We studied the changes in the heart rate variability of lame and non-lame dairy cows in response to transrectal examination and parlor milking. We hypothesized that lame cows experience greater stress, manifested in heart rate variability parameters, which would serve as an argument to promote more caution in the everyday handling of lame animals.In the study, we selected 55 lame (with lesions on at least one hoof, otherwise clinically healthy, with locomotion scores 4 and 5 of 5 point scores) and 55 non-lame (clinically healthy, with locomotion scores 1 and 2 of 5 point scores) cows. The heart rate (HR), root mean square of successive beat-to-beat intervals (RMSSD), the normalized unit of the high-frequency component (HF) of the spectral analysis and Poincaré measures (SD2/SD1) were compared between lame and non-lame cows during five distinct stages of transrectal examination (TRE) and seven stages of parlor milking. HR, RMSSD, and SD2/SD1 showed similar patterns during TRE and milking, while HF remained fairly constant during the studied phases. No distinct RMSSD, HF or SD2/SD1 changes were observed during the phases expected to elicit a stress response. Between-group differences were only observed in terms of HF. Baseline HF was higher in lame cows than in non-lame ones, and such a difference in direction was observed throughout the whole procedure. During milking and TRE, the HR, RMSSD, and SD2/SD1 values showed temporal changes in times of higher physical activity: moving to and waiting in the holding pen and moving into the milking stalls in the parlor for preparation in both lame and non-lame cows. The differences in baseline HF (normalised units) between lame and non-lame cows can not be fully explained based on available information. The lack of a distinct, stress-related change in heart rate variability parameters in both groups can originate in methodological challenges inherent in animal heart rate variability measurements in field conditions.

A promising strategy to improve newborn calf survival could be the administration of nonsteroidal antiinflammatory drugs (NSAID) especially in cases of low vitality calves born from difficult calvings. The objective of this clinical trial was to determine the effect of a single-dose meloxicam treatment [target dosage = 0.5 mg/kg ad usum veterinarium (A.U.V.) injection] on parameters of lying behavior of Holstein-Friesian dairy calves (n = 180) born to eutocic (n = 98) compared with dystocic dams (n = 82). Animal-based measures included newborn calf vitality (low, 1-6 scores; normal, 7-12 scores, scored immediately after birth), calf sex and birth weight, parity of the dam, and early maternal behavior (the time spent licking the calf). Parameters of the complex lying behavior were recorded during the first 48 h after delivery and included (1) the time spent standing, (2) lying down frequency, (3) the longest standing bout, (4) the average duration of standing bouts, (5) the first successful standing, and (6) the first attempt to stand. None of the parameters related to lying behavior were influenced by the parity of the dam, calf sex, or birth weight and maternal grooming behavior. The latency and the first attempt to stand were not influenced by the NSAID treatment; however, the time spent standing, the longest standing bout, and the average duration of standing indicated increased standing ability of meloxicam-treated calves with low vitality, but not in normal-vitality calves. The latency to stand and first attempt to stand were both increased by dystocia. Based on the present findings, sensory measurement of parameters of lying behavior during the immediate neonatal period might be useful to evaluate the efficiency of NSAID protocols. A single dose meloxicam treatment showed considerable promise for improving standing ability of low-vitality calves.

The intensity and the magnitude of saliva cortisol responses were investigated during the first 48 h following birth in newborn dairy calves which underwent normal (eutocic, EUT , n = 88) and difficult (dystocic, DYS , n = 70) calvings. The effects of parity and body condition of the dam, the duration of parturition, the time spent licking the calf, the sex and birth weight of the calf were also analyzed. Neonatal salivary cortisol concentrations were influenced neither by factors related to the dam (parity, body condition) nor the calf (sex, birth weight). The duration of parturition and the time spent licking the calf also had no effect on salivary cortisol levels. Salivary cortisol concentrations increased rapidly after delivery in both groups to reach their peak levels at 45 and 60 min after delivery in EUT and DYS calves, respectively supporting that the birth process means considerable stress for calves and the immediate postnatal period also appears to be stressful for newborn calves. DYS calves exhibited higher salivary cortisol concentrations compared to EUT ones for 0 ( P = 0.022), 15 ( P = 0.016), 30 ( P = 0.007), 45 ( P = 0.003), 60 ( P = 0.001) and 120 min ( P = 0.001), and for 24 h ( P = 0.040), respectively. Peak levels of salivary cortisol and the cortisol release into saliva calculated as AUC were higher in DYS than in EUT calves for the 48-h of the sampling period ( P = 0.009 and P = 0.003, respectively). The greater magnitude of saliva cortisol levels in DYS calves compared to EUT ones suggest that difficult parturition means severe stress for bovine neonates and salivary cortisol could be an opportunity for non-invasive assessment of stress during the early neonatal period in cattle.

