Two experiments were undertaken to determine the effects of cereal grain and fibre (hay or straw) supplements on the fatty acid composition of milk fat of grazing dairy cows in early lactation. In both experiments, grain supplements significantly increased (P<0·05) the proportion of the endogenously synthesized 10[ratio ]0–16[ratio ]0 fatty acids. Of the C18 acids, the proportion of 18[ratio ]0 and 18[ratio ]3 was significantly decreased (P<0·05) by grain supplementation, while that of 18[ratio ]2 was significantly increased (P<0·05). Irrespective of diet, 18[ratio ]1 trans-11 was the most dominant trans 18[ratio ]1 isomer in milk fat. In the first experiment, the proportions of the 18[ratio ]1 trans-11 isomer and conjugated linoleic acid (CLA, 18[ratio ]2 cis-9, trans-11) were highest for the pasture-only diets, and significantly (P<0·05) decreased with grain supplementation. The opposite result was observed in the second experiment, conducted in a different dairy region, suggesting that factors such as the quality of pasture on offer and the physiological state of the cow could affect the content of CLA and trans fatty acids in milk fat. In both experiments, there was a significant positive linear relationship between CLA and 18[ratio ]1 trans-11. Fibre supplements had little effect on the fatty acid composition of the milk.

Four lactating Holstein cows (average milk yield: 20±3 l/d) were used to develop and validate a method for estimating the size of udder cisterns (Sinus lactiferi) using ultrasonography. A sectorial transducer probe of 5 MHz, placed in contact with the teat in a parallel cranial position, was used to obtain vertical scans of the udder in two perpendicular planes with the teat canal axis as reference. Udder scans for each udder quarter were taken randomly at intervals of 4, 8, 12, 16, 20 and 24 h after milking. Glandular parenchyma (echogenic) and lumen of the cisterns full of milk (anechogenic) were evident in the scans, the calculated area of the anechogenic portion being defined as cistern area. Cistern areas measured in perpendicular scans were highly correlated. Immediately after each measurement, cisternal milk was removed from each quarter using a teat cannula after i.v. injection of an oxytocin-receptor blocking agent. Alveolar milk from each quarter was then obtained by machine milking after i.m. injection of oxytocin. Cistern area and cisternal milk volume increased with length of milking interval showing a curvilinear pattern with a plateau after 16 h. Correlations between cistern area and cisternal milk volume were positive and significant (P<0·001) at all intervals but showed the highest values with the smallest residual standard deviations at 8 h (r=0·88) and 12 h (r=0·84). Since 8 h has previously been identified as a suitable time at which to determine cisternal milk volume for the purposes of defining suitability for different milking strategies, we conclude that ultrasonography provides a satisfactory, non-invasive method for determination of milk storage characteristics in dairy cows.

Effects of two natural (retinol and retinoic acid, RA) and one synthetic N-(4-hydroxyphenyl) retinamide (4-HPR) retinoids on proliferation and expression of urokinase-plasminogen activator (u-PA) by bovine mammary epithelial cells were examined. The BME-UV1 established bovine mammary epithelial cell line was used as a model system. All retinoids tested (retinol, RA and 4-HPR) were effective inhibitors of cell proliferation. When cells were cultured in the absence of fetal bovine calf serum (FBCS), inhibition occurred at concentrations as low as 1 nM for all retinoids tested. The effect of retinoids on cell proliferation was not dose-related when cells were cultured in the absence of FBCS. All retinoids (retinol, RA, 4-HPR), when used in the range 1 nM–10 μM (noncytotoxic concentrations), were equally effective and had identical inhibition patterns. Inhibition of cell proliferation by RA was apparent by 6 h and was higher after 24 h in culture. In contrast, when cells were cultured in the presence of FBCS, the effect of RA and retinol on cell proliferation was dose-related. RA and retinol inhibited cell proliferation (P<0·01) when added to the culture medium in concentrations as low as 10 nM and 100 nM, respectively. 4-HPR was inhibitory (P<0·01) in concentrations as low as 1 nM. Higher concentrations of 4-HPR in the range 1 nM–1 μM had no further effect on cell proliferation. None of the retinoids tested, when added to cultures in the presence or absence of FBCS, could completely arrest cell proliferation at noncytotoxic concentrations. RA at 1 μM inhibited (P<0·05) insulin or IGF-I-induced cell proliferation but had no effect (P>0·05) on u-PA mRNA levels or u-PA activity. Furthermore, RA inhibited cell proliferation in the presence of FBCS but had no effect (P>0·05) on u-PA mRNA levels. Thus, retinoids are effective inhibitors of bovine mammary epithelial cell proliferation and this growth inhibition does not seem to correlate with any changes in u-PA mRNA or u-PA activity.

