The aim of the study was to establish in vitro a bovine mammary epithelial cell (MEC) clone, able to respond to mitogenic growth factors and to lactogenic hormones. Mammary tissue from a 200-d pregnant Holstein cow was used as a source of MEC, from which a clone was established through a process of limiting dilution. When plated on plastic, the cells assumed a monolayer, cobblestone, epithelial-like morphology, with close contact between cells. Inclusion of IGF-1 and EGF in the media significantly increased the number of cells 5 d after plating. All cells stained strongly for cytokeratin and moderately for vimentin at young and old passage stages, indicating the epithelial nature of this cell clone. When the cells were plated at a high density on a thin layer of a commercial extracellular matrix preparation (Matrigel), lobular, alveoli-like structures developed within approximately 5 d, with a clearly visible lumen. When cells were plated onto Matrigel in differentiation media (containing lactogenic hormones), detectable quantities of α-casein were present in the media and particularly on the lumen side of the structures. Omission of one of the lactogenic hormones (insulin, prolactin or hydrocortisone) reduced α-casein release to the limit of detection of the assay used. Lactoferrin was also produced when the cells were plated on Matrigel, again principally on the lumen side of the lobules, though this was independent of the lactogenic hormones. By passage 40, the cells had senesced, and it was not possible to induce α-casein or lactoferrin production. This study notes the establishment of a functional bovine mammary epithelial cell clone, which is responsive to mitogenic and lactogenic hormones and an extracellular matrix.

The contribution of the alternative pathway of complement activation to the capacity of normal milk to deposit C3 fragments on bacteria was tested by attempting to block C3 deposition with antibodies to the alternative pathway component factor B (fB). Factor B was purified and antibodies of the IgY class, which does not activate mammalian complement, were obtained from the egg yolk of immunized laying hens. These antibodies specifically inhibited the deposition of C3. This inhibition and the absence of deposition of C4 demonstrated that C3 deposition in normal milk resulted from the activation of the alternative pathway. Antibodies raised in rabbit were used to develop an ELISA for measuring fB concentrations in milk. The mean concentration of fB was 2·06 μg/ml (±0·18, SEM), 0·57% of the mean value found in serum (360 μg/ml). This proportion was comparable to that of serum albumin (0·63% of serum value) but less than the proportion of C3 in milk (2·71%). Nevertheless, fB was apparently not a limiting factor for the functioning of the alternative pathway, since addition of purified fB to normal milk did not improve C3 deposition. In serum, mild heat-treatment (56 °C for 3 min or 50 °C for 45 min) blocked the alternative pathway and destroyed fB, as shown by loss of antigenicity in ELISA. In milk, mild heat-treatment did not abrogate C3 deposition, and fB was protected, retaining its functionality and antigenicity. Heating at 56 °C for at least 45 min was necessary to completely inhibit C3 deposition in normal milk.

Transient vacuum drops in the milkline during one ordinary milking were recorded as a measure of vacuum stability in milking installations on 184 Norwegian dairy farms in the summer of 1997. An association between the frequency of vacuum drops and technical data including milkline diameter, length and slope, number of milking units and effective reserve was demonstrated. The direct connection between the transient vacuum drop and its cause could be established for most drops during milking observations. A high frequency of transient vacuum drops in the milkline was associated with a high level of mastitis and a high new infection rate as inferred from changes in somatic cell counts for individual cows. The frequency of vacuum drops during one milking is only a rough indication of the long-term vacuum stability in a specific installation, and must be interpreted with care. The dimensions and slope of the milkline and the effective reserve probably give as good information about the installation's ability to maintain a stable vacuum.

Data from 33 feeding trials, conducted on lactating riverine buffaloes from different institutes across India, were subjected to multiple regression analysis to derive nutritional requirements for dry matter (DM), total digestible nutrients (TDN), crude protein (CP) and digestible crude protein (DCP) for maintenance, milk production and body weight gain. Maintenance requirements for DM, TDN, CP and DCP were 59.9, 35.3, 5.43 and 3.14 g/kgW0.75, respectively; corresponding requirements for producing 1 kg 6% FCM were 688, 406, 90.3 and 55.2 g and for 1 g gain in body weight were 3.37, 1.97, 0.327 and 0.23 g. Regression equations had high R2 values (0.61, 0.66, 0.84 and 0.68 for prediction of DM, TDN, CP and DCP, respectively) and the equations (F-value) as well as coefficients were highly significant (P<0.001). Regressed values were used to derive feeding standards. Derived values matched well with the actual intake versus performance of animals under diverse feeding conditions. New standards predicted requirements and intake of nutrients for different production levels better than existing feeding standards. Because they are based on a more thorough analysis of data, the new feeding standards will be appropriate for use widely in India.

