Records of claw trimmings were analysed in seven organic and six conventional Danish herds (a total of 974 cows). The housing systems represented were tie stall systems, loose housing system with slatted floor (one organic herd), and deep litter systems (deep straw bedding). Occurrence of sole disorders was analysed separately for cows in first lactation and for cows in later lactations. Three different responses (acute haemorrhage, sole ulcer in one leg and sole ulcer in two or more legs) were analysed using three binomial logistic regression analyses for each group. Herd analysed as a fixed effect was a strong risk factor for all kinds of sole ulcer. Lactation stage was a risk factor for acute haemorrhage in both groups of cows, and for sole ulcer in first parity cows. In general, there was a strong positive association between the period 61–120 d post partum and the presence of sole disorders. Breed was associated with acute haemorrhage in cows in second and later parities, and sole ulcer in one leg only in first parity cows in an interaction with lactation stage in both conditions. Danish Friesian cows were strongly associated with sole disorder, although the combination of lactation stage from 61 to 120 d post partum in cows of other dual purpose breeds was positively associated with the presence of sole ulcer in one leg only in first parity cows. The time of year for claw trimming was a risk factor for acute haemorrhage in first parity cows, with the period from December to January most strongly associated with acute haemorrhage. Previous disease treatment was a risk factor for sole ulcer in two or more legs in second and later parities. Udder related disorders and disorders other than reproductive problems were positively associated with the occurrence of sole ulcer. Body weight at calving was associated with acute haemorrhage in cows in second and subsequent parities. Body weight lower than the mean herd level by >50 kg was negatively associated with acute haemorrhage.

Cows with subclinical intramammary infections were identified by milk bacteriology. The mastitis pathogens included Staphylococcus aureus (n=9), Streptococcus uberis (n=10) and coagulase-negative staphylococci (n=10). Samples of first fore milk, main flow milk and strippings milk fractions were collected from each quarter and laboratory measurements were made of electrical conductivity, milk fat concentration and somatic cell count. Conductivity measurements were corrected for milk fat concentration and within-cow inter-quarter conductivity ratios calculated. Repeatability estimates of all measurements between days were calculated. In the case of infected quarters, all conductivity values decreased markedly (P<0·05) from first fore milk to main flow milk fractions. Conductivity differences between quarters of infected cows were substantially lower during the main milk flow phase. For quarters infected with Staph. aureus an increase in conductivity was observed (P<0·05) from main flow to strippings fractions. For uninfected quarters, conductivity declined as milk fat concentration increased with successive milk fractions. Variation, both within and between milk fractions, was greater for somatic cell count than for conductivity. Differences in conductivity between milk fractions from individual infected quarters were not accounted for by changes in fat concentration and may result from the mixing of milk from infected and uninfected regions of the gland. Localized infection may produce a decrease in conductivity between fore milk and mid-flow fractions while differential drainage from an infection site in the secretory tissue may additionally produce an increase in conductivity from mid-flow to strippings fractions. Such changes may thus provide information on the location and magnitude of an infection. The results clearly demonstrate the importance of the milk fraction when using conductivity as a diagnostic of intramammary infection, the highest diagnostic sensitivity being achieved by using first fore milk samples.

The transport of L-glutamine by the lactating rat mammary gland has been investigated using rat mammary tissue explants and the in situ perfused rat mammary gland. L-glutamine uptake by both explants and the perfused mammary gland was via both Na+-dependent and Na+-independent pathways. It appeared that these pathways are situated on the blood-facing aspect of the mammary gland. L-glutamine uptake by both mammary preparations was markedly inhibited by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid in the absence of external Na+. This is consistent with L-glutamine uptake via system L. The Na+-dependent component(s) of L-glutamine uptake remains to be precisely identified. However, system A can be ruled out on the basis that L-glutamine was not inhibited by (methylamino)isobutyric acid. Mammary tissue concentrates L-glutamine with respect to both milk and plasma: we suggest that the Na+-dependent component(s) of L-glutamine uptake is responsible for generating the intracellular to extracellular concentration gradient.