We developed a simple, non-invasive method to measure the intramammary pressure (IMP) response in sows during natural milk ejection and after i.v. injections of oxytocin. The teats of sows were cannulated and the amplitude and duration of IMP changes were measured via a pressure transducer linked to a computer. During natural milk ejections, the amplitude of the response was 5·5±1·8 kPa (mean±SD) and the duration of response at half the peak amplitude was 6·8±1·6 s. In response to oxytocin a threshold response occurred at 60 mIU oxytocin with no further change up to 200 mIU. The amplitude of the response to an i.v. dose of 60–200 mIU oxytocin (5·5±2·2 kPa) and duration (6·4±1·7 s) were similar to that during a natural milk ejection. The responses of two galactophorous ducts of a teat measured simultaneously were similar in peak amplitude and duration. When the responses of four glands of a sow were measured simultaneously, there was a lag time in the onset of the IMP peak from the anterior to the posterior glands, and an increase in the peak amplitude from anterior to posterior. Both during natural milk ejections and in response to oxytocin injections, there were significant differences in peak amplitude between sows. We also aimed to determine whether the pressure or duration of milk flow during natural milk ejections was limiting piglet growth rates. IMP of nine sows and the growth rates of their piglets were measured. Both the peak amplitude of IMP response and the growth rate of piglets increased over the course of lactation, but the relationship was not causal. We found that the growth rate of piglets did not depend on IMP amplitude or duration, but we suggest that it may depend on the frequency of suckling and/or the ability of piglets to withdraw milk while it is available.

In five short-term experiments conducted in Victoria in 1997 and 1998, grazing dairy cows were given either pasture alone or pasture supplemented with high-energy concentrates, and the fatty acid profiles of milk fat were measured. We established the effects of these feeds on some aspects of milk fat of importance for human nutrition, but we specifically focused on the hypothesis that conjugated linoleic acid (CLA) concentrations in milk fat increase as pasture intake increases, and decrease as more concentrates are fed. In agreement with previous research, feeding fresh pasture alone resulted in high concentrations (1·0–1·8 g/100 g milk fat) of CLA. When the effect of level of pasture consumption on CLA content was examined, a significant positive relationship (r2=0·35; P<0·05) was obtained. When cereal grain concentrates were used to supplement pasture intake, the CLA content of milk fat generally declined (P<0·05), except where the amount of concentrates given led to a marked reduction in total milk fat concentration. The use of cereal grain concentrates also generally resulted in significant (P<0·05) increases in medium-chain saturated fatty acids, but always reduced the contribution of butyric acid to milk fat, from 4·5 to 3·9 g/100 g milk fat, on average.

