The influence of high altitude, alpine origin of the forage and roughage-only diets on milk protein content and composition, plasmin activity and cheesemaking properties was investigated. There were four treatment groups, each consisting of six dairy cows in early to mid-lactation. Two groups were fed only with hay ad libitum either at 2000 m or at 400 m a.s.l. One group, kept in the lowlands, was pair-fed to the alpine-site group and another group received a mixed diet of silages, hay and concentrates. Two hay types, harvested either at the alpine site or in the lowlands, were offered to all cows fed with hay alone, following a change-over design in three periods each of 3 weeks. In the respective third week, milk was sampled at every milking. Hay of alpine origin significantly reduced milk protein, in particular whey proteins, which is why the casein number increased. κ-Casein proportion in total casein was reduced and its glycosylation was increased by the alpine hay. The apparent plasminogen-derived activity was reduced when alpine hay was given, but apparent plasmin activity and rennet coagulation properties of the milk were not affected by hay type. Independent of hay type, the high altitude group showed a significantly reduced milk protein content, lower glycosylation of κ-casein and impaired rennet coagulation properties. For several of the traits, the trend was the same in the pair-fed group. There was no effect of altitude on apparent plasmin activity. Hay-alone v. the mixed diet resulted initially in marked declines in milk protein content but did not impair cheesemaking properties. Thus the extensive diet without concentrates, typical of high-alpine conditions, contributed less to the overall effect of extensive alpine v. intensive lowland feeding systems than hay quality and altitude did. In conclusion, certain positive influences of the alpine sojourn of cows on cheese processing quality are overruled by the major adverse impact of lower milk protein content.

Kefir is a fermented milk produced by the action of lactic acid bacteria, yeasts and acetic acid bacteria, trapped in a complex matrix of polysaccharides and proteins. Beyond its inherent high nutritional value as a source of proteins and calcium, kefir has a long tradition of being regarded as good for health in countries where it is a staple in the diet. However, published human or animal feeding trials to substantiate this view are not numerous. The aim of this work was to determine the immunomodulating capacity of kefir on the intestinal mucosal immune response in mice and to demonstrate the importance of dose and cell viability on this response. BALB/c mice were fed with commercial kefir ad libitum (diluted 1/10, 1/50, 1/100 or 1/200) or pasteurized kefir (diluted 1/6, 1/10, 1/50, 1/100) for 2, 5 or 7 consecutive days. At the end of each feeding period, the bacterial translocation assay was performed in the liver. Small intestine structure was studied by haematoxilin-eosin staining and light microscopy. The number of IgA+ and IgG+ cells was also determined. For the functional doses chosen, cytokines (IL-2, IL-4, IL-6, IL-10, IL-12, TNF-α and IFN-γ) were determined. Kefir and pasteurized kefir were able to modulate the mucosal immune system in a dose-dependent manner. Kefir was administred 10-times more diluted than pasteurized kefir, but it induced an immunomodulation of similar magnitude, indicating the importance of cell viabilty. The results suggest that a Th1 response was controlled by Th2 cytokines induced by kefir feeding. Pasteurized kefir would induce both Th2 and Th1 responses. This is the first study in vivo regarding the mechanisms involved in the immunomodulating capacity of the oral administration of kefir containing viable or heat-inactivated bacteria at different doses.

A yogurt mix (2 g fat and 17 g solids/100 g) was supplemented with an algae oil emulsion to provide 500 mg ω-3 fatty acids per 272 g serving of yogurt white mass. The emulsion was added to the yogurt mix either before or after the homogenization step and prior to pasteurization. It was then flavoured with a strawberry fruit base and fermented and stored for up to three weeks. The oxidative deterioration of the products was determined by hydroperoxide measurements and by trained and consumer sensory evaluations. The hydroperoxide content of the supplemented yogurts increased over the storage treatment and was unaffected by the stage of addition. The trained panel could distinguish a stronger fishy flavour in both of the supplemented yogurts after 22 days storage, but the consumer panel rated both control and supplemented samples similarly, as ‘moderately liked’.

