of multibacteria-induced diarrhea, however, the effect of pH on its antigen-binding activity upon heating should be carefully considered and designed.

Grazing cows could produce milk with a higher proportion of polyunsaturated fatty acids, which is beneficial to human health, compared with non-grazing cows, though grazing alone could compromise milk production. Under oceanic climate conditions, a study involving 15 dairy cows, fed total mixed ration (TMR) ad libitum in combination with different grazing times of 12 h (TMR12), 6 h (TMR06) and zero grazing time (TMR00) with the aim to evaluate different strategies on the fatty acids profile of milk and milk production. No differences were seen between the treatments with respect to milk yield (34·4±6·3 kg/d) or milk protein content (30·4±1·8 g/kg). The milk produced by the TMR12 cows had less total fat (36·2 vs. 38·2 g/kg) and saturated fatty acid (FA, 69·39 vs. 71·44 g/100 g FA) than that produced by the TMR00 cows. The concentration of vaccenic acid in the TMR06 and TMR12 milk was twice that of the TMR00 milk (4·22, 4·09 and 2·26 g/100 g FA respectively). Linear increases in conjugated linoleic (CLA) and linolenic acids were observed with increasing grazing time. Pasture was an important source of FA especially C18:3 for TMR06 and TMR12 cows. Under oceanic climatic conditions, the grazing of dairy cows as a complement to feeding with TMR can improve the FA profile of milk and increase its CLA content.

On most dairy farms teat dips are applied to the teats of cows either before or after milking in order to prevent pathogens from gaining access to the mammary gland via the teat canal. In the present experiments, a natural teat dip was developed using a fermentate containing the live bacterium Lactococcus lactis DPC 3251. This bacterium produces lacticin 3147, a two-component lantibiotic which was previously shown to effectively kill Gram-positive mastitis pathogens. Lacticin 3147 activity in the fermentate was retained at 53% of its original level following storage for 3 weeks at 4°C. In the initial experiments in vitro, 105 colony-forming units/ml (cfu/ml) of either Staphylococcus aureus, Streptococcus dysgalactiae or Streptococcus uberis were introduced into the lacticin-containing fermentate. Neither Staph. aureus nor Str. dysgalactiae could be detected after 30 min or 15 min, respectively, while Str. uberis was reduced approximately 100-fold after 15 min. Following these trials, preliminary experiments were performed in vivo on teats of lactating dairy cows. In these experiments, teats were coated with each of the challenge organisms and then dipped with the lacticin-containing fermented teat dip. Following a dip contact time of 10 min, staphylococci were reduced by 80% when compared with the undipped control teat. Streptococcal challenges were reduced by 97% for Str. dysgalactiae and by 90% for Str. uberis. These trials showed that the teat dip is able to reduce mastitis pathogens on the teats of lactating cows.

The aim of this study was to evaluate the milk quality of Massese ewes receiving diets with different forage:concentrate ratios (FC ratio), specially on milk fat globules characteristics and fatty acids composition. The diet is one of the main environmental factors that influence the lipidic content of milk. A trial was carried out on twenty ewes, which had been subdivided into two homogeneous groups and kept indoors at 25 days post partum. The experiment lasted 60 days, from 40 to 100 days post partum and the animals were fed two diets that differed in terms of the FC ratio: 60:40 and 40:60, as fed. The results obtained in this study showed that a greater proportion of forage, compared with an higher percentage of concentrate, led to an increase in the percentage of fat (+8·66%) and to a decrease in the percentage of milk fat globules with a size between 2 and 5 μm (−17·32%). However, the average diameter was not affected. There was also a decrease in the percentages of some medium chain fatty acids (C12:0, C14:0; −14·89% and −4·03 respectively) and an increase in mono and polyunsaturated ones such as trans11-C18:1 (+31·71%), total CLA (+22%), EPA (+18·18%) and DHA (+66·67%). In conclusion, a greater proportion of forage seem to improve the milk fatty acid profile by the increase of some fatty acid identified has being beneficial for human health.

