Dairy cow response to electrical environment fnial report part 3

Submitted To the Minnesota Public Utilities Commission Douglas J. Reinemann, Ph.D., Associate Professor, Biological Systems Engineering, Lewis G. Sheffield, Ph.D., Professor, of Dairy Science, University of Wisconsin- Steven D. LeMire, Research Assistant, Biological Systems Engineering, University of Morten Dam Rasmusssen, Ph.D., Senior Scientist, Danish Institute of Agricultural Sciences, Department of Animal Health and Welfare Milo C. Wiltbank, Ph.D., Professor of Dairy Science, University of Wisconsin-Madison ABSTRACT
Twelve lactating Holstein cows, housed in a stanchion barn, were exposed to 1 mA of 60Hz electrical current from front to rear hooves for two weeks. Twelve cows acted ascontrols. Immune function was assessed by analyzing blood samples taken twice a weekfor thirteen different response variables. The measures for lymphocyte blastogenesis(concanavalin A and phytohemagglutanin mitogens), and oxidative burst (PMA-inducedchemiluminescence) were chosen a priori as the best indicators of immune functionresponse. Immunoglobulin production and interleukin 1 and 2 were also assessed. Therewas no statistically significant difference between control and treatment cows for any ofthe main response variables. The difference between the control and treatment cows wasstatistically significant for one of the secondary response variables but did not appear tobe consistent with other observations. Collectively, these results suggest that exposure to1 mA of current for two weeks had no significant effect on the immune function of dairycattle.
The Minnesota Legislature authorized the Minnesota Public Utilities Commission toestablish a committee of science advisors in response to claims by some dairy farmersthat electric currents in the earth from electric utility distribution systems are somehowresponsible for problems with behavior, health and production of dairy cows. Amultidisciplinary group with expertise in the fields of agricultural engineering, animalphysiology, biochemistry, electrical engineering, electrochemistry, epidemiology, physics, soil science, and veterinary science were assembled to serve as science advisors.
The consensus of the science advisors was that currents in the earth can only interact withdairy cows through their associated electric fields, magnetic fields and voltages, and thatthese parameters should be the focus of analysis. Five possible mechanisms wereidentified by which the electrical distribution system could conceivably affect dairy cows.
A field study was conducted to investigate the magnitude of these hypothesized electricalfactors on 19 Minnesota dairy farms. The combined electrical data from the field studyindicated that while none of the five electrical hypotheses could be ruled out, only one ofthem was a priority for research. This hypothesis is that continuous or frequentlyrepeated contact of confined cows to sources of low level stray voltage may result inelectric fields inside the cow at levels high enough to produce biological effects withoutproducing observable or measurable behavior modifications. The front to rear hoof steppotential measured in the field study resulted in the continuous and longer-term exposurerequired to satisfy this low level voltage hypothesis. If a physiological response is tooccur in dairy cows, it is more likely to be produced by step potential exposures in thestalls rather than outside because: 1. step potentials in the stall are larger than outside,and, 2. step potentials in the stall last longer because of long periods of cow confinement.
A physiological response in dairy cows that are exposed to low level voltages (1-100mV) has not been specified. Various types of physiological responses (e.g., circulatinghormones or their metabolites) to electric and magnetic field exposures have been shownin the published literature to occur in various animals other than dairy cows. These areneither equivalent to, nor indicative of, pathological effects that cause poor health andproduction in dairy cows. Since it is not possible to extrapolate to dairy cows, furtherstudies were recommended that specifically examine exposure of dairy cows to steppotentials lower than those threshold levels already known to elicit behavioral responses.
There have been several studies that have investigated the physiological response of dairycows exposed to electrical current. Endocrine response experiments are summarized inthe previous sections of this report. Gorewit et al. (1992) reported that dairy cowsexposed to up to 4 V of 60 Hz while drinking, during the entire lactation, showed nodifference in milk yield, somatic cell count, cow health or reproductive performance.
Reinemann et al. (1996) reported that cows exposed to transient currents for three weeksshowed no significant treatment effect for the following parameters: sodium, albumin,potassium, enzymatic CO2, chloride, calcium, phosphorus, glucose, creatinine, andcreatine kinase. The absence of significant changes in these laboratory data in treatmentcattle over time (each cow serving as her own control), as well as the lack of differencebetween treatment and control cows, indicate that there was no alteration in circulatingvolume or acid-base balance, nor was there significant stress (as measured by glucoseconcentration) or muscle injury inflicted by the treatment. In both studies (Gorewit et al.
