Plasma concentrations of the oxytocin receptor blocking agent Atosiban were measured at 2, 4, 10, 15 and 20 min after injection of 5, 10, 20 and 50 μg Atosiban/kg body weight in six dairy cows. The half life of Atosiban was 18 min and the total body clearance was 3301 ml/min. Intramammary pressure (IMP) within the teat cistern was measured in six cows before and after i.v. injection of 0 or 20 μg Atosiban/kg body weight and repeated injections of 0·2 or 0·5 i.u. oxytocin. IMP was also measured in eleven cows after injection of 0, 10 or 50 μg Atosiban/kg body weight: in seven during oxytocin infusions, in four after oxytocin injections in successively increasing dosages (0·05, 0·1, 0·2, 0·5, 1 and 10 i.u.). The occurrence of milk ejection was indicated by a rise in IMP. After injection of 20 μg Atosiban/kg body weight, 0·2 i.u. oxytocin did not induce an IMP rise before 48 min, whereas 0·5 i.u. oxytocin induced an IMP rise within 4 min. The time from the start of infusion until the beginning of the IMP rise and the duration of IMP rise during oxytocin infusions both increased, whereas the IMP rise itself was diminished by increasing Atosiban dosages. The amount of injected oxytocin necessary to induce an IMP response increased with increasing Atosiban dosages. Atosiban was shown to have a powerful effect in inhibiting milk ejection in dairy cows.

The effects of replacing whole cottonseed by heated whole cottonseed at two levels of crude protein on amino acid (AA) utilization by the bovine mammary gland were investigated. Four Israeli Holstein cows were used in a 4×4 Latin square design with 2×2 factorial arrangements. Diets were formulated to contain two levels of crude protein (CP), normal (160 g CP/kg dry matter) and low (140 g CP/kg dry matter), and two levels of rumen undegradable protein (UDP), high (380 g UDP/kg CP) and low (350 g UDP/kg CP). Whole cottonseed was quantitatively substituted by heated whole cottonseed to formulate the high UDP diets. Intakes of dry matter and organic matter were similar for all treatments and averaged 15·9 and 14·4 kg/d respectively. Yields of milk and its constituents were similar for all treatments. Milk yield averaged 23·1 kg/d and this contained (per kg) 32·3 g CP, 25·4 g total casein and 47·5 g lactose. The milk fat content was lower in the cows given the high UDP diets, averaging 34·6 g/kg compared with 38·9 g/kg in the cows fed on the diets with low UDP. Plasma AA concentrations were similar for all treatments apart from Leu and Ile, which were higher in the cows given the high CP diets: 152 and 103 μm compared with 183·8 and 131·5 μm for the low CP diets. Net extraction and balance of essential AA across the half udder suggested that essential AA were supplied in amounts sufficient to meet milk protein requirements for all treatments. The surplus AA supplied as a result of feeding the high dietary CP were probably catabolized via an enhanced oxidative pathway, possibly in the liver, presumably as a passive response to their disposal.

Laminitis is a major cause of lameness in dairy cattle, and is widely attributed to a defect in the horny tissue that gives the hoof its mechanical strength. Defective horn is associated with, and may be preceded by, impaired keratin deposition in the hoof epidermis. The cause of abnormal keratin deposition is not easily identified but, like epidermal keratinization in other tissues, is likely to be controlled by hormones and the paracrine action of locally produced growth factors. The hormonal regulation of keratin synthesis and cell proliferation in the bovine hoof was studied using tissue explants in organ culture. As the highest incidence of laminitis is in early lactation, the study focused on insulin, cortisol and prolactin, three hormones implicated in lactogenesis and galactopoiesis. Incubation of tissue explants for 24 h in medium containing insulin (10–5000 ng/ml) stimulated protein synthesis measured by incorporation of 35 S-labelled amino acids. Histochemical examination showed that insulin binding co-localized with the site of protein synthesis. Insulin also stimulated DNA synthesis, an index of cell proliferation, which was measured by incorporation of [3H]methyl thymidine. Cortisol (10–5000 ng/ml) decreased protein synthesis, whereas prolactin (10–5000 ng/ml) had no significant effect on protein or DNA synthesis. Epidermal growth factor (10–200 ng/ml), a potent inhibitor of keratinization in other tissues, stimulated protein synthesis compared with untreated controls. Epidermal growth factor binding was located microscopically to the germinal and differentiating epidermal layers. SDS-PAGE and fluorography showed that the population of proteins synthesized in the presence of any hormone or growth factor combination did not differ from that in untreated controls and included the keratins involved in horn deposition. The results show that bovine hoof keratinization is under endocrine and growth factor control, and suggest that systemic changes in lactogenic hormones may act to inhibit keratin deposition.

