Thermoduric and Psychrotrophic Organisms on Poorly Cleansed Milking Plants and Farm Bulk Milk Tanks

S ummary . Poorly cleansed pipeline milking plants gave much higher total and thermoduric colony counts than the internal surfaces of refrigerated farm bulk milk tanks, higher counts being recorded for milking plants after circulation cleaning than after cleansing by the acidified boiling water (ABW) method. Psychrotrophic counts were highest for farm milk tanks and lowest for milking plants cleansed by the ABW method. There was much variation in the proportion of thermoduric and psychrotrophic organisms in the total bacterial content of milking plants determined at 30°, thermodurics being much more prevalent in 21% of the rinses and psychrotrophs in 16%. Both groups formed about equal proportions in 9% of the pipeline rinses. The bacterial content of the farm bulk milk tanks was characterized by many psychrotrophs and few thermodurics.     

Comparative bio-efficacy of nuclear polyhedrosis virus (NPV) and Spinosad against American bollwormm,Helicoverpa armigera (Hubner)

American bollworm (ABW), Helicoverpa armigera (Hubner), is considered as a major pest of cotton, Gossypium hirsutum, all over the globe. Due to its destructive feeding nature and continuous consumption of the same chemicals, it devolved resistant against many insecticides. Therefore, a combined application of bio- and synthetic-pesticide need to evaluate against this pest. The entomopathogenic viruses like nuclear polyhedrosis virus (NPV), a member of baculoviruses, can be the potential candidates for better control against ABW. The present study was conducted to assess the comparative efficacy of NPV and Spinosad 240SC (with the concentration of 250 mL · ha￣?) against ABW in the controlled environment. The ABW was treated with different concentrations of NPV and Spinosad separately and in a combination of NPV with 0.1% Spinosad. The results revealed that highest concentrations showed highest mortality (95%) followed by 95%, 92%, 84%, 82% and 78% mortality at 1 × 10⁹, 1 × 10⁸, 1 × 10⁷, 1 × 10⁶ and 1 × 10⁵ POBs, respectively. Spinosad when mixed in diet give 100% mortality at 0.8% followed by 50.87%, 42.10%, 29.82%, 26.31% and 22.80% mortality at 0.4%, 0.2%, 0.1%, 0.5% and 0.025% respectively. The results of this study revealed that microbial control of ABW through NPV is an effective tool. The repeated use of synthetic pesticides caused the resurgence of many insect pests, and this study results would provide useful insight to build a framework for future investigations for the management of many major insect pests.     

Endolithic microbial life in hot and cold deserts

Endolithic microorganisms (those living inside rocks) occur in hot and cold deserts and exist under extreme environmental conditions. These conditions are discussed on a comparative basis. Quantitative estimates of biomass are comparable in hot and cold deserts. Despite the obvious differences between the hot and cold desert environment, survival strategies show some common features. These endolithic organisms are able to switch rapidly their metabolic activities on an off in response to changes in the environment. Conditions in hot deserts impose a more severe environmental stress on the organisms than in the cold Antarctic desert. This is reflected in the composition of the microbial flora which in hot desert rocks consist entirely of prokaryotic microorganisms, while under cold desert conditions eukaryotes predominate.Proceedings of the Fourth College Park Colloquium on Chemical Evolution:Limits of Life, University of Maryland, College Park, 18–20 October 1978.     

Step behaviour and autonomic nervous system activity in multiparous dairy cows during milking in a herringbone milking system

Behavioural and cardiac responses of multiparous dairy cows (n=24) during milking in a 2×4 stall herringbone milking system were evaluated in this study. Heart rate (HR), parasympathetic tone index (high frequency component, HF) of heart rate variability and sympathovagal balance indicator LF/HF ratio (the ratio of the low frequency (LF) and the HF component) were analysed. Measurement periods were established as follows: (1) standing calm (baseline), (2) udder preparation, (3) milking, (4) waiting after milking in the milking stall and (5) in the night (2 h after milking). Step behaviour was recorded and calculated per minute for the three phases of the milking process (udder preparation, milking and waiting after milking). HR was higher during udder preparation and milking compared with baseline (P=0.03, 0.027, respectively). HF was significantly lower than baseline levels during waiting in the milking stall after milking (P=0.009), however, during udder preparation, milking and 2 h after milking did not differ from baseline (P>0.05, in either case). LF/HF during the three phases of the milking process differed neither from baseline levels nor from each other. Steps occurred more often during waiting after milking than during udder preparation (P=0.042) or during milking (23; P=0.017). Our results suggest that the milking procedure itself was not stressful for these animals. After milking (following the removal of the last teat cup and before leaving the milking stall), both decreased parasympathetic tone (lower HF) and increased stepping rate indicated a sensitive period for animals during this phase.     

