Location and animal management
The study was conducted at Vasen Dairy Farm in Nye, Jönköping county, Sweden, between June and August 2016. The farm maintains approximately 450 dairy cows, mainly Swedish Holstein and Swedish Red breeds, with an average rate of 50 births per month. In accordance with the management of newborn animals at the farm, the calves were separated from the dam between the first and the third day of age. Before transfer to single pens, the calves were fed by suckling the dam and by using a calf bottle with a rubber teat two to three times per day. Calves in the single pens were fed milk twice per day, at 7:00 h and at 16:00 h, suckling from a teat bucket. The teat buckets were removed after each feeding session.
Testing method
Testing was conducted daily at the single calf pens between 12:00 and 15:00 h, for a total of 10 consecutive days per calf. Calves transferred to the single pens on the morning of a testing day received a brief health check from a veterinarian. Calves requiring veterinary treatment on the day of transfer were excluded from testing. The calves were then assessed for their motivation to stand up voluntarily and suckle in the single calf pen by stimulating the suckling reflex. This was achieved by rubbing a finger against the calf’s lips or by inserting a finger in the calf’s mouth for a few seconds. Calves that suckled the finger, subsequently stood up voluntarily without assistance and continued suckling or sought to suckle were marked as “motivated”. The rest of the calves were marked as “unmotivated”. “Motivated” calves were assigned to the taste and odor tests, while “unmotivated” calves were assigned to the suckling reflex test, as described below. All “unmotivated” calves were controlled for their ability to stand and for body temperature before their participation in the suckling reflex test; all of the animals could stand without assistance and had regular body temperature (38.6–39.4 °C) on the testing day.
Taste and odor tests
The taste and odor tests were conducted once per day for each calf. “Motivated” calves were tested for ten consecutive days (testing sessions 1–10), while “unmotivated” calves were tested for nine consecutive days (testing sessions 2–10) after having participated in the suckling reflex test on day one. These tests were performed in order to assess whether the calves showed a preference for suckling a plain (control) dry teat versus a dry teat that was modified with a presumably attractive taste or odor. Calves that required veterinary treatment were excluded from testing on that testing day, and the testing session was marked as "did not participate".
A two-choice test apparatus was used that could be adapted to the requirements of each test and that allowed the simultaneous presentation of the control and the modified teat to the calf (Fig. 1). The apparatus was made of stainless steel and consisted of:
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a)
An “inverted L-shaped” bar (50 × 2 × 6.5 cm, 2 mm thick), with four holes of 3.8 cm diameter each. The adjacent holes had a distance of 10 cm from one another, measured at the center of each hole. The two centermost holes were used for positioning the teats for the taste test. The two outermost holes were used for positioning the teats for the odor test. Thus, the teats for the taste test had a distance of 10 cm from one another, allowing the calf to perceive and taste both with ease. The teats for the odor test had a distance of 30 cm from one another. This distance created sufficient space between the odor stimulus placed above one of the teats (as described below) and the blank stimulus placed above the other teat in order for them to be distinguishable, and at the same time allowed the calf to perceive both teats.
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b)
Two mesh cases (9.5 × 2.3 × 9.5 cm, 1 mm thick) with holes of 5 mm diameter. The mesh cases were placed above the outermost holes of the metal bar where the teats for the odor test were positioned. Open petri dishes containing odorized or non-odorized filter papers were inserted in the mesh cases, with the open side facing the mesh for stimulus dispersal towards the teat (as shown in Fig. 1, Panel c).
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c)
Two cylindrical handles (3.9 × 25 cm) positioned at the outermost parts of the “inverted L-shaped” bar used by the experimenter to hold the apparatus. Each handle bore a rectangular surface (7 × 5 cm) allowing the attachment of a stopwatch. The stopwatches were used for measuring suckling time for each teat.
For the taste test, the modified teat consisted of a plain teat dipped in water and then coated with glucose ("Just 100% Dextro", 13: e Protein Import AB, Skogås, Sweden), using a shaker to apply approximately 0.70 g of the substance as evenly as possible on the teat surface. Glucose was selected as the presumably attractive taste stimulus according to the findings of Hellekant et al. [17] regarding behavioral responses of calves to sweeteners, and due to the frequent availability of the substance in dairy farms for the preparation of electrolyte solutions for calves.
It has been clearly demonstrated that the taste of milk elicits suckling in calves of at least three weeks of age [18,19,20], who should by that time be fully efficient in suckling from an artificial teat. As our testing group for the current study involved efficient drinkers, we opted against using milk as a presumably attractive substance due to its known effects on such a group. However, to our knowledge, it has not been investigated if the taste and smell of milk on the artificial teat have similar effects on the suckling motivation of newborn dairy calves immediately after birth and up until the point where they become efficient drinkers a few days later. In October 2017 we conducted a preliminary online survey asking dairy calf caretakers in Sweden about newborn calves having difficulty using the artificial teat on the first 24–72 h after birth. The responses of the survey, available in the supplementary material of this study [see Additional file 1], suggest that the taste of milk alone is not always successful in motivating newborn dairy calves to use the artificial teat. For this reason, it would be beneficial to initially investigate the effects of other potentially attractive substances that imitate or belong to the repertoire of tastes and odors that a calf encounters either during gestation or during the early days of life.