The mammary gland is an example of a tissue of epidermal origin that depends for the development of its characteristic morphology on underlying mesenchymal cells. The interaction between mesenchyme and epithelium appears to be mediated by polypetide growth factors. In situ hybridization has been used to study, in the mammary gland of female sheep fetuses, the distribution of mRNA for the mammary mitogens, insulin-like growth factor (IGF)-I and IGF-II, and the IGF-I receptor, from 10 to 20 weeks of intrauterine life (term is ∼22 weeks). At 10 weeks, secondary ducts had formed from the primary duct. By week 20, the gland had increased in volume and complexity, showing primitive lobules embedded in intralobular connective tissue disposed around main ducts. IGF-I and IGF-II mRNA were expressed in cells of the intralobular connective tissue underlying the epithelium, while the IGF-I receptor was expressed in epithelium. Quantitation by absorbance measurements showed that mRNA expression increased with pregnancy stage for IGF-I and IGF-II, but not significantly for the IGF-I receptor, and that IGF-II was more highly expressed than IGF-I. A role for the IGF system in mediating mesenchymal–epithelial interactions in mammary development is indicated.

Intravenous infusions of glucose and/or glucagon-like peptide-1(7–36)-amide (GLP) or somatostatin-28 (S28) were administered to dry and lactating sheep and changes in plasma glucose and serum insulin were followed before, during and after infusion. Basal serum insulin concentrations were significantly lower in lactating sheep but there was no significant difference in plasma glucose concentrations. During glucose infusion, changes in serum insulin concentrations were diminished by comparison with those in dry animals. GLP stimulated insulin secretion during hyperglycaemia in both dry and lactating sheep but, proportionately, the response was significantly greater in the lactating animals. S28 inhibited glucose-stimulated insulin secretion in both dry and lactating sheep and there was no significant difference in the extent of the inhibition between the two physiological states. S28 infusion also inhibited the secretion of GLP from the intestinal tract and this effect was significantly greater in the lactating animals. The results demonstrate a difference in response in the lactating animal to GLP compared with S28. A possible explanation of the difference is a decreased sensitivity (i.e. increased Km) of the pancreas to the insulinotropic effects of GLP but an increased sensitivity to the inhibitory effects of S28 at tissues other than the pancreas in lactation.

The peptide Val–Arg–Arg–Pro–Asn–Leu–His–Pro–Ser–Phe–Ile–Ala–Ile–Pro–Pro–Lys–Lys–Ile, which corresponds to residues 84–101 of human κ-casein, has been synthesized and its conformation preferences determined by 1H-nuclear magnetic resonance spectroscopy in dimethyl sulphoxide. The peptide adopted a largely extended chain conformation in solution and there was evidence for the presence of a β-turn involving residues Pro87–His90 of human κ-casein. The presence of a turn in this position would make the physiologically significant Arg85 residue of human κ-casein (which is equivalent to Arg97 in bovine κ-casein) unavailable for interaction with Asp249of bovine chymosin, and may partly explain why human κ-casein is hydrolysed more slowly than its bovine counterpart by bovine chymosin.

Changes in the distribution of minerals in skim goats' milk by high pressure (400 MPa) and/or heat (85°C for 30 min) treatment have been studied. Heat treatment caused reduced solubility of the calcium, magnesium and phosphorus, and this increased with the severity of heating. In contrast, high pressure released different levels of micellar elements into the soluble phase without causing appreciable changes in pH or ionic calcium concentration. The levels of soluble salts returned to their original values when the heated samples were subjected to high pressure. However, heating pressurized milk resulted in concentrations of soluble minerals that were lower than in control milks, and close to values found in heated milks. The salt balance in goats' milk was less affected by high pressure treatment at 75°C than was that of cows' milk. These results are discussed in relation to the effects of high pressure and heat treatment on mineral equilibrium and micellar structure.