Twenty-two Murciano-Granadina dairy goats were used to investigate the effects of organic Zn supplementation of a diet containing a high level of inorganic Zn. Goats were kept in pens, machine milked once a day throughout lactation and fed a diet based on a dehydrated mixture of whole-plant maize and alfalfa ad libitum, alfalfa pellets, barley grain and a concentrate mixture. Treatments were: (1) control, and (2) supplemented with 1 g/d Zn-Methionine (Zn-Met) included in the concentrate mixture. After parturition, goats were blocked in week 3 and dietary treatments were applied until week 23. From weeks 3–20, feed intake, milk yield, milk composition, milk somatic cell count (SCC), and udder health were measured. In week 21, all goats were injected intraperitoneally with 1 g/d DL-methionine for 5 d to establish the effects of methionine under the conditions of udder stress induced by hand milking on the second day. During weeks 22 and 23, diet digestibility, and N and Zn balance were determined. Dry matter intake, milk yield, and milk contents of total solids, fat, total and true protein, and casein did not differ between treatments, but whey protein and non-protein nitrogen contents were significantly lower for the Zn-Met group. Milk SCC tended to decrease as a result of Zn-Met supplementation but differences between treatments were not significant when halves with persistent infection were excluded. Hand milking increased SCC in both groups, but udders of supplemented goats showed a lower reaction. Apparent absorption of N significantly increased and Zn retention tended to increase in Zn-Met supplemented goats. We conclude that Zn-Met supplementation can enhance resistance to udder stress in dairy goats. Effects were attributed to the organic Zn and not to the methionine component. Zn retention and protein utilization were also improved by the Zn-Met supplement.

Effects of atropine on blood plasma amino acid profile and on the yields and concentration of milk components were investigated in 12 Friesian cows in early lactation. Cows were housed indoors and fed with cut pasture ad libitum. Each cow received four treatments over 12 d during a replicated 4×4 Latin square experiment. Treatments were: control (saline); low dose (L; 30 μg atropine/kg body weight (BW)); medium dose (M; 40 μg atropine/kg BW); and 2×L dose, 2 h apart (2×L). On each of four treatment days, cows were milked at about 7.00, after which treatments were administered by subcutaneous injection. Cows were milked again at 2 h, 6 h and 10 h after injection. Milk samples were collected at each milking. Immediately after the 2 h milking, blood samples were drawn from each cow and the second injection was given for the 2×L treatment. Atropine reduced hourly milk yield, and concentrations and hourly yields of total protein, casein, whey protein, α-casein, β-casein, κ-casein, β-lactoglobulin and α-lactalbumin, but by differing amounts. Milk concentrations of bovine serum albumin and immunoglobulin G were increased by atropine, and overall yields of these proteins were mostly unchanged. Atropine lowered concentrations of most, but not all, amino acids in blood plasma, with essential amino acids reduced more than non-essential amino acids. Concentrations of α-amino N in whole blood, and glucose and insulin in blood plasma, fell after atropine injection. There was no difference between the L and M doses of atropine, but the 2×L dose had greater effects on milk composition than the single doses. For yields of milk and milk components, the effect of the 2×L dose was also more persistent. The results highlight the differential synthesis of individual milk proteins, and suggest that atropine might be useful for evaluating the mechanisms regulating milk protein composition.

Samples of foremilk were collected from 261 clinically normal glands of 150 ewes, and tested using the California mastitis test (CMT). Further samples were collected from 195 of these glands for determination of automated somatic cell counts (SCC), and from 60 of these glands for bacteriological assessment. The sensitivity and specificity of CMT for detecting samples with SCC above different threshold levels and for CMT and SCC in determining bacteriological status were evaluated using two-graph receiver operating characteristics (TG-ROC). Milk samples were obtained subsequently from ten CMT positive, and five CMT negative first- and second-lactation ewes. Samples were cultured using a variety of media, incubation temperatures and atmospheric conditions, immediately after collection, and 1 week after storage at 4°C and −21°C. Results suggested that CMT is best used as a diagnostic test for ovine subclinical mastitis (SCM) with a cut-off of 3 (distinct gel formation), and that automated SCC thresholds of >1200×103 cells/ml are appropriate, especially where low prevalences are expected (e.g. <5%). Additionally, this study showed that routine bacteriological methods were appropriate for isolation of most species of pathogen responsible for ovine SCM, but storage of samples prior to culture, either at 4°C or −21°C, was detrimental to the isolation of several of these organisms.

