In line with our hypothesis, different stroking styles (“reactive” vs. “ventral neck”) elicited differences in the heifers’ ear positions. However, no other behaviours differed significantly in reaction to stroking with different stroking styles. Independently of the stroking style, the heifers reacted with longer durations of neck stretching and contact and decreased durations of the ear positions centre and forward up during STR compared with PRE, supporting our hypothesis of a positive perception of stroking. We did not confirm the predicted changes in HR and HRV, but instead found a slightly increased mean HR during stroking, and no changes in HRV parameters.
4.1. General Effects of Gentle Tactile Interactions on Behaviour and Cardiac Parameters
We found a significant effect of phase on behaviours indicating a positive affective state during STR for both stroking styles. For instance, the duration of neck stretching increased from PRE to STR. Neck stretching is shown by cattle during intraspecific social grooming [13
] after they actively solicited it, and during stroking by humans [9
] after they voluntarily approached them. It can thus be assumed that the situation is perceived as positive and neck stretching can be interpreted as a sign of enjoyment. The animals also established physical contact with the stroker for longer durations in STR than in PRE. This concurs with other studies where calves leaned against the brush during brushing by a human [17
] and heifers approached and proactively offered body parts to a human during positive tactile contact [16
]. Following the concept that animals seek out situations of positive valence [2
], seeking proximity to humans indicates that our stroking treatment was perceived as positive.
We expected to induce a low-arousal state during STR. Surprisingly, the mean HR of the animals was significantly higher during STR than PRE; however, the increase was low with less than 2 bpm on average. Although this finding contradicts our hypothesis of a decrease of arousal through stroking, it is in line with the slightly accelerated HR found in animals licked by conspecifics while lying [15
]. Since the animals were lying for a minimum of five minutes before we started a trial, we can assume that they were already in a low-arousal state. This is reflected in the low values of baseline HR (raw data, mean ± SD: “reactive” 75 ± 9 bpm, “ventral neck” 77 ± 8 bpm) that were found in PRE and fall below the reported HR of standing cattle that reacted with HR decreases to allogrooming [15
]. Such low baseline values might have caused a physiological floor effect, where a further decrease of HR is quite impossible, even if stroking is perceived as calming. Additionally, compared to resting in PRE, any physical reaction to the stroking treatment (such as neck stretching, seeking contact to the stroker or presenting body parts) would lead to an increase of HR and might therefore mask the calming effect of stroking. In conclusion, our hypothesis that both stroking styles would elicit a positive, low-arousal state can only be confirmed with regard to valence, but not to arousal. Although there was an effect of phase on HR, there was none on HRV parameters that would have surpassed the effect of the phase on HR. Thus, the stroking of lying heifers did not seem to exert an additional psychophysiological effect on the autonomous nervous system, likely due to the already existing low-arousal, relaxed state and the dominance of vagal regulation during rest [30
To meaningfully compare our results regarding ear positions with previous findings, differences in the definitions of ear positions need to be considered. Often, specific discrete ear postures are defined [26
] and their frequency or duration is recorded, which means that ear positions divergent from the predefined postures might not be recorded or analysed. It is not reported which degree of divergence from the definition is allowed for an ear position to still be included in that definition. To cover the continuous spectrum of possible ear positions, we described them according to their position along the vertical and the horizontal axis. This resulted in nine different ear positions: back up, back centre, back down, centre up, centre, centre down, forward up, forward centre and forward down, which were then analysed for their duration, plus ear flicking and ear hanging. This different way of defining ear positions, in our opinion, better reflects the continuous nature of ear positions, but leads to a reduced comparability of our findings with previous studies.
Looking at the proportions of the individual ear positions, we found a decrease of centre and forward up during STR. Erect ears and ears directed forwards have been associated with heightened attention or high-arousal states in dairy cows [28
]. A decrease of these positions might indicate reduced vigilance or a decrease in arousal during the stroking phase. The graphs show a similar pattern for changes of ear positions and ear flicking, which have numerically lower values during STR. Frequent changes of ear positions were found in reaction to a presumably negative, high-arousal situation in sheep [55
] and in dairy cows [27
], but also during a positive, presumably low-arousal stroking situation [32
]. Ear flicking is a behaviour that is mostly associated with negative affective states [56
] or reactions to insect attacks [58
]. Changes of ear positions and ear flicking should therefore be investigated further as indicators of emotional state.
The effects of the treatment on behaviour and cardiac activity that we saw in STR were not observed in POST, indicating that the positive effects of stroking in lying heifers did not last long enough for carry-over effects to be observed. Some of the observed behaviours (such as neck stretching) are more immediate reactions to positive stimuli and do not allow observation of longer-lasting changes in affective states.