1992; Reinemann et al. 1996) cows were exposed to electrical current only whiledrinking, not continuously.
Physiological responses of farm animals to electrical environment have also been studied.
Burchard et al. (1998) reported that nocturnal melatonin concentrations in dairy cows didnot show any variation that could be attributed to exposure to a vertical electric field of10 kV/m and a uniform horizontal magnetic field of 30 µT. Thompson et al. (1995)reported that cortisol concentrations, weight gain, and wool fiber length and diameter did not differ between the controls and ewes exposed to a mean electric field of 6 kV andmean magnetic field of 40 mG.
Physiological responses of farm animals to stresses other than electrical exposure havebeen studied. Cummins and Brunner (1991) reported that housing in metal pensdecreased cortisol, plasma ascorbate, IgG and specific antibody titres in dairy calvesrelative to calves housed in hutches. Elvinger et al. (1992) reported that the major effectof heat stress on immune function of dairy cows was decreased migration of leukocytesto the mammary gland after chemotactic challenge. In a study by Minton et al. (1995),reduced lymphocyte proliferative responses (PHA, Con A, PWM) were reported forlambs subjected to restraint and isolation stress for 6 h on three consecutive days.
Treatment did not affect IL2 or MHCII.
The specific objective of these experiments was to test the hypothesis proposed by theScience Advisors to the Minnesota Public utility Commission by measuring immunefunction response of dairy cows to continuously applied hoof-hoof voltage exposurebelow the level that would produce a behavioral response. Assays were chosen as rapid,routine measures to provide important initial information on immune system function.
Test facilities were constructed for groups of 8 cows. Treatment animals were exposed to1 mA of current flow for a period of 2 weeks. Each replicate used 4 control and 4treatment animals. Blood samples were taken from all 8 cows twice a week for oneweek before electrical exposure and for the 2 weeks of electrical exposure. The changein immune function measures was compared between treatment and control groups.
Three replicates of 8 cows each were performed using a total of 24 cows. Treatment andcontrol cows had identical stall conditions except for the current treatment. The treatmentand control stalls were selected in the systematic pattern shown in Figure 1. Cows wererandomized to the stalls and hence the treatment conditions. The cows for this trial wereselected on the following criteria: Lactation number no less than 2 and no greater than 4 (multiparous).
Days in milk (DIM) greater than 150 (mid lactation).
Somatic Cell Count (SSC) less than 150,000 (no mastitis infection).
Days Carrying Calf (DCC) greater than 40 (confirmed pregnant).
The cows in this research herd normally receive BGH injections every 2 weeks. BGHwas not administered during this trial so all cows would have missed one scheduledinjection during these experiments. The information for the cows used in this study isgiven in the appendix.
The cows were released from their stalls for milking at approximately 5:30 a.m. and 5:30p.m. After each milking, the cows were let out into an exercise yard. Cows were returnedto the test stalls within 1 hour of being released.
Twelve cows were exposed to 1 mA of current for two weeks (treatment group) and 12cows were not (control group). The statistical analysis method defined a priori was totake the difference between response variables measured on day 21 (at the end of thetreatment period) minus the average of days 3 and 7 (during the pre-treatment period) foreach cow. The response is, therefore, the difference from baseline for each cow with theexperimental unit defined as an individual cow. The differences of the treatment cowswere compared to the differences of the control cows using an independent t-test.
Test Stalls
The test stalls were constructed to allow precise control and measurement of electricalstimuli to individual cows and to eliminate interference from other electrical stimulioccurring in the cow environment. The test stalls consisted of a wooden framework filledwith two 120x76 cm (48x30 in.) concrete pads (Figure 2). A 15x15 cm (6x6 in.) weldedgrid of 9.5 mm (3/8 in.) reinforcing steel was embedded in each pad. There is a 9 cm airgap between the front and rear pads. Cows were secured with head-locking stanchionssupported on a wooden framework. When a cow stood in the stall, the front hooves wereon the front concrete pad and the rear hooves were on the rear pad.