Secretory IgA found in external secretions are constituted by polymeric IgA (pIgA) bound to the extra-cellular part of the polymeric immunoglobulin receptor (pIgR). The receptor mediates transcytosis of pIgA across epithelial cells. The aim of the present study was to analyse the evolution of pIgR expression in the sheep mammary gland during the development of the mammary gland and to analyse its hormonal regulation. Gene expression of the pIgR was analysed in sheep mammary gland during pregnancy and lactation. By Northern Blot analysis, we observed that low levels of pIgR mRNA are expressed until day 70 of pregnancy. Accumulation of pIgR mRNA started during the third part of pregnancy and intensified 3 d after parturition to reach highest levels during established lactation (day 70). In situ hybridization analysis was used to confirm the increase in pIgR gene expression per mammary epithelial cell. In order to examine the hormonal regulation of the pIgR expression, virgin ewes were hormonally treated. Treatment with oestradiol and progesterone increased pIgR mRNA levels slightly. Subsequent addition of glucocorticoids induced a significant accumulation of pIgR mRNA in the mammary gland of the treated animals. Immunohistochemical analysis was performed to verify that the increase of pIgR mRNA level was associated with enhancement of the pIgR protein in mammary cells. No increase of pIgR mRNA levels were observed if PRL secretion was blocked by bromocryptine injections throughout the hormonal procedure. In conclusion, the present experiments suggest that the enhancement of pIgR levels during lactation result from combined effects of both prolactin and glucocorticoids.

Thirty-two lactating Karagouniko ewes were allocated at random to four groups for 6 weeks, to examine the effect of four diets: C (control treatment, ration without algae); LA (ration with low level of algae); MA (ration with medium level of algae) and HA (ration with high level of algae); containing 0, 23·5, 47 and 94 g algae, respectively, on the enrichment of milk and dairy products. Addition of algae reduced (P<0·001) DM intake for treatments MA and HA. Milk yield did not differ between treatments but milk composition was significantly affected by dietary inclusion of algae. Milk fat content was significantly increased (P<0·001) for treatment HA whereas milk protein content was significantly increased (P<0·001) for all treatments containing algae. Milk from treatments LA, MA and HA was significantly enriched in the following PUFA: C20[ratio ]5 (n-3) (0·4–2·1%), C22[ratio ]5 (n-6) (0·8–4·1%), C22[ratio ]6 (n-3) (4·3–12·4%) (P<0·001) and C22[ratio ]5 (n-3) (2·1–3·1%) (P<0·05), which were not detected in control milk. Feta cheese and yogurts produced from the enriched milk had identical composition with the milk, and would be characterized as healthy foods. The ratio of n-6 to n-3 fatty acids was 2·5–4·5.

Forty lactating Comisana ewes were either exposed to or protected from solar radiation and fed either in the morning or afternoon during summer in a Mediterranean climate. Individual milk samples were taken on days 7, 21 and 42 of the study period to determine fatty acid composition by gas chromatography. Exposure to solar radiation resulted in higher proportions of short-chain and saturated fatty acids in milk, primarily because of increased contents of caproic, capric, lauric, myristic and stearic acids (by 3–18%), and decreased contents of oleic, linoleic and linolenic acids (by 2–9%). As a consequence, the long to short chain and the unsaturated to saturated fatty acid ratios were significantly higher by 4 and 13% respectively in the milk of the protected ewes compared with that of the exposed animals. Provision of shade also led to an increase in the 18[ratio ]0+18[ratio ]1 to 16[ratio ]0 ratio, and to a decrease in the 12[ratio ]0+14[ratio ]0+16[ratio ]0 fatty acid group, which are regarded as reliable indexes of the nutritional property of dietary fat in reducing cholesterol levels in human plasma. Feeding time had little impact on milk fat. Our findings suggest that high ambient temperature may markedly modify the lipid composition of ewe milk and that provision of shade, but not feeding management, can improve the milk fatty acid profile in dairy sheep raised in hot climates.