4.2. Effects of Stroking Style
Responding to the animals’ signals in the “reactive” stroking style resulted in the longest duration of stroking on the areas of dorsal neck, cheek, ventral neck, lateral neck and jaw (order according to descending duration). This distribution between the neck and head during “reactive” stroking is quite similar to the one found during allogrooming ([21
]: neck 65%, head 25% of total duration), which may indicate that the stroker correctly identified the animals’ preferences.
Nevertheless, we found only limited support for our hypothesis that “reactive” stroking would elicit a more positive emotional state than stroking the ventral neck only. The two different tactile stimuli did not lead to significant differences in behaviours or cardiac parameters, except for ear positions. Animals stroked in a “reactive” style showed an increase in low ear positions and in back centre during STR, while animals stroked at the ventral neck showed a significant and strong increase in back up with a concurrent decrease in back centre.
The significant increase of low ear positions during STR in the “reactive” treatment partly confirmed our prediction of lower ear positions during the low-arousal state elicited during STR. However, there was no increase of ear low during “ventral neck” stroking. While we found a similar HR in “reactive” stroking as in “ventral neck” stroking, ear low only increased during “reactive” stroking, possibly indicating that low ear positions are reflecting not only arousal, but must be influenced by other factors as well, such as affective valence or attention. However, low ear positions generally occurred for small proportions of time and far less often than expected. In previous studies, dairy cows showed hanging ears for about 5%–65% of the time [9
]. Reasons for the short durations of low ear positions, especially ear hanging, in our experiment might be specific to our study population: unlike other studies, which were conducted on adult cattle, we worked with young stock, who might show shorter durations of low ear positions due to a higher reactivity [59
]. There are no studies yet investigating the relation of age and low ear positions in cattle.
By far the most common position in our study was the back up position. One factor that possibly influenced the position of the heifers’ ears was the location of the stroker. In our study, the stroker was kneeling beside the animal’s shoulder, possibly causing the heifer to turn her ears backwards and upwards while directing attention to the human. However, the stroker’s position was the same for both stroking styles, but back up increased significantly during the stroking phase only in the “ventral neck” treatment. When interpreting this position, the aforementioned differences in definitions across literature must be taken into account. Backwards ear positions have been found to be associated with both negative and positive affective states. They are part of the facial expressions shown by cattle in pain [60
]. However, in a study with a similar design [32
], more “back” positions occurred during a low-arousal, positive situation similar to our stroking phase, which might correspond to our result regarding the back up position; however, neither study differentiated the height of the ear in the “back” ear position and it is unclear what position was recorded if the ear was held both backwards and upwards.
One study that defined a position similar to our back up position found higher frequencies during positive states, such as using the brush or feeding compared to queuing to be milked, and suggested it might indicate a higher-arousal positive state than ears back down [26
]. The significant increase of back up and the concurrent decrease of back centre that we observed during stroking in the “ventral neck” treatment might thus indicate a higher-arousal state during “ventral neck” stroking than in “reactive” stroking. However, this was not supported by the HR values, which did not differ between the stroking styles. In general, this absence of differences in HR between treatments indicates that the different distributions of ear positions occurring with different stroking styles were probably influenced by factors other than arousal. Therefore, ear positions could be helpful indicators of subtle differences in the valence of affective states of cattle in the future. However, especially regarding the lack of significant differences of HRV or behavioural parameters between the two treatments in our study and the ambiguous findings of previous studies, more research is necessary before making clear interpretations towards the meaning of different ear positions with regard to valence.
One possible alternative explanation for the lack of other differences between the two stroking treatments could be that during “reactive” stroking, the ventral neck was also stroked. An individual might thus have also been stroked mainly at the ventral neck in the “reactive” treatment if it indicated such a preference, which could have led to the absence of obvious differences between the two treatments. However, over all trials of the “reactive” treatment, stroking was performed for the longest durations in the region of the dorsal neck, followed by the cheek, ventral neck, lateral neck, jaw and nose, indicating that there was a meaningful difference between the stroking treatments. Another explanation for the lack of differences lies in a potential masking effect: stroking on the neck might specifically elicit neck stretching as a direct reaction in the case of a positive perception, while stroking another region may induce other behavioural signs of positive perception as well. It is possible that animals perceived the interaction as more pleasant during “reactive” stroking than during “ventral neck” stroking, but because the neck was stroked for shorter durations of the overall treatment time, less neck stretching was induced and similar durations of neck stretching occurred with both stroking styles.
Regarding the behaviours evaluated at the descriptive level, the duration of resting head increased numerically over the three phases in the “reactive” treatment but not in the “ventral neck” treatment, which is comparable with the results of ear low. This behaviour should be recorded and evaluated statistically in future studies.
The conflicting results regarding the variability indicate that the relationship between the degree of standardisation of the treatment and the variability in the observed behaviour is more complex than expected or the standardisation has different effects on different parameters. The higher degree of standardisation in the “ventral neck” treatment did not necessarily lead to a reduction in variability and therefore should not be the sole criterion for the selection of stroking style for gentle human–animal interactions in experimental settings.