The solute carrier family 27 member 1 (SLC27A1) is an integral membrane protein involved in the transport of long-chain fatty acids across the plasma membrane. This protein has been implicated in diet-induced obesity and is thought to be important in the control of energy homeostasis. In previous reports, our group described the isolation and characterization of the bovine SLC27A1 gene. The bovine gene is organized in 13 exons spanning over more than 40 kb of genomic DNA and maps in BTA 7 where several quantitative trait loci for fat related traits have been described. Because of its key role in lipid metabolism and its genomic localization, in the present work the search for variability in the bovine SLC27A1 gene was carried out with the aim of evaluating its potential association with milk fat content in dairy cattle. By sequencing analysis of all exons and flanking regions 14 new single nucleotide polymorphisms (SNPs) were identified: 1 in the promoter, 7 in introns and 6 in exons. Allele frequencies of all the SNPs were calculated by minisequencing analysis in two groups of Holstein-Friesian animals with highest and lowest milk-fat content estimated breeding values as well as in animals of two Spanish cattle breeds, Asturiana de los Valles and Menorquina. In the conditions assayed, no significant differences between Holstein-Friesian groups were found for any of the SNPs, suggesting that the SLC27A1 gene may have a poor or null effect on milk fat content. In Asturiana and Menorquina breeds all the positions were polymorphic with the exception of SNPs 1 and 8 in which C allele was fixed in both of them.

The concentration of lactoferrin (LTF) in milk varies during lactation, rising sharply during involution. We proposed that LTF might have a regulatory role in involution and investigated its effects in vitro on the viability of bovine mammary epithelial cells (BMEC) and on casein expression in bovine mammospheres. Mammospheres capable of milk protein expression were formed by culturing primary BMEC on extracellular matrix in the presence of lactogenic hormones. Exogenously added LTF decreased β-casein and κ-casein mRNA expression in mammospheres while transfection of a short interfering RNA (siRNA) to suppress LTF expression resulted in increased casein mRNA expression. We believe that LTF exerts its effect on casein gene expression by up-regulating interleukin-1β (IL-1β) as IL-Iβ gene expression was elevated in mammospheres treated with LTF. LTF also decreased viability of BMEC grown as monolayers and as mammospheres. Interestingly, LTF was only effective in reducing casein mRNA expression and viability in mammospheres when added at concentrations found during early involution but was inactive when used at concentrations found in milk. We suggest that LTF has a regulatory role during early involution, decreasing casein expression and reducing BMEC viability.

The present study tested the hypothesis that supplemental dietary fatty acids (FA) affect the energy corrected milk yield in proportion to the milk production level of dairy cows, and increase both long chain FA proportion of milk FA and milk fat globule diameter. Sixteen Danish Holstein cows were divided into four 4×4 Latin squares with two squares of medium yielding cows (32·2 kg energy corrected milk (ECM)/d; 158 days in milk (DIM)) and two squares of high yielding cows (40·0 kg ECM/d; 74 DIM). Experimental length was 12 weeks, with three weeks for each of the four periods. The four treatments were no supplementation (17 g FA/kg dry matter (DM)) and three diets with supplemented FA (29, 40, and 52 g total FA/kg DM, respectively) obtained by substituting barley with Palm Fatty Acid Distillate (PFAD) fat. Diets were offered as total mixed rations with 63% grass/clover silage (DM basis). Dry matter intake decreased with increasing FA supplementation, but net energy intake was not affected. The general linear responses to 10 g/kg DM increase in FA level were 1·1 kg ECM (P<0·0001), 0·061 kg milk fat (P<0·0001), 0·012 kg milk protein (P=0·09) and 0·052 kg lactose (P=0·0002) per day, and linear responses in milk composition were 0·39 g fat (P=0·07), −0·71 g protein (P<0·0001) and 0·05 g lactose (P=0·3) per kg milk, and 0·092 μm (P<0·0001) in milk fat average globule diameter. Fatty acid supplementation decreased short- and medium-chain FA and C16:0 and increased C18:1 proportions of total FA in milk. Supplemental dietary FA increased ECM yield but not in proportion to production level as anticipated, and increased average FA chain length and milk fat globule diameter.