Thirty Holstein cows in mid-lactation (158±20 DIM) were given a total mixed ration based on grass silage, maize silage and rolled barley. After a preliminary period of 1 week, this diet was supplemented with nothing (control), unprotected fish oil (3.7% of dry matter, DM), or two levels of glutaraldehyde-protected microcapsules of fish oil (1.5% and 3.0% of DM, respectively). Unprotected and protected supplements contained, respectively, 74% and 58% of DM as lipids. Cows given the unprotected supplement reduced their feed intake by >25%. Consequently, these cows lost body weight and produced less milk. DM intake, body weight, and milk yield were unaffected by protected fish oil. Fish oil reduced both milk fat and protein percentages, and decreased the proportion of short-chain fatty acids, stearic, and oleic acids in milk fat. Milk trans C18[ratio ]1 fatty acids increased in cows given both unprotected and protected fish oil. Milk fat content of very-long-chain n3 polyunsaturated fatty acids, including C20[ratio ]5 and C22[ratio ]6, increased with fish oil in the diet. Accordingly, the peroxide index increased and a taste panel was able to detect unusual taste in milk from cows consuming the higher level of protected fish oil and disliked the milk from cows given unprotected fish oil. In conclusion, when lactating cows consumed fish oil, milk concentration of long-chain n3 fatty acids increased and mammary de novo synthesis of fatty acids decreased, but milk yield and milk protein content were reduced, and the milk was more susceptible to oxidation and its taste was adversely affected.

Changes in urokinase-plasminogen activator (u-PA) and u-PA receptor (u-PAR) expression at the protein and mRNA level in resting neutrophils and in neutrophils activated by phorbol myristate acetate (PMA) were examined. Low amounts of u-PA were found intracellularly or membrane-bound in resting neutrophils. However, incubation of resting neutrophils with purified exogenous u-PA (10 IU/ml) revealed extensive binding of u-PA to cell membranes. Excess amino-terminal fragment of the u-PA molecule, a proteolytically inactive fragment of u-PA (amino acids 1–135) blocked binding of exogenous u-PA to the cell membrane. These results, collectively, indicate that the binding of u-PA is specific and that resting neutrophils have unoccupied u-PA receptors on their cell membrane. Addition of PMA led to an increase (P<0.01) in total cell-associated, membrane-bound u-PA activity and u-PA mRNA expression by bovine neutrophils. In contrast, PMA increased u-PAR mRNA levels but this was accompanied by a decrease (2.5-fold; P<0.01) in free, unoccupied u-PA binding sites. No significant effects on total cell-associated or membrane-bound u-PA were found when neutrophils were treated with 4-phorbol 12,13 didecanoate, a phorbol ester that does not activate protein kinase C (PKC). Furthermore, addition of 1-(5-isoquinolinesylphonyl)-2-methylpiperazine dihydrochloride (H-7), a potent PKC inhibitor, blocked the effect of PMA on total cell-associated u-PA activity. Thus, PKC plays a role in the modulation of u-PA and u-PAR by PMA in bovine neutrophils.

A nephelometric immunoassay, with a detection range of 0·3 to 5 g IgG1/l, was developed for the determination of immunoglobulin in bovine milk. The assay exhibited no significant cross-reactivity with αS1-casein, αS2-casein, β-casein, κ-casein or β-lactoglobulin and 39% cross-reactivity with IgG2. The nephelometric assay was compared with ELISA and RID (24 h and 48 h incubations) assays using 105 duplicate milk samples covering IgG1 values ranging from 0·45 to 1·8 g1. The results obtained from all assays showed good agreement with the exception of those obtained by the RID assay (24 h incubation) which gave lower results in samples containing more than 1·2 g IgG1/l. It was concluded that the nephelometric assay is a reliable, rapid and convenient method suitable for the quantification of IgG1 in milk. The assay can be configured for routine high-throughput milk quality assurance for IgG1 in dairy laboratories.