Local and systemic changes in the acute phase proteins, haptoglobin and serum amyloid A (SAA), were studied in six dairy cows during the acute and chronic phases of experimentally induced Staphylococcus aureus mastitis. Haptoglobin and SAA were measured in serum, and in milk from infected and healthy control udder quarters within each cow. Concentrations of haptoglobin and SAA increased rapidly in both serum and milk during the acute phase of mastitis and followed a similar pattern. Significantly raised milk concentrations of SAA were also found during chronic subclinical mastitis. Serum concentrations of SAA also tended to be higher during the chronic phase than pre-infection. Increases in milk haptoglobin and SAA were specific for the infected udder quarters. In conclusion, measurement of SAA in milk samples could be a useful tool in diagnosing mastitis.

The aim of the study was to determine the role of opioids modulating the release of prolactin (PRL) in response to milking in sixteen Brown-Swiss dairy cows. Two experiments were carried out to measure the dose-related effect of morphine and the effect of the opioid antagonist naloxone (NAL), with or without morphine. In the first experiment, six cows were injected (via catheter) on 3 consecutive days after the control milking (0 mg) with 21, 70 and 210 mg morphine-HCl 10 min before the start of the morning milking. The second experiment was divided into two parts. In the first part, four cows were injected after control morning milking with 210 mg morphine, 10 min before the start of the following morning milking. This was followed on the next day by an application of 210 mg NAL (15 min before the start of milking) and 210 mg morphine. In the second part, after control milking for 1 d, six cows were injected with 210 mg NAL 10 min before milking. Morphine at the highest dose tended to stimulate basal PRL levels in the first and second experiments (P<0·10). PRL increased in response to machine milking but morphine did not stimulate its release further. NAL alone, or when given with morphine did not influence the release of PRL in response to machine milking. However, NAL was effective in suppressing the increase in basal levels of PRL caused by morphine. In conclusion, although morphine tended to stimulate basal levels of PRL before milking, the release of PRL during milking seemed not to be regulated by opioids.

Effective diagnostic tools for screening herds for mastitis pathogens are important in development and monitoring of mastitis control programmes. A multiplex polymerase chain reaction (PCR) assay for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis was used in preliminary studies to assess its applicability as an alternative method for monitoring mastitis caused by these organisms at the herd level. PCR was used to detect the presence of these organisms in bulk milk samples. Correlations with bulk milk somatic cell counts (BMCC), total bacteria counts and thermoduric bacteria counts were evaluated. A total of 176 bulk milk samples were collected from 42 herds on five consecutive occasions at approx. 10-d intervals. Str. uberis was the most common organism in these bulk milk samples. There was no relationship between presence of either Staph. aureus, Str. dysgalactiae or Str. uberis and BMCC, total bacteria counts or thermoduric bacteria counts. However, presence of Str. agalactiae was associated with high BMCC and total bacteria counts. The results of this study show that regular analysis of bulk milk using this multiplex PCR assay may be a useful tool for monitoring herd status with respect to Str. agalactiae, but is of less value for monitoring occurrence of Staph. aureus, Str. dysgalactiae and Str. uberis. Further investigations are needed to clarify the relationship between positive PCR results and the prevalence of infected cows in the herd.