The lactoperoxidase system is a naturally occurring antimicrobial system found in milk, with lactoperoxidase, thiocyanate and hydrogen peroxide as its components. The keeping quality of milk pasteurized at 72°C for 15 s was found to be better than that of milk heated at 80°C for 15 s. This agrees with previous findings and is usually attributed to heat shocking of spores. However, complete deactivation of lactoperoxidase occurred at 80°C–15 s, whereas at 72°C–15 s residual lactoperoxidase activity was ∼70%, which may provide an alternative explanation. Higher levels of hypothiocyanite (the major antimicrobial agent produced by the lactoperoxidase system) were also detected in milk processed at 72 than at 80°C, which supports the theory that the lactoperoxidase system has a role in the keeping quality of pasteurized milk. Of all the methods evaluated, titratable acidity and alcohol stability gave the most consistent estimates of keeping quality, while dissolved oxygen was a good indication of the onset of spoilage. Lactoperoxidase activity decreased with temperature more rapidly between 70 and 80°C than is usual for an enzyme over a 10 deg C range.

The two glycosylated N- and C-terminal lobes of buffalo lactoferrin have been produced by limited proteolysis using proteinase K. Lactoferrin is a single chain glycoprotein of molecular mass 80 kDa with two iron-binding sites and two structural lobes connected by a short peptide. Purified samples of lactoferrin, isolated from buffalo colostrum, were subjected to hydrolysis using trypsin, chymotrypsin, pepsin, subtilisin and proteinase K. The first three proteinases produced two major fragments of approximately 35 and 23 kDa together with small molecular mass peptides. Trypsin and chymotrypsin partly digested lactoferrin, while pepsin converted all the intact lactoferrin into fragments. Subtilisin hydrolysis produced fragments of 40 and 26 kDa together with low molecular mass peptides. However, SDS-PAGE of the proteinase K hydrolysis product gave a clear band at 40 kDa together with a band indicating a substantial quantity of low molecular mass peptides (<14·4 kDa). Upon ion-exchange chromatography this product gave two major fractions, which were further purified by gel filtration and identified as the C and N lobes from their N-terminal sequences. Thus, the 40 kDa band in SDS-PAGE of the proteinase K hydrolysis product contained two fragments of equal molecular mass. On further hydrolysis with proteinase K, the N lobe was completely hydrolysed into low molecular mass peptides, while only a small fraction of the C lobe was converted into small products. This suggested that an inhibitory fragment was present in the C lobe that was released on hydrolysis to small fragments and prevented complete digestion of the C lobe by high-affinity binding to the active site of proteinase K. This fragment was isolated from the lactoferrin–proteinase K complex and its sequence determined to be Val–Ala–Gln–Gly–Gly–Ala–Ala–Gly–Leu–Ala. Circular dichroism studies indicated a high α-helical content in the native lactoferrin while comparatively lower helical structures were present in the N and C lobes. In addition, the iron saturations of the N and C lobes appeared to be lower than that of the native protein.

Various methods were used to study 42 Gram-positive cocci belonging to the family of Micrococcaceae and isolated from dairy products. Only a few strains could be identified using the ATB 32 biochemical micromethod rapid gallery system (API). All strains were subjected to conventional biochemical tests. The results were then analysed by a numerical method using the Sokal–Michener similarity coefficient. The 42 strains were distributed into 10 clusters at the Euclidean distance of 3·2. Most (75%) of the isolates were identified at the species level, but Micrococcus luteus could not be differentiated from Mc. lylae within the Micrococcus genus. An identification method based on the analysis of ribotype patterns was then applied to the isolates and these were compared with the patterns of collection strains. Cellular DNA was cleaved by the restriction enzymes SacI and KpnI and probed with the peroxidase-labelled 16S+23S rRNA of Escherichia coli. Ribotype patterns were analysed using a Dice coefficient and the unweighted pair group method using arithmetic averages, making dendrogram construction possible. Of the strains investigated, >84% were characterized to the species level. The remaining strains were found to belong to the Arthrobacter group or were atypical Micrococcus species. Of the isolates, 52% belonged to Micrococcus luteus, 17% were identified as Kocuria varians and 7% were assigned to the Brachybacterium genus.

