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.

The transfer of ingested α-linolenic acid (ALA) and linoleic acid (LA) determines the nutritional quality of milk, but the factors determining this transfer are unclear. The present experiment investigated the influence of roughage to concentrate proportions and the effect of concentrate types on milk fat composition. Respectively, six lactating dairy cows were fed one of three isoenergetic (5·4±0·05 MJ net energy for lactation/kg dry matter; DM) and isonitrogenous (215±3·5 g crude protein/kg DM) diets, consisting of ryegrass hay only (33 g fatty acids/kg DM; ALA-rich, no concentrate), maize (straw, whole maize pellets and gluten; 36 g fatty acids/kg DM; LA-rich; 560 g concentrate/kg DM), or barley (straw and grain plus soybean meal; 19 g fatty acids/kg DM; LA-rich; 540 g concentrate/kg DM). The fatty acid composition of feeds and resulting milk fat were determined by gas chromatography. The ALA concentration in milk fat was highest (P<0·001) with the hay-diet, but the proportionate transfer of ALA from diet to milk was lower (P<0·001) than with the maize- or barley-diets. The LA concentration in milk fat was highest with the maize-diet (P<0·05, compared with hay) but relative transfer rate was lower (P=0·01). The transfer rates of ALA and LA were reciprocal to the intake of individual fatty acids which thus contributed more to milk fat composition than did roughage to concentrate proportions. The amount of trans-11 18:1 in milk fat was lowest with the barley-diet (P<0·001) and depended on the sum of ALA and LA consumed. The milk fat concentration of cis-9, trans-11 18:2 (rumenic acid) was more effectively promoted by increasing dietary LA (maize) than ALA (hay). Amounts of 18:0 secreted in milk were four (maize) to seven (hay) times higher than the amounts ingested. This was suggestive of a partial inhibition of biohydrogenation in the maize-diet, possibly caused by the high dietary LA level.

Milk is the normal secretion of the mammary gland, practically free of colostrum and obtained by the complete milking of one or more healthy animals. Mastitis is an inflammatory process of the mammary gland and it may cause alterations in the milk. The present work aimed to verify whether it is possible, by means of the counts of microorganism in the bulk raw milk in four selective culture media, to establish a correlation with the occurrence of mastitis and therefore, to monitor this disease in bovine dairy herds. The following selective culture media were used: KF Streptococcus Agar, Edwards Agar, Baird-Parker Agar, Blood Agar plus potassium tellurite. Spearman's correlation coefficient was calculated in order to compare the occurrence of mastitis (percentage) in each herd with respective selective culture media counts of microorganisms in bulk raw milk. Thirty-six possibilities were analysed (Tamis and CMT-positive rates were compared with the log-transformed count in four selective culture media) and there was a negative correlation between Tamis 3 and the Baird-Parker Agar plate count. The total results of microbiological tests showed that there were three correlations of the counts in selective culture media. Fifty-two possibilities were analysed and there was a negative correlation between no-bacterial-growth mastitis rates and log10 of KF Streptoccocus Agar plate count and there were two positive correlations between coagulase-positive staphylococci and log10 of Baird-Parker Agar plate count and Blood Agar plus potassium tellurite plate count.

Low somatic cell count (SCC) is a reliable indicator of high-quality milk free of pathogenic microorganisms. Thus, an important goal in dairy practice is to produce milk with low SCC. Selection for cows with low SCC can sometimes lead to extremely low SCC in single quarters. The cells in milk are, however, predominantly immune cells with important immune functions. To investigate the mammary immune competence of quarters with very low SCC, healthy udder quarters of cows with normal SCC of (40–100)×103 cells/ml and very low SCC of <20×103 cells/ml were challenged with lipopolysaccharide (LPS) from Escherichia coli. In the first experiment, SCC and cell viability after a challenge with 50 ng of LPS/quarter was investigated. In the second experiment, tumour necrosis factor α (TNF-α) concentration and lactate dehydrogenase (LDH) activity in milk, and mRNA expression of various innate immune factors in milk cells were measured after a challenge with 100 μg LPS/quarter. LPS challenge induced an increase of SCC. SCC levels reached were higher in quarters with normal SCC and maximum SCC was reached 1 h earlier than in very low SCC quarters. The increase of TNF-α concentrations in milk in response to LPS challenge was lower in quarters with very low SCC than in quarters with normal SCC. The viability of cells and the LDH activity in milk increased in response to LPS challenge, however, without a difference between the groups. The mRNA expression of IL-1β and IL-8 was increased in milk cells at 12 h after LPS challenge, whereas that of TNF-α and lactoferrin was not increased at the measured time points (12, 24 and 36 h after LPS challenge). No differences of mRNA expression of measured immune factors between normal and very low SCC samples were detected. The study showed that udder quarters with very low SCC responded with a less marked increase of SCC compared with quarters with normal SCC. This difference corresponded with simultaneously lower TNF-α concentrations in milk. However, the immune competence of the cells themselves based on mRNA expression of TNF-α, IL-8, IL-1β, and lactoferrin, did not differ. The results may indicate that very low SCC can impair the immune competence of udder quarters, because the immune response in udder quarters with lower SCC is less efficient as fewer cells contribute to the production of immunoregulators.