Damage to, or deterioration of, the keratinized horn tissue of the bovine hoof claw culminates ultimately in the development of solear ulceration. We have observed abnormal keratin distribution at the site of solear ulceration in the bovine claw that may be due to alteration of the positional cues of the keratinocytes. In this study we have characterized key cell biological changes associated with ulceration in the claw that may precipitate abnormal keratinization. Loss of basement membrane at sites of ulceration was found by immunofluorescent detection of laminin and integrins. In other tissues, basement membrane breakdown results from degradation by matrix metalloproteinases (MMPs). Similarly, elevated levels of MMPs 2 and 9 were observed in ulcerated bovine claw tissue both by zymography and, quantitatively, by assay of enzyme activity. In the sole of claws that contained an ulcer, tissue distal to the ulcer site also had elevated MMP 2 when compared with healthy sole tissue from the same animals, as did sole tissue of claws recovering from ulceration. Tissue inhibitor of metalloproteinase 2 (TIMP 2) was detected by ELISA in healthy tissue. TIMP 2 tended to be lower in diseased tissue distal to ulcer sites, and was significantly lower in ulcerated tissue. MMP 2 was located by immunofluorescence in the dermal and basal epidermal region of sole tissue, in the region of the basement membrane. Increased punctate staining of material in the dermis was associated with ulcerated material. ELISA of TIMP 2 in tissue extracts enriched for dermis or epidermis confirmed that the inhibitor was located predominantly in the dermis. To investigate a possible causal relationship between basement membrane anchorage and epidermal keratinization, the effect of function-blocking antibodies to laminins and integrins was tested in tissue explant cultures prepared from healthy sole tissue. Anti-integrin antibody treatment had no effect on either protein or DNA synthesis. In contrast, in the presence of anti-laminin antibody, protein synthesis was decreased in a concentration-dependent manner, a significant effect being observed at the highest concentration after treatment for 24 h. At this concentration, DNA synthesis was also decreased after 48 h of culture, an effect that may be relevant to a hibernal reduction in claw cell turnover, and the associated seasonal vulnerability of cows to claw damage. The results provide evidence for basement membrane disruption at ulcer sites, and an increased potential for disruption in the diseased claw, and a causal link between this and abnormal epidermal keratinization. Basement membrane disruption is in turn associated with reciprocal changes in MMPs and their inhibitors, favouring extracellular proteolysis. Whether MMP activation is the primary cause of dermal–epidermal deterioration and, if so, how MMP activation is triggered, remains to be determined.

A randomized, controlled field trial was performed in The Netherlands to determine the therapeutic efficacy of parenteral penethamate hydriodide (Leocillin®) against naturally occurring, chronic, streptococcal mastitis during lactation. Quarter milk samples were collected from subclinical cases of Streptococcus uberis or Streptococcus dysgalactiae mastitis to determine the effect of treatment on bacteriological cure and somatic cell count (SCC) at quarter level. A quarter was considered to be cured when the bacterial species, isolated prior to treatment, was not isolated from the quarter milk samples taken on days 10 and 20 post-treatment (bacteriological cure), or when a quarter milk SCC (QMSCC) was <250000 cells/ml on days 10 and 20 post-treatment (SCC cure). Longitudinal data analysis was performed to determine the effect of antibiotic therapy on SCC and milk yield at cow level. Bacteriological cure occurred in 59% of 29 treated quarters, while no cure was observed in any of the 21 untreated control quarters. Treatment resulted in a significant decrease in SCC at cow and quarter level in comparison with untreated controls. There was no significant effect of treatment on milk production. Antibacterial treatment of subclinical streptococcal infections during lactation also prevented clinical mastitis. Furthermore, the treatment may contribute to reduction of bulk milk SCC and to prevention of pathogen spread in dairy herds.

The vacuum in the mouthpiece chamber (MPC) was measured in a factorial trial with two liners (DeLaval 964008-01 and DeLaval 999007-03), two pulsator ratios (60 and 70%) and morning and evening milking over eight milkings of ten cows. Teat length and diameter were measured before and after milking, and maximum milk flow rate and depth of penetration of the teat into the teatcup were also recorded. The MPC vacuum at peak flow rate was classified as stable, at various levels, in 79% of the recordings. In the rest of the milkings the vacuum declined gradually, and this decline was associated with gradually deeper penetration of the teat into the teatcup. The DeLaval 964008-01 liner with the narrower bore and larger mouthpiece opening gave lower MPC vacuum at peak flow rate than did the wider bore DeLaval 999007-03 with the smaller mouthpiece opening. Morning milking gave higher MPC vacuum than evening milking. Increased maximum milk flow rate and increased length and diameter of the teat gave decreased MPC vacuum. The teats were longer before milking in the morning than in the evening. However, during peak flow rate, the teats had penetrated deeper into the teatcup in the evening than in the morning. A marked change in MPC vacuum in the transition to the period of flow rate can be used to identify the start of over-milking of an individual teat. A general model explaining the MPC vacuum as the outcome of the balance between air leakage past the mouthpiece lip and the leakage past the teat in the liner barrel is suggested.