The objective was to determine the effect of once-daily milking (ODM) and omitting one evening milking each week (13TWM), in late lactation on milk production, composition and processability. Seventy-two cows were assigned to three treatments (ODM, 13TWM and twice-daily milking [TDM]) from 4 October to 12 December. Cows were on average 218 d into lactation at the start of the trial, and all cows were managed similarly throughout the trial. Milk yields and gross milk composition of cows on all treatments were measured, and milk samples for detailed compositional and processability analysis were collected from TDM and ODM treatments at two consecutive milkings and at one milking each week, respectively. Milk yield was significantly reduced (P<0.001) and milk fat and protein concentrations were increased (P<0·01) with ODM compared with TDM. Milk yield and fat and protein concentrations of milk from TDM and 13TWM herds were similar. Casein concentrations in ODM and TDM milks were similar, but ODM milk had a higher (P<0·05) whey protein content. Somatic cell count of ODM and TDM milks was similar. Rennet coagulation time (RCT) and curd firmness (A60) of milk were not affected by milking frequency. However, rate of curd aggregation (K20) of ODM milk was reduced (P<0·05) compared with that of TDM milk. Plasmin activity in ODM milk was numerically higher than in TDM milk, but the effect was not significant. ODM milk had higher NAGase activity than TDM milk (P<0·01). In conclusion, once daily milking reduced milk yield by 29% and did not adversely affect the processability of milk. Moreover, one evening milking per week could be eliminated without adverse effects on milk yield or composition.

Sixteen Holstein cows in mid-lactation were used to determine whether alterations of mammary fatty acid metabolism are responsible for the milk fat depression associated with consumption of fish oil. Cows were given a total mixed ration with no added fish oil (control), unprotected fish oil (3.7% of dry matter), or glutaraldehyde-protected microcapsules of fish oil (1.5% or 3.0% of dry matter) for 4 weeks. Milk samples were taken once a week and a mammary biopsy was taken from a rear quarter at the end of the treatment period. Milk fat content was lower in cows given unprotected fish oil (26.0 g/kg), 1.5% protected fish oil (24.6 g/kg) and 3% protected fish oil (20.4 g/kg) than in cows fed the control diet (36.0 g/kg). This was mainly due to a decrease in the synthesis of short-chain fatty acids. Consumption of protected fish oil decreased the abundance of lipogenic enzymes mRNA in the mammary gland. Acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase mRNAs for cows given 3% protected fish oil averaged only 30%, 25% and 25% of control values, respectively. Dietary addition of unprotected fish oil slightly decreased mRNA abundance of these enzymes but markedly reduced the amount of lipoprotein lipase mRNA. Milk fat content was significantly correlated with gene expression of acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase but not lipoprotein lipase. These results suggest that fish oil reduces milk fat percentage by inhibiting gene expression of mammary lipogenic enzymes.

The amount of protein synthesis translational elongation factor 2 (eEF-2) was estimated employing diphtheria toxin-dependent ADP-ribosylation in samples prepared from small amounts of tissue from mammary gland, skeletal muscle and liver from lactating dairy cows. A very high level of ADP-ribosylatable eEF-2 was found in mammary gland, amounting to 20-times the level found in liver and 50-times the level found in skeletal muscle. This obviously reflects the high protein synthesis activity in mammary tissue. To our knowledge, similar high activities have previously been reported only for cancer cells. A close linear relationship was found between the amount of diphtheria-toxin catalysed ADP-ribosylated eEF-2 and protein and casein output in milk from cows in late lactation. This strongly suggests that eEF-2 may be a limiting factor in milk protein synthesis.

Milk from dairy cows never known to have had an intramammary infection with Streptococcus uberis can inhibit growth of Str. uberis for up to 7 h. This inhibition is abolished if milk is heated to 80 °C. Inhibition appears not to be related to immune defences as it occurs in skimmed milk (cell free), is unrelated to the concentration of immunoglobulin and survives heating to 56 °C. The effect is partly overcome by addition of selected amino acids and vitamins. It is suggested that the inhibition is caused by a restriction in the supply of essential nutrients part of which may require the conversion of plasminogen to plasmin.