The heart rate variability (HRV) parameters of dairy cows were monitored during parlour (PARL) and the later installed automatic (AMS) milking on a small-scale commercial dairy farm in Hungary. The aim of the study was to assess stress in relation to the type of milking and the frequency of human interaction. Parlour milking involved regular moving and crowding of the animals with frequent human interaction, which were much less frequent in automatic milking. The first phase of the study was conducted prior to the changeover [n = 27] and the second two months afterwards [n = 19 (of the cows from the first phase)]. Heart rate (HR) was recorded by the Polar RS800 CX recording system. HRV parameters indicative of sympathovagal balance were calculated for periods of lying and standing in the barn, waiting before milking and milking, respectively. Morning and evening faecal glucocorticoid concentrations were also measured. Fear of humans was tested by an avoidance distance test. Baseline HRV parameters showed no difference (P > 0.05) between the two systems. In the periods before, during and after milking a higher sympathetic tone was detected in cows in the PARL phase. Mean faecal glucocorticoid concentrations were higher at the time of parlour milking. The avoidance distance did not differ between the two phases. The results suggest that automatic milking might be less stressful for cows than parlour milking, possibly due to the shorter duration of restraint after milking and the less frequent human interaction.

We studied the adrenocortical and vagal tone responses to a single ACTH challenge in lame (n = 9) vs nonlame (n = 9) dairy cows. Cows were paired according to parity, days in milk, and milk yield. Plasma cortisol and dehydroepiandrosterone concentrations and cardiac vagal tone response (high-frequency component of heart rate variability) were compared after intravenous ACTH administration. Baseline, minimum or maximum, amplitude of the response and area under the response curve were compared. No difference was detected between groups in the cortisol response. Dehydroepiandrosterone was irresponsive to ACTH treatment, and concentrations did not differ between lame and nonlame cows. Vagal tone decreased in response to the ACTH treatment. High frequency component of heart rate variability was lower in the lame group at all sampling times. Lameness was associated with delayed return to baseline. We concluded that the adrenal response capacity is not influenced by lameness, which supports the concept of lameness being a chronic intermittent rather than a chronically persistent stressor. Dehydroepiandrosterone concentrations were not proven to be useful indicators of hypothalamus-pituitary axis dysfunction in cattle. A decreased vagal contribution to heart rate variability-possibly coupled with increased sympathetic modulation-was observed in lame cows, which suggests that lameness affects the mechanisms underlying the action of ACTH on cardiovascular activity.

Responses to heat stress have not been evaluated in dairy cattle by noninvasive techniques such as analysis of saliva cortisol concentrations. The aim of the present study was the assessment of saliva cortisol levels in Holstein bull calves with (n = 8) or without supplemental shade (n = 8) in response to acute heat stress. Measurements were carried out during a 5-d period [temperature, average/max (°C); day 1 (control, all calves shaded): 22.9/29.4, day 2 (heat stress day): 28.3/38.8, day 3: 26.2/33.5, day 4: 23.7/28.7, and day 5: 21.2/24.7]. The level of thermal stress was characterized with a temperature-humidity index (THI). Saliva cortisol levels did not differ between groups during the control day. On the heat stress day, saliva cortisol levels increased from 8:00 to 12:00 by 51% and 342% in shaded and nonshaded calves, respectively, and nonshaded calves showed higher cortisol concentrations at 12:00, 16:00, and 24:00. Saliva cortisol levels peaked at 12:00 on day 3 in both groups. On days 4 and 5, saliva cortisol did not show significant daytime elevations in either group; however, group differences remained significant until 20:00 on day 4. Based on our results, measurement of saliva cortisol concentrations is a promising approach to detect acute heat stress in dairy calves, which could be reduced by artificial shading.

