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.

Four different field studies including 64, 140, 850 and 180 herds were used to investigate the association between pulsator characteristics and udder health. The results indicate that the best udder health is found in herds with a d phase > 330 ms and a pulsation rate > 55 min-1. A d phase of < 250 ms was associated with significant elevation in bulk milk somatic cell count, a higher incidence of acute clinical mastitis and a higher frequency of cows having mastitis associated with major pathogens (mainly Staphylococcus aureus) as compared with a d phase of > 331 ms. There was also a significant positive association between a shorter d phase and a high incidence of teat lesions needing veterinary treatment. Herds having no such teat lesions had pulsators with significantly longer d phase than herds with teat lesions (300 ms ν. 288 ms). These results strongly suggest that in high-line milking machines the d phase should not be as short as 250 ms and should preferably be ∼ 300 ms, and the pulsation rate should be at least > 55 cycles min-1. There was a strong relationship between the effect of d phase and the rate of pulsation.

To test the hypothesis that propionate can reduce hepatic capacity to detoxify ammonia, effects of the inclusion of propionate in intraruminal infusions of urea on the concentrations of ammonia, other metabolites and insulin in peripheral blood were investigated in two experiments with non-lactating dairy cows. Both experiments were of a 4 × 4 Latin square design with four animals, four treatments and four experimental periods of 7 d; feed was given in two equal meals each day, all intraruminal infusions were given for 1 h at the time of the morning feed, and propionic acid was partly neutralized with NaOH. In Expt 1, the treatments were a basal diet of pelleted lucerne and chopped hay alone or with the following infusions (g/d): urea 80, propionic acid 350, urea 80 plus propionic acid 350. The inclusion of propionate in the urea infusion markedly increased (P < 0·001) the concentration of ammonia in plasma compared with infusion of urea alone. Moreover, the inclusion of urea with the propionate infusion abolished (P < 0·01) the increase in blood insulin level seen with the infusion of propionate alone. In Expt 2, less severe treatments were imposed, the aim being to reproduce metabolic loads of propionate and ammonia that might be expected from a diet of high-protein grass silage rich in lactic acid. The treatments were a basal diet of grass silage alone or with the following infusions (g/d): NaCl 145, NaCl 145 plus urea 50, propionic acid 200, urea 50 plus propionic acid 200. Effects were less pronounced than in Expt 1 but, in the period immediately after infusion, similar effects were seen. It is concluded that propionate–ammonia interactions may have potentially important effects on milk production especially for diets with high proportions of grass silage containing high levels of protein and lactic acid.

The requirement for accuracy in the counting of somatic cells in milk has been increased by the use of this measurement as a basis of payment for milk. This paper reports on several factors that can substantially affect efficiency of staining and cell detection in various types of fluorescent cell counters. These mordant factors include sample age, chemical preservative, other sample preservation treatments and reagent properties. Experiments were conducted in several laboratories with Fossomatic 215 and 360 cell counting instruments to measure the effect of these mordant factors on both cell fluorescence and cell count. Counting efficiency was as low as 20% for some instruments counting some particular samples, depending upon the combination of adverse mordant factors. The problem of poor counting efficiency was also not detected by calibration samples that had more favourable mordant treatments applied. The adverse mordant factors were shown to reduce the intensity of fluorescence of cells for all instruments. We suggest that a significant reduction in counting efficiency due to these adverse mordant factors is most likely to occur when the particular instrument already has an intrinsic inability to separate cell fluorescence properly from background noise. This paper also discusses quality control samples, the impact of various factors on discriminator curves and the use of appropriate measures to maintain good calibration adjustment.

