Inhibition of milk ejection in cows by oxytocin receptor blockade (Atosiban) and α-adrenergic receptor stimulation (phenylephrine) prior to prestimulation was compared with inhibition of milk ejection in unfamiliar surroundings. In addition, Atosiban and phenylephrine were administered after a 1 min prestimulation or 1 min after the start of milking. Oxytocin concentrations increased during milking in all treatments. The spontaneously removed milk fraction (before oxytocin was injected) was similar for Atosiban and phenylephrine treatments and in unfamiliar surroundings, but lower than in controls. Peak flow rates were similar in all treatments, but reduced as compared with controls when phenylephrine and Atosiban were administered before prestimulation. Peripheral (Atosiban, phenylephrine) and central (unfamiliar surroundings) inhibition of milk ejection reduced the amount of available milk similarly. Drug treatments resulted in similar peak flow rates; however, teats were contracted after phenylephrine administration but not after Atosiban. The inhibition induced by Atosiban could be abolished by oxytocin injection, but not that induced by phenylephrine, which was antagonized by α-adrenergic receptor blockade. These results indicate that inhibition of milk ejection through activation of α-adrenergic receptors is based on blockade of milk flow into the cistern, but not through the teats.

In a national survey of US dairy operations, 1685 dairy operations reported 47057 new dairy heifers (either births or acquisitions) and 4427 deaths (9·4%) of preweaned dairy heifer calves over a 3 month period. Stepwise logistic regression was used to identify management practices associated with high mortality among preweaned heifers in dairy operations where at least three dairy heifer calves were born alive or moved on to the operation. Analysis was done twice: once by separating all operations by size into high or low mortality; again using only operations with <2 and >10% mortality to eliminate dairy operations with intermediate levels of mortality from the comparisons. Results were similar. Dairy operations in the West were more likely to fall in the high mortality category than dairy operations in the rest of the country. In addition, the following dairy operation characteristics were associated with high death levels in both models: rolling herd average milk production <7710 kg, preweaned heifers placed in groups of seven or more, a male having primary responsibility for the care and feeding of preweaned heifers, calves not receiving hay or other roughages until >20 d old, calves fed on mastitic or antibiotic milk after colostrum and calves not given whole milk after colostrum.

Mammary cistern anatomy derived from ultrasound measurement, milk ejection in response to exogenous oxytocin and oxytocin release and milking characteristics with and without manual prestimulation in early (months 2–4) and late (months 5–8) lactation were investigated in Lacaune and Ostfriesian dairy ewes. Vertical ultrasound cisternal cross sections of the cisternal cavities did not differ in the two breeds, whereas the cisternal area fraction located lower than the exit into the teat channel was larger in Ostfriesian than in Lacaune sheep. The cisternal area enlarged within 1 min in response to i.v. oxytocin injection, indicating milk ejection. During milking, oxytocin concentrations in Lacaune generally increased dramatically within 0·5 min from the start of prestimulation or milking, whereas only slight or no oxytocin release was detected during milking without prestimulation in Ostfriesian ewes. Prestimulation induced oxytocin release in Ostfriesian sheep within 1–2 min after the start of milking, indicating delayed response to stimulation. Two peaked milk flow curves were observed when oxytocin release and milk ejection occurred only after removal of cisternal milk. This type of milk flow was more frequent in Ostfriesian than in Lacaune ewes and was reduced in both breeds by prestimulation, while the frequency of one peaked milk flow curves increased. During the course of lactation, milk yield, main milk fraction and milk flow rates decreased, while stripping yield was almost unchanged. Although milk yield was similar in both breeds, milk flow was lower and stripping yield was higher in Ostfriesian than in Lacaune ewes. We conclude that milk ejection in ewes occurred in response to elevated oxytocin concentrations. In Ostfriesian ewes reduced and delayed oxytocin response to teat stimulation resulted in milk ejection only during stripping. Therefore, and probably because a larger volume of the cistern was located below the teat exit in the Ostfriesian breed, stripping yield was higher in Ostfriesian than in Lacaune sheep.

