We have modified deproteinization methods and a number of spectrophotometric and bioluminescent methods in order to measure the concentrations of cellular metabolites in small volumes (< 0·3 ml) of sows' colostrum and milk. For the majority of the assays, recoveries ranged from 92 to 105%. The binding of ATP and UTP to a calcium phosphate–citrate–caseinate complex in milk, and the decrease in ATP (92%/h) and UTP (18·1 %/h) concentrations during in vitro incubation of the whey fraction suggested that it was unlikely ATP and UTP (both < 1 µM) could exist free in sows' milk. The mean concentrations (range) of cellular metabolites in milk (6–11 d post partum) were: glucose, 669 µM (220–1367); glucose 6-phosphate, 63·0 µM (27·6–101·4); glucose 1-phosphate, 18·3 µM (13·1–24·8); UDPglucose, 296 µM (170–494); UDPgalactose, 635 µM (230–945); lactose, 162 mM (124–187); UDP, 105 µM (85–130); UMP, 1760 µM (1326–2587); inorganic phosphate, 135 mM (1·4–29·3); ATP, <0·5 µM; ADP, 53·6 µM (10·5–171·25); AMP, 215 µM (61·6–491·6); cAMP, 22·3 µM (3·5–61·6); galactose, 198 µM (118–474) and fructose, 226 µM (172–283). Differences in the concentrations of glucose, glucose 6-phosphate, glucose 1-phosphate, UDPgalactose and cAMP between fore and hind milk samples indicated postsecretory changes in the concentrations of certain metabolites. Changes in the concentrations of metabolites during in vitro incubation of milk and of colostrum suggested that these postsecretory changes were probably due to the actions of enzymes present in mammary secretion. Collection of milk that has been most recently secreted (hind milk) will provide the most accurate indication of the intracellular concentrations of these metabolites. The concentrations of cellular metabolites in the milks from different species are compared.

The concentrations of lactose, glucose, glucose 6-phosphate, glucose 1-phosphate, UDPglucose, UDPgalactose, UDP, UMP, inorganic phosphate, ADP and AMP (metabolites involved in the lactose synthesis pathway), and cAMP, galactose and fructose were measured in the mammary secretion from sucked (n = 9) and unsucked (n = 4) mammary glands of nine sows during the first 5 d post partum. The concentrations of lactose, glucose, galactose and fructose were also measured in plasma during this time. The progressive increase in the concentration of lactose, and changes in the concentrations of cellular metabolites in the mammary secretion from sucked glands were consistent with an increase in the metabolic activity of those glands during lactogenesis II. In contrast, unsucked glands showed a progressive decrease in the concentration of lactose, while the concentrations of cellular metabolites in the milk generally remained unchanged. These results indicated that there was no increase in the metabolic activity of unsucked glands (no increase in lactose synthesis or utilization of glucose and ATP) and that the rate of lactose synthesis prior to milk removal was limited by the availability of glucose and/or UDPgalactose. Therefore, the removal of colostrum from the mammary gland was necessary for an increase in the rate of lactose synthesis (and probably de novo fatty acid synthesis) and implies that autocrine mechanisms are operating to control the rate of milk synthesis during lactogenesis in the sow. The low concentration of glucose in colostrum compared with that in plasma is discussed in view of the paracellular pathway.

The concentrations of lactose, glucose, glucose 6-phosphate, glucose 1-phosphate, UDPglucose, UDPgalactose, UDP, UMP, inorganic phosphate, ADP and AMP (metabolites involved in the lactose synthesis pathway), and cAMP, galactose and sodium were measured in the mammary secretion from four or five mammary glands on each of six sows during the first 5 d post weaning. The concentrations of lactose, glucose and galactose were also measured in plasma during this time. Following weaning, the rapid increase in the concentrations of glucose 6-phosphate and UDPgalactose suggested that the rate of lactose synthesis was regulated by the inhibition of hexokinase and/or lactose synthase, while the decrease in glucose and AMP indicated a subsequent decline in glucose and ATP utilization. The rapid increase in glucose 6-phosphate which plays a pivotal role as a substrate for both lactose and de novo fatty acid synthesis, and the rapid decrease in AMP which reflects ATP utilization, were good markers of decreased metabolic activity. These rapid changes in the metabolic activity of the mammary glands were not observed in a second weaning study when two piglets were removed from selected mammary glands for periods up to 5 h during established lactation. Since concentrations of lactogenic hormones remain elevated following partial weaning, but fall following total weaning (Rojkittikhun et al. 1991), these differences in mammary gland metabolism indicate that endocrine rather than autocrine mechanisms are controlling lactose and fat synthesis during the initial stages of total weaning.

