Detecting and predicting changes in milk homogeneity using data from automatic milking systems.

To ensure milk quality and detect cows with signs of mastitis, visual inspection of milk by prestripping quarters before milking is recommended in many countries. An objective method to find milk changed in homogeneity (i.e., with clots) is to use commercially available inline filters to inspect the milk. Due to the required manual labor, this method is not applicable in automatic milking systems (AMS). We investigated the possibility of detecting and predicting changes in milk homogeneity using data generated by AMS. In total, 21,335 quarter-level milk inspections were performed on 5,424 milkings of 624 unique cows on 4 farms by applying visual inspection of inline filters that assembled clots from the separate quarters during milking. Images of the filters with clots were scored for density, resulting in 892 observations with signs of clots for analysis (77% traces or mild cases, 15% moderate cases, and 8% heavy cases). The quarter density scores were combined into 1 score indicating the presence of clots during a single cow milking and into 2 scores summarizing the density scores in cow milkings during a 30-h sampling period. Data generated from the AMS, such as milk yield, milk flow, conductivity, and online somatic cell counts, were used as input to 4 multilayer perceptron models to detect or predict single milkings with clots and to detect milking periods with clots. All models resulted in high specificity (98-100%), showing that the models correctly classified cow milkings or cow milking periods with no clots observed. The ability to successfully classify cow milkings or cow periods with observed clots had a low sensitivity. The highest sensitivity (26%) was obtained by the model that detected clots in a single milking. The prevalence of clots in the data was low (2.4%), which was reflected in the results. The positive predictive value depends on the prevalence and was relatively high, with the highest positive predictive value (72%) reached in the model that detected clots during the 30-h sampling periods. The misclassification rate for cow milkings that included higher-density scores was lower, indicating that the models that detected or predicted clots in a single milking could better distinguish the heavier cases of clots. Using data from AMS to detect and predict changes in milk homogeneity seems to be possible, although the prediction performance for the definitions of clots used in this study was poor.

Homogeneity density scores of quarter milk in automatic milking systems.

Milk quality and clinical mastitis in dairy cows are monitored by detecting visually abnormal milk. A standardized method to evaluate clots in milk and studies of the incidence and dynamics of clots in milk at the quarter level are lacking. We validated a method to score clot density in quarter milk samples and describe the prevalence and dynamics of the density scores between consecutive samplings and periods in 4 farms with automatic milking systems. Using in-line filters, we collected quarter milk samples at each milking during 3 periods of 30 h each in each farm. Clot density was scored based on coverage of the filter area as 0 (negative), 1 (trace), 2 (mild), 3 (moderate), 4 (heavy), and 5 (very heavy). The score for a specific quarter and milking is referred to as the quarter milking score (QMS). Three assessors independently scored 902 images of filter samples with a Fleiss kappa value of 0.72. In total, 21,202 quarter milk samples from 5,398 milkings of 621 cows were collected. Of the quarter filter samples, 2.4% had visible clots, distributed as mild (1.4%), moderate (0.6%), heavy (0.3%), and very heavy (<0.1%, n = 8). Cases with a cow period sum of QMS ≥ 4, corresponding to 9.4% of all periods, harbored 86% and 94% of all QMS of 2 to 5 and 3 to 5, respectively. Of these cases, cows sampled in all 3 periods and clots in only 1 period had a quarter period sum score ≥ 1 in 1.8 different quarters in average. Corresponding numbers for the cows with clots or traces in 2 or 3 periods were 2.2 and 2.5 different quarters, respectively. A QMS of 2 to 5 in the preceding milking increased the chance of a QMS >1 in the following milking, with an average chance of 38%. The probability of a QMS > 1 increased with increasing previous QMS, a higher sum of QMS during the milking period, longer milking interval, and higher lactation number, but decreased with increasing days in milk. Our study showed that the method of clot-density scoring is feasible to perform and reproducible for investigating the occurrence and dynamics of clots in milk. Elevated clot-density scores clustered within certain cows and cow periods and appeared in new quarters of the cows over time. The low recurrence of QMS of 1 and 2 within quarters indicated that QMS 3 could be a reasonable threshold for detecting quarters with abnormal milk that require further attention.

Incidence and duration of increased somatic cell count in Swedish dairy cows and associations with milking system type.