Twelve multiparous British Friesian cows in early (40±23 d in milk; n=6) or late (216±17 d in milk; n=6) lactation were used to study the effects of milking frequency on yield, udder volume and milk storage within the udder. After a 2 week control period of twice daily milking, diagonally opposed udder halves within a cow were milked once or twice daily for 3 weeks. Milk yield was 28–38% lower from the halves that were milked once daily than from halves that were milked twice daily. The loss of milk yield, expressed as a decrease in the relative milk yield quotient (an index that accounts for pretreatment differences), was greater for cows in early than in late lactation (0·59 v. 0·68). Empty udder-half volume was not decreased by once daily milking, suggesting that no cell loss occurred. Instead, once daily milking reduced the secretion efficiency (units of milk per unit of empty udder-half volume) by 46 and 27% respectively in early and late lactation; thus, at least part of the loss was due to reduced metabolic activity of the mammary epithelium. There were positive correlations between the relative milk yield quotient and the proportion (r=0·804) or volume (r=0·644) of cisternal milk in the glands that were milked once daily. These results confirm that, during extended milking intervals, milk loss was smallest for cows that stored a larger proportion of milk in the gland cistern.

The effects of a daily supplement of 300 ml fish oil and 20 g rumen-protected methionine, alone or in combination, were investigated in mid-lactation cows receiving a maize silage-based diet ad lib. Fish oil significantly decreased feed intake, increased milk yield, decreased protein and casein concentrations, and especially fat concentration (by 13·1 g/kg) and output. Fat concentration decreased more in primiparous than in multiparous cows. Methionine supplementation increased protein and casein concentrations and outputs. No significant interaction between oil and methionine supplementation was found on milk composition. Treatments did not modify live weight or body condition changes, or lactose and non-casein nitrogenous compounds in milk. Oil plus methionine supplementation made it possible to decrease milk fat content without changing protein content.

Cortisol response to stress appears to differ between lactating and non- lactating animals. Lactating (14 d post partum) and non-lactating sheep were fitted with probes so that drugs and hormones could be infused directly into the posterior pituitary and paraventricular nucleus of the hypothalamus. The animals were also fitted with instruments to allow monitoring of heart rate, body temperature and blood cortisol levels. Their reactions to a source of acute stress (a barking dog) were then followed, with or without drug and hormone manipulation. Results in both lactating and non-lactating animals indicated shortcomings in the use of cortisol as a stress indicator. Infusing prolactin and oxytocin into either the posterior pituitary or the paraventricular nucleus of the hypothalamus suppressed cortisol responsiveness to stress in both lactating and non-lactating animals (the latter to a greater extent). In the absence of drugs, lactating animals had a slightly higher basal level of cortisol and a lower cortisol response to stress than their non-lactating counterparts. Despite suppression of cortisol responses, with or without drugs, other indicators of stress still changed with the presence of a barking dog, suggesting the complexity of control involved in stress responses.

The Dairy Herd Improvement Fund of Victoria recently identified a requirement for a simple and inexpensive in-line sampler to enable dairy farmers to collect representative milk samples for counting somatic cells. We found that the currently available simple in-line milk samplers, when connected to standard 35 ml collection vessels, terminate sampling early in a milking, and thus provide samples that are unrepresentative of the whole milking. We showed that cell count during a milking varies greatly, tending to be high for the first 1–2 l. Analyses of resulting samples will thus tend to overestimate cell counts if samplers are used in their traditional way. We found greater sampling rates in high-line compared with low-line milking systems, and consequently developed modified samplers suitable for both situations. Our samplers utilize low sampling rates (∼1–3%) and large collection vessels (450 ml). Compared with currently available simple in-line samplers, our type of sampler provided milk samples considerably more representative of the entire milking for the majority of cows. In conjunction with subsampling, they provided samples of appropriate size (12·5 ml minimum to 25 ml maximum) for testing fat, protein, lactose and cell count. Cell count results indicated that errors associated with the use of currently available simple in-line samplers could frequently be >200%. In contrast, we found that use of our samplers gave an estimate for cell count that was only slightly higher (mean 20%) than that from samples collected by an approved Tru-Test sampler.