Ca-Fe interactions are known, but no studies are available about the effects of Ca-enriched goat or cow milk on Fe status in nutritional ferropenic anaemia (NFA). To examine this matter, control and Fe-deficient rats were fed for 14 d with goat or cow milk diets containing either normal or high Ca content (5000 or 10 000 mg/kg diet), and different indices and parameters related to iron status were measured. The apparent digestibility coefficient (ADC) and the Fe retention/intake (R/I) ratio were higher in control and anaemic rats fed goat milk diet (G diet), despite high-Ca content. Ca enrichment decreased Fe stores in liver and sternum in anaemic rats fed cow milk diet (C diet), however G diet did not modify Fe content in the organs studied in control and anaemic rats. In anaemic rats, Ca-supplementation decreased haematocrit, but platelets and serum Fe were not affected, however, in control rats platelets increased except for Ca-enriched G diet, this fact reveals that Ca-Fe interaction is minimized with G diet. Serum ferritin was always higher in rats fed G vs. C diet, both in control and anaemic rats fed either normal or Ca-enriched diets. Ca-supplementation decreased ferritin levels in control and anaemic rats fed C diet and also, though to a lesser extent, in those given the G diet. This indicates that with this G diet there is a better recovery of body Fe stores in anaemic rats, despite Ca-supplementation. In this study it is noteworthy that despite high Ca content, a goat milk diet resulted in minimal Ca-Fe interactions and did not adversely affect Fe status in rats with NFA.

Sediment formation was investigated during UHT treatment of goats' milk, subjected to indirect treatment at 140°C for 2 s, with upstream homogenisation. Stabilisers evaluated were sodium hexametaphosphate (SHMP), trisodium citrate (TSC), disodium hydrogen orthophosphate (DSHP), and sodium dihydrogen orthophosphate (SDHP). With no added stabiliser, goats' milk produced a heavy sediment on UHT treatment. Addition of SDHP reduced pH, had little effect on ionic calcium and did not substantially reduce sediment. However, addition of SHMP, DSHP and TSC each reduced ionic calcium, increased ethanol stability and reduced sediment. Following stabiliser additions, there was a good correlation between ethanol stability and ionic calcium (R2=0·85) but not between ethanol stability and pH (R2=0·08). Overall, reducing ionic calcium reduced the amount of sediment formed for all these three stabilisers, although there was no single trend line between sediment formation and ionic calcium concentration. Sediment formation was not well correlated with pH for TSC or for SHMP, but it was for DSHP, making it the only stabiliser where sediment formation correlated well both with ionic calcium and pH, which might account for its effectiveness at higher ionic calcium levels. Sediment was much reduced when the temperature was reduced from 140°C to 125°C and 114°C. There were no further changes in sediment on storage for two weeks. Analysis of the sediment showed that it was predominantly fat and protein, with a mass ratio ranging between 1·43:1 and 1·67:1. Its mineral content was usually less than 5% of dry weight. The maximum amounts of P and Ca were found to be 2·32% and 1·63%, respectively.

Agents that increase natural protective mechanisms have been proposed for prevention and treatment of intramammary infections. The objective of this study was to describe the effects of a single intramammary infusion of a lipopolysaccharide (LPS)-based biological response modifier (BRM) on cellular death mechanism in uninfected and Staphylococcus aureus-infected bovine mammary glands during involution. Three groups of 12 cows, each one including 6 Staph. aureus-infected and 6 uninfected, were infused in two mammary quarters with BRM or placebo and slaughtered at 7, 14 and 21 d of involution. In infected quarters, BRM treatment produced a significant increase in percent of stained epithelial cells for the apoptosis-promoting protein Bax at every observation period. In addition, BRM produced a significant increase of immunostained stromal cells for Bax compared with placebo-treated quarters. BRM treatment produced an increase in percentages of epithelial cells staining with active caspase-3 at 7 d and 14 d of involution compared with placebo-treated quarters and a significant decrease in percentages of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive epithelial cells at 7 d and 21 d of involution. In addition, BRM treatment caused an increase in percentage of stromal cells immunostaining for active caspase-3 and TUNEL. An increase of active caspase-3 and TUNEL epithelial and stromal cell immunostaining was observed in Staph. aureus-infected compared with uninfected quarters. Cellular proliferation, determined by Ki-67 immunostaining, was increased in epithelial and stromal cells from Staph. aureus-infected compared with uninfected quarters at every observation period. These results provide new insights into the mechanism of mammary cell death in uninfected and Staph. aureus-infected bovine mammary gland during involution and illustrate the effects of LPS-based BRM on apoptosis and cell proliferation during mammary involution.