Prolactin (PRL) plays a key role in normal growth, development and differentiation of the mammary gland. Indeed, strong evidence suggests that the development of alveolar cells requires not only oestradiol and progesterone, but also PRL. In vitro, PRL has mitogenic activity on normal mouse mammary epithelial cells (reviewed in Das & Vonderhaar, 1997). In vivo, PRL also seems to be involved in such proliferative activity, although it is more difficult to distinguish the role of PRL from the influence of the hormonal milieu (Das & Vonderhaar, 1997). This physiological role of PRL in lobular development of the mammary gland is supported by results obtained from mice deficient for PRL (Horseman et al. 1997) or for its receptor (PRLR) (Ormandy et al. 1997). Although the infertility of females homozygous for the deletion of the PRLR gene (PRLR−/−) can be partially reversed by restoring progesterone levels close to normal, their mammary gland fails to differentiate during pregnancy, leading to lactation failure (Binart et al. 2000). In addition, heterozygous mice (PRLR+/−), who have half normal receptor levels, show impaired mammary gland development and fail to lactate following their first pregnancy, clearly indicating that signals mediated by the PRL/PRLR interaction have to achieve a certain level to permit mammary gland differentiation and lactation (Kelly et al. 2002). Since the pioneering work of Topper (Topper, 1970), who observed that PRL was necessary to induce casein synthesis, our understanding of the mechanism of such induction has greatly expanded. PRL appears to be the primary hormone involved in this activity, although other hormones such as insulin and glucocorticoids are also required for lactation.

The influence of three different ventilation regimens on air pollution in sheep houses and on the quality of ewe milk and of Canestrato Pugliese cheese was investigated during the summer season. The experimental treatments were low ventilation regimen (VR=35 m3/h per ewe) split in 30-min ventilation cycles (LOV-30); moderate ventilation regimen (VR=70 m3/h per ewe) split in 30-min ventilation cycles (MOV-30); moderate ventilation regimen (VR=70 m3/h per ewe) split in 60-min ventilation cycles (MOV-60). The LOV-30 milk had higher microbial load and bulk milk somatic cell count (BMSCC) and resulted in a weaker casein matrix in the curd compared with the MOV-30 and MOV-60 treatments. At 45 d of ripening, the LOV-30 cheeses had a lower casein content and higher non-casein nitrogen (NCN) and water-soluble nitrogen (WSN) contents than the MOV-30 and MOV-60 cheeses. Urea-polyacrylamide gel electrophoresis (urea-PAGE) of the pH 4·6-soluble N extract showed that the MOV-60 cheeses had fewer bands derived from casein (CN) hydrolysis than the LOV-30 or MOV-30 cheeses, despite its having exhibited the highest plasmin (PL) activity levels. Our results suggest that the ventilation regimen is critical in dairy sheep housing for optimizing the hygienic quality of ewe milk and the proteolytic processes occurring in Canestrato Pugliese cheese during ripening.

Staphylococcus aureus isolates produce several pathogenic factors. The combination of these products influences the pathogenic role of different isolates, but their specific effects are well known in the pathogenesis of udder infections. This study focused on the association of polymorphism of the coagulase gene, protein A gene, collagen-binding protein gene, and of fibrinogen-binding protein gene on somatic cell count (SCC) and on Staph. aureus growth rate. Fifty Staph. aureus isolates from 13 dairy cow herds, located in seven different provinces, were considered. The results showed a low frequency of cna gene, similar to the one observed in human isolates. Meanwhile, the high frequency of efb gene indirectly confirmed the role of this factor in bacterial pathogenesis, being associated with adhesion to epithelia. The association of these two single genes with SCC and growth rate showed to be not significant. The polymorphism of spa gene was confirmed to be significantly associated with inflammatory response and growth rate, albeit with a pattern different from the one suggested for human isolates. Sorting of isolates based on the clusters obtained by combining polymorphisms of spa and coa genes and the presence of cna and efb genes, showed that a single cluster (cluster V) was prevalent in the different herds and provinces, while the other six clusters identified were widely spread among the remaining 60% of the isolates. Results showed that clusters VI and VII had significantly higher growth rates at 3, 4, and 6 h in comparison with the other clusters. Meanwhile, quarters infected with these strains showed significantly lower SCC levels. The frequency of isolates from cluster V, suggested that they should possess pathogenic factors increasing their invasiveness, even if in the presence of a stronger inflammatory response. These results indirectly confirm previous findings on the different interactions between isolates and the udder immune system. They also suggest that isolates with higher growth rates and inducing a lower inflammatory response have better chances to spread among the herd. The relatively simple genomic method proposed in this study could be applied by an increasing number of diagnostic laboratories and could be useful in studying the epidemiology of Staph. aureus intramammary infections in dairy herds when collecting data from the field.