Warm summer episodes have a significant effect on the overall health and well-being of young cattle; however, it is not known which temperature measure should be used for estimating heat stress in dairy calves. In this study, generalized linear mixed-effects models were used to estimate the relationships between thermal comfort indices and animal-based heat stress indicators in sixteen Holstein bull calves that were housed in individual calf hutches. Data were collected under continental weather characteristics over a 5-day period: day 1 (lower-temperature day), days 2 and 3 (heat stress days), and a 2-day post-stress period. Relative humidity, ambient temperature, the heat index, the humidex and five different temperature-humidity indices (THI) were used as thermal indices. Physiological variables monitored included respiratory rate, rectal temperature, ear skin temperature and heart rate. The heat index and the humidex measuring human thermal comfort were more closely associated with physiological measures than were the ambient temperature or the THIs (in case of heat index: R2 = 0.87 for respiratory rate, R2 = 0.63 for rectal temperature, R2 = 0.70 for ear skin temperature, and R2 = 0.78 for heart rate, respectively; in case of humidex: R2 = 0.85 for respiratory rate, R2 = 0.60 for rectal temperature, R2 = 0.68 for ear skin temperature, and R2 = 0.75 for heart rate, respectively). Based on our results, parameters of human outdoor comfort seem better to estimate heat stress in dairy calves in a continental region than those of THIs or ambient temperature.

[This corrects the article DOI: 10.1371/journal.pone.0200622.].

We estimated thermal stress in 7-week old Holstein bull calves during a warm episode in summer to study acute physiological responses of calves to heat stress. Data were collected over a 5-day period: day 1 (control), day 2 (heat stress), and a 3-day post-stress period in shaded (n = 8) and unshaded (n = 8) thermal environments. On the control day, both groups were shaded. Thermal environment was characterized by relative humidity, ambient temperature, and the temperature-humidity index (THI). Physiological variables included respiratory rate, rectal temperature, ear skin temperature and heart rate. Correlations between animal-based and meteorological indices were calculated, and ambient temperature correlated slightly better with physiological measures than THI. Rectal temperature was the only animal-based parameter that showed stronger correlations with the thermal indices when calculated for the shaded than for the unshaded environment [r = 0.42 vs. r = 0.47, P = 0.032 (ambient temperature), r = -0.39 vs. r = -0.45, P = 0.012 P = 0.015 (relative humidity), r = 0.41 vs. r = 0.46, P = 0.022 (THI)]. No differences were found between groups during the control day for any of the physiological parameters. During days 2 and 3, average and maximal values of respiratory and heart rates were higher in unshaded calves than in shaded ones. Maximal respiratory rates were in average by 25.9, 17.8 and 10.1 breaths/min lower in shaded calves than in unshaded calves for days 2, 3 and 4, respectively (P < 0.001, P < 0.001 and P = 0.024). Maximal heart rate was 127.4 ± 8.5 vs. 99.2 ± 6.3 beats/min on the heat stress day (P < 0.001), and 121.0 ± 6.9 vs. 103.4 ± 7.7 beats/min on day 3 (P = 0.006) in unshaded and shaded calves, respectively. Maximal body temperatures were higher measured either in the rectum or on the ear skin in unshaded calves than in shaded ones (with 0.5 and 1.6°C, P = 0.040 and P = 0.018, respectively), but only on the heat stress day. Based on our results, shading of young calves may be adequate for alleviating acute heat stress in continental regions. Ambient temperature is appropriate to estimate acute heat stress in dairy calves.

[This corrects the article DOI: 10.1371/journal.pone.0200622.].