Mastitis is one of the most costly diseases in dairy cows worldwide. Increased somatic cell count (SCC) is an indication of mastitis, often subclinical, which implies bacterial infection without clinical signs of inflammation. The aim was to investigate the occurrence of elevated udder SCC (defined as ≥200,000 cells/mL) over the lactation period, and before and after the dry period, for cows of different parity. The aim was also to analyze the association between prevalence and incidence of increased udder SCC and information on cow and herd level, such as breed and milking system type. Data were extracted from the Swedish Official Milk Recording Scheme between January 1, 2008, and December 31, 2011, including all herds with a yearly average of >60 cows. The data include descriptive information on herd and cow level and the results from the systematic test milking. The data included the following: for 2009, 239,182 cows in 1,633 herds; for 2010, 251,852 cows in 1,680 herds; and for 2011, 247,746 cows in 1,596 herds. The results show a peak in elevated udder SCC during the late summer season and that the highest proportion of cases occurs during the first lactation month; the latter was most prominent for primiparous cows. Forty-seven percent of all cows with elevated SCC recovered during dry period (went from high to low SCC), whereas 34% of all cows with low SCC before the dry period had an elevated SCC at first testing after calving. For first lactation cows, 19% had an elevated SCC at first test milking. When the outcomes for the 3 consecutive years were reanalyzed, it was confirmed that the effect of fixed factors such as breed, milk yield, and parity did not change over time, whereas the effect of milking system type did. For the incidence of becoming a new case and the prevalence of cows with elevated udder SCC, automatic milking system (AMS) was associated with reduced SCC in 2009 but associated with increased SCC in 2011. Regarding the proportion of new cases of elevated SCC per cow and year, AMS appeared to be a risk factor for all 3 yr, but the effect decreased over time. The shift for AMS from protective to risk factor regarding incidence of new cases and number of recordings with elevated SCC might reflect a change of the AMS population over these years. The findings indicate the need for appropriate udder health management customized to the system.

Associations between calf mortality during days 1 to 90 and herd-level cow and production variables in large Swedish dairy herds.

The aim of the study was to describe large Swedish dairy herds with high and low mortality risk in calves during the first 90 d of life, using herd-level data, and to evaluate if high calf mortality risk is associated with other herd-level management variables that influence cow health. A total of 57 Swedish dairy herds met the inclusion criteria of affiliation to the Swedish official milk recording scheme, herd size of ≥140 and ≥160 cows in 2008/2009 and 2009/2010, and calf mortality risks, classified as high (HM; calf mortality risk at least 3.5% in 2008/2009 and 5.5% in 2009/2010; n=28) or low (LM; calf mortality risk less than <1.5% in 2008/2009 and 2009/2010; n=29), and were thus included in the study. The data used in this study were collected from the Swedish Dairy association during the milking year 2009/2010. For LM herds, the calf mortality risk ranged from 0 to 1.46 (median=0.66) in 2008/2009 and from 0 to 1.48 (median=0.67) in 2009/2010. For HM herds, the calf mortality risk ranged from 3.57 to 11.52 (median=6.15) in 2008/2009 and from 5.88 to 18.23 (median=8.39) in 2009/2010. Median age at death was 28 d for HM and 37 d for LM herds. Associations between type of herd (HM or LM) and the production variables were evaluated using multi-correspondence analysis and logistic regression models covering the areas "mortality and culling," "health," "herd/production variables," and "fertility." Herds with HM risks during d 1 to 90 were associated with higher on-farm mortality rate in cows, lower average milk yield, higher incidence of antibiotic treatment, and a higher proportion of purchased animals. These results indicate that herds with HM risk during d 1 to 90 have coexisting issues concerning cow management and health. Future research is needed to evaluate if identifying HM herds and working with advisory and preventive manners at these herds also can be positive for a reduction of on-farm mortality and antibiotic usage, which are important issues from a global perspective.

Farm characteristics related to on-farm cow mortality in dairy herds: a questionnaire study.