The front of the test stall was supported by a single 7.3 cm diameter PVC pipe section 5cm high, located at the center of the stall front end. The rear of the stalls were suspendedabout 3 cm off the barn floor by two hangers attached at the back corners of the woodenstall frame and metal posts anchored in the concrete. This arrangement providedelectrical insulation for all current other than the cow.
Several experiments were carried out to determine the best stall surface for maintainingcurrent exposure levels over extended periods. Single day trials with bare concrete andseveral different types of organic bedding proved unsatisfactory. The back of the stallsurface was periodically wetted with urine and then drained dry. This variable level ofmoisture in combination with accumulation of organic bedding on the animal hooveschanged the animal resistance by a factor of 1000 times or more. It was not possible tomaintain current exposure within +/- 10% unless very high source voltages were used.
The concrete surface of the pads were then covered with electrically conductive rubbermats 1.4 cm (9/16 in.) thick (American Health and Safety Inc., item number 1-786.3X5S). These resilient mats allowed the cows to be kept comfortably in the stallswithout the use of organic bedding and reduced the risk of injuring feet and legs. Theconductive mats with no bedding provided much better control of current exposure withcows both standing and lying than the bare concrete surface either with or withoutorganic bedding.
The stalls were maintained twice a day when the cows were let out of the test stalls formilking. At each of these times the cow contact current level was checked as describedbelow and recorded. Following this current check the stalls were cleaned by removingmanure and other foreign material from the stall surface as well as areas surrounding thestalls. The rubber stall surfaces were then washed with a disinfectant (Muliquat, No. 455,Hydrite Chemical Co). The cow contact currents were then rechecked. If the currentdeviated by more than 10% of the treatment current (1 mA), the current level wasadjusted by changing the source resistance. The water cups were also checked to makesure they were dispensing water properly.
Current Application
The intended treatment current was 1 mA through the cow’s body. Current was appliedcontinuously for two weeks in a 20-min cycle (10 min on, 10 min off). This cycledpattern was used because previous research suggests that the effects of electric fields maybe more pronounced for changing electric fields than for steady fields.
A source voltage of 240 V was created using 120 V output from an uninterruptible powersupply (UPS) with power conditioning capabilities and stepped up to 240 volts with anisolated transformer (Figure 3). Power was switched on and off in 10-min intervals usinga repeat cycle timer/relay (Syrelec #ODRU, Dallas, Texas).
Current to each of the four treatment stalls was controlled by an adjustable sourceresistance (decade box power resistor) for each stall. Each current application wire alsohad a 1k ohm resistor in series to measure the total current flow in that line by measuringthe voltage drop across this resistor. The return wire from the rear pad of each test stallwas grounded using a separately derived ground located just outside of the barn near thetest stalls.
The treatment current level was measured in each treatment stall just before and after thetwice-daily stall maintenance. The current exposure was measured using standardmethodology used in field investigations of stray voltage. Copper plates (9x9 cm) wereplaced over wetted paper cloth at the center of the front and rear stall pads. A 3.6 kgweight was placed on the copper plates and the voltage across the plates was measuredwith 1k ohm shunt resistor and a Fluke 87 true rms multimeter. Leakage current wasestimated by comparing the current measured at the 1k ohm resistor in the control boxwith the “cow contact” current measured at the 1k ohm resistor between the front andrear pads. The amount of leakage current is a function of the resistance of the intendedpath (pad-cow-pad) the resistance of alternate paths (debris bridging pads or from frontpad to ground and wood rails connecting pads).
Periodic measurements of the step potential in control stalls were also made during thesecond replicate of this study using this method. The range of the measured values was1.4 mV to 1.7 mV rms. The step potential values were much less than 5% of the 400 mVrange specified as the lowest limit of accuracy by the manufacturer. As specified by themanufacturer, the offset of the Fluke 87 meter was checked with the test leads shortedand found to be 1.4 mV. This is a result of internal amplifier noise in the meter. Withinthe accuracy of this meter, the step potential was not different from zero.
Magnetic Field measurement
Background magnetic field levels were measured using an EmdexC magnetic field meter.
This meter is designed to measure the resultant 3-axis 50-60 Hz magnetic field. Fieldreadings were taken directly in front of each stall, in the center of the stall, and directlybehind each stall at a height of 1 m from the floor. The average magnetic field at all teststall locations with all electrical devices in the barn running (lights and fans) was 0.3 mG.
