J. W. Blum

Real-time RT-PCR quantification of insulin-like growth factor (IGF)-1, IGF-1 receptor, IGF-2, IGF-2 receptor, insulin receptor, growth hormone receptor, IGF-binding proteins 1, 2 and 3 in the bovine species

Reverse transcription (RT) followed by polymerase chain reaction (PCR) is the technique of choice for analysing mRNA in extremely low abundance. Real-time RT-PCR using SYBR Green I detection combines the ease and necessary exactness to be able to produce reliable as well as rapid results. To obtain highly accurate and reliable results in a real-time RT-PCR a highly defined calibration curve is needed. We designed and developed nine different calibration curves, based on recombinant DNA plasmid standards and established them on a constant real-time PCR platform for the following factors: growth hormone receptor (GHR), insulin-like growth factor (IGF)-1, IGF-1 receptor (IGF-1R), IGF-2, IGF-2 receptor (IGF-2R), insulin receptor (INSR), and IGF-binding proteins (IGF-BP) 1, 2 and 3. Developed assays were applied in the LightCycler system on bovine ileum and liver total RNA and showed high specificity and sensitivity of quantification. All assays had a detection limit of under 35 recombinant DNA molecules present in the capillary. The SYBR Green I determination resulted in a reliable and accurate quantification with high test linearity (Pearson correlation coefficient r > 0.99) over seven orders of magnitude from <10(2) to >10(8) recombinant DNA start molecules and an assay variation of maximal 5.3%. Applicability of the method was shown by analysing mRNA levels in newborn calves: mRNA concentrations per gram tissue of mRNAs of IGF-1, IGF-1R, IGF-2, IGF-2R, GHR, INSR, and IGF-BP1, 2 and 3 were all different between in liver and ileum and the traits all exhibited individual differences.

Role of endotoxin and TNF-α in the pathogenesis of experimentally induced coliform mastitis in periparturient cows

Twelve cows were experimentally infected in two quarters with 1 x 10(4) cfu Escherichia coli per quarter and six cows were infused with 500 microg endotoxin into two quarters. Six cows infected intramammarily with Esch. coli were treated intravenously with a bactericidal antibiotic 10 h after infection and subcutaneously 20 h later. Blood and milk samples were collected from all cows at regular time intervals. Milk production decreased more rapidly, but was less pronounced, after endotoxin infusion than (during Esch. coli mastitis. The milk production losses in the noninflamed quarters were negligible in endotoxin mastitis, but were substantial during Esch. coli mastitis, probably due to more pronounced systemic effects. Reticulorumen motility was inhibited only during Esch. coli mastitis. Changes in plasma haptoglobin were more pronounced during Esch. coli mastitis, although they occurred sooner during endotoxin mastitis. No changes in plasma activities of enzymes such as lactate dehydrogenase, glutamic-oxaloacetic transaminase and gamma-glutamyl transpeptidase were observed. Concentrations of tumour necrosis factor-alpha increased in both types of mastitis. Absorption of these cytokines into the circulation was highest during Esch. coli mastitis, especially in the untreated control group. We found only minor differences between the treated and untreated Esch. coli groups, but there were larger differences between the Esch. coli groups and the endotoxin group. These differences were probably due to differences in kinetics, composition and amounts of different cytokines released in the mammary gland and subsequently absorption into the circulation. Endotoxin is probably not directly responsible for the systemic changes during coliform mastitis.

Milk removal in familiar and unfamiliar surroundings: concentrations of oxytocin, prolactin, cortisol and β –endorphin

Eight cows were machine milked either in an operating theatre or in their familiar barn. During the experiments, milk flow curves were recorded and blood samples were taken for determination of concentrations of oxytocin, prolactin, cortisol and beta-endorphin. The milking cluster was attached without udder preparation. After cessation of milk flow, air was blown into the vagina for 2 min. When milk flow had stopped again, 1 i.u. oxytocin and finally 10 i.u. oxytocin were injected to remove the remaining milk. After the start of milking, oxytocin remained basal in unfamiliar, but increased in familiar surroundings. Therefore, during normal milking only 9% of total milk was removed in unfamiliar, whereas 79% was available in familiar surroundings. In response to subsequent vaginal stimulation in the operating theatre, oxytocin increased transiently in five cows and 15-71% of the milk was removed in these animals. In the other three cows in the operating theatre, oxytocin remained basal during vaginal stimulation, and no more milk was available. After injection of 1 i.u. oxytocin, 56 and 11%, and after injection of 10 i.u. oxytocin, 13 and 8% of milk was removed in unfamiliar and familiar surroundings respectively. Concentrations of prolactin increased during the course of milking in both treatments. Premilking concentrations of cortisol and beta-endorphin were elevated in unfamiliar as compared with familiar surroundings. During the course of milking, cortisol increased slightly and beta-endorphin decreased in unfamiliar, whereas both hormones increased markedly during milking in familiar surroundings.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous recording of oxytocin release, milk ejection and milk flow during milking of dairy cows with and without prestimulation