The effects of prolonged (22 weeks) treatment of lactating goats with bovine somatotropin on the metabolism of adipose tissue and liver has been investigated. Somatotropin treatment resulted in smaller adipocytes, decreased rate of fatty acid synthesis and decreased total acetyl-CoA carboxylase activity of adipocytes, but with no change in the proportion of this enzyme in the active state. The rate of acylglycerol glycerol synthesis from glucose of adipocytes tended to decrease as did total glucose utilization by the tissue. Glucose conversion to lactate was unchanged by somatotropin treatment but glucose conversion to other products was decreased. Maximum response of adipose tissue to insulin was unchanged but the sensitivity to insulin decreased on somatotropin treatment. Treatment with somatotropin had no effect on basal lipolysis and decreased maximum response to the β-agonist isoproterenol, but this probably reflects the rate of isoproterenol-stimulated lipolysis varying with cell volume in adipocytes. No apparent change in response either to α2-adrenergic agonists or to adenosine was apparent. The number of β-adrenergic receptors was unchanged in adipocyte membranes but the number of α2-adrenergic receptors increased. The rate of hepatic gluconeogenesis in vitro, the activity of key gluconeogenic enzymes and the modulation of the rate of gluconeogenesis by butyrate were unchanged except for the effect of this latter agent on gluconeogenesis from propionate. Hepatic ketogenic activity, as indicated by the activity of carnitine palmitoyl-CoA-transferase-1 and the concentrations of carnitine and acyl carnitines, was unchanged by treatment. Thus at the end of a prolonged period of treatment with somatotropin in lactating goats, lipid synthesis in adipose tissue is still decreased but no effects on liver lipid and carbohydrate metabolism were apparent.

Plasma concentrations of oxytocin and cortisol, and milk yield and flow rates, were compared in three primiparous cows and two cows in their second lactation during suckling and subsequent machine milking. After calving, cows suckled their calves for 3–4 weeks and then the experiment was carried out over 4 d. Blood samples were taken prior to, during and after suckling or evening machine milking (EMM) on day 1 of the experiment (the last day of suckling), day 2 (first EMM) and day 4 (third EMM). After weaning and rehousing, cows were machine milked twice daily. During the first EMM, average milk yield and flow rate in the second minute of milking were significantly lower (P < 0·05) than corresponding values for the other 2 d. Plasma oxytocin concentrations were lower during the first than during the third EMM (P < 0·001) and suckling (P < 0·001), and more oxytocin was released during the third EMM than during suckling (P < 0·01). Cortisol concentrations were higher during and after the first EMM than during the third EMM. Thus in cows sucked for several weeks after calving and then separated from their calves and rehoused we found a transient decrease in oxytocin release, milk yield and flow rates during the first machine milking.

Heat-induced gelation of milk was studied using both rheological and structural techniques. The sample was a conventional skim milk, concentrated with an ultrafiltration membrane, which formed gels when heated at appropriate pH. We investigated some factors that are considered to affect the gelation, such as concentration, pH and rennet treatment. The gelation process was monitored with a high precision oscillatory shear rheometer and the structure of gels was evaluated with quasi-elastic laser light scattering. From these results the gelation and phase separation behaviour were determined. By combining the results for different concentrations a phase diagram was obtained, which indicated that skim milk had a two-phase region on the higher temperature side. The effects of pH and rennet treatment were also evaluated with the aid of this phase diagram. The results were discussed on the basis of concepts of the phase behaviour of polymers, which were successfully developed in polymer physics.

Clarification of whey by microfiltration (MF) can be achieved after appropriate pretreatment of the feed. A control pretreatment consists of a physicochemical process comprising increased ionic calcium and pH accompanied by heat (50 °C, 15 min) to cause aggregation of complex lipid–calcium phosphate particles, which are then separated by MF. This pretreatment process was modified by increasing the temperature to 55 °C and by maintaining the pH constant during heat treatment. This modification resulted in larger calcium phosphate particles and a lower content of soluble calcium and phosphate ions. As a consequence, a longer period of MF operation, better whey clarification and lower calcium and phosphate content of the filtrate were achieved. This suggests that a loosely structured deposit was formed on the membrane surface which was less resistant to filtration than that resulting from the control pretreatment. During MF, it was necessary to avoid zones of high shear in the retentate compartment that might cause physical alteration of the aggregates.