The effects of addition of EDTA on the dissociation of caseins and foaming properties of milks (100 g solids/l) reconstituted from skim milk powders given a low-heat (72°C for 30 s) or high-heat (85°C for 30 min) treatment during powder manufacture were determined. The EDTA-induced dissociation of caseins was independent of heat treatment but in high-heat milk was accompanied by release of denatured whey proteins. EDTA changed the proportions of individual caseins in the supernatant. EDTA addition improved both foam overrun and foam stability of low- and high-heat milks. The increase in serum protein on addition of EDTA contributed to the improvement in foaming properties of milks by increasing the availability of the proteins for formation of the air–water interface.

Four mid-lactation Holstein dairy cows (mean milk yield on day of experiments 26·1 kg/d) were used in a series of experiments to establish the contribution of non-insulin-mediated glucose uptake to total glucose uptake at basal insulin concentrations. A secondary objective was to determine whether somatostatin affects the action of infused insulin. In part I of the experiment a primed continuous infusion of [6,6-2H]glucose (45·2 μg/kg per min) was begun at time 0 and continued for 5 h. After 3 h of [6,6-2H]glucose infusion (basal period) a primed continuous infusion of insulin (0·001 i.u./kg per min) was administered for 2 h. Coincident with the insulin infusion, normal glucose was also infused in order to maintain the plasma glucose concentration at euglycaemia. Part II of the experiment was the same as part I except that somatostatin was infused for 2 h (0·333 μg/kg per min) instead of insulin. In part III of the experiment both insulin and somatostatin were infused for the final 2 h. Plasma insulin levels were increased by insulin infusion (to 0·1476 and 0·1290 i.u./l for parts I and III respectively) and were reduced by somatostatin infusion in part II (to 0·006 i.u./l) relative to the basal periods (mean 0·021 i.u./l). Glucose uptake during somatostatin infusion (2·50 mg/kg per min; part II) was 92·0% of that observed in the respective basal period (2·72 mg/kg per min). Circulating insulin levels were much lower than the dose of insulin that causes a half maximal effect on glucose uptake (0·06–0·10 i.u./l for ruminants); consequently insulin-mediated glucose uptake was probably absent in part II. Secondly, glucose uptake following insulin only infusion (4·05 mg/kg per min) was significantly lower than that observed when insulin plus somatostatin was infused (4·69 mg/kg per min), indicating that somatostatin either directly or indirectly enhanced the action of insulin on glucose uptake.

In an approach to develop a commercial-scale process for the production of casein phosphopeptides containing the cluster sequence −SerP−SerP−SerP−Glu−Glu–, we have studied the relationship between casein hydrolysis and phosphopeptide release. The degrees of hydrolysis (DH) of casein using Novo trypsin PTN 3.0 S and pancreatin 4NF independently, at enzyme to substrate (E[ratio ]S) ratios of 1[ratio ]50–1[ratio ]1600 (by weight), were determined using the pH-stat method. Casein phosphopeptides (CPP) were selectively precipitated using Ca2+ and ethanol from the acid-clarified hydrolysates. The precipitates were analysed by high performance capillary electrophoresis to calculate individual phosphopeptide yields based on extinction coefficients of the purified peptides. Individual peptides were purified by reversed-phase HPLC and anion-exchange FPLC4 and characterized by MALDI-TOF mass spectrometry and amino acid sequence analysis. For both enzymes, lowering the E[ratio ]S ratio resulted in reductions in the DH and the release of the CPP, and an increase in peptide chain length. The longer chain length offset the reduction in release such that the gravimetric yields of CPP preparations remained relatively constant. For Novo trypsin the highest yields of the major cluster peptides (β-casein(CN)f(1–25), αs1-CNf(59–79), αs2-CNf(1–21), αs2-CNf(46–70) and related peptides) in the selective precipitates were obtained at a casein DH of 17%. At lower DH values (9–15%), there was a decrease in yield of the peptides derived from αs1-CN and αs2-CN while the yield of the β-CN-derived cluster peptides remained relatively constant. The CPP produced using pancreatin were found to be truncated at all E[ratio ]S ratios, relative to the tryptic CPP, owing to higher levels of chymotryptic and carboxypeptidase activities in pancreatin. The highest yields of the truncated forms of the major cluster peptides using pancreatin were obtained at a casein DH of 19–23%.