The objective of this study was to determine the effects of stage of lactation (SOL) and time of year on plasmin-derived proteolytic activity in the milk of pasture-fed dairy cows in New Zealand. Four herds of 20 Friesian cows were used, one herd calving in each of January, April, July and October. Cows grazed ryegrass/white clover pasture only, except during June (winter) when all cows received supplementary pasture silage. Milk samples were collected on four occasions during the year (spring, summer, autumn and winter) from each cow in milk, to give a total of three samples per cow (early, mid and late lactation; c. 30, 120 and 220 days after calving, respectively). Milk samples were analysed for plasmin-derived proteolytic activity. There was no effect of either SOL or time of year on plasmin activity and therefore yields of plasmin followed patterns in milk yield (highest in early lactation and in summer). There were effects of both SOL and time of year on plasminogen-derived and total plasmin plus plasminogen-derived activity, both of which were highest in late lactation and in spring. Changes in plasminogen-derived activity and total plasmin plus plasminogen-derived activity due to SOL were not only due to the decrease in milk yield associated with advancing lactation, because enzyme yields were also increased with advancing lactation. Similarly, effects of time of year on plasminogen-derived activity and total plasmin plus plasminogen-derived activity could not be attributed solely to concomitant changes in milk yield, and may be influenced by the variation in the quality and quantity of feed during the year inherent in a pasture-based dairy system. Effects of SOL on proteolytic activity were greater than, and independent of, effects of time of year.

The decline in milk yield after peak lactation in dairy animals has long been a biological conundrum for the mammary biologist, as well as a cause of considerable lost income for the dairy farmer. Recent advances in understanding the control of the mammary cell population now offer new insights on the former, and a potential means of alleviating the latter. The weight of evidence now indicates that a change in mammary cell number, the result of an imbalance between cell proliferation and cell removal, is a principal cause of declining production. Further, it suggests that the persistency of lactation, the rate of decline in milk yield with stage of lactation, is strongly influenced by the rate of cell death by apoptosis in the lactating gland. Mammary apoptosis was first demonstrated during tissue involution after lactation, but has now been detected during lactation, in mammary tissue of lactating mice, goats and cattle. Those factors that determine the rate of cell death by apoptosis are as yet poorly characterized, but include the frequency of milking in lactating goats. Initial evidence suggests that nutrition also is likely to influence cell survival after peak lactation, an important factor being the degree of oxidative stress imposed by feed and the tissue's ability to deal with, and prevent damage by, reactive oxygen species. Comparison of cows in calf or not pregnant during declining lactation also indicates a likely influence of reproductive hormones, with oestradiol and progesterone acting to preserve mammary ductal and alveolar integrity during the dry period, while allowing a degree of apoptosis and cell replacement. In each case, the molecular mechanisms controlling mammary cell survival (or otherwise) are as yet poorly defined. On the other hand, more persistent lactations are likely to benefit animal welfare through fewer calvings and by placing less emphasis on maximal production at peak lactation, and modelling of persistent lactation with longer calving intervals indicates their likely economic benefits. In these circumstances, there is considerable incentive to elucidate the determinants of mammary apoptosis, and the factors controlling the dynamic balance between cell proliferation and cell death in the lactating mammary gland.

To establish whether fishy off-flavour observed in cows' milk in Sweden is due to abnormal concentrations of trimethylamine (TMA) in milk, 15 milk samples from 13 cows of the Swedish Red and White breed (SRB) were analysed for this compound using dynamic headspace (DHS) gas chromatography (GC). A mass selective detector (MSD) was used for the qualitative and a flame ionization detector (FID) for the quantitative analyses. Further confirmation was obtained using a solid phase microextraction (SPME) followed by GC-olfactometry (GC-O) and GC-NPD (nitrogen and phosphorus detector). Samples were also evaluated by a two-person organoleptic panel for the presence of fishy off-flavour. Results showed that the seven milk samples with a fish-like taint contained >1 mg TMA/kg milk, whereas, with a single exception, the seven samples that were judged to be normal did not contain this compound. Furthermore, there were indications of a dose-dependent relationship between TMA concentration and fishy off-flavour score.