Swiss-type cheeses such as Emmental are characterized by the successive development of thermophilic lactic acid bacteria (TLAB) and propionibacteria. The aim of this study was to determine whether the choice of TLAB strain influenced propionibacteria. TLAB and propionibacteria were cultured sequentially under the conditions prevailing in cheese. Firstly, 11 Emmental juice-like media were prepared by fermenting casein-enriched milk with pure or mixed cultures of TLAB (Lactobacillus helveticus, Lb. delbrueckii subsp. lactis and Streptococcus thermophilus), differing in their proteolytic activities. TLAB cells were then removed by microfiltration. Finally, five strains of Propionibacterium freudenreichii were grown on these media at 24°C under anaerobiosis and their growth characteristics and lactate consumption determined. The media mainly differed in their contents of peptides (1·9–5·3 g/kg) and free amino acids (1·0–5·6 g/kg) and the proportions of lactate isomers (42–92% of the L(+) isomer). Propionibacteria were significantly (P<0·05) influenced by TLAB strains (differences in doubling times of up to 20% and differences in lactate consumption after 600 h culture of up to 52%). The influence of TLAB was similar for all the propionibacteria tested, depended on the TLAB strains and could not be generalized to the TLAB species. Propionibacteria were stimulated by high peptide levels, low levels of free amino acids and NaCl, a low proportion of L(+)-lactate and other undetermined factors. However, variations due to TLAB were less than those between propionibacteria strains.

The reduced sulphur volatiles, methanethiol and dimethyldisulphide (but not dimethylsulphide), have been detected in the headspace of butter samples. Their concentrations in the butter itself were calculated by determination of the distribution coefficient (K) through spiking butter samples with known quantities of methanethiol and dimethyldisulphide. The K values obtained suggest that the dimethyldisulphide had a greater affinity than methanethiol for the lipid phase of the butter. Solid phase microextraction used in conjunction with gas chromatography–mass spectrometry has been shown to be an effective method for headspace analysis and was used for quantifying the butter headspace concentrations of the reduced sulphur compounds. Seasonal variation of both methanethiol and dimethyldisulphide were found in a series of butter samples, the highest concentration being in spring with an overall decrease through the New Zealand summer. Grass type and condition appeared to influence the concentrations of these compounds in the butter. Storage of the series of butter samples at 4°C for 5 weeks resulted in a significant decrease in the concentrations of both methanethiol and dimethyldisulphide. The dimethylsulphide concentration in all butter samples was below the detection limit of the analytical system used.

An automatic milking system (AMS) uses a robot to attach the teatcups and can be continuously available for cows to present themselves to be milked with minimal human intervention. Milking in an AMS is therefore a continuous rather than a batch process, and the conventional practice of cleaning a milking system after each batch of cows has been milked is not applicable.

Milk flow through an AMS is different in scale and in temporal distribution from that in a conventional system. These differences in flow patterns may be significant for hygiene. It has been suggested by Verheij (1992), for example, that milk will flush away much of the residue of the previous milking and that microbial growth will be retarded because of the bacteriostatic properties of fresh milk. Ordolff & Bölling (1992) reported experiments in which batches of milk were passed through a milking installation that was said to simulate an AMS. Their main conclusion was that milk residues left in the system for up to 60 min did not increase the bacterial contamination of the next batch of milk. However, the practical relevance of this conclusion is not clear because the design of the simulated AMS was not specified, so it can not be compared to a practical AMS. Moreover, mixtures of fresh and UHT milk were used, with unknown consequences. The purpose of the experiment reported in this paper was to strengthen the evidence on which to base recommendations for effective cleaning procedures for AMS.

The bacterial count of milk that had been passed through a system was used as the indicator of the cleanliness of the system. It was apparent from previous work by Ordolff & Bölling (1992) that this would produce results that varied widely. The experiment was therefore designed to be as simple as possible. In particular, a simple, easily defined laboratory rig was used rather than an AMS.

Rotavirus is the leading cause of infectious diarrhoea in infants and children throughout the world (Davidson, 1996). In developed countries, rotavirus is associated with significant morbidity, while it causes a high rate of infant mortality in developing countries (Blacklow & Greenberg, 1991).

At present there is no effective treatment or vaccine against human rotavirus. Although considerable effort is being put into developing such a vaccine (Molyneaux, 1995; Midthun & Kapikian, 1996), researchers are encountering difficulty in obtaining sufficiently broad protection against the great variety of human rotavirus serotypes (Kapikian & Chanock, 1985). Passive immunization has been tried as an alternative, and clinical trials have shown that oral intake of bovine colostrum containing anti-human rotavirus antibodies is effective in preventing rotavirus infection and transmission in experimental animals, infants and children (Davidson, 1996). Since cross reactivity has been found between bovine and human rotavirus strains (Kapikian & Chanock, 1985), a product containing specific antibodies against human or bovine rotavirus strains may exert a protective effect in human babies. Such a product would be a useful temporary measure while an effective vaccine is developed. Furthermore, these products could be valuable for immunocompromised patients in preventing rotavirus infections.