Danish organic dairy production is characterized by a low input of antibiotics for udder treatment and a high input of other mastitis control procedures. A study was conducted in 14 organic dairy herds with the objectives of obtaining a comprehensive description of clinical mastitis cases and identifying characteristic patterns in these results. Clinical signs, inflammatory reactions and microbiological identifications were obtained from 367 cases of clinical mastitis occurring over 18 months. Cow characteristics and preincident values such as milk yield and somatic cell count were obtained for each cow. Signs of previous udder inflammation were present in two-thirds of the clinical mastitis cases. Severe local inflammatory reactions were found in 21% of the cases and some indication of generalized signs such as fever and reduced appetite were found in 35% of the cases. Logistic regression analyses were performed based on the results of an initial (exploratory) multiple correspondence analysis. Coliform mastitis (6% of the cases) was rarely preceded by pathogen isolation or inflammatory reactions in the same quarter. Coliform mastitis cases usually occurred in one quarter only. Escherichia coli infections were typically (truly) acute cases. Bacteriologically negative mastitis (20% of the cases) showed strong similarities with clinical coliform mastitis. Staphylococcus aureus cases (18% of the cases) occurred most frequently in late lactation or around drying-off. Prior isolation of Staph. aureus and slight decreases in milk yield were two factors that interacted but both were strongly and positively related to clinical Staph. aureus. Staph. aureus mastitis typically had a subclinical debut, and increasing degrees and duration of inflammation decreased shedding of this pathogen. Streptococcus dysgalactiae (9% of the cases) mastitis was typically persistent, virulent and manifest in periods of lower cow resistance. More patterns of subclinical and clinical Str. uberis mastitis (23% of the cases) seemed to be present.

Dairy cows fitted with ruminal, duodenal and ileal cannulas were utilized to investigate the effects of feeding with Ca soaps (CaS) of palm fatty acids (FA) and rapeseed FA. Diets compared were control diet based on maize silage and concentrate, and two diets with 40 g CaS of palm oil FA or rapeseed oil FA/kg diet, replacing part of the concentrates of the control diet. Total digestibilities of dry matter, fibre and fat, and ruminal fermentation were not significantly altered by giving CaS; the extent of ruminal biohydrogenation of total unsaturated C18 FA was significantly reduced by both CaS diets. Apparent intestinal digestibility of FA was not different among diets, although the amount of FA absorbed with the CaS diets was twice that with the control diet. No difference among diets was observed for milk production, or fat and protein contents. Giving CaS diets decreased the proportions of 4[ratio ]0 to 14[ratio ]0 FA in milk fat, and increased cis-18[ratio ]1n−9, compared with control diet. The rapeseed diet lowered the content of 16[ratio ]0, and increased the contents of 18[ratio ]0 and trans-18[ratio ]1n−7. CaS diets did not result in a marked increase of polyunsaturated FA content in milk fat. Butter from cows fed on the CaS diets contained more liquid fat at 6 and 14°C than butter from the cows fed on the control diet. Incorporating CaS, particularly those from rapeseed, in dairy cows' diets increased C18 FA in milk and improved butter spreadability.

Polymorphism of caprine milk proteins was studied by capillary electrophoresis. Identification of casein (CN) fractions was effected by using isolated fractions from cation-exchange fast protein liquid chromatography. Genetic polymorphisms in caprine αs2-CN, αs1-CN, β-CN and κ-CN have been determined. κ-CN A and B, β-CN A and null, αs2-CN A, B and C, αs1-CN A, B, C and null, and other forms with intermediate and low αs1-CN content have been identified. The capillary electrophoresis method made it possible to analyse whole caprine milk using simple sample preparation and was rapid, automated and suitable for phenotyping studies. This method may also permit the quantitative study of different protein fractions.