Changes in non-invasive stress biomarkers were studied in shaded ( n = 8) and non-shaded ( n = 8) Holstein bull calves exposed to extreme heat load conditions in a continental region. Ambient temperature and humidity data were recorded for the S and NS hutch and exercise pen environments. Temperature-humidity-index (THI) was also calculated. Respiratory rate, rectal temperature, heart rate, and the root mean square of successive differences between R–R intervals (RMSSD) were recorded as animal-based indicators during three periods: (1) day 1, control day , during which all calves were shaded for 24 h (shade removal from non-shaded calves at 2400 h); (2) day 2, heat stress day , with shade over shaded calves; and (3) days 3–5, post-stress period , with shade over shaded calves. On the heat stress day, the maximum temperatures were 44.3 and 46.7 °C for the non-shaded hutch and pen environments, respectively. The temperatures were with 6.2 and 6.9 °C ( P = 0.015 and P = 0.008) and the THIs were with 5.9 and 4.2 units higher ( P = 0.020 and P = 0.032) in the non-shaded than in the shaded environment for the hutch and exercise pen, respectively. Shaded calves had with 42.3 ± 3.2 breaths/min higher respiratory rate than non-shaded ones on the heat stress day at 1200 h ( P = 0.001), which was moderated to 20.1 ± 2.4 breaths/min at 1600 h ( P = 0.023). Significant differences in respiratory rate occurred earlier than in any other animal-based parameter between shaded and non-shaded calves on day 2. The only significant group difference in rectal temperature was found at 1200 h on day 2 when THI exceeded 91 units, with 0.59 °C higher values for non-shaded calves ( P = 0.045). The heart rate on days 2 and 3 was higher for non-shaded calves than for shaded ones. Group differences were also significant at 0800 h (18.2 ± 1.2 beats/min, P = 0.008), 1200 h (22.3 ± 1.4 beats/min, P = 0.003), 1600 h (15.3 ± 0.8 beats/min, P = 0.012), and 2000 h (19.0 ± 1.1 beats/min, P = 0.010) on day 2. Following a rapid daytime reduction, RMSSD showed a nighttime overcompensation in non-shaded calves on day 3 (between 0000 and 0600 h) and day 4 (between 0000 and 0800 h), exceeding the levels recorded on day 1 (control) and the levels for shaded calves, thus suggesting a recovery of the autonomic nervous system from heat stress. Based on our results, shading effectively reduced heat stress as evidenced by heart rate and RMSSD in addition to traditional measures of heat stress. Respiratory rate and heart rates exhibited by non-shaded calves support that the well-being of pre-weaned calves can be impaired in continental regions during following a heat stress day without providing shade.

In this study, we used linear mixed models to determine the effects of season, time of sampling relative to birth (factors), duration of the delivery process, duration of maternal grooming, calf body weight (BW) at birth, and time of day (covariates) on values of venous blood gas, acid-base and electrolyte parameters, and l-lactate concentrations in dairy calves born to eutocic dams in summer (SUM, n = 101) and winter (WIN, n = 104). Neonatal vitality was assessed at 0, 1, and 24 h after delivery in a linear scoring system using muscle tone, erection of the head, muscle reflexes, heart rate, and sucking drive as criteria. Simultaneously with vitality scoring, venous blood samples were collected by jugular venipuncture. Blood was tested for pH, partial pressure of CO2 (pCO2; mmHg) and oxygen (pO2; mmHg), l-lactate (mmol/L), hemoglobin (Hb; g/L), ionized calcium (Ca2+; mmol/L), sodium (Na+; mmol/L), potassium (K+; mmol/L) and chloride (Cl-; mmol/L). Bicarbonate (HCO3-; mmol/L), base excess (BE; mmol/L), total carbon dioxide (TCO2; mmol/L), and anion gap (mmol/L) were calculated. Electrolyte parameters were affected by none of the factors or covariates. Time of day at birth did not affect any of the parameters of interest. Vitality score tended to increase over time and it showed higher values in WIN calves than in SUM calves. Concentrations of HCO3-, BE, and l-lactate indicated a higher degree of metabolic acidosis in SUM calves; however, pH was not affected by season. Concentrations of Hb were higher in SUM calves than in WIN calves; however, covariates did not affect Hb concentrations. Blood pH, concentrations of pO2, HCO3-, and BE decreased, whereas l-lactate concentrations and values of pCO2, TCO2, and anion gap increased with longer duration of delivery. A shift in acid-base balance was also linked to BW of the calf at birth, with lower values of blood pH, HCO3-, and BE in calves with higher BW compared with those with lower BW at birth, whereas TCO2 and l-lactate concentrations increased with higher calf BW at birth. Values of pO2 increased and pCO2 decreased with longer duration of maternal grooming. Blood pH, HCO3-, and BE increased, whereas l-lactate concentrations and anion gap decreased with longer duration of licking the calf. Our results indicate that prolonged delivery can impair acid-base status and can cause slight lactic acidosis, even in calves born from spontaneous or eutocic calvings, and that high BW at birth predisposes calves to acidosis. The positive effect of maternal grooming on neonatal acid-base status should be considered in parturition management. Season, duration of the delivery process, calf BW at birth, and duration of maternal grooming are recommended for consideration in future studies on blood gas and acid-base parameters in dairy calves in the immediate neonatal period.