The responses of five lactating East Friesian milk ewes to experimental mammary infection with Staphylococcus epidermidis and of five control ewes were examined over a period of 10 weeks. Infection caused an influx of neutrophils into milk, the numbers of which started to rise 4 h post infection and peaked 24 h after infection. The initial response was accompanied by mild fever and mild leucopaenia in blood (8 h after infection). No other signs of systemic infection were observed. Milk appeared normal at all times, although the milk yield of infected ewes tended to decline. Staphylococci were absent in milk from four ewes at 2 d and at 3 d after infection, but re-emerged intermittently in four of five ewes at subsequent samplings. Cytokines in milk were measured by ELISA. IL-8 was elevated in infected glands at 2 h and peaked at 8 h. In the four ewes intermittently shedding bacteria, IL-8 remained elevated until the final sampling at 10 weeks. IL-1β was transiently elevated at 1 d and 2 d and showed a pronounced peak in one sheep. Milk samples from this ewe were bacteriologically negative, somatic cell count (SCC) was within the normal range and the concentrations of IL-1β, as well as IL-8, were similiar to the control group (n=5) from 1 week after infection until the final sampling. Histological examination revealed leucocytic infiltrates in the four glands remaining infected at the end of the experiment, and a high level of CD5+ lymphocytes in three ewes. The results suggest that the relationship between the initial neutrophil influx and the proinflammatory cytokines may be responsible for determining the course of infection. Subclinical mastitis due to coagulase-negative staphylococci leads to minor changes in milk yield and milk constituents.

Here we report the entire cDNA sequences for equine αS1-, β- and κ-casein. Based on interspecies comparison, nine exons were found in equine β-casein and five in κ-casein. In equine αS1-casein cDNA the exon 5 was missing, which resulted in the total of 18 exons instead of 19 theoretically possible exons in αS1-casein cDNA. Comparison of DNA sequences representing exon 5 in other species with corresponding equine genomic region confirmed the presence of cryptic exon in horse genomic DNA. Equine αS1-casein mRNA was present in three forms in the lactating mammary gland and we showed that the two shorter forms were produced by skipping either the exon 8 or exon 15. In horse, as in some other mammals, β- and κ-casein are considerably more conserved (sequence identity 53% to 59% and 57% to 67%, respectively) than αS1-casein which appears as the most variable casein among species (sequence identity 40% to 54%). Interestingly, horse caseins resemble human much more than bovine caseins which may also explain the high dietetic value of mares' milk.

Chymosin, an aspartyl proteinase, is used for curdling of milk and manufacture of cheese. We report the purification and the physicochemical properties of chymosin isolated from the abomasal tissue of buffalo calves. The enzyme preparation extracted from buffalo abomasal tissues could be purified 29–fold using anion exchange and gel filtration chromatography. The molecular weight of the purified enzyme was 35·6 kDa on SDS-PAGE. Partial N-terminal amino acid sequence of the first eight amino acid sequences of buffalo chymosin was identical to the first eight amino acid sequences of cattle chymosin. Buffalo chymosin exhibited a skewed bell-shaped stability profile as a function of temperature with maximum activity near 55 °C. Milk clotting activity decreased gradually as pH increased. The enzyme became completely inactive, however, above pH 7·0. The ratio of milk clotting to proteolytic activity was 3·03. When compared with cattle chymosin, there were subtle differences in the stability and relative proteolytic activity of buffalo chymosin.