Six lactating dairy cows were used in a three period, part balanced changeover design experiment to investigate the effects of forage digestibility and concentrate composition on the efficiency of nutrient utilization in lactating dairy cows. Six treatments comprising three forage regimens and two concentrate types (starch υ. fibre) were examined in a 3 × 2 factorial design. The three forage regimens were high digestibility grass silage offered ad lib. (HA) or restricted to 6·5 kg dry matter/d (HR) and a low digestibility grass silage offered ad lib. (LA). Within each forage regimen animals were offered 10 kg·d of supplements containing either high-starch or high-fibre concentrations. Experimental periods lasted 28 d with a 10 d recording period, during which animal performance, ration digestibility and nitrogen and energy utilization were measured. Respiratory exchange measurements were made over a 72 h period using indirect open-circuit calorimetry. Throughout the experiment, there were no significant forage × concentrate interactions in any of the intake, production or nutrient utilization results. Milk yield was significantly influenced by forage regimen (24·1, 21·7 and 21·9 kg/d for HA, HR and LA respectively) and concentrate type (21·6 and 23·5 kg/d for high-starch and high-fibre respectively). Concentrate type also significantly influenced milk protein concentration (32·8 and 30·9 g/kg for high-starch and high-fibre respectively). Forage regimen significantly influenced the efficiency of utilization of metabolizable energy (ME) for milk production (κ1) with values of 0·62, 0·64 and 0·59 for HA, HR and LA respectively. Concentrate type had no significant effect on ME intake, heat production or κ1, although animals receiving the high-fibre concentrates synthesized proportionately 0·11 more milk energy per unit of available energy (ME intake – heat production) than those receiving the high-starch concentrates. Interpolation of the values obtained with the two high digestibility forage regimens indicated that at similar ME intakes there was a trend towards a higher κ1 with the diet based on high digestibility silage, and this was in line with the higher metabolizability of the overall diet with this silage.

Physical characteristics of the bovine teat canal were examined for their influence on susceptibility to intramammary infection. All quarters of 18 cows were inoculated with 2 × 105 cfu Streptococcus agalactiae (Trial 1) and 20 cows with 105 cfu Str. Uberis (Trial 2) 3–4 mm into the teat canal every 3 d for 12 d. Incidence of quarter infection was similar for both pathogens, 30/72 (42%) in Trial 1 and 32/80 (40%) in Trial 2. Logistic regression analysis showed that probability of infection by Str. Agalactiae increased significantly with an increase in quarter peak flow rate (P < 0·05) whereas probability of infection increased for Str. Uberis with a decrease in teat canal length (P < 0·05). A significantly higher (P < 0·001) incidence of infection by Str. Uberis was observed in quarters that contained a low wet weight (< 1·8 mg) of removable keratin compared with those that contained > 1·8 mg keratin, but there was no correlation between weight of keratin and length of the teat canal. Infections by Str. Uberis took significantly less (P < 0·05) time to show a rise in somatic cell count above 7·5 × 105 cells/ml than Str. agalactiae. The results provide evidence that these pathogens use different mechanisms to pass through the teat canal.

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.

Experiments were designed to test the hypothesis that milk ejection rate decreases during milking, thereby causing insufficient refill of the cistern and decreasing milk flow rate towards the end of milking. In a first series of experiments machine milking of the left front quarters of 11 cows was interrupted for 2 min after removal of 25, 50 or 75% of expected total milk yield, while milking was continued in the other three quarters. Milk flow was recorded during machine-on times. Intramammary pressure (IMP) was recorded during premilking teat stimulation and during interruption of milking. IMP during interruption of milking decreased with decreasing amounts of milk remaining in the udder. The IMP did not change during these interruptions when they occurred after 25 and 50% of expected total milk yield was removed. Thus, the ejection rate could keep up with the milk flow or removal rate. However, IMP increased during interruption of milking following removal of 75% of total yield, although significantly so only in cows with a high milk flow rate. Obviously, more milk was removed than was transported to the cisternal cavity. It is likely that a reduced ejection rate caused the decreased milk flow rate. In a second series of experiments the pulsation ratio of the milking machine was changed from the usual 70:30 to 50:50 with the aim of reducing the milk flow rate and thus adapting to the ejection rate at the end of milking. The changed pulsation ratio caused a reduced peak flow rate and a prolonged high milk flow period, whereas the main flow rate did not change significantly.

Milk yield and milking time were measured on one occasion for several daughters (n = 6–44) from 16 bulls at morning milkings. Blood from the bulls was collected, and platelets and mononuclear leucocytes were isolated. The α2-adrenoceptors on platelet membranes were identified by binding of [3H]rauwolscine, whereas for the determination of β2-adrenoceptors on intact mononuclear leucocytes, [3H]CGP–12177 was used. It was found that mean milk flow rate was highly correlated (P < 0·001) with the α2-adrenoceptor densities on blood platelets. No correlation was found with the β2-adrenoceptors on mononuclear leucocytes. It is concluded that estimation of the α2-adrenoceptors on blood platelets from bulls could eventually be used to investigate milking characteristics of cows, and might be useful in the future as a marker in genetic studies.