The objectives of this study were to explore changes of rumination time and reticuloruminal pH and temperature of dairy cows and heifers (means ± standard deviation; age = 5.8 ± 1.9; parity = 2.7 ± 1.4; body condition score = 3.2 ± 0.2) with eutocic (EUT, n = 10) and dystocic calving (DYS, n = 8). The recording period lasted from 3 d before calving until 7 d in milk. For the comparison of rumination time and reticuloruminal characteristics between groups, time to return to baseline (the time interval required to return to baseline from the delivery of the calf) and area under the curve (AUC; both for prepartum and postpartum periods) were calculated for each parameter. Rumination time decreased from baseline 28 h before calving both for EUT and DYS cows; after 20 h before calving, it decreased to 32.4 ± 2.3 and 13.2 ± 2.0 min/4 h between 8 and 4 h before delivery in EUT and DYS cows, respectively, and then it decreased below 10 and 5 min during the last 4 h before calving. Until 12 h after delivery, rumination time reached 42.6 ± 2.7 and 51.0 ± 3.1 min/4 h in DYS and EUT dams, respectively; however, AUC and time to return to baseline suggested lower rumination activity in DYS cows than in EUT dams for the 168-h postpartum observational period. Reticuloruminal pH decreased from baseline 56 h before calving both for EUT and DYS cows, but did not differ between groups before delivery. Reticuloruminal pH showed a decreasing tendency and clear diurnal variation after calving for both EUT and DYS cows, with slightly higher AUC values in DYS cows. In DYS cows, reticuloruminal temperature decreased from baseline 32 h before calving by 0.23 ± 0.02°C, whereas in EUT cows such a decrease was found only 20 h before delivery (0.48 ± 0.05°C). The AUC of reticuloruminal temperature calculated for the prepartum period was greater in EUT cows than in DYS cows. During the first 4 h after calving, reticuloruminal temperature decreased from 39.68 ± 0.09 to 38.96 ± 0.10°C and from 39.80 ± 0.06 to 38.81 ± 0.08°C in EUT and DYS cows, respectively, and reached baseline levels after 35.4 ± 3.4 and 37.8 ± 4.2 h after calving in EUT and DYS cows, respectively. Based on our results, continuous monitoring of changes in rumination time and reticuloruminal temperature seems to be promising in the early detection of cows with a higher risk of dystocia. Depressed rumination activity of DYS cows after calving highlights the importance of the postpartum monitoring of cows experiencing difficulties at calving. The effect of dystocia on postpartum reticuloruminal pH was not pronounced.