The objective of this clinical control trial was to examine the effectiveness of the classical homeopathic treatment strategy in cases of mild and moderate bovine clinical mastitis in comparison with antibiotic and placebo treatments. Owing to characteristics of the selected herds, only cases of clinical mastitis caused by environmental pathogens and clinical cases with negative bacteriological result in the pre-treatment milk sample were included in the trial. A total of 136 lactating dairy cows with 147 affected quarters from four herds in Germany were randomly allocated to three treatment groups. The cows were examined on days 0, 1, 2 and on days 7, 14, 28 and 56 post initial infection to assess clinical signs. Simultaneously, with the exception of days 1 and 2, quarter milk samples for laboratory examinations (bacteriology, somatic cell count) were collected to assess bacteriological and cytological cure rates. On days 28 and 56, treatment strategies did not differ significantly with respect to the clinical outcomes and the total cure rate in cases of bacteriological negative mastitis (n=56). In cases of pathogen-positive mastitis (n=91), the cure rate after 4 and 8 weeks was similar between the two treatment strategies, homeopathy and antibiotic treatment, but the difference between the homeopathic and the placebo treatment at day 56 was significant (P<0·05). The results indicate a therapeutic effect of homeopathic treatment in cases of mild and moderate clinical mastitis. However, independent of treatment strategy and bacteriological status, the total cure rate was on a low level, revealing limitations in the effectiveness of both antibiotic and homeopathic treatment strategies.

Impact of milk protein composition on casein (CN) retention in curd during the milk coagulation process was studied using a model cheese making system. Individual milk samples from 110 cows in mid lactation of the Swedish Red and Swedish Holstein breeds with known genotypes of β-casein, κ-casein and β-lactoglobulin were defatted, coagulated with chymosin, subjected to syneresis and subsequent pressing simulated by centrifugation. The results indicated that κ-casein concentration of milk plays an important role in the curd formation process and initial syneresis (whey after cutting), whereas an increased CN ratio was associated with less casein in whey after simulated pressing. Increased κ-casein concentration of milk also characterized the milk samples with no measurable loss of casein in whey, compared with milk samples with casein lost in whey, both after cutting and after simulated pressing. Concentrations of αs1-casein, β-casein, and total casein in milk were positively associated with fresh curd yield, which showed a strong correlation with amount of casein retained in curd. No effect of protein genotype on fresh curd yield or casein in whey was found. The β-lactoglobulin BB genotype was associated with increased casein retention in curd, most likely due to the association of this genotype with CN ratio.

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.

The impact of fresh legume types or silage on the composition of milk fatty acids and transcription of enzymes involved in the synthesis of milk fat in cows was studied. Three groups of cows grazed high proportions of white clover, red clover and lucerne, respectively. A fourth group of cows was fed maize/grass silage. The cows grazing high proportions of legumes produced significantly more 18:1 trans-11, 18:2 cis9-trans11, 18:2 trans10-cis12 and 18:3 fatty acids than cows fed silage. White clover and lucerne grazing resulted in significantly lower output of 18:1 trans9 in milk than red clover grazing and maize/grass silages. Transcription of stearoyl-CoA desaturase (SCD) in mammary tissue was significantly increased by grazing high proportions of legume whereas fatty acid synthase and acetyl-CoA carboxylase were not affected by type of feeding. Furthermore, average milk fat globule diameter was correlated to daily milk fat yield but was not affected by feeding. Although the fresh forage affected the transcription of SCD in mammary tissue, the largest effects were on the trans11-based fatty acids. It is concluded that type of forage, i.e. fresh or silage, had a greater impact on rumen fermentation pattern than on transcription of enzymes involved in the synthesis of milk fat.

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.