The objective was to evaluate the effect of β-lactoglobulin (β-lg) polymorphism and seasonality on milk composition (fat, lactose, total solids, milk urea nitrogen, total protein, true protein, casein and somatic cell counts) of Holstein and Girolando cows. Milk and blood samples from 278 Holsteins cows and 156 Girolando cows were taken during two dry seasons and two rainy seasons, for milk composition analysis and to determine β-lg genotypes, respectively. BB genotype was the most frequent for both breeds, followed by AA genotype for Holstein (BB>AA>AB) and by AB for Girolando cows (BB>AB>AA). No differences were found in milk compositional characteristics among genetic variants of β-lg (AA, AB and BB) either between Holstein or Girolando cows. No association between milk composition and β-lg genetic polymorphism was observed. During the dry season, independently of the breed considered, higher contents of lactose, true protein, casein and casein:true protein ratio were found.

In a previous study, ultrafiltration (UF) at 1·2×reduced residual chymosin activity and bitterness in exopolysaccharide (EPS)-positive reduced fat Cheddar cheese. The objective of this research was to study the effect of this level of concentration on the textural and functional characteristics of the reduced fat cheese. Ultrafiltration (1·2×) did not affect the hardness, cohesiveness, adhesiveness, chewiness, and gumminess of EPS-positive cheese. The 6-month old UF cheeses were springier than non-UF cheeses. However, the springiness of the EPS-positive cheese made from UF milk was much lower than that of the EPS-negative cheeses. Texture of the EPS-negative cheese was more affected by UF than that of the EPS-positive cheese. Differences were seen in the extent of flow between UF and non-UF cheeses at 1 and 3-months but not after 6 months ripening. Ultrafiltration increased the elastic modulus in the 6-month old EPS-positive cheeses. Higher body and texture scores were given to EPS-positive cheeses than the EPS-negative ones. Sensory panelists found the body of the UF and non-UF cheeses to be similar.

In this study, the effect of dahi containing probiotic Lactobacillus acidophilus NCDC14 and Lactobacillus casei NCDC19 (∼73×108 cfu/g) on progression of streptozotocin (STZ)-induced diabetes in rats (15 g/day/rat) for 28 days was investigated. Feeding of probiotic dahi significantly suppressed the incremental peaks and area under the curve and delayed reduction of insulin secretion during oral glucose tolerance test more than skim milk or control dahi. The feeding of milk products reduced the total cholesterol, triglycerides, LDL and VLDL-cholesterol and increased HDL-cholesterol levels (P<0·05). Moreover, probiotic dahi significantly suppressed STZ-induced oxidative damage in pancreatic tissues by inhibiting the lipid peroxidation and formation of nitric oxide, and preserving antioxidant pool such as glutathione content and activities of superoxide dismutase, catalase and glutathione peroxidase. The results suggest that the supplementation of probiotic Lb. acidophilus and Lb. casei with dahi cultures increased the efficacy of dahi to suppress STZ-induced diabetes in rats by inhibiting depletion of insulin as well as preserving diabetic dyslipidemia, and inhibiting lipid peroxidation and nitrite formation. This may empower antioxidant system of β-cells and may slow down the reduction of insulin and elevation of blood glucose levels.

A PCR protocol for detection of Lactobacillus helveticus bacteriophages was optimized. PCR was designed taking into account the sequence of the lys gene of temperate bacteriophage Φ-0303 and optimized to obtain a fragment of 222 bp using different Lb. helveticus phages from our collection. PCR was applied to total phage DNA extracted from 53 natural whey starters used for the production of Grana cheese and all gave the expected fragment. The presence of actively growing phages in the cultures was verified by traditional tests. Several PCR products of the lys gene were sequenced and aligned. The resulting sequences showed variable heterogeneity between the phages.

The level of gelatinases in surrounding body fluids of actively remodelling tissue is indicative of basement membrane and extracellular matrix degradation under various physiological and pathological circumstances. To elucidate the association of gelatinase with mammary tissue remodelling during gradual or acute involution, in the first trial, goats milked twice daily (lactation) and goats receiving decreased milking frequency (involution) served to provide a total of 12 milk samples and 11 mammary secretion samples, respectively. In the second trial, 6 cows served to provide samples of dry secretion in 3 consecutive weeks immediately following milk stasis. Gelatin zymography was applied for gelatinase phenotyping and quantification on milk, plasma and the degranulation medium/lysate of milk somatic cells. Results indicated that the most prevalent gelatinase subtype switched from gelatinase A in milk to gelatinase B in involution secretion. Mammary secretion of goats during involution contained marginally higher protein level, significantly lower casein ratio and greater specific capacity of gelatinase B compared with those of milk during lactation. Specific capacities of gelatinases A and B in plasma of goats were similar during lactation and involution, while gelatinase B capacity in degranulation medium/lysates based on unit number of goat somatic cell was significantly higher during involution than during lactation. Milk stasis of cows induced a significant increase in specific capacity of gelatinase B, but not gelatinase A, of dry secretion up to the third week. Results of both trials agree that regional selective accretion of gelatinase B in milk might have played a role in mammary tissue remodelling during involution induced by either decreasing milking frequency or milk stasis. It is suggested that infiltrated polymorphonuclear neutrophils are one of the potential contributors responsible for the accumulation of gelatinase B during involution.