Peripartal autonomic nervous system function and early maternal behavior were investigated in 79 multiparous Holstein-Friesian cows. Animals were allocated into four groups based on the technology of calving management: 1) unassisted calving in a group pen (UCG; N=19), 2) unassisted calving in an individual pen (UCI; N=21), 3) assisted calving with appropriately timed obstetrical assistance (ACA; N=20), and 4) assisted calving with premature obstetrical assistance (ACP; N=19). Heart rate, the high frequency (HF) component of heart rate variability (HRV) as a measure of vagal activity and the ratio between the low frequency (LF) and HF components (LF/HF ratio) as a parameter of sympathetic nervous system activity were calculated. Heart rate and HRV parameters were presented as areas under the curves (AUC) for the following periods: 1) prepartum period (between 96h before the onset of calving restlessness and the onset of restlessness), 2) parturition (between the onset calving restlessness and delivery), and 3) postpartum period (during a 48-h period after delivery). Pain-related behaviors were recorded during parturition (i.e., the occurrence of vocalization and stretching the neck towards the abdomen) and during a 2-h observation period after calving (i.e., the occurrence of vocalization, stretching the neck towards the abdomen and the duration of standing with an arched back). Early maternal behavior was observed during the first 2h following calving as follows: 1) latency and duration of sniffing calf's head/body, and 2) latency and duration of licking calf's head/body. No difference was found across groups in autonomic function before the onset of calving restlessness. Area under the heart rate curve was higher in ACP cows during parturition (39.6±2.5beats/min×h) compared to UCG, UCI and ACA animals (AUC=13.1±0.9beats/min×h, AUC=22.3±1.4beats/min×h and AUC=25.0±2.1beats/min×h, respectively). Area under the heart rate curve did not differ across the UCG, UCI and ACA groups during the postpartum period (AUC=65.2±16.7beats/min×h, AUC=58.0±14.2beats/min×h and AUC=62.9±12.1beats/min×h, respectively) but it was higher in ACP cows compared to the former groups (AUC=269.1±36.3beats/min×h). During parturition, area under the HF curve reflected a lower vagal tone (AUC=-30.5±1.6n.u.×h) in cows with premature obstetrical assistance than in animals that calved individually without farmer assistance (AUC=2.7±0.4n.u.×h) or with appropriately timed assistance (AUC=3.2±1.2n.u.×h). During parturition, LF/HF ratio showed greater sympathetic activity in ACP cows than in animals from any other group. Area under the HF curve was similar across UCG, UCI and ACA cows (AUC=-232.1±42.0n.u.×h, AUC=-163.4±35.6n.u.×h and AUC=-331.4±56.2n.u.×h, respectively) during the postpartum period and was the lowest in ACP cows (AUC=-1025.6±44.2n.u.×h) reflecting a long-term stress load in the latter group. During parturition, both vocalization and stretching the neck towards the abdomen occurred more often in UCG cows than in cows from any other groups, and the incidence of both behaviors was statistically higher in ACP cows than in UCI and ACA animals. There were no significant differences across groups in these behaviors during the 2-h postpartum observation. UCG cows had a shorter latency and a longer duration of maternal grooming during the first 2h following delivery compared to any other groups. UCI and ACA dams spent more time with licking the calf within the 2-h period after calving and had a shorter latency to sniff and lick the offspring compared to cows that received premature assistance. Group calving is less stressful for cows than calving in an individual pen either with or without obstetrical assistance. Calving in a group or with appropriately timed farmer assistance supports the expression of early maternal behavior and lead to a rapid postpartum recovery of the autonomic nervous system. Premature obstetrical assistance means stress for cows during parturition, leads to a prolonged postpartum recovery of the autonomic nervous system and inhibits the expression of early maternal behavior.

This investigation was conducted to examine circadian and seasonal rhythms of heart rate and heart rate variability (HRV) by means of hour-by-hour recordings over 24 h in a large population of non-lactating Holstein–Friesian pregnant cows [N = 56, summer (June–July); N = 61, winter (November–December)]. Data were collected during a 5-day period from each animal. Besides parameters of cardiac autonomic function [the high-frequency (HF) component of HRV and the ratio between the low-frequency (LF) and the HF components (LF/HF ratio)], the RR triangular index and Lmax were calculated. A clear circadian profile was observed for every parameter in summer. Heart rate elevated gradually with the course of the day from 7:00 to 17:00 o'clock and then slightly decreased from 18:00 to 6:00. Sympathovagal balance shifted towards sympathetic dominance during the daytime (increased LF/HF ratio), whereas parasympathetic activity was predominant during the night (increased HF). Lmax reflected a chaotic behavior of heart rate fluctuations during the afternoon in summer. Decreased values of RR triangular index indicated a sensitive period for cows between 14:00 and 16:00 o'clock in summer. During winter, except for the RR triangular (RRtri) index reflecting a high overall variability in R–R intervals between 12:00 and 23:00 o'clock, heart rate and HRV showed no periodicity over the 24-h period. The results suggest an impaired cardiac autonomic function during daytime in summer. HF, Lmax and RRtri index showed seasonal differences for both daytime and nighttime. Heart rate was higher in summer than in winter during the daytime, whereas the LF/HF ratio was higher in winter during the nighttime. Circadian and seasonal rhythms of cardiovascular function are presumably related to the differing temperature, and animal activity associated with summer and winter. As all of the investigated parameters are commonly used in bovine HRV research, these findings have practical implications for behavioral, physiological and welfare studies on dairy cattle.