The present study was conducted to assess the interrelation between teat anatomy and machine milking in dairy buffaloes raised in Switzerland. A 3-min pre-stimulation induced milk ejection before cluster attachment in most cases and caused an optimal milk removal during machine milking. In an additional experiment, longitudinal cross-section ultrasound was obtained before and after a 3-min pre-stimulation. Teat wall thickness, teat diameter, cisternal diameter and teat canal length were evaluated. It was observed that 3-min pre-stimulation dramatically reduced teat canal length whereas all the other anatomical parameters remained unchanged. The vacuum needed to open the teat canal was also measured before and after a 3-min pre-stimulation by using a special teat cup with only the mouthpiece of the liner remaining on the top of the teat cup (no liner, no pulsation). Without pre-stimulation but after wetting the teat canal by stripping one squirt of milk out of the teat, no milk could be withdrawn with a vacuum up to 39 kPa. However, after pre-stimulation, milk flow occurred in all buffaloes at a vacuum between 16 and 38 kPa. In the last experiment, the teat tissue was examined in slaughtered buffaloes and compared with teat tissue of cows. No difference was noted in histological sections and teat canal length was similar in cows and buffaloes. Proximal to the teat canal, the teat did not pass into an open cistern but the lumen was collapsed. In conclusion, buffaloes need to be well pre-stimulated because the tissue above the teat canal provides additional teat closure before milk ejection. Therefore, milk can only be obtained after pre-stimulation.

Social rank in cervids and other mammals is not entirely predicted by body weight, but in most cases influences access to food directly. Milk provisioning depends on maternal weight and on daily food intake. Usually, body weight, body condition, age and social rank are inter-correlated making it very difficult to discern the relative importance of each variable to milk production. This study used path analysis to assess direct versus indirect effects of these variables on milk production of 62 Iberian red deer hinds (Cervus elaphus hispanicus). Once the known direct effects of body weight and body condition were set as fixed, hind age and social rank did not affect milk production directly. In contrast, they exerted an indirect influence through the correlation both with hind body weight and body condition. Body weight exerted an effect on milk production nearly twice as great as that of body condition. This study shows, for the first time in a wild mammal, the relative importance of social rank, body weight, body condition and age in affecting milk production ability.

The objective of this study was to investigate whether there is a difference in chemical composition and particularly in fatty acid (FA) profile, with emphasis on cis-9, trans-11 CLA, of milk obtained from conventional and organic dairy sheep and goats farms under the farming conditions practiced in Greece. Four dairy sheep and four dairy goat farms, representing common conventional production systems and another four dairy sheep and four dairy goat farms, organically certified, representing organic production and feeding systems were selected from all over Greece. One hundred and sixty two individual milk samples were collected from those farms in January–February 2009, about three months after parturition. The milk samples were analyzed for their main chemical constituents and their FA profile. The results showed that the production system affected milk chemical composition: in particular fat content was lower in the organic sheep and goats milk compared with the corresponding conventional. Milk from organic sheep had higher content in MUFA, PUFA, α-LNA, cis-9, trans-11 CLA, and ω-3 FA, whereas in milk from organic goats α-LNA and ω-3 FA content was higher than that in conventional one. These differences are, mainly, attributed to different feeding practices used by the two production systems. The results of this study show that the organic milk produced under the farming conditions practiced in Greece has higher nutritional value, due to its FA profile, compared with the respective conventional milk.

Lactoferrin (Lf) is a molecule naturally present in bovine milk that affects the availability and transport systems of iron. Lf also binds endotoxin (lipopolysaccharide, LPS) of Gram-negative bacteria and modulates the immunological response. In the present study, concentrations of bovine Lf (bLf) and citrate in milk were determined in early (EL) and late (LL) lactating dairy cows, using an experimentally induced endotoxin mastitis model and a crossover design. Nine clinically healthy Finnish Ayrshire cows were challenged twice with 100 μg endotoxin infused into one udder quarter. Milk samples were collected from the challenged and control quarters of each cow before and after endotoxin infusion during 3 d, and bLf and citrate concentrations were measured. In all cows, clinical signs of mastitis were seen at both times of challenge, but the response was more severe in EL than in LL. Concentration of bLf in the milk started to rise approximately 8 h after endotoxin infusion and was still higher than normal on the third day, especially in the late-lactating cows. In milk of the LL group, concentrations of bLf were significantly higher than in the EL group. In contrast, concentrations of citrate were higher in milk of the EL cows compared with the LL cows. Concentration of bLf and citrate varied substantially among cows. The molar ratio of citrate to bLf before and after challenge was significantly higher during the EL period. The results of this study partly explain why cows in early lactation are more susceptible to intramammary infections and why mastitis is more severe in them.