αs1-, αs2-, β- and κ-caseins from Somali camels (Camelus dromedarius) were purified by acid precipitation at pH 4·4, crudely separated into an α-CN and a β-CN fraction and further purified by reversed-phase HPLC. Fragments of tryptic digests were sequenced. Amino acid patterns obtained were used to screen a cDNA library constructed from mRNA from lactating udder tissue. Full length clones corresponding to the four caseins were sequenced. The numbers of residues in the sequences deduced were αs1-CN 207, αs2-CN 178, β-CN 217, κ-CN 162. Percentage similarity to bovine proteins was αs1-CN A 39, αs2-CN 56, β-CN 64, κ-CN 56. Acid-precipitated casein of pooled milk was separated by reversed-phase HPLC and monitored at 220 nm, and its composition, estimated from peak integration, was (g/kg total casein) αs1-CN 220, αs2-CN 95, β-CN 650, κ-CN 35. Degrees of phosphorylation and glycosylation were determined by laser ionization mass spectrometry and sequence pattern analysis. Molecular masses determined were (kDa) αs1-CN A, 24·755 and 24·668; αs1-CN B, 25·293; αs2-CN 21·993; β-CN, 24·900; κ-CN 22·294–22·987. The pH values of the most probable isoelectric points were: αs1-CN A 6P 4·41, αs1-CN B 6P 4·40, αs2-CN 9P 4·58, β-CN 4P 4·66, κ-CN 1P, with ten sialic acid residues bound, 4·10.

Bovine αs1-casein F (αs1-CN F) was found in a genetic resource of Deutsches Schwarzbuntes Niederungsrind cows at a frequency of 0·009. Biochemical characterization of this new variant was obtained by automated sequencing of reversed-phase HPLC-separated tryptic peptides of αs1-CN F and αs1-CN B. αs1-CN F was found to be a subtype of αs1-CN B with a single amino acid substitution (SerP/Leu) in position 66. DNA sequencing revealed a C/T transition in position 8418 of the gene. Sequence-specific primers were designed to perform an allele-specific polymerase chain reaction for detection of αs1CnF. Typing of artificial insemination sperm samples included in the genetic resource sperm pool identified one sire heterozygous for αs1CnF.

From hydrolysis experiments carried out on αs1-caseins A and F at pH 5·2 in the presence of 30 g NaCl/l, i.e. the conditions encountered in many young goats' cheeses, it was found that minima of 19 and 9 bonds were sensitive to chymosin in variants A and F respectively. Variant A was hydrolysed faster than variant F and the proteolytic pattern (reversed-phase HPLC and polyacrylamide agarose gel electrophoresis) differed between the variants. Hydrolysates from both variants had a number of cleavage sites in common (Leu20–Leu21, Phe23–Ala24 and Phe32–Arg33 in both variants, Leu101–Lys102 and Leu64–Lys65, Leu120–His121 and Leu83–His84, Leu142–Ala143 and Leu105–Ala106, Leu149–Phe150 and Leu112–Phe113, Leu156–Asp157 and Leu119–Asp120, Trp164–Tyr165 and Trp127–Tyr128 in variants A and F respectively), while other bonds were split only in variant A (Leu16–Asn17, Glu18–Asn19, Phe28–Pro29, Ile44–Gly45, Tyr80–Ile81, Gln82–Lys83, Tyr91–Leu92, Tyr94–Leu95, Leu109–Glu110 and Phe179–Ser180). Major cleavage sites appeared to be at Phe23–Val24, Leu142–Ala143 and Trp164–Tyr165 for variant A, and Phe23–Val24 and Leu64–Lys65 for variant F. Cleavage site Phe23–Val24 could be the origin of the first breakdown product from goat αs1-caseins A and F visible in polyacrylamide agarose gel electrophoresis.

Four minor protein components were detected in whey from Romagnola cows' milk by polyacrylamide gel isoelectric focusing and two dimensional gel electrophoresis. Individual protein spots were transferred by electroblotting on to a polyvinylidene difluoride membrane and isolated by cutting out the relevant area. After in situ trypsinolysis, a portion of the digest was analysed directly by matrix-assisted laser desorption ionization–time of flight mass spectrometry. The mass profile allowed us to establish a correlation between β-lactoglobulins A and B and the four minor whey protein components. They were identified as C-terminally truncated β-lactoglobulin A and B variants with missing N-terminal peptides, beyond residues in the range 100–123 and 136–147 respectively. Two of the minor components were related to β-lactoglobulin A and two to β-lactoglobulin B.