Sixty samples of raw goat milk intended for Caprino cheese-making were collected from ten farms in the Bergamo area over a 6-month period. Analyses of main microbial groups, somatic cell count (SCC) and pH were performed to determine the effect of origin (farm) and lactation period (April – September) on microbial composition and the incidence of pathogens in milk. Overall mean values were: standard plate count (SPC), 5.0×104 cfu/ml; yeasts, 2.5×102 cfu/ml; coliforms, 9.1×102 cfu/ml; Escherichia coli, 2.9 cells/ml; enterococci, 1.1×102 cfu/ml; lactococci, 3.4×103 cfu/ml; lactobacilli, 3.0×103 cfu/ml; halotolerant bacteria, 8.2×103 cfu/ml; spores of mesophilic aerobic bacteria, 11 cfu/ml; SSC, 9.9×105 cells/ml; pH, 6.63. Moulds and spores of sulphite-reducing clostridia were found intermittently. Neither Salmonella spp. nor Listeria monocytogenes was detected, while Esch. coli O157[ratio ]H7 was isolated from one milk sample (an incidence of 1.7%). Staphylococcus aureus was discovered at a level >102 cfu/ml in 26 samples (43%) with an overall mean of 1.2×103 cfu/ml, whereas coagulase-negative staphylococci were found in 54 samples (90%) with an overall mean of 1.3×103 cfu/ml. Of Staph. aureus strains, 23% proved to be enterotoxinogenic with a prevalence of enterotoxin C producers. Staph. caprae was the coagulase-negative species most frequently isolated; none of the coagulase-negative staphylococci strains synthesized any of the enterotoxins tested for.

Sample source was the major factor affecting the microbial composition of goat milk: significant differences (P<0.01) were observed among samples from different farms for SPC, coliforms, lactococci, lactobacilli and halotolerant bacteria. Period of lactation had a significant effect (P<0.025) on SCC and pH. SPC correlated well with coliforms, lactococci and lactobacilli; SSC did not reveal positive interactions with any microbial groups or pH.

A total of 7492 test-day observations for mean contents of fat, protein, casein, serum protein and lactose and individual laboratory cheese yield (ILCY) were obtained, at approximately monthly intervals, from 1119 ewes belonging to eight Churra dairy flocks. The effect of various factors on these variables was examined and phenotypic correlations among all traits were estimated. Least squares analyses showed significant effects of flock test-date, stage of lactation, age of ewe, and number of lambs weaned on almost all variables. Protein content and composition were not affected by the number of lambs weaned. ILCY had an unadjusted mean (26·55 kg cheese/100 l milk) close to those reported for real cheese yield in dairy ewes and was affected similarly to the main milk components. Fat, protein, casein, and serum protein contents, and ILCY, showed a generally increasing trend as lactation progressed. These components reached a minimum at 1 month into lactation, when milk yield was highest, and increased for the remainder of the lactation. ILCY depended mainly on fat, protein and casein contents. Protein and casein contents were closely related and equally correlated with ILCY. An increase in somatic cell count (SCC) was associated with decreased milk yield and decreased lactose content.

In the context of identifying intrinsic time temperature integrators (TTIs) for evaluating heat processing of milk, the extent to which milk fat content has an effect on alkaline phosphatase (ALP) and lactoperoxidase (Lpo) inactivation and on β-lactoglobulin (β-lg) denaturation kinetics was studied. Inactivation and denaturation kinetics were analysed in whole, semi-skimmed and skimmed milk. In previous experiments (isothermal and non-isothermal heating conditions), heat inactivation of ALP and Lpo and heat denaturation of β-lg were found to follow first order kinetics. This allowed experimental design to be simplified. Data analysis was performed by non-linear regression and results were evaluated by construction of joint confidence regions. The possible effect of milk fat was illustrated by temperature time tolerance (TTT-) diagrams. Although initial ALP activity was lower in skimmed milk compared with semi-skimmed or whole milk, kinetics were comparable and fat content did not seem to substantially affect the ALP test result for pasteurized milk. Unlike ALP, Lpo inactivation and β-lg denaturation kinetics differed significantly in milk with different fat content. Differences between Lpo inactivation kinetics were relatively small and acceptable in the context of quantifying the process impact. Denaturation of β-lg, on the other hand, seemed to be enhanced at higher milk fat content (>72 °C).