Behavior, hypothalamic-pituitary-adrenal axis, and cardiac autonomic nervous system (ANS) activity were evaluated in response to transrectal examination in nonlactating Holstein-Friesian cows with different behavioral reactivity. According to behavioral reactions shown to the procedure of fixing the heart rate (HR) monitors, the 20 cows with the highest and the 20 cows with the lowest behavioral reactivity were involved in the study (high responder, n=20; and low responder, n=20, respectively). Activity of the ANS was assessed by HR and HR variability parameters. Blood and saliva were collected at 5 min before (baseline) and 0, 5 10, 15, 20, 30, 40, 60, and 120 min after the examination to determine cortisol concentrations. The examination lasted for 5 min. Cardiac parameters included HR, the root mean square of successive differences between the consecutive interbeat intervals, the high frequency (HF) component of heart rate variability, and the ratio between the low frequency (LF) and HF parameter (LF/HF). Following the examination, peak plasma and saliva cortisol levels and the amplitude of the plasma and saliva cortisol response were higher in high responder cows than in low responders. Areas under the plasma and saliva cortisol response curves were greater in high responder cows. Plasma and salivary cortisol levels correlated significantly at baseline (r=0.91), right after examination (r=0.98), and at peak levels (r=0.96). Area under the HR response curve was higher in low responder cows; however, maximum HR and the amplitude of the HR response showed no differences between groups. Minimum values of both parameters calculated for the examination were higher in high responders. Following the examination, response parameters of root mean square of successive differences and HF did not differ between groups. The maximum and the amplitude of LF/HF response and area under the LF/HF response curve were lower in low responder cows, suggesting a lower sympathetic activation of the ANS. Although changes in behaviors indicated that the procedure was painful for the animals, no differences were observed either in vocalization or in attendant behavior between groups during the examination. Our results demonstrate that behaviorally more reactive animals exhibit increased plasma and salivary cortisol concentrations and higher cardiac autonomic responsiveness to transrectal examination than less reactive cows. Salivary cortisol may substitute for plasma cortisol when assessing response of cattle to stress.

