Mice in mid lactation (n=6 per group) were fed a control diet (1·19% fat), or diets containing safflower oil (25% w/w) or olive oil (25% w/w) for 7 d. Mammary and liver stearoyl CoA desaturase (SCD) mRNA levels and mammary SCD activities were higher in lactating mice fed the control diet than in those fed the oil-supplemented diets. Further, mammary SCD mRNA was directly related to mammary SCD activity. Milk fat composition was influenced by dietary fat composition. The olive oil diet, high in 18[ratio ]1, led to high levels of this fatty acid in milk and the safflower oil diet, high in 18[ratio ]2, resulted in a milk fat with high levels of 18[ratio ]2. These results show that there is regulation of SCD at the transcriptional level, associated with changes in enzyme activity and in milk fat composition.

The objective was to measure the activities of all the enzymes essential for hepatic gluconeogenesis in dairy cows with induced fatty liver. We aimed to induce severe fatty liver in ten experimental cows by overfeeding them during the dry period while seven control cows were maintained on a restricted diet. To induce a marked negative energy balance, the experimental cows were deprived of feed for 8 h immediately after parturition. In addition, the experimental cows were given a restricted amount of diet during the first 5 d of lactation. Liver samples were collected 1 week before and 1, 2 and 4 weeks after parturition. Before parturition, liver triacylglycerol concentrations did not differ between the two groups. After parturition, the experimental cows developed marked fatty liver as indicated by a higher level of triacylglycerols in the liver compared with the control cows.

Before parturition, all gluconeogenic enzymes in the liver were lower in experimental cows than in control cows. Phosphoenolpyruvate carboxykinase, pyruvate carboxylase and propionyl-CoA carboxylase were significantly lower and fructose 1,6-bisphosphatase and glucose 6-phosphatase tended to be lower in the experimental cows. The activities of two crucial enzymes for gluconeogenesis in ruminants, i.e., phosphoenolpyruvate carboxykinase and propionyl-CoA carboxylase, remained low throughout the sampling period post partum. Activities of pyruvate carboxylase and glucose 6-phosphatase in the experimental cows post partum were upgraded to values similar to those of the control cows. The results showed that the capacity for hepatic gluconeogenesis before parturition was lower in cows with induced fatty liver than in control cows. After parturition, the low activities of crucial gluconeogenic enzymes indicated insufficient production of glucose. It is suggested that the low gluconeogenic capacity leads successively to low blood glucose concentrations, low insulin levels and high rates of mobilization of fatty acid, causing severe hepatic lipidosis.

An increase in the proportion of unsaturated fatty acids in milk is considered desirable for human health. A prerequisite for the manipulation of milk fat composition is a co-ordinated understanding of the complex interactions in its biosynthesis. It has been suggested that an increase in the expression of mammary stearoyl-CoA-desaturase (SCD) would enrich mono-unsaturated fatty acids in milk, and therefore improve its nutritional properties. To investigate the potential effects of changes in expression of mammary enzymes and substrate availability on milk fat composition, we constructed, parameterized and evaluated a mechanistic mathematical model of fatty acid biosynthesis and milk-fat triglyceride assembly. The objective was to describe changes in the amount and composition of milk fat produced by bovine mammary cells due to changes in nutrition. Using the model we found that a 50% up-regulation in SCD activity increased the molar fraction of milk triglyceride 18[ratio ]1 from 0·30 to 0·33 and 16[ratio ]1 from 0·04 to 0·06. Up-regulation of SCD therefore did not appear to be the optimal method for increasing the content of unsaturated fatty acids in milk fat. The model was also used to determine the likely rate-limiting processes for the incorporation of unsaturated fatty acids into milk fat. Halving the concentration of glycerol 3-phosphate increased the molar fraction of milk triglyceride 18[ratio ]1 from 0·30 to 0·35 and decreased the molar fraction of milk triglyceride 16[ratio ]0 from 0·30 to 0·22. This achieved the desirable outcome of producing more unsaturated low-fat milk. Our model also predicted that a K232A mutation in the bovine mammary DGAT1 gene that is linked with an increase in milk fat yield would be consistent with a 120% increase in the DGAT acylation rate and also would be associated with a decrease in milk mono-unsaturated fatty acids.