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.

Optimization of the cultivation conditions and medium components for folate production by the highest folate producing Streptococcus thermophilus strain RD102 was carried out using a 23 central composite design and surface modelling method of response surface methodology. As folate production was observed to be growth-associated, hence the desired responses selected for the optimization were both folate production and growth. The selected factors based on preliminary investigations were incubation period, concentrations of p-amino benzoic acid (PABA) the growth precursor, and lactose as carbon source. The optimum concentrations of PABA and lactose were found to be 300 μm and 3% respectively at 72 h incubation. The optimized conditions resulted in an increase in folate production by 26% compared with control conditions (0% PABA and lactose at 37°C). Using the method of experimental factorial design and response surface analysis, it was possible to determine optimal operating conditions to obtain a higher folate production by Strep. thermophilus. Therefore, this study constitutes a step in developing strategies to modulate the folate level to a higher level.

The adaptation of a colorimetric technique for the analysis of cholesterol in raw milk is presented. Performance quality was satisfying (mean intra-assay coefficient of variation (CV) 4·8, inter-assay CV 9·1%, linearity between 0 and 7 mm, recovery of spiked cholesterol into raw milk 98·1 and 106·3%). However, the milk fat extraction must be carried out within the 48 hours following milk sampling. When performing sampling, the significant variation of milk cholesterol composition during the milking process has to be taken into account.

This field study focused on the possible effects of increased milking frequency (milking four times a day in comparison with milking twice a day) on clinical and bacteriological cure rates of clinical, antibiotically treated mastitis cases. Parameters tested were clinical, microbiological and full (cytomicrobiological) cure as well as the development of milk yield after the clinical mastitis episode. Cows from a large dairy herd meeting the study criteria (n=93) were assigned to two treatment groups by a systematic randomization scheme (blocked by body temperature <=or >39·5°C). Both groups were randomly divided by experimental treatments: a) antibiotic intramammary treatment and milking 2-times a day; b) antibiotic intramammary treatment and milking 4-times a day. Treatments were initiated before the culture results were known. Cows were surveyed and evaluated on days 1–6, 24 and 31. No significant differences between treatment and control groups regarding clinical cure, microbiological cure, full cure and milk production could be established. Applying a 4-times a day milking regime did not lead to any significant effect, either positive or negative. Therefore, the results suggest that milking 4-times a day as a supporting therapy for mild, moderate and severe antimicrobially treated mastitis cases cannot be recommended.

Concentrations of IgA and IgM in milk from normal lactating cows were measured to determine whether they were affected by lactation number, lactation stage, daily milk production, milk composition and somatic cell score (SCS). A total of 284 Chinese Holstein cows showing no signs of mastitis were selected from more than 1600 cows from one farm. Concentrations of IgA and IgM in normal milk were determined by ELISA. Results revealed that average milk IgA and IgM concentrations were 235±11 and 32±3 μg/ml, respectively. IgA and IgM concentrations in milk were found to have an association with lactation number, stage of lactation, daily milk production and SCS. Both SCS and daily milk production were found to be strong significant (P<0·01) direct factors correlating to IgA concentration. Stage of lactation and SCS were found to be strong significant (P<0·01) direct factors correlating to IgM concentration.

Lactoperoxidase (LP) was isolated from whey protein by cation-exchange using Carboxymethyl resin (CM-25C) and Sulphopropyl Toyopearl resin (SP-650C). Both batch and column procedures were employed and the adsorption capacities and extraction efficiencies were compared. The resin bed volume to whey volume ratios were 0·96:1·0 for CM-25C and ⩽0·64:1·0 for SP-650 indicating higher adsorption capacity of SP-650 compared with CM-25C. The effluent LP activity depended on both the enzyme activity in the whey and the amount of whey loaded on the column within the saturation limits of the resin. The percentage recovery was high below the saturation point and fell off rapidly with over-saturation. While effective recovery was achieved with column extraction procedures, the recovery was poor in batch procedures. The whey-resin contact time had little impact on the enzyme adsorption. SDS PAGE and HPLC analyses were also carried out, the purity was examined and the proteins characterised in terms of molecular weights. Reversed phase HPLC provided clear distinction of the LP and lactoferrin (LF) peaks. The enzyme purity was higher in column effluents compared with batch effluents, judged on the basis of the clarity of the gel bands and the resolved peaks in HPLC chromatograms.

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.