An analytical procedure, based upon HPLC, has been used to determine the degree of isomerization of retinol (vitamin A) in various dairy products. In raw milks, which are not subjected to thermal processing, there was no conversion of the predominant all-trans-isomers to cis-isomers in samples from various species. Pasteurized milks with mild heat treatment (high quality milk) had an average 13-cis[ratio ]all-trans ratio of 2·6%, while pasteurized milk treated for 15 s at temperatures ranging from 72 to 76°C had an average ratio of 6·4%. Milk subjected to more severe heat treatments had a higher degree of isomerization (UHT milk, 15·7%; sterilized milk, 33·5%), consistent with increased thermal conversion of the retinol isomers. In pasteurized and UHT cream, the increase in 13-cis-isomer was also a consequence of heat treatment (pasteurized cream, 3·0%; UHT cream, 14·4%). The presence of cis isomers in fermented milk suggests that fermentation processes, directly or indirectly, can induce cis–trans isomerization. In the cheeses analysed, the extent of retinol isomerization ranged from 7·6 to 35·0%. Our results confirm the importance of individual isomers in evaluating the vitamin A status of dairy products.

HPLC size exclusion chromatography experiments showed that during inactivation at 40–70°C of the extracellular proteinase from Pseudomonas fluorescens 22F small molecular mass fragments were formed, indicating that autoproteolysis was at least one of the major causes of inactivation. The formation of small molecular mass fragments and the reaction order indicated that intermolecular autoproteolysis was more likely than intramolecular autodigestion. This was confirmed by computer simulations. The rate constants and the activation enthalpy (ΔH[Dagger]) and entropy (ΔS[Dagger]) for the reactions of the intermolecular autoproteolysis model were derived from computer simulations. ΔH[Dagger] and ΔS[Dagger] of the unfolding reaction were 504 kJ mol−1 and 1252 J mol K−1 respectively. ΔH[Dagger] and ΔS[Dagger] of the refolding reaction were strongly temperature dependent. The estimates for the enthalpy (ΔH0) and entropy (ΔS0) difference between the folded and unfolded state as derived from the reaction rate constants of unfolding and refolding were subject to large deviations, owing to accumulation of errors in the estimation of the kinetic characteristics.

To study the possible presence of staphylococcal enterotoxin A in raw goats' milk lactic cheese, milk was inoculated with an enterotoxigenic Staphylococcus aureus strain to a final concentration of 4, 5 and 6 log(cfu/ml). Cheese was prepared following industrial specifications and ripened for 42 d. Detection of the enterotoxins was by the Vidas Staph enterotoxin test (BioMérieux) and by an indirect double-sandwich ELISA technique using anti-enterotoxin monoclonal antibodies. Staphylococcal counts declined markedly after draining, and by the end of ripening they had disappeared from some cheeses. In contrast, aerobic mesophilic organisms grew well. The level of staphylococcal enterotoxin A recovered varied from 1 to 2·5 ng/g cheese made with an initial population of 105 or 106 cfu/ml. Only traces of enterotoxin A (0·5 ng/g) were detected in cheeses made with the lowest Staph. aureus inoculum used in this study. Enterotoxin A was also detected in cheeses from which Staph. aureus had disappeared.

Lactoferrin (Lf) is a milk iron-binding glycoprotein that plays a role in iron transport and acts as both a bacteriostatic and a growth modulating agent. The aim of this study was to investigate the nature of immune responses induced by repeated oral administration of bovine milk Lf in mice. Groups of ten female BALB/c mice were fed daily for 4 weeks with two doses of protein antigen: a low (0·05 mg/g body weight per d) or high (1 mg/g body weight per d) dose of Lf, or water as a control. A fourth group was immunized intramuscularly with 0·01 mg Lf in complete Freund's adjuvant. Anti-Lf IgA and IgG were detected in the intestinal fluid and serum of mice given Lf. Total immunoglobulins were higher in the intestinal fluid in Lf groups than in the control group. No difference could be detected in the serum. IgA and IgG secretion was enhanced in Peyer's patches and spleen from Lf-fed mice, in comparison with controls. [3H] thymidine uptake into Peyer's patch and spleen cells from both control and Lf-fed mice was enhanced by 75 μg Lf/ml in vitro, but Lf groups had a greater proliferation rate than the control group. These findings suggested that Lf could act as an immunostimulating factor on the mucosal immune system and that activation of the mucosal immune system is dependent on the ability of Lf to bind to the intestinal mucosa.