The magnetic field levels were between 0.14 and 0.4 mG at all locations except at thefront of stall 1, which had readings of up to 0.54 mG.
Immune Function Assays
Blood samples were collected by tail bleeding twice weekly for assessment of immunefunction. Samples were collected for one week before exposure and for the two weeks ofexposure. A sample was allowed to clot and resulting serum analyzed forimmunoglobulin content by ELISA and IL1 and IL2 by bioassay (Wudhwa et al., 1991).
Remaining blood was used to collect leukocytes, as previously described (Lohuis et al.,1990). Hypotonic lysis was used to remove red blood cells, and percoll gradientcentrifugation was used to enrich target leukocyte populations. Leukocytes were usedimmediately for lymphocyte blastogenesis, antibody production and oxidative burstassays.
For lymphocyte blastogenesis (Lane et al., 1979), cells were diluted in Fisher’s mediumand 50 µL containing 105 cells plated onto 96-well culture dishes. Responses to standardmitogens, including S. aureus, phytohemagglutinin, pokeweed mitogen and concanavalinA were determined. Phytohemagglutanin and concanavalin A activate largely Tlymphocytes, pokeweed mitogen T and B lymphocytes and S. aureus cells Blymphocytes. After 72 hours, 1 µCi 3H-thymidine was added, cells incubated anadditional 4 hours and cells harvested using a 96-well plate harvester. Incorporation of3H-thymidine into DNA was used as an index of mitogenesis.
To assess immunoglobulin production (Lane et al., 1979), 3x106 cells were suspended in300 µL media. Cells were treated with or without pokeweed mitogen for 5-10 days andimmunoglobulin production assessed by ELISA, using antibodies against specific bovineimmunoglobulins.
To assess oxidative burst (Trush et al., 1978), chemiluminescence in response to standardactivators of macrophage and neutrophil function was used. Leukocytes (106) wereplaced in 0.5 mL phenol red free Dulbecco’s Modified Eagle’s Medium (DMEM)containing 100 mg/mL luminol. Baseline luminescence was assessed after 10 minutesincubation. Next, 0 or 10 ng/mL phorbol myristate acetate (PMA) was added, cellsincubated 1 minute and light emission determined again. The difference was used toestimate PMA-induced chemiluminescence.
The measures for lymphocyte blastogenesis using concanavalin A, andphytohemagglutanin mitogens and oxidative burst as measured by PMA-inducedchemiluminescence were chosen a priori as the best indicators of immune functionresponse. These questions were selected from the response variables to control the Type Ierror for the experiment’s most important questions.
Other Responses
In addition to the blood measures, daily water volume and feed consumed, cowtemperature and daily milk production were monitored. Each test stall was equipped witha water meter that was read once daily during the morning milking. Feed intake wasmonitored for each cow by measuring daily feed supplied minus leftover feed found inthe feed bins. The amount of feed supplied was intended to keep some feed in the bins 24hours a day. The milk meters in the milking parlor (BouMatic - Perfection), recordedmilk yields.
The time and pattern of standing and lying were recorded on one of the control days andagain near the end of the treatment period during the third replicate. The time for cows toreenter stalls after milking was measured. If the voltage/current exposure wereperceived, the time and pattern of lying or time to enter stalls could be changed.
Current Application
The results of the twice-daily measurements of cow contact current are summarized inTable I. The average cow current was within the +/- 10% target value for all cowsexcept 4262 in replicate II. This cow was fistulated and leaking rumen fluid caused thestall surface to remain wet and created a leakage path. The average value of 0.6 mA isprobably an under-estimate of the true average as these measurements were taken at theend of each 12-hour observation period, when the stall condition was likely at its worst.
Immediately after these measurements were taken, the stalls were cleaned and the currentlevels readjusted to 1 mA.
Table I. results of the twice-daily measurements of cow contact current.
Further measurements were done to estimate the stability of the cow current in the timebetween the twice-daily cow current measurements. Tests were done periodically usingshunt resistor values of 0.5k, 1k, 5, and 10k ohms. The cow contact current was within+/- 0.1 mA for all resistance values except the 10k resistor, which fell just outside the 10% deviation with an average cow contact current of 0.89 mA. The test stalls were thusable to maintain a cow contact current within +/- 10% for the practical range of cow andcontact resistances.