Considering that several health promoting properties are associated with kefir consumption and a reliable probiotic product requires a complete identification of the bacterial species, the present work evaluates several proved markers of probiotic potential of eleven isolates of homofermentative lactobacilli isolated from kefir grains and molecular identification and genotypic diversity. Using restriction analysis of amplified ribosomal DNA (ARDRA) and analysis of the 16S–23S rRNA internal spacer region we confirmed that all homofermentative lactobacilli belong to the species Lactobacillus plantarum. RAPD-PCR analysis allowed the discrimination of lactobacilli in five clusters. All isolates exhibited high resistance to bile salt. High survival after one hour of exposure to pH 2·5 was observed in Lb. plantarum CIDCA 8313, 83210, 8327 and 8338. All isolates were hydrophilic and non autoaggregative. Isolate CIDCA 8337 showed the highest percentage of adhesion among strains. All tested lactobacilli had strong inhibitory power against Salmonella typhimurium and Escherichia coli. Seven out of eleven isolates showed inhibition against Sal. enterica and five isolates were effective against Sal. gallinarum. Only CIDCA 8323 and CIDCA 8327 were able to inhibit Sal. sonnei. We did not find any correlation between the five clusters based on RAPD-PCR and the probiotic properties, suggesting that these isolates have unique characteristics.

The aim of this research was to study the effects of dairy maturation on the physico-chemical characteristics and technological properties of milk used for Parmigiano-Reggiano cheese manufacture. Three different operating conditions (CF1, CF2 and CF3) were considered. Full cream milk from the evening milking was stored on the farm and delivered to the cheese factory in churns (CF1) or in thermoregulated tanks at a temperature not lower than 18°C (CF2 and CF3). The natural creaming (10–12 h overnight) was performed in a traditional large flat vat containing 10–12 hl (CF1 and CF2) or in thermoregulated large flat vats containing 60 hl at about 15°C (CF3). Twenty-four, 24 and 22 maturation trials were performed in CF1, CF2, and CF3, respectively, during 2 consecutive years. A significant increase (P⩽0·05) in pH during the maturation of milk was observed in CF1 and CF2. The increase of pH was higher (P⩽0·05) in CF1 than CF2 and CF3. The values of titratable acidity were higher (P⩽0·05) in partially skimmed evening (PS) milk than in full cream (FC) milk in each operative condition. The increase observed in CF2 was higher than those reported in CF1 and CF3. Compared with FC milk, PS milk showed lower values (P⩽0·05) of casein and casein number and higher contents (P⩽0·05) of whey proteins and, particularly, proteose-peptone. The increase of proteose-peptone – per 100 g SNF or 100 g casein – was significantly higher (P⩽0·05) in CF1 than in CF2 and, in particular, than in CF3. A higher increase (P⩽0·05) of resistance to compression was observed in CF1 with respect to CF3. CF2 variation was not different with respect to either CF1 or CF3. Variation of the difference between PS and FC milks (PS-FC) in pH, TBC and fat were clearly lower in CF3 than CF1. This means that the control of milk temperature throughout the whole maturation phase offers a greater control of both microbial development and extent of creaming.

Subclinical mastitis is a costly disease and its diagnosis is difficult. Besides the somatic cell count (SCC) and bacteriology, the differential inflammatory cell count (DICC) is a meaningful tool for mastitis detection. As microscopy is very subjective because of the low number of events to be counted, flow cytometry has often been proposed for the differentiation of milk cells. The objective of this study was to determine whether it is possible to identify subclinical mastitis in cattle at an early stage by a simple and fast flow cytometric method. The aim was to identify the main leucocyte populations in flow cytometric dotplots (polymorphonuclear neutrophils (PMN), lymphocytes and macrophages) and, with these, to elaborate a method of mastitis prognostics. Milk from 15 German Holstein cows was sampled in cross-sectional studies and SCC determined. After preparation, the milk cells were incubated with different specific antibodies that bind to different cell types and also to propidium iodide (PI), which differs between viable and non-viable cells. This procedure made it possible to localize cell types in a flow cytometric dot plot and to differentiate between viable and non-viable PMN. Percentages of viable PMN can be determined by a procedure consisting of a simple centrifugation, incubation with PI, and flow cytometric measurement. So it is possible to quickly determine the stage of the inflammation even in quarters with a low SCC.