We partially sequenced the mitochondrial D-loop region in 47 individuals from eleven Spanish and foreign goat breeds. Phylogenetic analysis of these sequences allowed us to identify a particular D-loop haplotype shared by individuals from the Palmera, Majorera and Tinerfeña Canarian breeds. Genotyping of 281 goats from 17 different breeds by PCR-HpaII RFLP evidenced that the geographical distribution of this haplotype is restricted to the Canary Islands. This ancestral mitochondrial haplotype might originate in the domestic goat herds brought by the native Canarian inhabitants approximately 3000 years ago. Although we observed other miscellaneous D-loop haplotypes in the Palmera, Majorera and Tinerfeña breeds, any of them allowed us to group individuals from these three populations in a single cluster, a feature that suggests that these haplotypes might have diverse origins. The remarkable degree of phylogeographic structure of the Canary goat breeds with regard to other Spanish and foreign populations might be attributed to the isolation of these breeds in the Canary Islands for approximately 2500 years, without exposure to the migratory movements and commercial trading events that probably affected the genesis of most domestic goat breeds worldwide. The Canarian D-loop haplotype can be efficiently genotyped by using DNA isolated from milk and cheese samples, which paves the way for the future establishment of a Canary breed identity test for these dairy products.

Mammary epithelial cells, which express and secrete leptin into milk, accumulate triacylglycerol (TAG). We examined effects on the accumulation of cytosolic TAG of addition of short- (acetate and butyrate) or medium- (octanoate) chain fatty acids to the medium bathing bovine mammary epithelial cells (bMEC). Octanoate stimulated the accumulation of TAG in a concentration-dependent manner from 1 to 10 mM and increased lipid droplet formation and mRNA expression of CD36 (a fatty acid translocase). Additionally, expression of a peroxisome proliferator activated receptor (PPAR) γ 2 protein that is a lipid-activated transcription factor, was increased by the addition of acetate or octanoate. However, leptin mRNA expression was significantly reduced by addition of acetate or butyrate. Both short- and medium-chain fatty acids inhibited acetyl coenzyme A carboxylase (ACC) activities, which is pivotal in lipid synthesis, but elevated expression of uncoupling protein 2 (UCP2) mRNA, which is important in energy expenditure. These results suggest that octanoate induces cytosolic TAG accumulation and the formation of lipid droplets, and that acetate and butyrate inhibit leptin expression and lipid synthesis in bMEC.

Xanthine oxidoreductase (XOR) was purified from goats' milk. The u.v.–visible absorption spectrum was essentially identical to those of the corresponding bovine and human milk enzymes and showed an A280/A450 ratio of 5·20±0·12, indicating a high degree of purity. Like bovine and human milk XORs, enzyme purified from goats' milk showed a single band on SDS-PAGE corresponding to a subunit with approximate Mr 150000. On Western blotting, mouse monoclonal anti-human XOR antibody cross-reacted with purified caprine and bovine XORs. The specific xanthine oxidase activity of goats' milk XOR, however, was very much lower than that of bovine XOR, although NADH oxidase activities of XOR from the two sources were similar. In these respects, the caprine milk XOR mirrors the human milk enzyme, in which case the kinetic effects have previously been attributed to relatively low molybdenum content. The molybdenum content of goats' milk XOR also was shown to be relatively low, with 0·09 atoms Mo per subunit, compared with 0·55 atoms Mo per subunit for the bovine enzyme. A parallel purification of human milk XOR showed 0·03 atoms Mo per subunit. The possible physiological significance of the low molybdenum content of the caprine milk enzyme and of its correspondingly low enzymic activity is discussed.