The protein fraction of milk contains several components with physiological significance for the development of the newborn. Among them, immunomodulatory peptides and lactoferrin exemplify the complexity of biologically active substances of milk. Immunomodulatory peptides have latent activity within the native protein and are generated after proteolysis during gastrointestinal transit. Once they are generated, they modulate mucosal immunity, possibly by guiding the local immune system until it develops its full functionality. Lactoferrin is another milk bioactive compound with nutritional and health promoting properties; it modulates the microbial intestinal environment, displays anti-microbial activity against various pathogens and stimulates the establishment of beneficial microflora. The following overview focuses on the importance of immunomodulatory peptides and lactoferrin for the maturation of intestine and immune system that are functionally immature in the newborn.

The economic effect of lactational antibiotic treatment of chronic subclinical intramammary infections due to Streptococcus uberis or Streptococcus dysgalactiae was explored by means of partial budgeting. Effects at cow level and herd level were modelled, including prevention of clinical mastitis episodes and the prevention of transmission of infections. Input variables for our deterministic model were derived from literature or based on 2002/2003 dairy prices and farming conditions in The Netherlands. Sensitivity analysis was used to examine the effect of uncertainty around input variables or changes in price estimates. On farms where pathogen transmission was prevented through proper udder health management, 3-d antibiotic treatment during lactation resulted in an average net profit of €+11·62 over no treatment while 8-d antibiotic treatment had an average negative net result of €−21·83. Sensitivity analysis showed that profitability depends on the probability of treatment-induced cure, pathogen transmission rates, culling rate, retention pay-off, and costs of antibiotic treatment. Three-day antibiotic treatment of chronic subclinical streptococcal mastitis is economically profitable over a range of input values for cure probabilities, transmission rates and losses due to culling. In contrast, 8-d lactational treatment is only profitable for very valuable animals, on farms where the risk of pathogen transmission is high and/or when the farmer is likely to cull a high percentage of cows with subclinical mastitis. Because bacterial flora, cow characteristics and management differ widely between farms, the economic outcome of lactational treatment of chronic subclinical streptococcal mastitis may be highly farm-dependent.

The goat calcium-sensitive caseins (αs1, β and αs2) represent, over many years, an excellent model for demonstrating that the major part of the variability observed in the content of these proteins in goat milk is mostly due to the presence of autosomal alleles at single structural loci (CSN1S1, CSN2 and CSN1S2 respectively) clustered on a 200 kb segment of chromosome 6; furthermore, CSN1S1 and CSN2 are convergently transcribed and are only 12 kb apart (Rijnkels, 2002).

Genetic polymorphisms in CSN3 gene in Pag (Croatia), Sarda (Italy) and Pramenka (Serbia) sheep breeds were investigated. A single nucleotide polymorphism (SNP) was localized by sequence analysis (sequence submitted to GenBank under accession AY237637) relying on an original primer pair. Primers for sequencing (κ-casF and κ-casR) were designed on the available CSN3 sequences to amplify the genomic region encoding the major part of the mature protein (exon 4). An SNP was detected at position 237 of the sheep κ-casein mRNA (reference sequence: GenBank X51822), where a thymine was substituted for a cytosine. The SNP was typed by conventional PCR and SYBR Green I-based real-time PCR. C and T alleles were discriminated using a dedicated set of primers that consisted of one common forward primer (SNP-TC) and two reverse primers (SNP-T and SNP-C), the latter two differing in the 3′ end base and in the presence of a 12 bp poly-G tail in SNP-C. The SNP was found in both the heterozygous and the homozygous state in Sarda and Pramenka breeds, and in the heterozygous state only in the Pag breed. The observed allelic frequencies of the SNP were 0·12 in Pag, 0·27 in Sarda and 0·45 in Pramenka.