The effects of the form in which amino acids are presented to the abomasum on the milk production of dairy cows receiving a basal diet of grass silage and a barley-based supplement were examined in two experiments. Effects of abomasal infusions of sodium caseinate were compared with the effects of corresponding levels of either an enzymic hydrolysate of casein (Expt 1) or a corresponding mixture of free amino acids (FAA; Expt 2). In Expt 1, although the yield of protein in milk increased progressively with each level of infusion, the yields of protein were greater for the caseinate than for the hydrolysate. Again, in Expt 2, for milk protein yield, sodium caseinate was superior to FAA at the lower level of infusion. In both experiments, the hydrolysate and FAA treatments were associated with higher concentrations of fat in the milk. There were indications of differences in the pattern of secretion of glucagon between the caseinate and FAA treatments. It is concluded that the differences between treatments relate either to the kinetics of absorption of amino acid residues or to the action of bioactive peptides released during digestion of casein.

A dynamic mathematical model of blood flow regulation in the mammary glands of a lactating cow was constructed from a principle of local vasodilator release in response to changes in intracellular adenylate charge. An equation was derived to predict uptake of the milk precursors acetate, glucose, amino acids and fatty acids, as affected by mammary blood flow rate. Metabolism of the precursors to milk components and CO2 was simulated with a set of empirically derived equations. Relative rates of ATP production and utilization regulated both the number of perfused capillaries and the conductance of arteriolar segments in the mammary glands. The model simulated local control phenomena of functional and reactive hyperaemia, and simulation of autoregulation under changing arterial pressure suggested a predominance of precapillary sphincter regulation. It was predicted that an increase in blood flow without the mammary capacity to utilize blood metabolites efficiently would be detrimental to milk synthesis. Conversely, increased blood flow through changes in mammary activity resulted in predictions of higher milk production. It was proposed that the equation for uptake,

be used in analysis of mammary arteriovenous differences.

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.

The effects of culture supernatants conditioned by the growth of Staphylococcus aureus M60 on in vitro growth and functional properties of bovine mammary myoepithelial cells were examined. Myoepithelial cell proliferation was reduced by Staph, aurexis M60 culture supernatants. Exposure of myoepithelial cells to culture supernatants of isogeneic mutants of Staph, aureus M60 that produced either α or β toxins reduced proliferation, but to a lesser extent than supernatants from the wild type strain. Thus, α and β toxins may play some role in affecting myoepithelial cell proliferation. Of the cells tested, 42% contracted following addition of oxytocin (10–7 M) in the culture medium. Treatment of myoepithelial cells for 15 min with Staph, aureus M60 supernatants, prior to addition of oxytocin in the culture medium, increased the number of cells that contracted to 92%. Exposure of cells for 3 h to the same supernatant, prior to addition of oxytocin in the culture medium, abolished oxytocin responsiveness, had no effect on immunolocalization of actin and vimentin, but affected the localization of α-actinin within myoepithelial cells. Treatment of myoepithelial cells for 3 h with a combination of purified staphylococcal proteinases XVI and XVII-B abolished oxytocin responsiveness and mimicked the effect of the Staph, aureus culture supernatant. We conclude that Staph, aureus M60 culture supernatant affected proliferation and functional properties of myoepithelial cells.

The udder health of a research herd of between 160 and 220 Friesian cows run on a commercial basis has been monitored closely, including detailed bacteriological study, over 5 years. The five point mastitis control plan had been in use for several years prior to this study and was continued with minor alterations to the management of the plan, more detailed bacteriological monitoring and increased encouragement to apply it. It has proved possible to make a substantial improvement in the udder health of the herd. The percentage of infected cows fell from 21·9 to 12·0 and the percentage of infected quarters from 7·3 to 3·3. The main benefit has been a drastic reduction in the rate of clinical and subclinical mastitis caused by coagulase-positive staphylococci. However the total incidence of clinical mastitis did not change substantially, averaging around 30 cases/100 cows per year. This was largely because environmental mastitis organisms were responsible for 65 % of all clinical cases. The results showed marked differences in the patterns of infection due to the environmental mastitis pathogens, Gram-negative bacteria and aesculin-hydrolysing streptococci, suggesting different mechanisms of invasion of the gland.