A method for the collection of mammary biopsies developed previously was refined and used to study the endocrine regulation of bovine milk protein gene expression. Our surgical biopsy method used real-time ultrasound imaging and epidural analgesia to enable recovery of a sufficient quantity of mammary tissue from late-pregnant dairy cows for explant culture in vitro. The time of biopsy was critical for prolactin-dependent induction of milk protein gene expression in mammary explants, as only mammary tissue from cows nearing 30 d prepartum was hormone-responsive. This suggests that during the later stages of pregnancy a change in the responsiveness of milk protein gene expression to endocrine stimuli occurred in preparation for lactation. This may relate to the diminution of a putative population of undifferentiated cells that were still responsive to prolactin. Alternatively, the metabolic activity of the tissue had increased to the level whereby the response of the tissue was no longer assessable using this model in vitro.

We examined the physical and chemical changes in milk during early lactation, and how these changes were affected by leaving one quarter unmilked in either the first or second milking, with the purpose of discriminating between colostrum and normal milk. Milk samples were collected from each quarter of 17 cows during the first 5 d after calving and then after about 7 d and 14 d. Samples were analysed for somatic cell count (SCC), fat, protein, casein, lactose, IgG1, colour, plasmin, pH and coagulation properties. Large variations occurred in both chemical and physical properties throughout the study period. Within six milkings, the concentration of casein decreased by 60%, IgG1 by 94%, and lactose increased by 34%. At milking number 6, rennet coagulation time was lowest and curd firmness was highest. The pH increased from 6·4 to 6·7 over the period of the experiment, and the colour changed from yellow (reddish) to white. Coagulation properties and the pH fell within the range of normal milk after five milkings. Measurement of colour and density appeared to be a potential method for detection of milk unsuitable for the dairy factory. Effects of omitting one quarter in one milking differed between milk components, but seemed to be of little importance to the physical properties.

A differential allele-specific accumulation of κ-casein mRNA that is not linked to the κ-casein protein variants is described in Holstein cows. Actually, cows genotyped κ-casein AB were a mixed population. For the first group of κ-casein AB cows, allele A-specific κ-casein mRNA contents within mammary epithelial cells were lower than the allele B-specific ones (cows LH), suggesting that the allele A-specific κ-casein gene was expressed with lower efficiency in mRNA. For the other group of κ-casein AB cows, allele A- and B-specific κ-casein mRNA accumulated to a similar level within mammary epithelial cells (cows HH). The objective of this study was to determine whether the accumulation of allele-specific κ-casein mRNA remained constant throughout lactation for the two groups of cows. Quantitative RT-PCR was used to monitor Holstein cows κ-casein AB genotyped HH and LH throughout lactation for the proportion of allele B-specific mRNA accumulation relative to the total κ-casein encoded mRNA within mammary epithelial cells: RNA was extracted from milk somatic cells known to contain a small proportion of mammary epithelial cells. Mean values of allele B-specific mRNA content were 50·6±0·5 and 54·0±0·9%, for cows HH and cows LH, respectively, and did not vary during lactation (P>0·10). This suggests that the phenotypic expression of the genetic mutation that causes the differential allele-specific accumulation of κ-casein mRNA was not affected by physiological and environmental factors, which tend to vary considerably throughout lactation.

Three galactopoietic stimuli, frequent milking (4X), bovine somatotrophin (bST) and thyroxine (T4) were used in an additive stair-step design to achieve maximum output (metabolic capacity) in six peak-lactation cows of high genetic merit (HT) and six of low genetic merit (LT). A further six of each merit were untreated controls (HC, LC). Milk yield was increased significantly by 4X, increased further by the combination of 4X and bST and increased further still and significantly by the full combination of 4X, bST and T4. The magnitude of the yield response to the sequence of treatments did not differ significantly between HT and LT. The yield response to 4X and bST was sustainable without significant loss of body weight or body condition score for the 6 weeks during which these stimuli were administered. The response to the full combination, which included T4, was accompanied by significantly elevated heart rate and significant loss of body weight and condition compared with the combination of 4X and bST. As a result, treatments were discontinued, on an individual cow basis, before completion of this 6-week phase. Time on experiment did not differ between HT and LT. The results do not support the commonly held belief that selective breeding of dairy cows for high milk production has rendered them markedly more susceptible to metabolic disturbances.