A study was carried out to evaluate how the energy level of the diet can affect milk production and quality in Girgentana lactating goats in relation to polymorphism at the αs1-casein (CSN1S1) genotype locus. Twenty-seven goats, homogeneous for milk production (1·5±0·3 kg/d), days of lactation (90±10 d) and body weight (35·8±5·5 kg) were selected on the basis of their CSN1S1 genotype, as follows: nine goats homozygous for strong (AA) alleles, nine goats homozygous for weak alleles (FF) and nine goats heterozygous (AF). The goats were used in a 3×3 factorial arrangement of treatments, with three genotypes (AA, FF, AF) and three diets at different energy levels (100%, 65% and 30% of hay inclusion). The experiment consisted of three simultaneous 3×3 Latin squares for the three genotypes, with one square for each level of hay inclusion in the diet. All the animals were housed in individual pens. Each experimental period lasted 23 d and consisted of 15 d for adaptation and 8 d for data and sample collection, during which the goats received the scheduled diet ad libitum. The animals were fed three different diets designed to have the same crude protein content (about 15%) but different energy levels: a pelleted alfalfa hay (H100) and two feeds including 65% (H65) and 30% (H30) of alfalfa hay (respectively 1099, 1386 and 1590 kcal NE for lactation/kg DM). All the diets were ground and pelleted (6 mm diameter). AA goats were more productive than AF and FF goats (respectively: 1419 v. 1145 and 1014 g/d; P=0·002). Indeed the interaction energy level×genotype was significant (P=0·018): in fact AA goats showed their milk increase only when fed with concentrates. Differences in protein and in casein levels between the three genotypes were in line with results expected from the different allele contribution to αs1-casein synthesis. Milk urea levels were significantly lower in AA goats compared with AF and FF genotypes (respectively 32·7 v. 40·4 and 40·4 mg/dl; P=0·049) and significantly lower when goats were fed with 65H and 30H diets than with 100H diet (respectively 37·4 and 34·3 v. 41·7 mg/dl; P<0·001). Indeed, a significant interaction genotype×diet (P=0·043) occurred for milk urea, which was significantly lower in AA goats but only when fed with concentrates (65H and 30H). Blood concentrations of energy indicators (glucose, non-esterified fatty acids and beta-hydroxybutyric acid) were not influenced by genotype. The results confirm that strong alleles are associated with a greater efficiency of feed utilization and seem to show that a high energy level of the diet can further improve this efficiency.

In order to contribute to the breeding programmes of Asian water buffalo, the aim of this study was to analyse the influence of genetic effects in the stayability of Murrah dairy buffaloes. The stayability trait (ST) was defined as the female's ability to stay in the herd for one (ST1), two (ST2), three (ST3), four (ST4), five (ST5) or six years (ST6) after the first calving. The same trait was also considered as continuous and was designated stayability in days up to one (STD1), two (STD2), three (STD3), four (STD4), five (STD5) or six years (STD6) after the first calving. Data from 1016 females reared in nine herds located in the State of São Paulo, Brazil, were analysed. Statistical models included the additive genetic effect of the animal and the fixed effects of the buffalo breeding herd, birth year and birth season. Additive effects for ST were estimated by approximate restricted maximum likelihood using a threshold model, while for STD, the additive effects were estimated by restricted maximum likelihood. Heritability estimates were lower for ST, except for ST1, (0·11±0·07, 0·17±0·06, 0·23±0·06, 0·16±0·08, 0·14±0·09 and 0·16±0·10 for ST1, ST2, ST3, ST4, ST5 and ST6, respectively) when compared with STD (0·05±0·06, 0·18±0·08, 0·40±0·10, 0·49±0·11, 0·41±0·11 and 0·30±0·13, for STD1, STD2, STD3, STD4, STD5 and STD6, respectively). Considering the values of heritability and owing to the serial nature of STD to a specific age, selection for STD3 should have a favourable influence on STD to other ages.