The primary structures of ovine α>s1-casein variants A, C and D (formerly called Welsh variant) were determined. Separation of variants from whole casein was achieved using a fast and reliable reversed-phase HPLC method. Extended structural characterization of the purified proteins using electrospray mass spectrometry, automated Edman degradation and peptide mapping by means of HPLC-fast atom bombardment-mass spectrometry demonstrated that the mature protein was a mixture of two molecular species that differed in the deletion of residues 141–148 and were therefore 199 and 191 residues long respectively. The 199 residue peptide chain, which accounted for ∼ 80% of the entire translated αsl-casein, was as long as its caprine and bovine counterparts, and had a 98 and 89 % degree of identity with those two proteins respectively. Nine serine residues (positions 12, 44, 46, 64 to 68 and 75) were fully phosphorylated in αsl-casein A, whereas Ser115 and Ser41 were phosphorylated by ∼ 50 and ∼ 20% respectively. The differences between the three genetic variants A, C and D were simple silent substitutions, which however involved the degree to which the protein was phosphorylated. Variant C differed from variant A in the substitution Ser13 →> Pro13 which determined the loss of the phosphate group on site 12 of the protein chain, SerP12→>Ser12. A further substitution, SerP68 →> Asn68 caused the disappearance of both phosphate groups in the phosphorylated residues Ser64 and Ser66 in variant D; in this last casein variant there was no evidence of phosphorylation at Ser41.

The whole N fraction of six samples of hard and semi-hard pressed cheeses was analysed using PAGE, polyacrylamide gel isoelectric focusing and immuno blotting with polyclonal antibodies against β- and αs1-casein. The origin of some electrophoretic bands corresponding to peptides produced from the enzymic degradation of the casein fractions was established. A number of these peptides were also present in the in vitro hydrolysates of casein with plasmin and chymosin. Thus, it was also possible to determine which casein was the source of each peptide and which enzymes were active in cheese. Compared with the traditional Coomassie staining procedures, immunoblotting is more sensitive and specific, making the interpretation of each electrophoretic profile easy. Thus, it was also possible to obtain a clear picture of the state of each casein fraction in a cheese variety. Two main peptides were isolated from the pH 4·6-insoluble N fraction of Parmigiano-Reggiano using DEAE-cellulose chromatography and identified, from the amino acid sequence of the N- and C-terminal ends, as γ3-casein (β-casein(f108–209)) and αs1-PL1 (αs1-casein(f80–199)). In both cases, a Lys–X bond was hydrolysed, indicating the action of a trypsin-like enzyme in β- and αs1-casein hydrolysis during the ripening of this variety of hard pressed cheese.

Different electrophoretic techniques have been applied to the study of ovine whey protein polymorphism. Isoelectric focusing (IEF) over the pH gradient 3·5–9·5 was a suitable method for resolving both genetic variants of ovine β-lactoglobulin (β-lg); using this type of IEF, ovine β-lg A seemed to be defined by a major and a minor ‘satellite’ band. Approximate isoelectric point, relative molecular mass, amino acid composition and N-terminal sequence of this minor band were determined. A good separation of ovine β-lg was achieved using ultrathin layer isoelectric focusing (UTLIEF) over the pH gradient 2·5–7·0. However, addition of urea in UTLIEF gels led to some differences in the patterns of the ovine whey proteins when compared with those obtained by IEF on gels not containing urea. Two dimensional electrophoretic techniques provided a considerable separation of ovine whey proteins. Immunoblotting applied to a two dimensional separation permitted the identification of the bands belonging to β-lg and α-lactalbumin fractions.

The minimal hormonal requirements for inducing the ovine β-lactoglobulin gene have been investigated using mammary gland explants from ewes in the first half of pregnancy. Quantification of β-lactoglobulin mRNA showed that a combination of insulin, Cortisol and prolactin was required to stimulate the expression of the gene and that this response could not be enhanced by the addition of oestrogen and thyroid hormone to the culture medium. Explants cultured in the presence of insulin, Cortisol and prolactin also demonstrated the capacity to synthesize the protein. Progesterone did not inhibit the induction of the gene, which is consistent with the increase in β-lactoglobulin mRNA observed in vivo in the mammary gland during the final 2 months of pregnancy when the circulating level of progesterone is elevated.