A novel electrophoretic α-lactalbumin (α-la) variant was detected in the Italian water buffalo breed. The isoelectric point of the variant, labelled A, was lower than the most frequent variant B. It presented an allelic frequency of 0·5% compared with the 97·1% of the BB allele. From Liquid Chromatography-Electrospray Ionization/Mass spectrometry, the molecular mass of the two α-la A and B variants were measured as 14235·1±0·8 and 14236·1±0·9 Da, respectively. The two proteins were sequenced and differentiated from one another by a single amino acid substitution, Asn45(B)→Asp45(A). As this amino acid substitution altered the N-glycosylation sequence consensus Asn45–X–Ser46 it may be deduced that the protein glycosylation level of the α-la A would decrease.

Fifty-six Holstein dairy cows from a commercial dairy herd in the Northern part of Greece were used to determine the effect of vitamin E supplementation on immune parameters, milk composition and milk quality. Cows were assigned to one of two experimental groups: control (no vitamin E supplementation) and vitamin E supplementation. Supplementation of vitamin E started 4 weeks prior to and continued up to 12 weeks after parturition. Supplementation included daily oral administration of vitamin E at 3000 i.u./cow prepartum and was reduced to 1000 i.u./cow post partum. Blood samples were collected weekly for 8 weeks starting 4 weeks before parturition, neutrophils were isolated and the following parameters were determined in neutrophils activated by phorbol myristate acetate: total cell-associated and membrane-bound urokinase plasminogen activator (u-PA) activity and superoxide production. Milk samples were collected weekly and fat, protein, lactose, somatic cell count (SCC), plasmin and plasminogen-derived activity were determined. Activated neutrophils isolated from cows that received supplemental vitamin E had higher (P<0·01) total and membrane-bound u-PA activities during the first 3 weeks after parturition and higher (P<0·01) superoxide production during week 1 prepartum and week 1 post partum compared with the corresponding values of activated neutrophils isolated from control cows. Vitamin E supplementation had no effect (P=0·28) on plasminogen-derived activity in milk. Milk obtained from cows that received supplemental vitamin E had SCC lower by 25% (P<0·05) and plasmin lower by 30% (P<0·01) than corresponding values in milk obtained from control cows. The reduction in plasmin as a result of vitamin E supplementation is very beneficial to the dairy industry because plasmin reduces the cheese-yielding capacity of milk, affects the coagulating properties of milk and its overall ability to withstand processing during cheesemaking. In conclusion, vitamin E supplementation had positive effects on the function of bovine neutrophils and milk quality in a commercial dairy herd.

Economic decisions on animal health strategies address the cost-benefit aspect along with animal welfare and public health concerns. Decision tree analysis at an individual cow level highlighted that there is little economic difference between the use of either dry cow antibiotic or an internal teat sealant in preventing a new intramammary infection in a cow free of infection in all quarters of the mammary gland at drying off. However, a potential net loss of over £20 per cow might occur if the uninfected cow was left untreated. The only economically viable option, for a cow with one or more quarters infected at drying off, is antibiotic treatment, although a loss might still be incurred depending on the pathogen concerned and the cure rates achievable. There was a net loss for cows with quarters infected with Corynebacterium spp. at drying off, for both the teat sealant and untreated groups (£22 and £48, respectively) with only antibiotic-treated cows showing a gain.

We examined the effects of administering recombinant bovine cytokines to non-lactating dairy cows and measured mammary gland leucocytes and the involution process. After the final milking, groups of cows were given an intramammary infusion of cytokine in two quarters. These cytokines were recombinant bovine interleukin-2 (rboIL-2) (2×105 units, n=6), recombinant bovine granulocyte-macrophage colony stimulating factor (rboGM-CSF) (500 μg, n=4) and recombinant bovine interleukin-1β (rboIL-1β) (10 μg, n=10). Each animal also received an infusion of phosphate-buffered saline (PBS) in the other two quarters as controls. The rboIL-2 and rboGM-CSF were produced in a yeast expression system, while rboIL-1β was produced in Escherichia coli. Leucocyte numbers, bactericidal activity of leucocytes, and concentrations of citrate and lactoferrin in quarter secretion samples were monitored after infusion of cytokine or PBS. Infusion of rboIL-2 had minimal effect on leucocyte numbers and concentrations of citrate and lactoferrin. Both rboGM-CSF and rboIL-1β induced a rapid increase in the number of neutrophils and macrophages compared with control PBS quarters. Concentrations of lactoferrin in secretions were increased by rboGM-CSF and rboIL-1β compared with control PBS quarters. In addition, infusion of glands with rboIL-1β lowered the citrate[ratio ]lactoferrin molar ratio compared with PBS control quarters. The results indicate that intramammary infusion of either rboGM-CSF or rboIL-1β at cessation of milking immediately increased the number of phagocytic cells in the gland. These cytokines, in particular rboIL-1β, also increased the rate of mammary gland involution during the early dry period.