Ovipositional preferences and larval survival of annual bluegrass weevil,Listronotus maculicollis,on Poa annua and selected bentgrasses (Agrostis spp.)

The annual bluegrass weevil (ABW), Listronotus maculicollis Kirby (Coleoptera: Curculionidae), is a serious and expanding pest of short‐cut turfgrass on golf courses in eastern North America. Increasing problems with the development of insecticide resistance in this pest highlights the need for more sustainable management approaches. Plant resistance is one of the most promising alternative strategies. Bentgrasses are the dominant grass species on golf course fairways, tees, and putting greens in the areas affected by ABW. But Poa annua L. (Poaceae), a highly invasive weed, often constitutes a large percentage of turf stands in short‐mown golf courses and is thought to be particularly susceptible to ABW. We studied resistance to ABW in four cultivars of creeping bentgrass, Agrostis stolonifera L., and two cultivars each of colonial bentgrass, Agrostis capillaris L., and velvet bentgrass, Agrostis canina L. (Poaceae), in comparison with P. annua by addressing the three major components of resistance: antixenosis (adult ovipositional and feeding preferences), antibiosis (larval survival and growth), and grass tolerance (grass damage). Our findings suggest that antixenosis/non‐preference is at least partially involved in bentgrass resistance to ABW. Even though oviposition was observed in all tested grasses, females laid significantly fewer eggs in Agrostis spp. than in P. annua. Compared to P. annua, Agrostis spp. were also less suitable for larval development with lower numbers of ABW immatures recovered and larvae weighing less and being less advanced in development. Resistance levels to ABW larvae varied significantly among Agrostis spp. and cultivars. Agrostis canina was least preferred by females for oviposition and A. stolonifera was the least suitable for larval survival and development. Agrostis spp., especially A. stolonifera, were more tolerant to ABW feeding than P. annua. Our findings suggest that reduction in P. annua and replacement with Agrostis spp., especially A. stolonifera, wherever feasible should be integral to more sustainable approaches to ABW management.     

Microflora associated with the internal surfaces of rubber and stainless steel milk transfer pipeline

Sterile sections of rubber and stainless steel milk transfer pipeline were inserted sequentially into a milking installation and soiled with fresh raw milk over a period of 5 d. The resultant adherent microbial population was removed and the generic composition of mesophilic and psychrotrophic types was determined. In all cases Acinetobacter spp. were found to predominate (59.5–75.6%). The generic composition of the raw milk used to soil the milking unit (with inserted pipe section) was determined once during each 5-d soiling period. In general the milk was found to contain a mixed flora in which Gram-positive organisms predominated.     

Bacteriological Examination of Pipeline Milking Machines by Rinsing the Entire System

The results of bacteriological examination of rinses of the entire systems of 12 pipeline milking machines were compared with a similar examination of the milk collected while they were used for milking. The machines were cleaned in three different ways and showed varying degrees of complexity. There was a close correlation between the results when the milk was collected with the maximum of cleanliness during milking. This is attributed to the localization of most of the micro-organisms present in the milking machine in the junctions and complex parts of the machine and not on the surface of the equipment, so they are removed with the same efficiency by cold water or warm milk. The number of micro-organisms retrieved by water during successive rinses (or by the milk during milking) decreased at a rate which depended either on the level of initial contamination of the equipment, its design or on the types of micro-organisms present. Recommendations are made for the interpretation of the results of rinsing.     

Microflora associated with the internal surfaces of rubber and stainless steel milk transfer pipeline

Sterile sections of rubber and stainless steel milk transfer pipeline were inserted sequentially into a milking installation and soiled with fresh raw milk over a period of 5 d. The resultant adherent microbial population was removed and the generic composition of mesophilic and psychotropic types was determined. In all cases Acinetobacter spp. were found to predominate (59.5-75.6%). The generic composition of the raw milk used to soil the milking unit (with inserted pipe section) was determined once during each 5-d soiling period. In general the milk was found to contain a mixed flora in which Gram-positive organisms predominated.     