A batch fermenter modified to simulate the physical conditions of an inflamed v. uninflamed mammary gland was utilized to evaluate the effect of oxygen tension on killing of Escherichia coli P4 by bovine polymorphonuclear neutrophil leucocytes. Leucocytosis was simulated in vitro 4 h after inoculation with Escherichia coli by adding bovine neutrophils isolated from peripheral blood to the culture medium. Experiments were conducted at 39°C in UHT treated milk. At micro-aerophilic oxygen tension (oxygen partial pressure, 3·11kPa), bacterial numbers declined during the first hour following addition of the neutrophils. Oxygen tension declined rapidly following PMN addition. Once oxygen was depleted, neutrophil activity was presumably diminished and Esch. coli numbers began to increase. Under anaerobic conditions (oxygen partial pressure, 0·17kPa), no reduction in population was observed. Photomicrographs taken at the time of neutrophil addition and at subsequent time intervals demonstrated a specific association between neutrophils and the pathogen. Subsequent lysis of neutrophils associated with Esch. coli growth was seen coincident with oxygen depletion.

The modulating effect of bovine milk casein components and their digests on the proliferative responses of mouse spleen lymphocytes and rabbit Peyer's patch cells induced or not induced by mitogens has been studied with a colorimetric assay using 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. All the casein components and their digests tested had little mitogenic effect on the proliferative responses of mouse spleen lymphocytes and rabbit Peyer's patch cells. Intact κ-casein significantly inhibited the proliferative responses of mouse spleen lymphocytes and Peyer's patch cells induced by mitogens such as lipopolysaccharide from Salmonella typhimurium, concanavalin A, phytohaemagglutinin and pokeweed mitogen. In contrast, intact αsl-casein and β-casein had little effect. κ-Casein had an inhibitory effect after digesti on by pancreatin or trypsin, but not after pepsin or chymotrypsin digestion. Both pancreatin and trypsin digests of αsl-casein and -casein significantly inhibited the proliferative responses of mouse spleen lymphocytes and rabbit Peyer's patch cells induced by mitogens, whereas pepsin and chymotrypsin digests of both caseins were without effect. Moreover, the trypsin digest of each casein component had an inhibitory effect on mouse spleen lymphocyte proliferation in the absence of mitogen. Since trypsin is a major proteinase in pancreatin, the substrate specificity of trypsin seems to be important for the formation of the inhibitory peptides from casein components. These observations suggest that intact κ-casein and some peptides formed from milk casein components by the action of trypsin may suppress the immune responsiveness of neonates.

Bovine κ-caseinoglycopeptides (i.e. residues 106–169, CGP) were prepared from κ-casein digested with rennin and a commercial whey protein concentrate. CGP from whey protein concentrate was further divided into seven CGP fractions having different carbohydrate compositions using FPLC. Unfractionated CGP inhibited lipopolysaccharide (LPS)- and phytohaemagglutinin (PHA)-induced proliferative responses of mouse spleen cells and rabbit Peyer's patch cells. The unfractionated CGP also inhibited antibody responses to sheep red blood cells in mouse spleen cell cultures. However, seven CGP fractions having zero to five N-acetylneuraminic acid (NANA) residues had different inhibitory effects on both LPS- and PHA-induced proliferative responses of mouse spleen cells. The inhibitory effect on PHA-induced proliferative responses increased with increasing numbers of NANA residues, whereas that on LPS-induced proliferation was highest with the CGP fraction having two NANA residues. Both inhibitory effects decreased significantly after neuraminidase or chymotrypsin digestion. These findings indicate that both the carbohydrate (particularly the NANA residues) and the polypeptide portions are essential for inhibitory effects on LPS- and PHA-induced proliferative responses of mouse spleen cells.

Studies on the immunomodulatory properties of dietary whey proteins in mice are reported. Ingestion of bovine milk whey proteins, either as a supplement in an adequately balanced commercial diet or as the only protein source in a balanced diet, consistently enhanced secondary humoral antibody responses following systemic immunization with ovalbumin, when compared with other protein sources such as soyabean protein isolate and ovine colostral whey proteins. After 5–8 weeks of feeding, dietary milk whey proteins enhanced cell-mediated immune responses as revealed by footpad delayed-type hypersensitivity responses, and concanavalin A-induced spleen cell proliferative responses. To monitor nutritional effects of milk whey proteins, live weight, leucocyte counts and clinical changes of diet-fed mice were examined. The present results confirm other previous results that dietary bovine milk whey proteins have immunoenhancing properties in mice and these properties are unlikely to be related solely to the nutritional effects.