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.

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.

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.

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.

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.

A total of 1304 goat udder halves were sampled monthly during an entire lactation (6262 samples) with the aim of identifying factors affecting milk somatic cell count (SCC). Bacteriological analyses for identification of mastitis pathogens were carried out on all samples and SCC was also determined. All animals were examined for infection by caprine arthritis-encephalitis virus (CAEV) using a commercial ELISA test kit. Results obtained were arranged in two databases (whole-lactation average half-udder database and monthly half-udder database) and two mixed models were applied. Random effects of half udder nested into flock and fixed effects of flock, intramammary infection (IMI) status, number of kids born, length of lactation and interaction of parity with IMI status were significant for the first database. CAEV infection and its interaction with IMI status was not significant. Milk SCC was significantly increased for infected udder halves and milk from udder halves infected with minor pathogens had lower SCC than udder halves infected with major pathogens. For healthy udder halves, SCC was higher in older animals but this effect was not evident in halves with IMI. Multiple birth and short-duration lactation were factors associated with elevated milk SCC. The second mixed model considered repeated measures in time for consecutive samplings throughout lactation (stage of lactation) which was also a significant factor with increasing stage of lactation. The influence of all these factors should be taken into account in the establishment of more reliable diagnostic SCC thresholds for IMI.

A study was undertaken in cattle to evaluate changes in milk L-lactate in relation to mastitis. A healthy, rear quarter of the udder of each of ten cows in mid-lactation was infused with 1000 colony-forming units (cfu) of Streptococcus uberis following an afternoon milking. Foremilk samples were taken at each milking from control and treated quarters and antibiotic treatment was applied following the onset of clinical mastitis or after 72 h. One cow did not become infected. Six quarters showed clinical symptoms of mastitis within 24–40 h and this was associated with a more than 30-fold increase in milk L-lactate (to 3·3 mM) and an increase in somatic cell count (SCC) from 4·5×103 to 1×107 cells/ml. Three cows were subclinical, with cell counts ranging from 1·5×106 to 1×107 cells/ml. In these animals, milk lactate ranged from 0·7 to 1·5 mM in the infected quarters up to 40 h post-infection, compared with less than 0·1 mM in control quarters. Milk was examined from 137 cows in mid-lactation which were known to have mastitis. Foremilk samples were taken aseptically from control and infected quarters of cows on commercial farms. Mean milk L-lactate concentrations and SCC were 0·14±0·02 mM and 1·85±0·3×105 cells/ml, respectively, in control (bacteriologically negative) samples. However, L-lactate concentrations exceeded 2·5 mM in the presence of some types of infection, the level of the lactate response being closely related to the impact of the infection on SCC. L-Lactate concentrations were relatively elevated in milk samples taken post partum, declining from 0·8 to 0·14 mM over the first few days of lactation. In conclusion, milk L-lactate has potential as an indicator of clinical and subclinical mastitis in dairy cows.

As a result of multiple QTL-mapping projects in recent years, a quantitative trait locus for milk fat percentage and milk yield has been described on BTA14. Recent reports name the acyl-CoA[ratio ]diacylglycerol acyltransferase (DGAT1) gene on BTA14 as a potential candidate gene, with a nonconservative substitution of lysine by alanine (K232A) producing a major effect on milk composition and yield. DGAT1K appears to be the ancestral allele and the K232A substitution probably occurred after the divergence of the Bos indicus and Bos taurus lineages. These findings prompted us to genotype 1748 DNA samples of 38 different Bos taurus and Bos indicus cattle breeds from 13 countries on five continents (Europe, Africa, Asia, North America and South America), to examine the occurrence of the DGAT1 polymorphism and characterize the K232A substitution in cattle breeds of different origins and selected for different purposes (e.g., beef, dairy and dual purpose). Calculating pairwise FST values for pooled subpopulations showed least divergence for Bos indicus breeds with high milk fat percentage. Fixation of DGAT1A was found in some Bos taurus breeds and fixation of DGAT1K in one Bos indicus breed. Breeds of no known organized breeding background from the Near East domestication centre of Bos taurus and taurine African N'Dama cattle were found to possess intermediate frequencies of DGAT1K. While beef breeds tended to harbour higher DGAT1A levels, dairy cattle showed everything from very low levels of DGAT1K to unexpectedly high frequencies of this allele.