The distribution pattern of myoepithelial cells in the bovine mammary gland was investigated by an immunohistochemical technique, using monoclonal antibodies against cytokeratins 5, 6 and 18 and cytokeratins 8 and 14 and against α-smooth-muscle actin filaments. Myoepithelial cells were shown to be present as a continuous basal cell layer in the intralobular ducts, as discontinuous cell rows in the basal cell layer of the interlobular ducts, and as single cells dispersed in the basal cell layer of the quarter cisterns, while they were apparently absent in the teat cisterns. Unlike the case with myoepithelial cells of the human breast, anti-cytokeratin 14 was less specific as a marker of bovine myoepithelial cells than was anti-α-smooth-muscle actin.

The physicochemical characteristics and the renneting properties of milks from Lacaune ewes are reported. The ewes belonged to divergent genetic lines termed ‘low’ or ‘high’ and were selected on a combination of fat and protein yield. The 179 second to fifth parity ewes were sampled three times during lactation at about 50, 135 and 175 d in milk. Physicochemical and renneting variables were principally influenced by days in milk and somatic cell count, whereas β-lactoglobulin genotype, parity and divergent lines had relatively few effects. Fat and protein content increased throughout lactation, whereas rennet gel firming rate decreased. Salt distribution and micellar characteristics were also significantly affected by days in milk. Milks with high somatic cell count were characterized by high values of pH and soluble nitrogen content and rennet coagulation properties were significantly affected at >3×105−5×105 cells/ml milk. Divergent lines had a strong effect on milk yield, but no effect on milk composition consistent with the selection criteria. Gel firming rate was slightly higher in milks from high yield ewes. Protein content, Ca concentration, pH and micelle characteristics were the main factors related to variations in the renneting properties of the milks.

The transepithelial potential difference (TEPD) and resistance (TER) in the alveoli, and the pressure in the mammary gland were measured in lactating mice at different times after they had finished suckling their young. Oxytocin administration caused a rise in TEPD, the amplitude of which decreased with time after cessation of the pups feeding. At 2·5 h after cessation, oxytocin administration caused a short-term decrease in TER, but at 5 h there was a two phase reaction: a fall in TER followed by a rise. At 20 h there was a one phase reaction, a rise in TER. TEPD in the mammary gland alveoli increased from its initial value of 18±1 mV to 25±1 mV at 2·5 h, and thereafter fell to zero by 15 h after suckling. The initial value of TER, 114±1 kΩ, increased to 131±4 kΩ at 2·5 h and then decreased by 5 h after suckling, but unlike TEPD it did not reach zero. These experiments show that extending the period after cessation of suckling does not cause a rise in intramammary pressure and that oxytocin injections cause a short-term rise in milk pressure. From these results, we propose a hypothesis of reducing ionic concentration gradients between intracellular fluid, cytoplasm of the secretory cell and milk at extended periods after cessation of suckling, starting at 2·5 h.

Polymorphism of ovine αs1-caseins was studied by capillary electrophoresis at pH 3·0±0·1. Individual caseins (CN) were selected according to their genetic variants, as determined by PAGE and isoelectric focusing. The ovine caseins, containing different genetic variants of αs1-CN and αs2-CN, were fractionated by cation-exchange FPLC®. The αs1-CN variants A, B and C, and the fast moving αs2-CN variant were identified by a capillary electrophoresis method. The fast moving αs2-CN variant, so-called for its behaviour in PAGE, also had a faster electrophoretic mobility than the common αs2-CN when analysed by the present technique. The capillary electrophoresis method gave excellent, rapid, automated separation of αs1-CN and αs2-CN variants and was suitable for screening studies.