The average source resistance, recorded twice daily was 196k ohms. Values werebetween 170k and 230k ohms for all tests except for cow 4262 in replicate II (leakyfistula) in which case the source resistance was typically 100k to 150k ohms. Theresistance of the rest of the circuit (pads, mats, cow and contact resistance) was between10k and 70 k ohms with a standard deviation of individual stalls between 1k to 3k ohms(or less than 2 % of the total circuit resistance). The only exception to this was the cowwith a leaking fistula in which case the standard deviation increased to 32k ohms or 13% of the total circuit resistance.
The current measured at the 1k ohm resistor in the control box was monitored for 24hours on all test stalls during the third replicate. The 12 hour average current wascompared to the last 10 minute interval (corresponding to the twice-daily cow-currentchecks). The ratio of the 12 hour average current to the last 10 minutes was between 94and 99 % with standard deviations between 8 and 10 %. The voltage between the wiresconnected to front and rear pads was also monitored for 24 hours for each stall duringreplicate III. The expected range of voltages for this measurement is 10 to 70 Vcorresponding to the source voltage and 10 to 70k ohm resistance measured for this partof the circuit. The 24 hour average measured pad to pad voltage was 28 V with a standarddeviation of 18 V. Less than 1% of the data points were in excess of 76 V. These valuesare within the expected range and indicate that the current exposure was stable during thetreatment periods.
The combination of these measurements show that the average cow contact current waswithin the design range of 1 mA +/- 0.1 mA except for the cow with a leaking fistula inwhich case the average current exposure was probably about 0.8 mA +/- 0.3 mA.
Immune Function Responses
The summary statistics for the 3 replicates of current exposure experiment are given inTable II. Box plots of the main response variables are given in Figures 4-7. Statisticalanalysis was done after taking the natural log of all immune response data. This logtransform yielded a more normal distribution of the data. The difference from baselinelevel for each measure was used as the response variable for each cow. The differencevalues for the treatment animals were then compared to the difference values for thecontrol cows using an independent, two-tailed t-test. The questions for this work havebeen divided into two groups--the main questions and other questions. The comparison-wise Type I error for the main questions was p=0.05.
Table II. Summary statistics for immune function measures. The main questions areindicated in Bold. Data analyzed as difference of natural logs, n of controls = 12, n oftreatments = 12, DPM = Disintegration per minute, RLU = Relative Light Units Conconavalin A
Positive Control
An experiment was done to validate the immune assays using the well-know immuneresponse of cows to dexamethasone as a positive control. Four non-pregnant cows wereinjected with dexamethasone for 4 days. Each of the treatment cows received twoinjections of 15 mg of Dexamethasone (Dexamethasone, Sodium phosphate, SterisLaboratories Inc. Phoenix, Arizona 85043 USA) per day at 12-hour intervals for fourdays (Monday, Tuesday, Wednesday, and Thursday, approximately 7 a.m. and 7 p.m.).
Blood samples were taken prior to the injection on Monday and at 7 a.m. Friday.
The 3 control cows received a placebo shot of the saline solution only. These shots weregiven at the same time that the treatment cows receive their shots. The cows hadidentical stall conditions. Blood samples were taken prior to the injection on Mondayand on Friday for the control cows as well. Cows were handled in the same way as in thecurrent exposure experiments except that no current was applied during this study. Sevencows were available for this trial, 4 were randomly selected as treatments and 3 ascontrols. Cows were selected on the following criteria: Lactating and no less than 2 and no greater than 4 if possible.
Information on the cows used for this trial is given in the appendix. Summary statistics ofthe positive control experiment are given in Table III and raw data in Figure 8.
One of the control cows (2336) injured her right front teat on the morning of 5/9/99 andsubsequently developed a mastitis infection. She was treated and stayed in theexperiment. This cow showed a reduction in all 3 of the main immune functionresponses.
Table III. Summary statistics for immune function measures for positive controlexperiment, dexamethazone injection. The main questions are indicated in Bold.