USE OF THE DCB TECHNIQUE FOR EXTRACTION OF HYDROUS IRON OXIDES FROM ROOTS OF WETLAND PLANTS

Replicate samples of Phragmites communis roots coated with hydrous iron oxide were analyzed for internal and external Fe using the dithionite-citrate-bicarbonate technique (hot DCB), a modified DCB technique (cold DCB), Ethylenediamine tetraacetic acid (EDTA), Diethylenetriamine pentaacetic acid (DTPA), and a total Fe analysis. EDTA and DTPA were found to be inefficient in extracting FeOOH while both DCB techniques were efficient. Concentrations of Fe extracted (8.93% and 9.37% by weight) and percent of total Fe extracted (98.3% and 98.6%) by the hot and cold DCB techniques (respectively) were not significantly different. Results of the total Fe analysis (8.58% Fe by weight) were the same as extracted Fe and total Fe determined by the DCB techniques. This may reflect the large external Fe : internal Fe ratio of the sample roots. Despite equivalent results, the cold DCB technique is preferred over the hot DCB technique due to a reduced likelihood of structural damage to roots.     

Relationship of goat milk flow emission variables with milking routine,milking parameters,milking machine characteristics and goat physiology

The aim of this paper was to study the relationship between milk flow emission variables recorded during milking of dairy goats with variables related to milking routine, goat physiology, milking parameters and milking machine characteristics, to determine the variables affecting milking performance and help the goat industry pinpoint farm and milking practices that improve milking performance. In total, 19 farms were visited once during the evening milking. Milking parameters (vacuum level (VL), pulsation ratio and pulsation rate, vacuum drop), milk emission flow variables (milking time, milk yield, maximum milk flow (MMF), average milk flow (AVMF), time until 500 g/min milk flow is established (TS500)), doe characteristics of 8 to 10 goats/farm (breed, days in milk and parity), milking practices (overmilking, overstripping, pre-lag time) and milking machine characteristics (line height, presence of claw) were recorded on every farm. The relationships between recorded variables and farm were analysed by a one-way ANOVA analysis. The relationships of milk yield, MMF, milking time and TS500 with goat physiology, milking routine, milking parameters and milking machine design were analysed using a linear mixed model, considering the farm as the random effect. Farm was significant (P<0.05) in all the studied variables. Milk emission flow variables were similar to those recommended in scientific studies. Milking parameters were adequate in most of the farms, being similar to those recommended in scientific studies. Few milking parameters and milking machine characteristics affected the tested variables: average vacuum level only showed tendency on MMF, and milk pipeline height on TS500. Milk yield (MY) was mainly affected by parity, as the interaction of days in milk with parity was also significant. Milking time was mainly affected by milk yield and breed. Also significant were parity, the interaction of days in milk with parity and overstripping, whereas overmilking showed a slight tendency. We concluded that most of the studied variables were mainly related to goat physiology characteristics, as the effects of milking parameters and milking machine characteristics were scarce.     

Synthesizing Oligomers from Monomeric Nucleotides in Simulated Hydrothermal Environments

Dimers and trimers of adenosine monophosphate(AMP) were synthesized from AMP in environmentssimulating hot vents on the sea floor of the primitiveEarth. The simulated environments were made in the flowreactor, in which an aqueous solution of reactants wascirculated from the hot to the cold region repeatedly. Theoligomerization proceeded most significantly when the hotreaction solution at about 110 °C was abruptly ejected into the cold environment maintained at about 0 °C.     

Biotechnology: impact on biological warfare and biodefense

Advances in biological research likely will permit development of a new class of advanced biological warfare (ABW) agents engineered to elicit novel effects. In addition, biotechnology will have applications supporting ABW weaponization, dissemination, and delivery. Such new agents and delivery systems would provide a variety of new use options, expanding the BW paradigm. Although ABW agents will not replace threats posed by traditional biological agents such as Bacillus anthracis (anthrax) and Variola (smallpox), they will necessitate novel approaches to counterproliferation, detection, medical countermeasures, and attribution.     