Fatty acids are the major source of energy for most tissues during periods of negative energy balance; however, fatty acids can, in some circumstances, have pathological effects. Fatty acids are stored as triacylglycerols (TAG), mostly in the various adipose tissue depots of the body. However, if blood unesterified fatty acid (NEFA) levels are elevated for prolonged periods, as may occur during lactation or obesity, TAG can accumulate in other tissues including liver and muscle cells (myocytes), and this can have pathological consequences such as the development of ketosis (Grummer, 1993; Drackley et al. 2001) or type 2 diabetes (Boden & Shulman, 2002; McGarry, 2002).

A total of 120 milk samples were collected from Comisana ewes throughout lactation. The ewes were ranked into two somatic cell count (SCC) categories: normal milk (N Milk) with SCC lower than 5·00×105/ml and high somatic cell milk (HSC Milk) with SCC higher than 1·00×106/ml. Milk samples were analysed in triplicate for pH, fat and protein contents, renneting parameters, and plasmin and plasminogen activities. The peptide profile due to total proteolytic activity (endogenous and exogenous enzymes) on α- and β-CNs were determined using urea-PAGE on sodium caseinate (pH 8·0 and pH 5·0) incubated at 37 °C for 4 d after sampling. The peptide profile due to non-plasmin enzyme activities at pH 5·0 was also determined using urea-PAGE. Plasmin activity was higher in the HSC milk than in the N milk throughout the study period. A decrease in plasmin activity was observed in the N milk during mid-lactation, which was probably related to decrease in pH, and in the HSC milk during late lactation, which may be ascribed to an enhanced influx of plasmin inhibitors from the blood stream. Proteolytic patterns in Comisana ewe milk were mainly affected by plasmin activity that increased with the SCC in milk. Also non-plasmin proteolytic activity was strongly enhanced by elevated SCC and resulted in a higher degradation of α-casein than of β-casein. In general, plasmin activity did not increase with the advancement of lactation and exhibited a different trend in HSC and N milk, suggesting that physiological factors did not play a key role in regulating the plasminogen-plasmin system in ewes' milk. Plasmin activity, detected with the colorimetric assay was consistent with proteolytic activity on sodium caseinate shown in urea-PAGE electrophoregram.

Indigenous L-lactate dehydrogenase (LDH) in milk originates mainly from somatic cells, leucocytes and invading microorganisms. Its activity may be used for detection of mastitis. However, existing methods to measure LDH activity in milk both need pretreatment of the samples and still suffer from methodological problems. The present paper describes a fast, reliable method for determination of LDH activity, suitable for milk samples. The method is based on fluorometric determination of enzyme kinetics when L-lactate is converted to pyruvate. The assay uses raw milk without pretreatment and the method is easily adjustable to large-scale analyses on micro assay plates. Detection is based on (straight line) linear response within 4–7 min of initiation of the reaction. A substrate concentration of 35 mM in the reaction mixture was considered to be optimal for the assay. Intra plate assay precision was approx. 6% (CV) and the inter plate precision approx. 10%. Known inhibitors of LDH activity (oxidative direction), i.e., oxalic acid, oxamate, and pyruvate, were tested in different concentrations in order to verify the specificity of the response. The detailed kinetics of samples analysed indicated that the isoenzyme composition may have differed between milk samples, and that this composition may have been altered in high activity samples.