We determined the effects of feeding canola oil or infusing it into the abomasum on rumen fermentation, nutrient digestibility, duodenal flows of fatty acids, and milk composition in Holstein cows. Five ruminally and duodenally cannulated Holstein cows in late lactation were used in a 3×5 incomplete Latin square design. Treatments were 1) Control: basal diet (CON), 2) Control+supplementation of canola oil at 1 kg/d in the feed (FED), and 3) Control+abomasal infusion of canola oil at 1 kg/d (INF). Compared with CON, feed intake, ruminal fermentation characteristics, ruminal and total tract digestibilities of nutrients were not significantly affected by FED treatment but duodenal flows and milk concentrations of fatty acids (FA) such as trans-11 18[ratio ]1 and cis-9 trans-11 18[ratio ]2 (conjugated linoleic acid, CLA) were increased. In contrast to the effects of FED, INF reduced feed intake, total VFA production, intestinal flows of nutrients, FA digestibility and yields of milk and milk fat. Both FED and INF significantly reduced the proportions of saturated and medium-chain FA, and increased cis 18[ratio ]1 in milk. Concentrations of 18[ratio ]2n-6 and 18[ratio ]3n-3 in milk were increased nearly 2-fold with INF relative to CON. Dietary or postruminal supplementation of canola oil to late-lactation cows reduced saturated FA and increased unsaturated C18 in milk but nutrient digestion was adversely affected with abomasal infusion of canola oil.

Cell proliferation and apoptosis were measured in bovine mammary tissue around the time of peak milk production in three heifers, and compared with changes in the expression of bcl-2-related intracellular signals and apoptosis-inducing factor (AIF). Cell proliferation and apoptosis were relatively constant during the study period with no significant change in the incidence of either event. However, the ratio of apoptotic to proliferating cells tended to change from 0·99 on day 45 of lactation to 1·82 on day 63 (P=0·064), suggesting that in the course of the study, a dynamic balance may have been succeeded by net cell loss. Average milk production recorded 6 d after biopsy was correlated with the estimated number of cells (r=0·898; P<0·01) but not with the apoptosis to proliferation ratio (r=−0·224, P>0·05). Turnover of the cell population was associated with relatively constant expression of anti-apoptotic bcl-2. Competitive PCR also indicated expression of bax, in contrast to observations in lactating rodent mammary tissue. Bax expression was relatively low compared with that of bcl-2, but immunohistochemical staining for bax protein, which was not detectable on day 45, was observed on day 53 and, more intensely, on day 60 when the protein appeared to be membrane-associated. A partial coding sequence for bovine AIF was identified and AIF expression was evaluated by in situ hybridization. The results indicated that AIF was expressed in luminal alveolar cells and that, in concert with a change in bax to bcl-2 ratio, they might contribute to signalling of a change in the dynamic balance of the cell population as lactation progresses.