Early Campanian Grünbach flora of Austria

The Grünbach flora from the Grünbach Formation of the Grünbach-Neue Welt Basin, Austria, is dated to the Early Campanian on the basis of foraminifers found in this formation and marine fossils serving as stratigraphic markers in the underlying (Maiersdorf Formation) and overlying (Piesting Formation) deposits. The Grünbach flora enumerates 53 species assigned to the Equisetopsida (1 species), Polypodiopsida (11 species), Cycadopsida (1 species), Pinopsida (4 species), Liliopsida (6 species), and Magnoliopsida (30 species). These plants constituted several plant communities, among which the following are more or less reliably defined: aquatic, swamp/semiaquatic, juglandaceous and palm wetland forest, riparian, and mesophytic forest. The Grünbach flora grew in a humid subtropical frost-free climate with a hot summer and a short and relatively dry, but not arid, period during the year.     

Characterization of the cold and hot natures of raw and processed Rehmanniae Radix by integrated metabolomics and network pharmacology

BackgroundThe processing of Chinese materia medica (CMM) is one of the characteristics and advantages of traditional Chinese medicine (TCM). Occasionally, the processing of CMM might reverse the cold/hot nature of CMM. For example, the nature of raw Rehmanniae Radix (RR) is cool, while the processed Rehmanniae Radix (PR) by steaming is hot. Because the cold/hot nature of CMM is defined by the body's response to CMMs, a metabolomics approach, allowing the monitoring of the fluctuation of endogenous metabolites related to an exogenous stimulus, might be an ideal tool to uncover the cold/hot nature of different forms of Rehmanniae Radix.PurposeAn integrated strategy combining metabolomics and network pharmacology was applied to illuminate the different natures of raw and processed Rehmanniae Radix.Study designMice were orally administered RR and PR once daily for ten days. The entire metabolic changes in the plasma of mice were profiled by ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF MS). Furthermore, network pharmacology analysis was performed to identify the underlying targets related to iridoids that significantly changed during the processing.ResultsThe metabolomics analysis results demonstrated a clear separation of the metabolic phenotypes among the control, RR and two PR groups in both the positive and negative modes. Nine lysophosphatidylcholines (LysoPCs), LysoPC (16:0), LysoPC (18:2), LysoPC (18:1), LysoPC (22:6), LysoPC (20:2), LysoPC (18:0), LysoPC (16:1), LysoPC (20:4) and LysoPC (20:5), that decreased in the RR-treated group, but increased in the PR-treated group, were identified to be potential biomarkers related to the natures of RR and PR. The network pharmacology results indicated that four iridoids in Rehmanniae Radix, 8-epiloganic acid, 6-O-p-coumaroyl ajugol, 6-O-p-hydroxybenzoyl ajugol and ajugol, might play important roles in the different natures of raw and processed Rehmanniae Radix.ConclusionsThere might be a strong connection between the cold/hot nature of different forms of Rehmanniae Radix and LysoPC metabolism. This study offers new insight into the cold/hot nature of Rehmanniae Radix.     

Rapid Counting of Bacteria in Rinses of Milking Equipment by a Membrane Filtration - Epifluorescent Microscopy Technique

Membrane filtration and epifluorescent microscopy were used for the rapid enumeration of bacteria in rinses of milking equipment. Rinse solution (10 ml) was filtered through a 0.6 µm pore size Nuclepore membrane filter and the bacteria retained stained with acridine orange. The clump count of orange fluorescing bacteria on the membrane correlated well with the corresponding plate count ( r =0.83). The technique is rapid, taking approximately 10 min, and is sufficiently sensitive to detect 1000 bacteria ml rinse (equivalent to 5 × 10³ bacteria/m² of milking equipment surfaces by the method currently in use).     

World-wide latitudinal and longitudinal seaweed distribution patterns and their possible causes,as illustrated by the distribution of Rhodophytan genera