Ataxia-telangiectasia (AT) is a rare autosomal recessive early childhood disorder, characterized by progressive neuronal degeneration, immunological deficiency, radiosensitivity and an increased risk of cancer caused in most cases by mutations in the AT-mutated gene (ATM). Epidemiological studies on AT families have shown that AT heterozygous women have an increased risk of developing breast cancer (BC). The ATM protein plays a central role in the recognition and repair of DNA double-strand breaks and the subsequent activation of cell-cycle checkpoints. Whilst AT is a rare disease, 0·5–1% of the general population are estimated to be AT mutation carriers, thus any increases in the risks of cancer associated with ATM carrier status are of public health relevance. The main results of our published studies on the risk of BC in 34 French AT families according to heterozygote status, type of ATM mutation and exogenous factors are summarized here. The risk of BC was higher in ATM heterozygous (HetATM) women and did not differ significantly according to the type of ATM mutation (missense vs truncating) carried by the AT family members but appeared associated with the position of some truncating mutations in certain binding domains of the ATM protein. The effect of exogenous factors, such as reproductive life factors and exposure to ionizing radiation, on the risk of BC according to ATM heterozygote status was assessed. There was no evidence for interaction (except for age at first full-term pregnancy). These findings does not appear to justify a separate screening program from that already available to other women with a first-degree relative affected by BC, as their risks have similar amplitude. Chest X-rays did not appear to be a risk factor for BC in our study population. More powerful studies, using data sets pooled from international sources are being set up to confirm these observations.

Unidentified constituents in fresh pasture increase milk fat cis-9, trans-11 conjugated linoleic acid (CLA) concentration, and prevent milk fat depression, even though ruminal conditions conducive to reducing milk fat synthesis exist. One possible explanation is vitamin E (α-tocopherol), a constituent high in fresh pasture, but naturally low in conserved/dried forages and cereal grains. Twenty late-lactating dairy cows previously consuming a total mixed ration (TMR) were randomly allocated to one of two dietary treatments for 21 d: TMR (control; n=10); and TMR plus an additional 10000 i.u. α-tocopherol/d (VIT E; n=10). These cows were simultaneously compared with 13 late-lactation dairy cows previously grazing fresh pasture (PAS) balanced for age, parity and genetic merit. Average daily α-tocopherol intakes were approximately 468, 10520 and 1590 i.u./cow for the control, VIT E and PAS treatments, respectively. Dietary α-tocopherol supplementation (VIT E v. control) slightly increased milk fat content by 0·23 percentage units, but did not significantly alter milk fatty acid composition. Plasma trans-11 18[ratio ]1 (VA) content tended to increase and trans-10 18[ratio ]1 levels numerically declined following α-tocopherol supplementation suggesting possible changes in rumen biohydrogenation products. In addition, increased α-tocopherol intake in TMR-fed cows decreased serum urea levels and tended to alter milk fat 15[ratio ]0 suggesting changes in rumen microbial populations. However, when compared with cows grazing pasture, TMR-fed cows supplemented with α-tocopherol, still produced milk with lower cis-9, trans-11 CLA and VA, and higher trans-10 18[ratio ]1 concentrations suggesting α-tocopherol is not a primary reason for milk fatty acid profile differences between pasture and TMR-fed cows. Therefore, additional unknown pasture constituents favour production of fatty acids originating from the cis-9, trans-11 instead of the trans-10, cis-12 CLA biohydrogenation pathways.

This paper presents an alternative method to estimate somatic cell count (SCC) in cows' milk. SCC is an important indicator in the detection of inflammatory reactions within the udder in cows and Direct Optical Microscopy (DOM) is the present reference method for SCC but, owing to its dependence on human operators, it is extremely costly, time-consuming and potentially subjective. The industrial method of choice is Epifluorescence (EF), which has the potential for impressive throughput and acceptable precision, but requires huge inversions and handling of highly toxic reactives and waste. In this paper, an advantageous method that involves application of a low-cost Video Microscopy (VM) system is analysed and discussed, including a comparison between DOM and VM, and an example of application of both methods to evaluate EF counts. We conclude that VM is sufficiently precise and very cheap to implement and operate.