The lactational variation in milk protein composition and renneting properties and their relationship to the cow's body condition at calving were investigated in 39 Danish Holstein first lactation cows fed on a well balanced standard diet. All milk characteristics measured were significantly affected by stage of lactation (P<0·01). Casein as a proportion of total milk nitrogen reached a maximum in mid lactation. The proportion of αs- and κ-casein in total casein decreased and the proportion of β-casein increased systematically during lactation while the proportion of γ-casein was lowest in mid lactation. The α-lactalbumin content of milk and its proportion of total whey proteins decreased during lactation. Renneting time was highest and curd firmness lowest in mid lactation. These results appeared to reflect a low degree of proteolysis in late-lactation milks compared with several other investigations, probably because of the good nutritional state of the cows. The body condition at calving affected proteolysis and the renneting properties of milk. A good body condition increased the content of whey protein in total milk nitrogen and of γ-casein in total caseins (P<0·05); in addition, curd firmness was improved (P<0·01) and aggregation time was reduced (P<0·05). We suggest that these effects were related to the fat metabolism and energy status of the cows during lactation. The interrelationships between the milk characteristics were evaluated by factor analysis to support the interpretation.

The aim of this work was to determine by ion-exchange liquid chromatography and isotope ratio mass spectrometry the specific 15N enrichment of amino acids in casein and plasma proteins in cows receiving three successive daily oral doses (300, 150 and 150 g) of (15NH4)2SO4 (10 atom per cent isotopic enrichment) and to examine the 15N enrichments obtained with regard to nitrogen transport and metabolism in the lactating cow. To investigate the 15N distribution in amino acids in casein and in plasma proteins, samples of 15N-labelled casein and plasma proteins were extracted either from a pool of several milkings (36–96 h after starting to administer the tracer) or from pooled venous blood (removed on the fourth day after the start of administration) from the four lactating cows. 15N enrichments of the proteins studied, expressed as atoms percent excess, were 0·2509 for casein and 0·0577 for plasma protein. Chromatographic fractionation of the amino acid mixture (protein hydrolysates) resulted in nine groups containing between one and four amino acids: Asp, Ser and Thr; Glu; Pro; Gly, Ala, Val and Met; Ileu and Leu; Tyr; Phe; His and Lys; and Arg. High 15N incorporation was demonstrated in all individual or groups of amino acids studied. In both proteins, Glu appeared to be the most enriched amino acid, Phe and Arg the least enriched. Most aliphatic molecules with a single amino group were highly enriched. The much lower (3·5–7·7-fold) enrichments in plasma protein compared with casein suggest considerable intracellular dilution at the site of liver protein synthesis. Finally, the amino acid separation methods are discussed and suggestions for improving them considered.

The importance of milk proteins and the positive effect of administration of growth hormone (GH) on milk production, and the presence in some dairy cattle lines of greater GH concentrations prompted us to examine the presence of restriction fragment length polymorphism at the GH gene using the restriction enzyme TaqI and to investigate associations between this polymorphism in Simmental cows and bulls, as well as milk protein variants in Simmental cows, and milk production traits. Blood and milk were sampled from 279 Italian Simmental cows and semen was collected from 148 bulls of the same breed. Two fragment bands, denoted A and B, of 6200 and 5200 bp respectively, were examined and three patterns, AA, AB and BB, were found in both animal samples. All variants previously reported in other studies, for κ, β, and αs1-caseins, and β-lactoglobulin, were found in the cows' samples. For the cows' samples, a BLUP (Best Linear Unbiased Predictor) analysis of results was performed using a REML (Restricted Maximum Likelihood) program and known heritabilities, whereas for bulls we have performed a General Linear Model analysis. The effect of GH gene polymorphism, using TaqI restriction enzyme, on milk production traits was not significant, but bulls of BB pattern had a higher breeding value for milk yield than AA bulls (P<0·05). For the κ-casein genotypic effects, cows of AB genotype gave milk with 1·53±0·70 g/kg less fat than cows of AA genotype. In addition, breeding values for milk protein content were significantly higher in BB bulls, with 0·87±0·32 and 0·71±0·34 g/kg more milk protein than AA and AB bulls respectively. Thus, our results revealed a GH gene polymorphism and indicated significant effects of milk protein polymorphisms on milk production traits in the Italian Simmental breed.