The degree of similarity between red algal generic floras in each pair of 22 climatically defined biogeographic regions was established on a world-wide scale by Jaccard's similarity index and by an hierarchical clustering with an agglomerative centroid method. Two clusterings were carried out, the first one on the basis of all 637 genera, and the second one on the basis of genera not occurring in the tropics and non-endemic to any one of the 22 regioms (145 genera). This latter clustering served to detect better the relationships among non-tropical floras. The results indicate the following division of the earth's rhodophytan seaweed floras: (1) A rich tropical-warm temperate "Tethyan" group including the rich tropical Indo W Pacific and W Atlantic floras, and the rich warm temperate NW Pacific and NE Atlantic floras; (2) the depauperate extensions of the above group (the tropical E Pacific and E Atlantic floras, and the warm temperate NW and SW Atlantic floras); (3) a cold temperate and a warm temperate N Pacific group; (4) an Arctic-cold temperate N Atlantic group and a NE Atlantic warm temperate flora; (5) an Antarctic-cold temperate southern hemisphere group including the cold temperate SE Pacific, SW Atlantic, SE Atlantic floras, and the Antarctic flora; (6) the two highly individual, but slightly related warm temperate SE Atlantic flora (S. Africa) and SW Pacific flora (Southern Australia and Northern New Zealand); (7) the depauperate warm temperate SE Pacific flora. Although the northern and southern hemisphere temperate and polar floras are quite unrelated (on the basis of genera lacking in the tropics), they share nonetheless a number of cool water genera which apparently have succeeded in passing the adverse tropical belt. The rich tropical-warm temperate group is thought to consist of vicariant portions of a formerly continuous Tethyan flora. The N Pacific and N Atlantic temperate floras are thought to have developed independently since the Oligocene (~ 40.10⁶ y) deterioration of the climate and to have partially mixed their cool water genera only after the Pliocene inundation (2.10⁶ y) of the Bering Land Bridge. The warm-temperate floras of S Africa and southern Australia probably owe their richness and individuality to a very long isolation (already at the start of the Cenozoicum) and a continued residence in warm temperate conditions with small seasonal fluctuations.Paper presented at the Seaweed Biogeography Workshop of the International Working Group on Seaweed Biogeography, held from 3–7 April 1984, at the Department of Marine Biology, University of Groningen (The Netherlands). Convenor: C. van den Hoek.     

Reduced cerebrospinal fluid levels of immunoreactive pro-opiomelanocortin related peptides (including beta-endorphin) in anorexia nervosa

The discovery that the endogenous opioid peptides contribute to the modulation of appetitive behavior and neuroendocrine function has raised questions as to whether disturbances of opioids contributes to the pathophysiology of eating disorders. To assess central nervous system (CNS) beta-endorphin in patients with anorexia nervosa we measured cerebrospinal fluid (CSF) beta-endorphin concentrations before, and at intervals after weight correction. In addition, we measured three sister peptides (beta-lipotropin, adrenocorticotropic hormone (ACTH), and the N-terminal fragment) derived from the same precursor molecule, pro-opiomelanocortin (POMC) to determine whether possible disturbances might extend to sister peptides. Underweight anorectics (58 +/- 5% of average body weight (ABW), n = 10) had significantly lower CSF concentrations of all 4 peptides compared to healthy controls (102 +/- 10% ABW, n = 11). CSF concentrations of all 4 POMC-related peptides were found to be significantly increased when the same anorectics were restudied 4 to 6 weeks after weight gain (83 +/- 4% ABW). After weight gain, levels of CSF beta-endorphin, beta-lipotropin, and ACTH were similar to controls, whereas levels of CSF N-POMC remained significantly less than controls. Another group of women, previously underweight with anorexia nervosa, but weight-restored (93 +/- 11% ABW, n = 12) for greater than 1 year had CSF concentrations of all 4 POMC-related peptides that were similar to controls. We conclude that underweight anorectics have state-associated disturbances of CNS beta-endorphin as well as other POMC-related peptides. These abnormalities are part of the neurobiological syndrome of anorexia nervosa and may contribute to the characteristic alterations in behavior and neuroendocrine function.     

Is there a massive glacial–Holocene flora continuity in Central Europe?

The prevailing paradigm about the Quaternary ecological and evolutionary history of Central European ecosystems is that they were repeatedly impoverished by regional extinctions of most species during the glacial periods, followed by massive recolonizations from southern and eastern refugia during interglacial periods. Recent literature partially contradicts this view and provides evidence to re-evaluate this Postglacial Recolonization Hypothesis and develop an alternative one. We examined the long-term history of the flora of the Carpathian (Pannonian) Basin by synthesising recent advances in ecological, phylogeographical, palaeoecological and palaeoclimatological research, and analysing the cold tolerance of the native flora of a test area (Hungary, the central part of the Carpathian Basin). We found that (1) many species have likely occurred there continuously since before the Last Glacial Maximum (LGM); (2) most of the present-day native flora (1404 species, about 80%) can occur in climates as cold as or colder than the LGM (mean annual temperature ≤+3.5°C); and (3) grasslands and forests can be species-rich under an LGM-like cold climate. These arguments support an alternative hypothesis, which we call the Flora Continuity Hypothesis. It states that long-term continuity of much of the flora in the Carpathian Basin is more plausible than regional extinctions during the LGM followed by massive postglacial recolonizations. The long-term continuity of the region's flora may have fundamental implications not only for understanding local biogeography and ecology (e.g. the temporal scale of processes), but also for conservation strategies focusing on protecting ancient species-rich ecosystems and local gene pools.     