The effects of manual teat stimulation compared with stimulation by the liner of a conventional milking machine and the influence of milk ejection on the course of milk flow during the entire milking were investigated. Twelve cows were milked with or without prestimulation on three quarters and milk flow was recorded while intramammary pressure (IMP) was recorded in the left front quarter and blood samples for the determination of oxytocin (OT) were taken at 1 min intervals. OT concentrations increased very similarly in response to manual teat stimulation or liner stimulation and remained elevated during the entire milking procedure. Milk flow curves were generally bimodal without stimulation and not bimodal during milking after prestimulation. Milk yield was not significantly lower in milkings without than with stimulation, whereas machine-on time was prolonged and peak milk flow rate was reduced during milking without stimulation. Time to reach milk flow plateau, time to reach peak flow rate and IMP profiles were similar in both treatments if time was calculated from the start of stimulation. IMP increased similarly in response to manual or liner teat stimulation. In milkings with and without prestimulation IMP did not reach its maximum until the start of milking. In bimodal milk flow curves the second increase of milk flow occurred concomitantly with the start of IMP increase; ejection IMP generally occurred concomitantly with the milk flow plateau. In some cases during milking without prestimulation only small and transient CT release was observed. In response, IMP did not increase to its maximum and increased further in response to a stripping-related CT release. During the course of milking IMP decreased slightly because of the emptying of the three milked quarters. After the end of milking, and with CT concentrations decreasing to the premilking values, IMP again decreased slightly to another plateau, probably owing to myoepithelial relaxation. Thus the course of milk ejection, i.e. the IMP profile, influenced milk removal markedly during early milking.

First ovulation and ketone body status in the early postpartum period of dairy cows.

The effect of ketone body status on occurrence of first ovulation during early lactation was assessed in 84 multiparous dairy cows under field conditions. Animals were equally distributed across 8 farms and were controlled by the same herd fertility monitoring program. Cows were visited twice antepartum and 6 times postpartum at weekly intervals between 5:30 and 8:30 AM. On these occasions, body condition scores and milk yields were measured, blood and milk samples were taken, cows were gynecologically examined, and parameters of reproduction were determined. The onset of first ovulation was specified by milk progesterone determination and rectal palpation. Cows starting postpartum ovarian cyclicity within or after 30 d were classified as early and late responders (ER and LR, respectively). Resumption of the estrous cycle within 30 d postpartum is considered optimal under practical conditions, and classification based on this threshold value resulted in groups of equal size and equal distribution of ER + LR cows within farms. Ketone bodies measured were beta-hydroxybutyrate in serum and acetoacetate and acetone in serum and milk. Blood serum and milk ketone body concentrations during the first 6 wk of lactation were higher in LR than in ER, whereas plasma glucose and nonesterified fatty acid and milk fat, protein and urea concentrations did not differ between groups. Maximal concentrations of ketone bodies from parturition to first ovulation were better predictors of the onset of the estrous cycle than mean or minimal concentrations over the same period. Milk acetone and serum beta-hydroxybutyrate concentrations provided the most reliable information with regard to resumption of ovarian activity of all ketone bodies.

Continuously elevated concentrations of oxytocin during milking are necessary for complete milk removal in dairy cows

The importance of elevated concentrations of oxytocin (OT) during the entire milking period was investigated in seven primiparous dairy cows with inherent disturbed milk ejection and in sixteen healthy cows with disturbed milk ejection induced by placing them in an operating theatre. Disturbance of milk removal in both groups has previously been demonstrated to be exclusively due to central blockage of the expected OT release in response to teat stimulation and milking. However, milk ejection can be induced by exogenous OT. OT (0.2 i.u.) was injected i.v. before milking and 49 +/- 6% of the total milk was removed. When plasma OT decreased, milk flow stopped. In response to a second and third injection of 0.2 i.u. OT, 30 +/- 4 and 7 +/- 2% of the milk were removed respectively. The remaining milk was removed with 10 i.u. OT. The lag time from injection of OT to the start of milk flow was inversely correlated with the amount of milk actually removed in response to the OT injection. If 0.2 i.u. OT was injected during intramammary pressure (IMP) recording, IMP immediately increased to its maximum value. After 2.5 +/- 0.3 min, IMP decreased to an intermediate IMP (between preinjection and maximum IMP). After two additional injections of 0.2 i.u. OT and after injection of 0.5 i.u. OT, IMP increased to a similar maximum. However, after injection of 0.5 i.u. OT, maximum IMP lasted longer (2.9 +/- 0.3 min; P < 0.05) than after injections of 0.2 i.u. If OT was continuously infused (0.15 i.u./min) during milking, milk flow lasted until the udder was completely emptied. IMP increased during OT infusion to a maximum which remained stable until infusion was stopped after 10 min. The same IMP maximum was reached after the first individual OT injection (0.2 i.u.), but when plasma OT decreased towards basal concentrations, milk flow ceased and IMP decreased to an intermediate level. Thus continuously elevated OT concentrations such as those during infusion or during normal milking are necessary for complete milk removal.