Dairy cow mortality is an important animal welfare issue that also causes financial losses. The objective of this study was to identify farm characteristics and herd management practices associated with high on-farm cow mortality in Swedish dairy herds. A postal questionnaire was sent to farmers that had either high or low mortality rates for 3 consecutive years. The questionnaire consisted of five sections: 'About the farm', 'Milking and housing', 'Feeding', 'Routines' and 'Lame and sick cows'. A total of 145 questionnaires were returned (response rate=33%). Ten of the 77 characteristics investigated met the inclusion criteria for multivariable analysis. The final logistic regression model included: herd size, breed, use of natural service bull, bedding improvement frequency and pasture system. Herds with Swedish Holstein as the predominant breed (odds ratio (OR) 22.9, 95% confidence interval (CI) 5.2 to 101.8) or with mixed breeds (OR 5.5, 95% CI 1.7 to 17.5) had a greater risk of being high mortality herds than herds that were predominantly Swedish Red (OR 1). Herds larger than 100 cows (OR 19.6, 95% CI 3.5 to 110.4) and herds with 50 to 99 cows (OR 13.8, 95% CI 3.2 to 60.6) had greater risk of mortality than herds numbering 35 to 50 cows (OR 1). Being a high mortality herd was also associated with having cows on exercise lots during the summer season (OR 3.6, 95% CI 1.3 to 9.9) compared with on pasture. A missing answer on the question of bedding improvement frequency was associated with high mortality herds. Overall, this study suggests that characteristics that are related to intensification of the dairy industry are also associated with high on-farm mortality of dairy cows.

Associations between decreased fertility and management factors, claw health, and somatic cell count in Swedish dairy cows.

The aim of this retrospective single-cohort study was to investigate if a rapid change in feeding, management, or housing or an increasing incidence of claw diseases or udder health problems is associated with decreased reproductive performance. Data on individual cows and herds were retrieved from the Swedish official milk recording system and questionnaire data on feeding system was obtained from the regional dairy associations. In total, 63,561 cows in 759 herds were included in the study. The associations between the probability of pregnancy at first insemination and number of inseminations per animal submitted for artificial insemination and potential predictor variables were investigated using a logistic regression model and a Poisson regression model, respectively. The results indicated that cows with severe claw lesions or an increasing somatic cell count after calving had a lower probability of pregnancy at first insemination and had a higher number of inseminations per animal submitted for artificial insemination than healthy cows. Variables representing a change in housing, production system, or milking system within the period from 6 mo before calving until establishment of a new pregnancy were significantly associated with decreased reproductive performance. No differences in fertility were observed between cows milked in an automatic milking system compared with cows milked conventionally. The results indicate that a change of system, rather than the actual type of milking or housing system negatively affects reproductive performance. Special attention should therefore be paid to the fertility of cows when the herd management is changing. It is also important to prevent claw lesions and increasing cell counts after calving to avoid a decrease in reproductive performance.

Herd-level risk factors associated with cow mortality in Swedish dairy herds.

An increase in on-farm mortality (euthanasia and death) in dairy herds has been reported in several countries in the last decade. This does not only imply possible problems with animal welfare, but it also causes economic losses to the farmer. The objective of this study was to evaluate time trends in on-farm dairy cow mortality in Sweden and identify potential herd-level risk factors. Data were retrieved on all Swedish dairy herds enrolled in the milk recording scheme between 2002 and 2010. Herds with a herd size of <20 cows or a mortality rate (MR) of >40 dead or euthanized cows per 100 cow-years were excluded. Two different models were used: 1 multiple-year analysis, which included 6,898 herds during the period 2002 to 2010 and 1 single-year analysis including 4,252 herds for the year 2010, where other variables that were not present during the entire multiple year study were analyzed. The outcome variable was the number of euthanized and dead cows per year and season. A negative binomial regression model, adjusted for clustering within herd, was applied to both models. Fixed effects in the multiple-year analysis were breed, calving interval, herd size, milk yield, region, season, pasture period, and year. The fixed effects in the single-year analysis were breed, calving interval, conventional versus organic farming, herd size, housing system, milk yield, region, and season. The results demonstrated that MR gradually increased from 5.1 to 6.6 events per 100 cow-years during the study period. Swedish MR are consequently on par with, or even greater than, MR among dairy herds in other comparable countries. Higher mortality was associated with larger herd size, longer calving intervals, and herds that had Swedish Holstein as the predominant breed. Lower mortality was observed in herds with a higher herd average milk yield, during the fall and winter, and in organically managed herds. There were regional differences in mortality. An interaction between herd size and season was found in both models. Also, an interaction between housing system and milk yield was found in the single-year analysis. This first assessment of on-farm mortality in Swedish dairy herds confirmed that the MR has increased over the last few years. The study also identified some herd-level risk factors.

Supplementation of RRR-alpha-tocopheryl acetate to periparturient dairy cows in commercial herds with high mastitis incidence.