We assessed polymorphisms in exon IV of the κ-casein gene (CSN3) in ten different breeds of domestic goat (Capra hircus) from three continents and in three related wild caprine taxa (Capra <ibex> ibex, Capra <ibex> sibirica and Capra aegagrus). Thirty-five DNA samples were sequenced within a 558 bp fragment of exon IV. Nine polymorphic sites were identified in domestic goat, including four new polymorphisms. In addition to four previously described polymorphic positions, a total of 13 polymorphisms allowed the identification of 13 DNA variants, corresponding to 10 protein variants. Because of conflicting nomenclature of these variants, we propose a standardized allele designation. CSN3*A, CSN3*B, and CSN3*D were found as widely distributed alleles in European goat breeds. Within Capra ibex we identified three variants and showed that the sequence of Capra aegagrus is identical to the most common Capra hircus variant, consistent with Capra aegagrus being the wild progenitor of domestic goats. A dendrogram was drawn to represent the molecular network between the caprine CSN3 variants.

The link between oxidation and increased proteolysis in raw milk was studied. To accelerate oxidation, H2O2 (1 mM) was added to raw milk, resulting in enhanced proteolysis by up to 11·2% after 24 h incubation at 5 °C. Addition of Cu2+ (10 μM) to milk or exposure of milk to light (60 min) likewise increased proteolysis. To explain the mechanism responsible for increased proteolysis as a result of oxidation, the effect of lipid oxidation products on plasmin-induced proteolysis was tested. Addition of malondialdehyde to skim milk increased the formation of γ-caseins, a proteolysis product from plasmin hydrolysis of β-casein. The same observation was made in a model system containing 4·5 g β-casein/l sodium tetraborate buffer at pH 8 and plasmin. Addition of a plasmin inhibitor blocked the formation of γ-casein. The results indicate that aldehydes accumulated from lipid oxidation can modify β-casein and thereby increase susceptibility of the proteins to proteolysis. Furthermore, the data suggest that proteolysis in raw milk may be connected to oxidative processes.

The objective of the present work was to identify the compositional parameters of raw milk that affected ethanol stability at natural pH when natural milk conditions were not modified. Heat stability, measured as coagulation time (CT), was included in the analysis to verify relation to alcohol test. Statistical models were proposed for alcohol and heat (CT) stabilities. Milk samples of good hygienic quality from dairy farms were classified in two groups according to their alcohol stability. Unstable samples to ethanol (72%, v/v) presented lower values of pH, somatic cells count, casein and non-fat-solids relative to ethanol stable samples (ethanol at 78%, v/v or more); whereas freezing point, chloride, sodium and potassium concentrations were higher in the unstable group. Logistic regression and multiple regression were applied to modelling alcohol and heat stability behaviour respectively. Chloride, potassium, ionic calcium and somatic cell count were included in the alcohol regression model, whereas calcium, phosphorous, urea, pH and ionic calcium were part of CT model. Ionic calcium was the only measured variable that contributed to both models; however coagulation time was noted to be more sensitive to ionic calcium than alcohol. The relation between ionic strength and casein was found to contribute to the alcohol model but not to the CT model. However, the interaction calcium plus magnesium plus phosphorous and casein contributed only to CT model.

Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) were used to monitor changes in the secondary structure and thermal stability of β-lactoglobulin A and B in the presence of sodium dodecyl sulphate (SDS), N-ethylmaleimide (NEM), urea and cysteine. An increase in the thermal stabilities of both proteins was noted in the presence of 10 mM-SDS. In the presence of 50 mM-SDS, there was extensive denaturation of both variants. In general, the β-strand/β-sheet regions in the secondary structure of both variants were very susceptible to denaturation by SDS and cysteine, suggesting that these regions may be held by hydrophobic and disulphide bonds. At ambient temperature and physiological pH, a notable difference was observed in the 1636 and 1627 cm−1 regions of the FTIR spectra of the two β-lg variants. The results suggest possible differences in the nature of the β-sheet/β-strand distribution/content of the two proteins. Urea and NEM at a concentration of 50 mM, had little effect on the secondary structure and denaturation of both variants. New findings are presented which further indicate that although the β-lg B variant showed greater thermal stability than the A variant in all the cases studied, its denaturation temperature and secondary structure were affected to a greater extent by the protein perturbants than β-lg A.

In this study, a two-plasmid system for enhanced and consistent biosynthesis of the model lactococcal bacteriocin lactococcin A in non-producing Lactococcus lactis hosts was developed. The system comprised a plasmid carrying the genes lcnA and lciA under the control of the nisin-inducible nisA promoter, and a second plasmid harbouring the lcnC and lcnD genes. The introduction of both plasmids into two strains containing the nisRK genes required for nisin-controlled expression, Lc. lactis FI5876 (a nisin A-producer strain) and FI7847, resulted in production of extracellular lactococcin A at a higher level than that for the parental strain, Lc. lactis WM4. In addition, transformation of the nisin-producing host with both plasmids led to a high-level production of both lactococcal bacteriocins, which may provide a means to exploit their complementary properties in cheese ripening.