However, such products would be subjected to processes such as pasteurization or ultra high temperature treatments, so it would be necessary to know the effect of heat on the biological activity of the antibodies. We have previously studied the effect of heat on the structure of bovine colostrum immunoglobulins IgG, IgA and IgM and evaluated the kinetics of heat denaturation by measuring the loss of their ability to bind to specific antibodies directed against them (Mainer et al. 1997). In the present study we have investigated the effect of different heat treatments on the biological activity of immunoglobulins from bovine colostrum by measuring the virus-neutralizing activity (VNA) of heat-treated colostrum containing anti-rotavirus immunoglobulins against a bovine strain. In addition, we have studied the distribution of the VNA between the different classes of immunoglobulins of the anti-rotavirus colostrum.

Ion exchange is used commercially in such processes as water softening, chemical purification, separation of ionic from non-ionic forms and analytical applications. Van Kreveld & van Minnen (1955) reported a method for the determination of Ca2+ and Mg2+ activity in milk using an ion-exchange resin. From this early work, it was established that with suitable resins exchange of salts was possible in milk, permeate and whey. This included the exchange of Ca and Mg from micelles by using Na+ or K+. Cationic resins of the Zerolit 236 type have been used successfully in the production of Ca-reduced single cream (Anderson et al. 1976). Such treatment prior to UHT processing was shown to increase physical stability when it was used as a coffee whitener. There is evidence that reduction of Ca2+ would also reduce the deposit formation that may occur during storage of UHT milk. Thus there is a need to establish commercial techniques to produce Ca-adjusted milks that could then be used as raw materials for industrial applications or product development work.

The present study investigated the use of ion exchange to reduce Ca in milk with particular reference to understanding the exchange of salts in Ca-reduced milk and the changes to some of its physical properties.

As the demands for food products of high quality increase, it will become more important to be able to modify the properties of food proteins. One possibility is to use enzymic modifications to improve functional properties and nutritional values. A highly efficient class of enzymic crosslinkers are transglutaminases (TG, EC 2.3.2.13), calcium-dependent enzymes that catalyse an acyl transfer reaction between protein-bound glutaminyl residues and primary amines (for review, see Aeschlimann & Paulsson, 1994). When protein-bound lysyl residues act as acyl acceptors, the reaction leads to the formation of intramolecular and/or intermolecular isopeptide bonds. Although a wide variety of amines such as putrescine, cadaverine or lysyl residues may act as amine donors in this reaction, only a limited number of glutamine residues in certain proteins will act as amine acceptors (Gorman & Folk, 1980).

Ikura et al. (1981, 1985) reported that TG can be used to introduce methionine into casein and soyabean proteins and lysine into wheat gluten, thereby showing that TG can also be utilized to improve the nutritional value of food proteins. There have been a number of reports concerning the TG-mediated polymerization of food proteins such as α-lactalbumin and β-lactoglobulin (Aboumahmoud & Savello, 1990; Færgemand et al. 1997), soyabean proteins and casein components (Ikura et al. 1980a, b) and pea legumin (Larré et al. 1993). Recently, improved protein foaming capacity and stability have been demonstrated in TG-catalysed polymers of soyabean protein and whey protein isolate (Yildirim et al. 1996).

Bovine PP3 (for review, see Girardet & Linden, 1996) is a phosphorylated glycoprotein isolated from the proteose peptone fraction of milk (Sørensen & Petersen, 1993a). The primary structure of PP3 has been determined (Sørensen & Petersen, 1993b) and comprises a polypeptide backbone of 135 amino acid residues containing five phosphorylated serines, two threonine-linked O-glycosylations, and one N-glycosylation. Immunological studies have shown that PP3 is present in the milk fat globule membrane, and that PP3 forms multimeric aggregates in bovine milk (Sørensen et al. 1997). Several functions have been suggested and investigated for bovine PP3 and fractions enriched with PP3, including emulsification (Shimizu et al. 1989), inhibition of lipolysis (Girardet et al. 1993) and mitogenesis (Mati et al. 1993).

Previously, we have localized the potential TG-reactive glutamines in the four bovine caseins (Christensen et al. 1996) and in milk osteopontin (Sørensen et al. 1994). In the present study we have shown that component PP3 is a substrate for guinea-pig liver TG containing both reactive glutamine and lysine residues. In addition, we have localized the glutamine residues that act as amine acceptors.