Dietary vitamin E supplementation of dairy cows around calving has been associated with improved udder health. However, few studies have been performed in Europe and in commercial dairy herds. Therefore, the main aim of the study was to evaluate whether extra daily dietary supplementation of 1,610 mg of alpha-tocopherol, in the form of RRR-alpha-tocopheryl acetate, to dairy cows around calving could improve the udder health in commercial herds with a high incidence of veterinary-treated clinical mastitis. In addition, the effects of supplementation on other cow diseases, fertility, milk production, culling, and stillbirth were also studied. Cows in 21 herds in southeast Sweden were assigned to 1 of 2 groups, 1) daily supplementation with 1,610 mg of RRR-alpha-tocopherol from 4 wk before to 2 wk after calving (n = 196), or 2) no extra supplementation (n = 203), on top of the normal diet containing average vitamin E supplementation (on average, 386, 664, and 957 mg/d to cows at 1 mo prior to calving, at calving, and at 15 d after calving, respectively). Multivariable generalized linear mixed models were used for statistical analyses of the effects of supplementation on the cumulative incidence risk of veterinary-treated clinical mastitis and of veterinary-treated diseases other than mastitis within 1 and 2 mo after calving, culling (due to udder health or other reasons) within 2 and 9 mo after calving, the risk of stillbirth or calf death within 24 h after calving, time from calving to first insemination, number of inseminations per service period, and cow composite natural logarithm of somatic cell count and daily milk production at the first monthly testing after calving. A significant effect of supplementation was observed only for stillbirth or calf death within 24 h after birth, which was significantly lower in the supplemented group. The reasons for this finding require further study.

Metabolic parameters and blood leukocyte profiles in cows from herds with high or low mastitis incidence.

The objective of this study was to determine whether there were differences in metabolic parameters and blood leukocyte profiles between cows in herds with high or low yearly mastitis incidence. In this study, 271 cows from 20 high yielding dairy herds were examined. According to the selection criteria, all herds had low somatic cell counts. Ten of the selected herds represented low mastitis treatment incidence (LMI) and ten herds had high mastitis treatment incidence (HMI). The farms were visited once and blood samples were taken from each cow that was in the interval from three weeks before to 15 weeks after parturition. The eosinophil count was significantly lower among cows from the HMI herds in the period from four weeks to 15 weeks after parturition. The plasma concentrations of beta-hydroxybutyrate, glucose, insulin and urea did not differ between groups, but the concentration of nonesterified fatty acids was significantly higher among HMI cows during the period three weeks after parturition. The concentration of the amino acid tryptophan in plasma was significantly lower among the HMI cows prior to parturition. Glutamine was significantly lower in cows from HMI herds during the first three weeks after parturition. Arginine was consistently lower in HMI cows, although the decrease was only significant during the period from four to fifteen weeks after parturition. The results suggest that there were differences in the metabolism and immune status between herds with high or low yearly mastitis treatment incidence indicating an increased metabolic stress in HMI cows.

Effects of certain inflammatory mediators on bovine neutrophil migration in vivo and in vitro.

Migration of bovine neutrophils towards endotoxin, recombinant bovine interleukin-1 beta (rBoIL-1 beta), recombinant human tumor necrosis factor-alpha (rhTNF-alpha), platelet-activating factor (PAF), complement factor C5a, leukotriene B4 (LTB4), and recombinant human interleukin-8 (rhIL-8) was studied in vivo, using the teat cistern model, and in vitro using the modified Boyden chamber method. Infusion of endotoxin, rBoIL-1 beta, rhTNF-alpha, PAF, or C5a into the teat cistern induced significant accumulation of leukocytes, mainly neutrophils, during the sampling period. Endotoxin was, on a molar basis, the most potent inducer of cell accumulation in vivo, followed by rBoIL-1 beta, while C5a, PAF and rhTNF-alpha were less potent. No significant cell accumulation was observed after infusion of LTB4 or rhIL-8. A significant migration of cells into the teat cistern was first observed 2 h after the infusion of endotoxin or rBoIL-1 beta, the rBoIL-1 beta-induced response started somewhat earlier. The first significant cell accumulation after infusion of PAF or C5a was observed already 1.5 h post infusion. The largest numbers of cells were reached 2.5-4.5 h after the infusion of endotoxin, rBoIL-1 beta, rhTNF-alpha, PAF or C5a. In vitro, significant migration of bovine blood neutrophils was observed towards C5a or rhIL-8, and to a lower extent towards LTB4, while no chemotactic response to endotoxin, rBoIL-1 beta, rhTNF-alpha, and PAF was observed. Possible roles of the different substances as inducers of neutrophil migration into the bovine teat are discussed.