Difference of natural logs, n of controls = 4, n of treatments = 3, DPM = Disintegrationper minute, RLU = Relative Light Units Conconavalin A
Other Responses
The standing and lying behavior of cows was analyzed in two ways. First the percentage
of time spent standing was calculated for each of the control and treatment cows during
the pre-exposure period. The change in this value for each cow was compared for control
and treatment cows measured again at the end of the current exposure period. The same
analysis was done using the percentage of periods in which cows changed status (from
standing to lying or from lying to standing). The results of these tests are summarized
A two tailed t-test indicated that the difference between control and treatment cows wasnot significant (p= 0.95) A two-tailed t-test indicated that the difference between control and treatment cows wasnot significant (p= 0.35) The time required for the cows to move from the center alley into the stalls was measuredon 3 consecutive days near the end of the exposure period of the third replicate. None ofthe cows showed any hesitation to enter the stalls. The mean of the treatment cows was3.5 s with a standard deviation of 1.0 s. The mean of the control cows was 4.2 s with astandard deviation of 1.6 s. The difference between the control and treatment animalswas not significant (p=0.46).
Data for cow temperature, daily milk weights, water consumption, and twice-dailycurrent measurements are given in the appendix.
Lymphocyte mitogenesis (blastogenesis) is a well-documented response to lectins and isgenerally recognized as a useful measure of systemic immune function (Lohuis et al.,1990). Chemiluminescence is widely used as a measure of respiratory burst inphagocytic cells, a key event in phagocytosis and intracellular killing of bacteria (Thrushet al., 1978). These two measures together provide important measures of lymphocyteand phagocyte function in response to various treatments. These measures representseveral of the major immunological processes and are the most likely to be altered ifsystemic immune function is suppressed by the treatments. The two-week exposureperiod appears justified as previous work on housing stress in farm animals has shownsignificant immune system response within 3 days (Minton et al., 1995) and one of the control cows in this experiment showed a change in immune function within 4 days inresponse to a mastitis infection.
The assays used in the present study are standard methods of assessing immunologicalfunction in mammals. Lymphocyte blastogenesis in response to concanavalin A andPhytohemagglutanin measures activation of T lymphocytes, while S. aureus measures Blymphocyte activation and pokeweed mitogen measures both T and B lymphocyteactivation (Lane et al., 1979). Of these measures, only S. aureus-induced blastogenesiswas significantly affected by 2 weeks of voltage exposure. This response would suggesta change in responsiveness of B cells (cells that eventually differentiate to produceimmunoglobulins). However, no other measures, including pokeweed mitogen-inducedblastogenesis, pokeweed mitogen-induced immunogolbulin production or in vivoantibody concentrations were affected. In addition, the difference in S. aureus wascaused by an increase in the control cows while the treatment cows showed no change.
Thus, it is possible that this response was a type I error. Concanavalin A-inducedblastogenesis was inhibited in positive controls cows (dexamethasone treated).
Chemiluminescence is a widely used measure of respiratory burst, a key event inintracellular killing of bacteria (Thrush et al., 1978). The present study found no effect ofvoltage exposure on chemiluminescence, suggesting that bactericidal activity ofcirculating phagocytic cells was unaffected by treatment. Treatment with dexamethasone(a glucocorticoid used as a positive control) significantly inhibited chemiluminescence.
In addition, immunoglobulin levels in vivo and in vitro in response to pokeweed mitogenwere measured as indices of immune function. As indicated earlier, these responses wereunaffected by voltage exposure of cattle. However, dexamethasone significantlyinhibited pokeweed mitogen-induced antibody production in vitro.
Two major cytokines regulating immune function, interleukin 1 and 2, were measured.
Measurements included both serum concentrations and pokeweed mitogen-inducedinterleukin production in vitro. Interleukin 1 concentrations in serum were slightlyelevated upon voltage exposure for 2 weeks (P<0.07), but serum interleukin 2concentration and interleukin 1 and 2 production in vitro were unaffected. IL1 change inthe combined date (all 3 replicates) appeared to be strongly influenced by one replicate(replicate 2, p < 0.06), with minimal change in the other two replicates (p = 0.96 and p =0.46). The bioassays used do not differentiate between a and b forms of interleukin 1(Wudhwa et al., 1991), so the possibility that one isoform was selectively affected cannotbe excluded. In positive controls, dexamethasone decreased interleukin 1 production.
Collectively, these results suggest that exposure to 1 mA of 60 Hz electrical current fortwo weeks had no significant effect on immune function of dairy cattle. One of 13response variables was statistically significant but did not appear to be entirely consistentwith other observations.