The impact of dietary components on the immune system is gaining increased attention in the effort to develop safe food products, some even with health-promoting potential, as well as to improve the basic understanding of the immunomodulatory potential of common food components. In such studies, which are mainly based on experiments in vitro, it is important to be able to differentiate nonspecific activation of immune cells induced by dietary components from ex vivo restimulation of antigen-specific cells that might be present in cell cultures owing to prior dietary exposure to the antigens in cell donors. Focusing on the immunostimulatory potential of cows' milk proteins and peptides, we studied the impact of prior dietary exposure to cows' milk on proliferation of murine immune cells upon ex vivo stimulation with bovine milk proteins. Nonspecific proliferation induced by β-casein peptides was further assessed on cells from mice bred on a cows'-milk-free diet. Regarding the dietary effect, we found that prior oral intake of cows' milk proteins affected cell proliferation induced by culturing with cows' milk proteins in vitro, as spleen cells from mice fed a milk-containing diet showed a significantly greater proliferative response than did cells from mice bred on a cows'-milk-free diet. Studies of immune enhancing potentials of β-casein peptides showed that some peptides stimulate proliferation of immune cells nonspecifically. In conclusion, these findings stress the importance of employing immune cells from mice unexposed to cows' milk for studies of the immunomodulating capacity of cows' milk proteins and peptides, in order to rule out the interference caused by antigen-specific immune responses. By using such cells, we here show that some β-casein peptides possess the potential to induce proliferation in immune cells in a nonspecific manner.

Sixty-nine bovine mastitis streptococci belonging to the species Str. agalactiae (n=13), Str. dysgalactiae (n=16), Str. canis (n=22), Str. uberis (n=20) and Str. parauberis (n=4) and six reference strains of the five streptococcal species were examined for their tRNA gene intergenic length polymorphism (tDNA-ILP) fingerprint pattern. Epidemiologically unrelated isolates from bovine mastitis cases were selected by macrorestriction analysis with pulsed-field gel electrophoresis (PFGE). Their results were compared with those obtained from biochemical and serological studies and with those obtained by PCR-mediated identification amplifying species-specific gene segments of the five streptococcal species. According to the present results tDNA-ILP allowed a correct identification of all Str. agalactiae, Str. uberis and Str. parauberis strains investigated also including the reference strains of each species showing species-specific banding pattern. However, all Str. dysgalactiae ssp. dysgalactiae and all Str. canis strains appeared with an undistinguishable pattern which did not allow an identification of the species.

Test day records of milk yield (38765), fat and protein contents (11357) of Sarda goats (the most numerous Italian goat breed) were analysed with mixed linear models in order to estimate the effects of test date (month and year of kidding for fat and protein contents) parity, number of kids born, altitude of location of flocks (<200 m asl, 200–500 m asl, >500 m asl), flocks within altitude and lactation stage (eight days-in-milk intervals of 30 d each) on milk production. All factors considered in the models affected milk traits significantly. Milk yield was lower in first parity goats than in higher parities whereas fat and protein contents showed an opposite trend. Goats with two kids at parturition had a higher milk yield than goats with one kid and tended to have lower fat and protein percentages. Repeatability between test days within lactation was 0·34, 0·17 and 0·45 for milk yield, fat content and protein content, respectively. Lactation curves of goats farmed at different altitudes were clearly separated, especially for milk yield. Results of the present study highlight differences in milk production traits among the three subpopulations that have been previously identified within the Sarda breed on the basis of the morphological structure of animals and altitude of location of flocks.

The fur seal is a mammal with an unusual ability to turn its milk production on and off without significantly altering the gross morphology of the mammary gland. This atypical lactation cycle is due to the fact that maternal foraging and infant nursing are spatially and temporally separate (Bonner, 1984). Maternal care involves the suckling of offspring over a period of at least 4 months, but lactation can extend to more than 12 months. Following a perinatal fast of approximately 1 week, females depart the breeding colony to forage at sea and, for the remainder of lactation, alternate between short periods ashore suckling their young with longer periods of up to 4 weeks foraging at sea. Whilst foraging at sea, milk production in the fur seal mammary gland either ceases or is reduced (Arnould & Boyd, 1995b).