A periodate-sensitive anti-phagocytic surface structure, induced by growth in milk whey, on Staphylococcus aureus isolated from bovine mastitis.

The phagocytic and chemiluminescent activity of purified bovine neutrophils in response to two Staphylococcus aureus strains isolated from mastitic bovine milk and grown in milk whey was studied. The activity was significantly reduced compared with the response elicited by the same strains grown in tryptic soy broth (TSB). A mild periodate treatment of the milk whey-grown strains resulted in a significant increase of both chemiluminescence and phagocytosis, whereas trypsin, subtilisin or papain treatment had no effect. The decreased binding of complement factor C3 to milk-whey-grown bacteria was restored to the level of TSB-grown homologous organisms by periodate treatment. Moreover, this treatment, but not treatment with trypsin, increased the surface hydrophobicity of milk-whey-grown bacteria. The chemiluminescent activity was as high towards heat-killed as towards live bacteria. Also, incubation of heat-killed TSB-grown bacteria in milk whey did not alter the chemiluminescent response, indicating that the reduced neutrophil activity towards milk-whey-grown bacteria was not due to binding of milk components to the microorganisms. These results strongly suggest that bovine mastitis S. aureus strains grown in milk whey produce an anti-phagocytic surface structure. This structure is heat- and protease-resistant and renders the bacterial surface hydrophilic. The anti-phagocytic material is altered or, more likely, released from the bacterial surface on periodate treatment and is probably of carbohydrate nature.

A rapid technique for the isolation of highly purified, functionally intact bovine neutrophilic granulocytes.

A new method for the isolation of bovine neutrophilic granulocytes from peripheral blood based on centrifugation in a discontinuous metrizamide gradient has been developed. The procedure is rapid, taking only about 2 h, and gives highly purified (greater than 90%) neutrophils in a high yield (approximately 85%). The function of the cells, as measured by chemiluminescence and migration assays, is not significantly influenced by the isolation procedure. Eosinophils can also be isolated by a slight variation of the method. Initial applications of the separation procedure indicate its usefulness in clinical studies of bovine neutrophil function. A variation between individuals in the function of the cells was thus demonstrated. Moreover, the chemiluminescence of neutrophils from infected animals was found to be greatly increased, and neutrophil migration was shown to be stimulated by in vivo ACTH treatment.

The conformational changes of alpha 2-macroglobulin induced by methylamine or trypsin. Characterization by extrinsic and intrinsic spectroscopic probes.

The conformational changes around the thioester-bond region of human or bovine alpha 2M (alpha 2-macroglobulin) on reaction with methylamine or trypsin were studied with the probe AEDANS [N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid], bound to the liberated thiol groups. The binding affected the fluorescence emission and lifetime of the probe in a manner indicating that the thioester-bond region is partially buried in all forms of the inhibitor. In human alpha 2M these effects were greater for the trypsin-treated than for the methylamine-treated inhibitor, which both have undergone similar, major, conformational changes. This difference may thus be due to a close proximity of the thioester region to the bound proteinase. Reaction of trypsin with thiol-labelled methylamine-treated bovine alpha 2M, which retains a near-native conformation and inhibitory activity, indicated that the major conformational change accompanying the binding of proteinases involves transfer of the thioester-bond region to a more polar environment without increasing the exposure of this region at the surface of the protein. Labelling of the transglutaminase cross-linking site of human alpha 2M with dansylcadaverine [N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide] suggested that this site is in moderately hydrophobic surroundings. Reaction of the labelled inhibitor with methylamine or trypsin produced fluorescence changes consistent with further burial of the cross-linking site. These changes were more pronounced for trypsin-treated than for methylamine-treated alpha 2M, presumably an effect of the cleavage of the adjacent 'bait' region. Solvent perturbation of the u.v. absorption and iodide quenching of the tryptophan fluorescence of human alpha 2M showed that one or two tryptophan residues in each alpha 2M monomer are buried on reaction with methylamine or trypsin, with no discernible change in the exposure of tyrosine residues. Together, these results indicate an extensive conformational change of alpha 2M on reaction with amines or proteinases and are consistent with several aspects of a recently proposed model of alpha 2M structure [Feldman, Gonias & Pizzo (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5700-5704].