Two groups of eight multiparous cows with different calving periods (November or February) were managed in the same way during lactation. During four 4 week experimental periods distributed over 12 months, the cows were fed on a diet composed of hay and concentrate (70[ratio ]30) in a restricted and controlled amount which varied according to their lactation stage, so as to cover the animals' requirements correctly. The animals' average lactation stage varied according to period from 26 to 298 d. Milk from each group was processed on two occasions during each period to make Saint-Nectaire-type cheese; the cheesemaking conditions were the same throughout. Lactation stage had an important effect on milk fat, protein and calcium contents but not on the casein[ratio ]protein ratio or phosphorus content. The milk pH and the urea content were higher in late lactation. The calcium concentration of milk was higher in late lactation but the soluble fraction was higher in early lactation. Despite higher protein contents, the maximal firmness of the coagulum of late-lactation milk was not different from that of early or mid-lactation milks. pH was higher in cheeses from late-lactation milks compared with those from early and mid-lactation milks. Cheeses from early lactation milks were more yellow than the others and had a lower dry matter fat content. In sensory analysis the odour of cheeses from early and late-lactation milks was less pleasant than that of those from mid-lactation milks. Cheeses from late-lactation milks were more melting and less firm than those from early or mid-lactation milks. Their taste was more intense and more persistent. At tasting, they were less appreciated than the others. These differences were linked to increased proteolysis in the cheeses made with late-lactation milks.

Low-moisture part-skim Mozzarella cheeses were made, at pilot scale (450 kg), on five occasions at weekly intervals from milks containing κ-casein AA, AB or BB genetic variants. Compared with κ-casein A variant milks, the κ-casein B variant milks were associated with higher concentrations of casein (P<0·001), whey protein (P<0·02) and total protein (P<0·001), superior curd-forming properties (P<0·05) and increased cheese yields (P<0·05). The moisture-adjusted (to 465 g/kg) cheese yields for the κ-casein AA, AB and BB cheeses were 91·5, 100·6, and 102·5 kg/1000 kg milk respectively. κ-Casein variant had no significant effect on the proteolysis and ripening of uncooked cheese or on the functionality (melt time, flowability and stretchability) of the cooked cheese during the course of a 90 d ripening period.

The volatile concentrate obtained from Grana Padano cheese by vacuum distillation was fractionated by continuous liquid–liquid extraction into neutral and acid fractions. Both were analysed by high resolution gas chromatography (HRGC), HRGC–mass spectrometry, and HRGC–olfactometry. A total of 67 components were identified in the neutral extract (22 esters, 13 alcohols, 12 ketones, 6 aldehydes, 5 nitrogen-containing compounds, 3 lactones and 6 miscellaneous compounds) and 16 in the acid extract. Esters were the predominant constituents of the neutral fraction, whose major components were ethyl butanoate and ethyl hexanoate. HRGC–olfactometry of the neutral compounds demonstrated that 23 were odour-active: ethyl butanoate, 2-heptanol, 3-methylthiopropanal, 1-octen-3-ol, ethyl hexanoate and nonanal being the most potent odorants. n-Butanoic and n-hexanoic acids were the main volatile free fatty acids identified in the acid extract as having an important odour with a high olfactometric index. The backbone of Grana Padano cheese aroma seemed to consist of these acids and 14 potent neutral odorants imparting fruity, green, nutty and coconut notes. The concentration of volatile components responsible for the fruity and green notes was inversely proportional to the length of ripening, whereas the concentration of volatile agents with spicy, nutty and earthy notes tended to increase during maturation. In a comparison of the olfactometric profile, the Grana Padano cheese aroma was found to be more complex than an imitation Grana Padano cheese produced with similar technology but outside the area of the genuine cheese. Some of the main metabolic pathways for the biosynthesis of cheese aroma are reviewed briefly to indicate the possible origin of the compounds identified.

Elevated free fatty acid (FFA) levels in milk can impair the flavour quality and shelf life of milk and milk products and thus have implications for the dairy industry. An increase in milk FFA is induced when the protective fat globule membrane is disrupted and the fat exposed to the action of lipoprotein lipase (EC 3.1.1.34). Various aspects of FFA development have been reviewed (Deeth & Fitz-Gerald, 1976; Fleming, 1979; International Dairy Federation, 1980). Mechanical factors that contribute to FFA development are directly related to poor milking systems and procedures (O'Halloran et al. 1975; Judge et al. 1977; Fleming, 1991). Mechanical abuse of milk in many modern milking systems arises from air admission to the milking system, height of milklines and pump operation. Surveys and experience (Fleming et al. 1996) showed that the quality of the components used and the overall maintenance of many milking machines was inadequate. In continental Europe low-level milking systems are generally recommended in preference to higher lines. However, low-level systems are considerably more expensive as units have to be doubled up compared with the mid-level system.