In the present study we report for the first time the presence of S-layer proteins in Lactobacillus kefir and Lactobacillus parakefir isolated from kefir grains. Soluble whole-cell protein profile obtained either by mechanical disruption (X-press) or by a combined treatment with lysozyme and SDS on whole cells, showed a significant band of apparent molecular mass of 66–71 kDa as measured by SDS–PAGE. The intensity of this band was considerably reduced when cells were treated with 5 M-LiCl. The above mentioned proteins were recovered in the LiCl extracts. After dialysis and concentration, the proteins extracted were able to reassemble in a regular array. Negative staining of these protein preparations were analysed by transmission electron microscopy and a paracrystalline arrangement was seen. Thin sections of bacteria analysed by transmission electron micrographs showed an outermost layer over the bacterial cell wall, that was lost after the LiCl treatment. The production of this surface structure under different culture conditions was also evaluated. Finally, the relationship between the presence of S-layer proteins and surface properties (e.g. adhesion to Caco-2 cells, autoaggregation, and hemagglutination) was investigated.

The objective of this work was to describe the diversity of lactic acid bacteria in traditional raw milk Salers cheeses at the species and strain levels. The characterization of 381 strains isolated during ripening and various strain collections was investigated using physiological analysis and molecular techniques: Rep-PCR, species and genus specific amplifications and the sequence analysis of 16S rDNA for strain typing and taxonomic identification. The strains belonged to Lactobacillus plantarum, Lactobacillus paracasei, Lactococcus lactis, Lactococcus garviae, Enterococcus faecalis, Enterococcus faecium, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Streptococcus salivarius, Streptococcus millieri, Streptococcus macedonicus and Pediococcus pentosaceus. A wide phenotypic and genomic heterogeneity was observed within the different species (Lactobacillus plantarum, Lactobacillus paracasei and Leuconostoc mesenteroides) according to the origin and the time of ripening. The natural microflora was different from strain collection and each method must be combined to identify and characterize natural microflora. This study revealed the low selectivity of selective media used for the isolation of different groups of lactic acid bacteria except the Facultatively Heterofermentative lactobacilli medium selecting mesophile lactobacilli and SB medium selective for Enterococcus. The study reveals, for the first time, the microbial lactic acid bacteria community of Salers cheese and its diversity. A better knowledge of microbial flora will be useful to improve understanding of sensory quality of cheeses.

The effect of the hygienic quality of milk on changes in microbial counts and biogenic amine content was evaluated during ripening of goat cheeses manufactured from pasteurized and raw milks at 1, 14, 30, 60 and 90 d. The original milk, rennet, curd and whey were also included in the study. The pH, salt content and extent of proteolysis in the cheese were also evaluated. Spermidine and spermine were the main amines in raw milk, while they were minor amines in cheeses. Other amines increased markedly during ripening, tyramine being the main amine in cheese made from raw milk and cadaverine and putrescine in those produced from pasteurized milk. Enterobacteriaceae counts decreased during ripening whereas those of lactic acid bacteria increased, especially lactobacilli and enterococci. Cheese made from raw milk showed higher microbial counts during ripening than those made from pasteurized milk, especially for Enterobacteriaceae and enterococci, counts being 2 or 3 log units higher. Raw milk cheese showed remarkably higher biogenic amines compared with pasteurized milk cheeses. Therefore, pasteurization of milk causes a decrease in final biogenic amine content of cheese as a result of the reduction of its microbial counts.

The teat canal is an invagination of the outer teat surface. Its epithelial constitution is, however, highly specialized as indicated by its high turnover rate, its unique function in trapping bacteria, and in sealing the teat canal between milkings. The keratin of the teat canal is in a dynamic state of generation and degradation. Repeated removal during milking of keratin contaminated with or colonized by bacteria plays a significant role in preventing mastitis (Murphy, 1959; Capuco et al. 1992). To study the biology of the keratin lining, e.g., its turnover, and its relation to mastitis defence, reliable methods of collecting keratin in vivo for quantitative and qualitative analysis are necessary. Bright et al. (1990) compared methods for keratin collection in vivo and suggested that a tapestry needle was a suitable tool for collecting repeatable, representative samples of keratin for lipid analysis from single teat canals of living cows.