Burchard, J. F., D. H. Nguyen, and E. Block, 1998. Effects of Electric and MagneticFields on Nocturnal Melatonin: Concentrations in Dairy Cows 1. J. Dairy Sci. 81:722-727.
Cummins, K. A., and C. J. Brunner, 1991. Effect of Calf Housing on Plasma Ascorbateand Endocrine and Immune Function. Journal of Dairy Science 74, no. 5:1582-88.
Elvinger, F., R. P. Natzke, and P. J. Hansen, 1992. Interactions of Heat Stress and BovineSomatotropin Affecting Physiology and Immunology of Lactating Cows. Journal ofDairy Science 75, no. 2: 449-62.
Gorewit, R.C., A.J. Aneshansley, and L.R. Price, 1992. Effects Of Voltages On CowsOver A Complete Lactation: 2. Health And Reproduction. J. Dairy Science 75:2726-2732.
Lane, H.C., G. Whalen and A.S. Fauci, 1979. In vitro evaluation of human lymphocytefunction. In: Immunological Methods, 3rd Edition. I. Lefkovits and B. Pernis, Eds. NewYork: Academic Press, pp. 6.1-66.14.
Lohuis, J.A.C.M., Y.H. Schukken, P.A.J. Henricks, R. Heyneman, C. Burvenich, J.M.H.
Verheijden, A.S.J.P.A.M. Van Miert and A. Brand, 1990. Preinfection functions of bloodpolymorphonuclear leukocytes and the outcome of experimental Escherichia coli mastitisin the cow. J. Dairy Sci. 73:342-350.
Minton, J. E., J. K. Apple, K. M. Parsons, and F. Blecha, 1995. Stress-AssociatedConcentrations of Plasma Cortisol Cannot Account for Reduced Lymphocyte Functionand Changes in Serum Enzymes in Lambs Exposed to Restraint and Isolation Stress.
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MPUC, 1996. Progress Report of the Science Advisors to the Minnesota Public UtilitiesCommission, 1996, Minnesota Public Utilities Commission, St. Paul, Minnesota MPUC, 1998. Research Findings and Recommendations Regarding Claims of PossibleEffects of Currents in the Earth on Dairy Cow Health and Milk Production. Final Reportof the Science Advisors to the Minnesota Public Utilities Commission, July 31,1998,Minnesota Public Utilities Commission, St. Paul, Minnesota Reinemann, D.J., L.E. Stetson, J.P. Reilly, N.K. Laughlin, S. McGuirk, S.D. LeMire,1996. Dairy Cow Sensitivity and Aversion to Short Duration Transient Currents. PaperNo. 963087, Written for presentation at the 1996 International Meeting sponsored byASAE: the Society for Engineering in Agriculture, Food and Biological Systems,Phoenix, Arizona, USA, 14-18 July 1996.
Thompson, J. M., F. Stormshak, J. M. Lee Jr., D. L. Hess, and L. J. Painter, 1995.
Cortisol Secretion and Growth in Ewe Lambs Chronically Exposed to Electric andMagnetic Fields of a 60-Hertz 500 Kilovolt AC Transmission Line. Journal of AnimalScience 73 (1995): 3274-80.
Trush, M.A., M.E. Wilson and K. Van Dyke, 1978. The generation ofchemiluminescence (CL) by phagocytic cells. Methods Enzymology 57:462-494.
Wudhwa, M., C. Bird, A. Tinker, A. Mire-Sluis and R. Thorpe, 1991. QuantitativeBiological Assays for Individual Cytokines. In: Cytokine: A Practical Approach. F.R.
Balkwill, Ed. IRL Press, New York, pp. 309-330.
Figure 1. Location of treatment and control stalls in the barn. .
Figure 3. Schematic of electrical exposure circuit.
Figure 4. Box plots of the main response variables. The horizontal white line is the meanof the data. The box includes +/- 25% of the data from the median. The horizontal blacklines are the maximum and minimum values. Current exposure started on day 8.
Control and Treatment (Difference Values) Control and Treatment (Difference Values) Control and Treatment (Difference Values) Figure 8. Main Response variables for positive control experiment.
Difference of ln of Response Variable -6
Cow Number (C=control, T=Treatment)

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