B-mode ultrasonography of mammary glands of cows, goats and sheep during α- and β-adrenergic agonist and oxytocin administration

Teat and gland cistern of the mammary glands of five dairy cows, five goats and five sheep were scanned in a water bath during alpha- and beta-adrenergic agonist and oxytocin administration. A 5 MHz linear array scanner was used to create vertical cut pictures with the scan plane longitudinally through the teat channel. The i.v. injection of the alpha-adrenergic agonist phenylephrine (30 micrograms/kg) induced diminution of the section area through teat and gland cistern by 38 +/- 17% on average within 1 min in all three species. In contrast, the i.v. injection of the beta-adrenergic agonist isoproterenol (1 microgram/kg) did not change the cisternal areas. The i.v. injection of oxytocin (2.0 x 10(-3) i.u./kg) elicited an enlargement of teat and gland cistern area by 48 +/- 12% on average. Ultrasonography proved to be a valuable system for visualizing changes of the cisternal volume. Smooth muscle contractions in response to phenylephrine administration are thought to cause area reduction, whereas an expected smooth muscle relaxation after injection of isoproterenol could not be observed by ultrasonography. Milk ejection as induced by oxytocin administration caused dramatic enlargement of the cistern area in all three species.

Inhibition of milk ejection in cows by oxytocin receptor blockade, α-adrenergic receptor stimulation and in unfamiliar surroundings

Inhibition of milk ejection in cows by oxytocin receptor blockade (Atosiban) and alpha-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 similarity. 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 induced by phenylephrine, which was antagonized by alpha-adrenergic receptor blockade. These results indicate that inhibition of milk ejection through activation of alpha-adrenergic receptors is based on blockade of milk flow into the cistern, but not through the teats.

Effects of feeding colostrum and a formula with nutrient contents as colostrum on metabolic and endocrine traits in neonatal calves.

Colostrum provides high amounts of nutrients and non-nutrient substances to neonates. To study differences between effects of nutritional and non-nutritional components on growth, health status and metabolic and endocrine traits, a formula was created based on bovine milk components which contained similar amounts of nutrients as bovine colostrum during the first 3 days of lactation, but only trace amounts of growth factors (such as insulin-like growth factor I) or hormones (such as insulin) in whey. Calves were fed either pooled colostrum of milkings 1 to 6, obtained during the first 3 days of lactation (GrC, n = 7) or a formula in the same amounts as colostrum (GrF, n = 7) for the first 3 days, followed by a milk replacer up to day 7. Pre- and postprandial blood samples were taken on days 1, 2, 3 and 7 for the determination of metabolic and endocrine traits and on day 5 we measured intestinal absorptive capacity by testing xylose absorption. Plasma concentrations of total protein and immunoglobulin G and γ-glutamyltransferase activity were lower (p < 0.05), whereas albumin and urea concentrations were higher (p < 0.05) in GrF than GrC during the first week of life. Plasma glucose concentrations were variably affected. Plasma triglyceride, phospholipid and cholesterol concentrations were higher (p < 0.05) in GrC than GrF on days 3 and 7. Insulin and growth hormone concentrations were higher (p < 0.05) in GrC than GrF on days 2 and 3 and on days 1 and 2, respectively, and glucagon concentrations were higher (p < 0.05) in GrC than GrF on day 1 and higher (p < 0.05) in GrF than GrC on day 3. Cortisol concentrations were higher (p < 0.05) on days 2 and 3 in GrF than GrC. Plasma xylose concentrations rose more markedly (p < 0.05) in GrC than GrF. In conclusion, feeding only trace amounts of bioactive substances appears to impair intestinal absorptive capacity and protein and fat metabolism and exert effects on endocrine systems in neonatal calves.

Subclinical mastitis in dairy cows in Swiss organic and conventional production systems

The objective was to compare the prevalence of subclinical mastitis (SM) and of udder pathogens in 60 Swiss organic (OP) and 60 conventional production systems (CP). Cows (n=970) were studied for SM prevalence and udder pathogens at median 31 d and 102 d post partum. Cows showing a >or=1+ positive California Mastitis Test (CMT) in at least one quarter were considered to have SM. Cow-level prevalences of SM for visits at 31 d and 102 d post partum (39% and 40% in OP and 34% and 35% in CP) were similar, but quarter-level prevalences of SM were higher (P<0.02) in OP than CP (15% and 18% in OP and 12% and 15% in CP). Median somatic cell counts in milk at 31 d post partum were higher (P<0.05) in OP than CP cows (43000 and 28000 cells/ml, respectively), but were similar at 102 d post partum in OP and CP cows (45000 and 38000 cells/ml, respectively). In milk samples from quarters showing a CMT reaction >or=2+ the prevalences of coagulase negative staphylococci were lower (P<0.05) at 102 d post partum, whereas prevalences of non-agalactiae streptococci were higher (P<0.05) in OP than in CP cows at 31 d and 102 d post partum. In conclusion, under Swiss conditions, subclinical mastitis is a greater problem in organic than in conventional production systems, but differences are not marked.