Raw ewes' milk semi-soft cheeses (RESS-cheeses) are important products in Portugal and in several European regions. Creamy texture is an essential attribute of these cheeses, which results from structural properties that are not always well characterized. Here, the structural changes occurring during the ripening period of a traditional RESS-cheese, known as Serpa cheese, were analysed through small amplitude oscillatory shear (SAOS). Rheological data was complemented with other physical and chemical parameters, that were monitored during ripening, in order to estimate Serpa cheese ripening time using multiple linear regression (MLR). Mechanical spectra indicated a relatively strong structure, comparable to a gel, with a low dependence on frequency at the beginning of ripening and a weak structure, comparable to a concentrated suspension, with a crossing point (G″=G′) at the left of the graphic and with both moduli highly dependent on frequency, at the end of ripening. Good correlations (P<0·05) were obtained between structural (hardness and storage modulus) and proteolysis indicators. Using a combination of chemical, colour and rheological parameters we were able to obtain a multiple linear regression (MLR) which allows the estimation of Serpa cheese ripening time with an estimation error of 1·7 d (adjusted R2=0·98, P<0·0001).

Interest in selective dry cow therapy (SDCT) has been increasing owing to concerns over development of antimicrobial resistance. Implementation of SDCT, however, requires a quick and cost-effective on-farm method for identifying cows for treatment and cows that can be left without treatment. The objective of the present study was to evaluate the use of clinical mastitis (CM) history and somatic cell counts (SCC) from monthly Dairy Herd Improvement (DHI) records in identification of infected and uninfected cows at dry-off. A total of 647 Holstein cows were classified as uninfected or infected at dry-off based on CM history and varying number of monthly SCC records (with three different SCC cut-offs). Cows were considered uninfected based on the following criteria: (1) SCC <100 000 cells/ml and no CM during the lactation; (2) SCC <200 000 cells/ml and no CM during the lactation; (3) as criterion two, but additionally a cow was also considered uninfected if it experienced a case of CM during the first 3 months of the lactation and the SCC was <100 000 cells/ml for the rest of the lactation; (4) SCC <300 000 cells/ml and no CM during the lactation; otherwise they were considered infected. Infected and uninfected cows at dry-off were most efficiently identified using three months' SCC records with a threshold of 200 000 cells/ml for cows without CM during the lactation and a threshold of 100 000 cells/ml during the rest of lactation for cows with CM during the first 90 days in milk. Moreover, this criterion also most efficiently identified cows infected with major pathogens only at dry-off. The success of the criteria used for identifying infected and uninfected cows will, however, depend on herd characteristics, such as prevalence of infection and type of pathogens present in the herd.

N-Chlorotaurine (NCT) is a promising endogenous agent for topical treatment of infections. We tested the tolerability and pharmakokinetics of NCT in the bovine mammary glands in a phase 1 study. Three concentrations of NCT in water (0·1%, 1·0%, 2·0%) were administered intramammarily in each of two cows. Into two quarters of the udder 100 ml NCT was injected into each twice daily for 5 d, while 0·9% NaCl was injected into the other two quarters in a randomized and blinded manner. Samples of milk were taken to determine the number of leucocytes and the activity of NCT, and samples of urine and blood to determine the taurine and chloride concentration. Chloride concentrations in serum samples were determined by an ISE-Unit of a Modular-System of the Roche Diagnostics company. The udder was monitored clinically for signs of inflammation. Oxidative activity could be detected in the milk after single irrigations for 15 min (0·1% NCT) and for maximally 5 h (1% and 2% NCT), respectively. On day 2, leucocytes increased to 4×106/ml in the NCT group, while they remained ⩽1×106/ml in the saline group. However, on days 3–5 they increased to (5–7)×106 in both the NCT and control group without any statistical difference. One day after the end of dosing the number decreased significantly and reached the baseline (<1×106/ml) on day 10. The decrease was similar in both groups. Except for sporadic slight induration of single quarters in both groups and slight reduction of milk performance no disorders occurred. Taurine levels in blood and urine did not change. Irrigation of the bovine mammary gland with both NCT and saline caused a transient increase of leucocytes in the milk, but no severe side effects. The absence of residues and decay products may be a great advantage of NCT over other antimicrobial agents.