We describe a case of listerial mastitis in a flock of 130 sheep. The animals were housed at a farm where the bulk raw ewe milk was processed to produce raw milk soft cheese. List. monocytogenes was shed from the right mammary complex. Shedding was observed over a period of 99 d. A mean level of 4·56×104 cfu (colony forming units) Listeria monocytogenes/ml was recovered from the raw milk originating from the infected udder. The numbers ranged from 9×101 to 2·95×105. The bulk milk was contaminated by approx. 5·7×103 cfu/ml. In the cheese product, 2·0×102 cfu List. monocytogenes/g were constantly detectable for a period of 7 d post manufacture. The starter culture used for coagulation had a pivotal influence on the behaviour of List. monocytogenes during cheesemaking. Using the same mesophilic buttermilk culture as used by the farmer allowed numbers of Listeria to increase 60-fold within 12 h owing to a delayed acidification of the bulk milk. Addition of a thermophilic yogurt culture reduced the numbers of Listeria within 8 h of incubation.

Alpha (α)- and beta (β)-adrenergic receptors in the bovine mammary gland are mainly present in the teat muscles and in the region where large milk ducts reach the cisternal cavities. The aim of the study was to test the hypothesis that the region of the large mammary ducts is the most important location of α- and β-adrenergic receptor stimulation affecting milk ejection and milk removal. Effects of α- and β-adrenergic receptor stimulation and of oxytocin (OT) receptor blockade on milking characteristics were tested in six cows. Milk flow was measured before and after the distal part of one teat, including the teat canal and teat sphincter, had been partly amputated. Before the operation, milk yield and peak flow rate decreased during α-adrenergic receptor stimulation and during the OT receptor blockade, and increased during β-adrenergic stimulation. After removal of the teat tip, relations of milk yield and peak flow rates after administration of α- and β-agonists and after application of an OT receptor blocking agent were similar to those before operation. Only total milk yield had decreased in the teat-amputated quarter owing to unhindered flow of cisternal milk before cluster attachment. Since responses to α- and β-adrenergic receptor stimulation as well as to OT receptor blockade do not differ with or without the teat sphincter, it is concluded that milk flow is mainly influenced by the muscle tone of the large mammary ducts.

Probiotic microorganisms, especially lactic acid bacteria, are effective in the treatment of infectious diarrhoeal diseases and experimental colitis. Although the mechanisms by which these organisms exert their anti-inflammatory effects are largely unknown, immunomodulating effects are suggested. The objective of this study was to examine the effect of a 5-week oral administration of Lactobacillus rhamnosus subspecies GG (Lb. GG) on the cellular immune response to intestinal microorganisms in ten healthy volunteers. Peripheral blood cells (PB) were stimulated with either ‘self’ or ‘non-self’ preparations of faecal samples and isolated Bacteroides fragilis group-organisms (Bfg) or Escherichia coli (Esch. coli), and pro- and anti-inflammatory cytokines (IL-10, IL-4, IL-6, IFN-γ, TNF-α) were measured in the culture supernatant. CD4+ T-lymphocyte activation was determined by measurement of intracellular ATP following lysis of the cells. The activational response of CD4+ T-lymphocytes towards isolated and heat-inactivated intestinal organisms was increased after the probiotic treatment. Additionally, TNF-α, IL-6 and in part IFN-γ cytokine secretion by PB cells following stimulation with whole stool preparations and single members of the flora was significantly decreased, whereas the IL-10 and in part IL-4 cytokine secretion was increased at the end of the study. In contrast, the activational response of CD4+ T-lymphocytes following stimulation with whole ‘non-self’ intestinal flora was higher than by ‘self’ intestinal flora, but both responses showed a trend towards a reduction at the end of the study. This study documents a direct effect by Lb. GG on the cellular immune system of healthy volunteers and offers a promising tool to investigate systemic immunomodulation due to oral administration of probiotic microorganisms.