Two experiments were conducted to test the hypothesis that responses of milk production to the abomasal infusion of casein or hydrolysates of casein can be influenced by the proportion of peptide-bound amino acids in the infusate. Eight lactating cows were used in two Latin square experiments with period lengths of 10 d. In Expt 1, the four cows were at a late stage of lactation and the four treatments were a basal diet of silage and a barley–soya supplement alone or with infusions into the abomasum of 180 g/d of sodium caseinate, an enzymic hydrolysate of casein or an acid hydrolysate of casein; all treatments supplied equivalent amounts of all the amino acids, this being achieved by addition of free amino acids as required. As infused, the three treatments contained 89 (caseinate), 40 (enzymic hydrolysate) and 15 (acid hydrolysate) % of their amino acids as peptides. The increase of milk production in response to infusion was small and there were no significant differences between the infusion treatments. In Expt 2, the cows received a basal diet of grass silage and a supplement containing feather meal as the main source of protein. Treatments were the basal diet alone and with three infusion treatments as in Expt 1, except that the amount infused was 230 g/d. The response to infusion was greater in this experiment, there were significant differences between infusions and the yield of milk protein with the infusion treatments was linearly related (P = 0·001) to the proportion of peptide-bound amino acids in the infusate. Moreover, there was a close inverse relation (P = 0·007) between the proportion of peptide-bound amino acids in the infusate and the concentration of total peptidebound amino acids in blood plasma.

Five British Saanen goats were milk sampled during the first 39 weeks of lactation to determine changes in casein composition. Caseins were separated by anion- and cation-exchange FPLC to determine the relative amounts of the individual caseins. Acid, alkaline and SDS-PAGE were used to determine possible genetic polymorphisms and observe any lactational changes. Total casein nitrogen was determined using a micro-Kjeldahl method and this allowed the concentrations of individual caseins to be calculated. The milk of one animal, which had the deduced genotype αs1-CnAB, showed higher concentrations of both total and αs1-casein. The remainder of the group were either heterozygous αs1-CnBE or, more probably, homozygous αs1-CnE and produced milk of a generally lower protein concentration. Both FPLC and PAGE results showed that the relative amounts and concentrations of αs2-casein decreased with stage of lactation, consistent with its susceptibility to proteolysis. The relative amounts of the breakdown products of plasmin attack on β-casein, γ-caseins, were highly negatively correlated with milk yield (r = –0·942, P < 0·001) in the declining phase of lactation, reflecting the gradual involution of the gland at this time. The relative amount of κ-casein increased by ∼ 50% after peak lactation and its concentration almost doubled near the end of lactation. These compositional changes may alter the processing qualities of goats' milk in relation to cheese production.

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.

Aseptic udder quarter secretion samples (n = 1112) from 200 mastitic and 65 non-mastitic control heifers from 160 different farms were collected for bacteriological examination and in vitro antimicrobial susceptibility testing. Coagulase-negative staphylococci (CNS) were the most frequently isolated bacteria (57·8%). The percentage of Staphylococcus aureus was 20·1, of streptococci 11·3 and of other pathogens 10·8. Staph. simulans, Staph. hyicus, Staph. xylosus and Staph. chromogenes were the most common CNS. Staph. simulans and Staph. hyicus were most frequently found in clinical samples after parturition, whereas other CNS were equally or more often found in control samples. Most CNS infections detected in the prepartum period were eliminated spontaneously or with antibiotic treatment during early lactation, but the infected quarters were more susceptible to new infection by other pathogens. Milk N-acetyl-β-glucosaminidase activities were measured to monitor changes in inflammation. Among the CNS infections, N-acetyl-β-glucosaminidase activities were highest for Staph. hyicus and lowest for Staph. xylosus in the lactating mammary gland of heifers. CNS were resistant to trimethoprim–sulphonamide, ampicillin and erythromycin. Staph. aureus was more resistant than CNS to β-lactamase-sensitive penicillins.

Peptides that inhibit calmodulin-dependent cyclic nucleotide phosphodiesterase were isolated from a pepsin digest of α-casein. Analysis of these peptides showed that they corresponded to the αs2-casein sequences 164–179 (Leu–Lys–Lys–Ile–Ser–Gln–Arg–Tyr–Gln–Lys–Phe–Ala–Leu–Pro–Gln–Tyr), 183–206 (Val–Tyr–Gln–His–Gln–Lys–Ala–Met–Lys–Pro–Trp–Ile–Gln–Pro–Lys–Thr–Lys–Val–Ile–Pro–Tyr–Val–Arg–Tyr) and 183–207 (C-terminus, Val–Tyr–Gln–His–Gln–Lys–Ala–Met–Lys–Pro–Trp–Ile–Gln–Pro–Lys–Thr–Lys–Val–Ile–Pro–Tyr–Val–Arg–Tyr–Leu). These peptides inhibited calmodulin-induced cyclic nucleotide phosphodiesterase activity over the range 1–50 μM without affecting the basal enzyme activity. These results demonstrated that the affinities of these peptides for calmodulin are comparable to the affinities of certain endogenous neurohormones and proteins that interact with calmodulin.