Some of the machine factors contributing to excessive FFA development in milk can be eliminated by proper design, installation, maintenance and operation; however, the benefits of design changes remain a source of debate. The purpose of this investigation was to determine the relative effects on milk FFA of various components of a modern mid-level milking system (a pipeline milking system with the milkline 1·2–1·5m above cow standing level) and then to compare the optimal mid-level system with direct-to-line and low-level milking systems.

Marker-assisted selection may provide the opportunity to make significant genetic gains in the improvement of economically important traits in livestock (Soller & Beckmann, 1983; Smith & Simpson, 1986). Implementation of this approach will first require identification of candidate genes or anonymous gene markers associated with the traits of interest. Candidate genes are those with a known relationship between physiological or biochemical processes and an economically important trait. In dairy cattle, genes associated with mammary gland growth, development and function are excellent candidate genes for milk production traits.

The polyamines are low molecular mass polycations that influence cell proliferation and growth (Tabor & Tabor, 1984; Pegg, 1986). Ornithine decarboxylase (ODC, EC 4.1.1.17) catalyses the conversion of ornithine to putrescine, the rate-limiting step in polyamine biosynthesis (Pegg, 1986). The level of ODC is induced in quiescent cells exposed to a wide variety of stimuli such as growth hormone, corticosteroids, testosterone and growth factors (Tabor & Tabor, 1984). There is ample evidence that growth factors influence morphogenesis and differentiation of the mammary gland (Imagawa et al. 1994). Trophic hormones that are associated with lactation, such as prolactin and growth hormone, are also required to induce differentiation of bovine mammary epithelial cells (Huynh et al. 1991; Flint & Gardner, 1994), and both mitogenic and lactogenic effects of prolactin, insulin and hydrocortisone appear to be mediated through the polyamine pathway (Rillema et al. 1977; Bedford & Zadworny, 1990; Golden & Rillema, 1993). Strange et al. (1992) have shown that ODC is involved in synthesis of a milk component; ODC has elevated expression in the lactating mammary gland, which declines sharply after weaning. It is therefore possible that particular variants of ODC could be associated with increased mammary gland function and thus influence milk related traits.

The objectives of the present study were to estimate allelic frequencies of ODC polymorphisms in Holstein bulls and to evaluate further the genotypic effects of ODC variants on milk production traits.

Previously we have examined the inactivation of unpurified extracellular proteinase from Pseudomonas fluorescens 22F diluted in demineralized water (Schokker & van Boekel, 1998) in the range 40–70°C. It appeared that the inactivation was most probably caused by intermolecular autoproteolysis, which is the hydrolysis of unfolded proteinase molecules by native (not yet unfolded) molecules. It has been reported that purification of proteinases from Pseudomonas spp. enhances the susceptibility of the proteinase to autoproteolysis (Barach et al. 1976; Griffiths et al. 1981; Leinmüller & Christophersen, 1982; Kroll, 1989; Kumura et al. 1991). On the other hand, when the proteinase is heated in milk or when proteins are added to the enzyme solution, the rate of inactivation by autoproteolysis diminishes (Barach et al. 1978; Kroll & Klostermeyer, 1984; Stepaniak et al. 1991). Apparently, proteins stabilize the proteinase against inactivation by autoproteolysis.

Substrate or other ligands stabilize many enzymes against limited proteolysis. Binding of these substances to the enzyme molecule, either to the catalytic centre or to amino acid residues on the enzyme molecule surface, may impose steric difficulties so that the susceptible peptide bonds are protected against proteolysis (Mihalyi, 1978). Such binding may also cause a conformational change of the enzyme molecule, such that susceptible peptide bonds cannot be attacked or that the conformation is stabilized against unfolding (Mihalyi, 1978). In the latter case an increase in the denaturation temperature (Td) would be expected.

In the case of proteinases, addition of substrate to the enzyme solution may protect the enzyme by a third mechanism. Besides autoproteolysis of the proteinase, the added proteins can be digested. An enzyme molecule digesting a protein is not available at the same time for autoproteolysis, so that the substrate may act as a competitive inhibitor against autoproteolysis.

The aim of this study was to determine the mechanism of protection of the proteinase from Ps. fluorescens 22F by sodium caseinate.