Tracheal vascular and smooth muscle responses to air temperature and humidity in dogs

Twenty-five dogs were anesthetized, paralyzed, and artificially ventilated. Their cranial tracheal arteries were perfused bilaterally with blood at constant flow, and the perfusion pressures (Patr) were measured. Tracheal smooth muscle function was assessed by recording changes in external diameter (delta Dtr). The perfused segment of the trachea was exposed to air at a constant unidirectional airflow of 25 l/min. Group 1 (n = 6) was exposed to cold dry air, ambient room air, and hot dry and hot humid air, each for 10 min with exposures starting from zero flow. The tracheal vascular responses to all four conditions were small vasodilations (delta Patr from -2 to -6%) followed by recovery or small vasoconstrictions. In group 2 (n = 19), exposures to cold dry and hot humid air were preceded and followed by body-temperature fully humidified air. Cold dry air caused a sustained vasodilation (delta Patr -9.0 +/- 1.1%), and hot humid air usually caused a biphasic response: a vasoconstriction (delta Patr 4.4 +/- 1.0%) followed by a vasodilation (delta Patr -5.7 +/- 1.9%). The warm humid air after cold dry air or hot humid air caused a further vasodilation, which lasted a short time after cold dry air (delta Patr -3.7 +/- 0.4%) but greater than 10 min after hot humid air (delta Patr -13.8 +/- 1.4%). In both groups, all exposures that cooled the trachea (cold dry air, ambient room air, and hot dry air) caused smooth muscle contraction, and hot humid air that warmed the trachea caused relaxation.     

The effect of omitted milking on the behaviour of cows in the context of cluster attachment failure during automatic milking

In robotic milking there is always a slight chance of failure to attach the milking cluster. Attachment failure is most likely for cows whose udder conformation is less convenient for robot attachment. In general, after milking failure cows try to revisit the milking robot if they are not sent to a separate area. Since it is difficult to estimate the effect of milking failure on such a cow and her welfare in conditions of robotic milking, a specific 16-day trial was conducted on 12 cows. These cows were milked in a milking parlour with six milking stalls. Each afternoon milking, three cows were not milked. All the cows were closely observed in the cubicle house for 1 h after the afternoon milking. Thereafter, all cows were brought to the milking parlour the third time and the three unmilked cows were milked. In total, each cow was observed 12 times after milking and four times after omitted milking. The following behavioural traits were registered: time budget for the 1 h, occurrence and time until eating, drinking, lying, urination and defecation, and aggressive interactions. Milking order was defined on the basis of how often a cow came to the milking parlour in the first batch of six cows. Moreover, the data related to the milk yield and the use of the automatic feeding installation with the complete diet were analysed. After omitted milking, only the cows from the first batch stood longer in cubicles (14.2 min of 1 h) and lay less (5.4 min of 1 h) than milked cows of the same batch (respectively 7.0 min and 16.3 min for standing and lying in cubicles) (P<0.01). After omitted milking, cows urinated earlier and more frequently (64.5%) than milked cows (36.3%) (P<0.002) (both batches). There were no statistically significant differences in eating time and feed intake after milking and omitted milking. Milk yield per cow averaged 24.9 kg during days with omitted (delayed by 1 h) milking and 25.3 kg during the days without omitted milking (P<0.05). It was concluded that cows show some signs of discomfort after omitted milking (urination); this discomfort seemed to be greater in cows coming earlier to the milking parlour (afterwards they preferred to stand rather than to lie). The 60% of cases of milk leakage found after omitted milking indicates that failed cluster attachment can be accompanied by an extra risk factor for the occurrence of mastitis. However, omitted milking as a treatment did not influence feeding and aggressive behaviour or milking order when unmilked cows were brought to the milking parlour the third time together with the milked cows. Our methods and results can be useful for estimating the effects of robot milking failures on a cow. Future studies should pay particular attention to high-yielding cows and to longer periods of delayed milking.