Behavioural changes before calving can be monitored on farms; however, predicting the onset of calving is sometimes difficult based only on clinical signs. Heart rate (HR) and heart rate variability (HRV) as non-invasive measures of autonomic nervous system (ANS) activity were investigated in Holstein–Friesian cows (N=20) with unassisted calvings in the periparturient period to predict the onset of calving and assess the stress associated with calving. R–R-intervals were analysed in 5-min time windows during the following three main periods of measurement: 1) between 0 and 96h before the onset of calving restlessness (prepartum period); 2) during four stages of calving: (I) early first stage; between the onset of calving restlessness and the first abdominal contractions; (II) late first stage (between the first abdominal contractions and the appearance of the amniotic sac); (III) early second stage (between the appearance of the amniotic sac and the appearance of the foetal hooves); (IV) late second stage (between the appearance of the foetal hooves and delivery of the calf), and 3) over 48h following calving (postpartum period). Data collected between 72 and 96h before calving restlessness was used as baseline. Besides HR, Poincaré measures [standard deviation 1 (SD1) and 2 (SD2) and SD2/SD1 ratio], the root mean square of successive differences (RMSSD) in R–R intervals, the high-frequency (HF) component of HRV and the ratio between the low-frequency (LF) and the HF components (LF/HF ratio) were calculated. Heart rate increased only following the onset of the behavioural signs, peaked before delivery of the calf, then decreased immediately after calving. Parasympathetic indices of HRV (RMSSD, HFnorm and SD1) decreased, whereas sympathovagal indices (LF/HF ratio and SD2/SD1 ratio) increased significantly from baseline between 12 and 24 before the onset of calving restlessness. The same pattern was observed between 0 and 1h before calving restlessness. Following the onset of behavioural signs, parasympathetic activity increased gradually with a parallel shift in sympathovagal balance towards parasympathetic tone, which was possibly a consequence of oxytocin release, which induces an increase in vagus nerve activity. Parasympathetic activity decreased rapidly between 0 and 0.5h following calving and was lower than measured during all other stages of the study, while sympathetic activity peaked during this stage and was higher than measured during any other stages. Between 0 and 4h after calving vagal tone was lower than baseline, whereas sympathovagal balance was higher, reflecting a prolonged physiological challenge caused by calving. Vagal activity decreased, whereas sympathovagal balance shifted towards sympathetic tone with increased live body weight of the calf during the late second stage of calving, suggesting higher levels of stress associated with the higher body weight of calves. All HRV indices, measured either at the late second stage of calving and between 12 and 24h after calving, were affected by the duration of calving. Our results indicate that ANS activity measured by HRV indices is a more immediate indicator of the onset of calving than behaviour or HR, as it changed earlier than when restlessness or elevation in HR could be observed. However, because of the possible effects of other physiological mechanisms (e.g. oxytocin release) on ANS activity it seems to be difficult to measure stress associated with calving by means of HRV between the onset of calving restlessness and delivery. Further research is needed to enable more precise interpretation of the prepartum changes in HR and HRV in dairy cattle.

Interest in the monitoring of heart rate variability (HRV) has increased recently, as it gives more detailed and immediate information about the level of stress than traditional behavioral or hypothalamus-pituitary-adrenal measures. In this study, we evaluated heart rate (HR) and parasympathetic HRV parameters to monitor cardiac stress responses to palpation per rectum (PPR) in lactating (LACT; n = 11) and nonlactating (NLACT; n = 12) dairy cows. Heart rate and HRV were recorded from 40 min before PPR until 120 min after it was completed. Heart rate, the root mean square of successive differences (RMSSD), and the high-frequency component (HF) of HRV were analyzed by examining 5-min time windows. To compare cardiac responses to PPR between groups, changes in HR and HRV parameters were calculated as area under the curve (AUC) for LACT and NLACT cows. An immediate increase in HR was detected during PPR in both LACT (+21.4 ± 2.4 beats/min) and NLACT cows (+20.6 ± 2.3 beats/min); however, no differences were found between groups on the basis of parameters of AUC. The increase in HR in both groups along with a parallel decrease in RMSSD (LACT cows: -5.2 ± 0.4 ms; NLACT cows: -5.1 ± 0.4 ms) and HF [LACT cows: -10.1 ± 0.8 nu (where nu = normalized units); NLACT cows: -16.9 ± 1.2 nu] during PPR indicate an increase in the sympathetic, and a decrease in the parasympathetic tone of the autonomic nervous system. The increase in RMSSD (LACT cows: +7.3 ± 0.7 ms; NL cows: +17.8 ± 2.2 ms) and in HF (LACT cows: +24.3 ± 2.6 nu; NLACT cows: +32.7 ± 3.5 nu) immediately after PPR indicated a rapid increase in parasympathetic activity, which decreased under the baseline values 10 min following PPR. The amplitude and the maximum RMSSD and HF values were greater in NLACT cows than in LACT animals, suggesting a higher short-term cardiac responsiveness of NLACT cows. However, the magnitude and the duration of the stress response were greater in LACT cows, as indicated by the analysis of AUC parameters (area under the HRV response curve and time to return to baseline). Cow response to the PPR was more prominent in parasympathetic HRV measures than in HR. Based on our results, the effect of PPR on the cows' cardiac stress responses may have an impact on animal welfare on dairy farms, and investigating the effect of lactation on the cardiac stress reactions could prove useful in modeling bovine stress sensitivity. Further research is needed to find out whether the differences due to lactation are physiological or management related.