The internal arrangement of the mammary gland cavity system, cisternal and alveolar milk fractions and the characteristics of milk ejection were investigated in buffaloes. Twenty-four Murrah buffaloes in three different stages of lactation and of two age groups were used. Continuous ultrasound cross-sections during milk ejection induced by exogenous oxytocin were performed to record the latency period of milk ejection. Buffaloes had small cisterns and the cavity area in the teat and gland regions were not significantly different (P>0·05). The animals had long teat canals (3·1±0·1 cm), longer in the hind than fore quarters. Cisternal milk yield was low (0·17±0·01 kg) and cisternal fraction was only 4·9±0·1% of the total milk. The cisternal area (cm2) was 69·6±4·6, 51·61±4·8 and 26·01±4·8 while the cisternal yield (kg) was 0·32±0·05, 0·18±0·05 and 0·05±0·05 in early, mid and late lactation, respectively. A close correlation (r=0·87, P<0·05) existed between the ultrasound cisternal area and cisternal milk yield. The latency period of induced milk ejection was similar to that reported for cows (25±1 s) and was negatively correlated with milk yield (r=−0·75, P<0·05). Milk ejection occurred shortly after elevated oxytocin concentrations were present. Delayed milk ejection reported earlier in this species must therefore be due to the absence of cisternal milk and delayed oxytocin release. An increase in teat length and circumference at milk ejection was also evident in the ultrasound cross sections.

We determined the relative importance of cholecystokinin (CCK), leptin, and fatty acid concentrations in plasma in mediating the satiety effects of supplemental fat in lactating cows. Five ruminally and duodenally cannulated Holstein cows in late lactation were used in a 3×5 incomplete Latin square design with three treatments: 1) Control: basal diet (CON), 2) Control+supplementation of canola oil at 1 kg/d in the feed (FED) and 3) Control+abomasal infusion of canola oil at 1 kg/d (INF). Relative to CON, feed intake was reduced by INF but not by FED. We provide evidence that both FED and INF treatments stimulated CCK gene expression in the duodenum and elevated plasma CCK concentrations. However, our results did not support a role for CCK in mediating satiety through an endocrine mechanism of action. We speculate that CCK might be acting either through paracrine and/or neurocrine routes to influence feed intake in cattle. Both FED and INF had no effect on the mRNA abundance of leptin, lipoprotein lipase, or acetyl-CoA carboxylase in adipose tissue. Plasma concentrations of leptin, insulin and IGF-I were not altered by FED or INF, indicating that these signals may not be involved in mediating short-term hypophagic effects of dietary fat. Plasma concentrations of 18[ratio ]1n-9 and 18[ratio ]2n-6 were significantly greater for INF than for FED or CON. We conclude that the hypophagic effects of supplemental fat in cattle depend on the amount of unsaturated fatty acids reaching the intestine and that this satiety effect is mediated through CCK, oleic acid and (or) linoleic acid, but leptin is not involved.

Casein micelles contain stabilized amorphous calcium phosphate that is bioavailable to the neonate (Holt & Sawyer, 1988). Tryptic phosphopeptides formed from casein digestion associate with amorphous calcium phosphate forming stable nanocomplexes that have been described as calcium phosphate delivery vehicles (Holt et al. 1996; Reynolds et al. 1999, 2003; Farrell et al. 2002).

Four milking regimes using automatic cluster removers (ACR) were tested over a 19-week period, from mid to late lactation. Each treatment group consisted of 16 slow-milking cows. The milking regimes used were: an ACR setting of 200 ml/min (Control); raised ACR setting from 200 to 500 in steps of 100 ml/min (Raised ACR); raised ACR as above in conjunction with pre-milking teat stimulation (Raised ACR+Stim); and terminating the milking when an ACR threshold of 200 ml/min was reached or when a predetermined maximum milking duration was reached (Timer). All incremental treatments were applied in blocks of 6 or 7 weeks duration. ACR thresholds were raised from 200 to 500 ml/min without observed loss of milk production when compared with controls. However, even up to an ACR setting of 500 ml/min there was little reduction in the group milking duration even when used with teat stimulation. In contrast, the Timer treatment resulted in a 34% reduction of the maximum milking duration for the group without significant loss of milk yield. For all groups, including Control, strip yield was occasionally very high and highly variable. Willingness of cows to enter the milking platform, behaviour during milking, teat condition and incidence of mastitis were similar for all treatment groups. The results indicated that simple truncation of milking at a predetermined maximum duration could be a most potent and inexpensive method of milking a herd more quickly. Such a method could be employed by using a simple timer in any dairy regardless of the level of sophistication of the milking system.