For the odor test, the modified teat consisted of a plain teat with an open petri dish positioned vertically above it which contained a filter paper impregnated with 1 mL of the odor "Freshly Cut Grass Fragrance Oil" (Mystic Moments, Alderholt, UK). The odor was selected as a presumably attractive olfactory stimulus according to the fact that grass is an important part of cattle diet [3, 21] and volatile compounds from grass feed are transferred to milk [22,23,24] that calves consume at least during the first days of life. As control odor, a petri dish containing a filter paper impregnated with 1 mL of water was placed above the plain (control) teat. The petri dishes were secured behind a mesh, so that the calves could smell but not come in direct physical contact with or lick the substances (see Fig. 1).
The majority of the calves were tested while standing and facing the experimenter, with the apparatus held at the head level, ensuring equal ease of access to both teats (Fig. 1, Panel c). On the few occasions that a calf was unable to or refused to stand, it was tested while lying on its chest and facing the experimenter. The calf was then allowed to interact with the teats for a period of 2 min. The rationale for using this period of time was that Ventorp and Michanek [25] defined a period of at least 0.5 min as successful suckling in calves.
The following data were recorded for each individual and test session:
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a)
investigating behavior consisting of sniffing, touching with the lips or licking the tip of a teat, or immediately suckling a teat,
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b)
order of teat investigation, i.e. which of the teats was investigated first (control teat first / modified teat first / did not investigate the teats), and
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c)
suckling time for each teat, in seconds, with the use of stopwatches.
Four latex rubber teats were used throughout the study (Foga Försäljning AB, Sweden). During a single testing day, one pair of teats was used for all the taste tests and one pair was used for all the odor tests, with each teat being randomly assigned the role of control or modified teat throughout the testing day. In order to avoid preference bias due to predictable teat placement (right or left side), the positioning of the control and the modified teat was pseudo-randomized throughout the testing days, but was always opposite for the two tests on a given testing day (e.g. modified teat positioned right for the taste test, and left for the odor test). After testing a calf, each teat was rinsed with water and, if necessary, wiped with a paper towel before being used for the next calf.
On each testing day, all calves were first tested with the taste test in order to avoid potential contamination of the apparatus from the test odor. Before conducting the odor test, the apparatus was wiped with a wet paper towel or, if necessary, rinsed with water, in order to remove potential glucose remnants. The time interval between the two tests for a single calf was at least five minutes.
At the end of each testing day, the apparatus and the teats were thoroughly cleaned with hot water and fragrance-free soap and were left to dry until the next day.
Suckling reflex test
The suckling reflex test was conducted only during the first testing day of “unmotivated” calves in order to assess whether a calf would successfully suckle a plain teat as well as the modified teats (taste and odor) described in the previous section. The same apparatus (Fig. 1) was used for this test. During the test, a plain teat was first presented to the calf in front of its muzzle, then inserted into the calf’s mouth for approximately five seconds, and the suckling response was recorded (suckled / did not suckle). The same procedure was repeated with a glucose-coated teat. For the teat that was modified with an odor, the calf was first presented with the "Freshly Cut Grass" odor by placing the mesh of the test apparatus bearing the odor stimulus directly in front of its muzzle and then the teat was inserted into the calf’s mouth, following the same procedure as with the other two teats. A “licking lips” behavioral response was also recorded during the presentation of each teat to the calf (performed / not performed). The behavior consisted of the calf licking the lower part of its muzzle, with the tongue being visible, for approximately one to three repetitions.
Sample size and age
In total, 78 newborn dairy calves (n = 78) were sampled for the study. Fifty-seven of these calves (n = 57) were marked “unmotivated” on the first day of testing and thus successfully participated in the suckling reflex test. Fifty-one out of 78 calves (n = 51), of which 29 male and 22 female, successfully completed the series of taste and odor tests. Successful completion of the taste and odor tests was achieved if a calf successfully participated in at least six out of nine tests (“unmotivated” calves) or seven out of ten tests (“motivated” calves), for the taste and the odor test, respectively. Participation was considered successful if a calf suckled at least one of the two teats for at least one second. The age of the calves when entering the testing group varied between one and five days old. Thus, all measurements took place within the calves’ first two weeks of age. Figure 2 summarizes the criteria used for inclusion of a calf into the study and the flow of events throughout the study.
Data analysis
The Chi-squared test was used to assess whether the “licking lips” behavior was performed by a significant proportion of calves during the suckling reflex test. The two-tailed binomial test was used to assess whether the calves showed a significant preference for the order of investigation of a teat (control or modified) during the odor test and the taste test. The Wilcoxon signed-ranks test for related samples was used to assess if there were significant differences between the suckling time for the control teat (STC) and the suckling time for the modified teat (STM) in the odor test and the taste test, as well as between total suckling times (STT = STC + STM) for the odor test versus the taste test. For this statistical test, an average suckling time per calf for each type of teat or test was first calculated (sum of suckling times per teat for all testing sessions of an individual, divided by the number of testing sessions, when comparing the control teat and the modified teat; sum of STT for all testing sessions of an individual, divided by the number of testing sessions, when comparing the odor test and the taste test). The Mann-Whitney U-test for independent samples was used to assess if there were significant differences regarding suckling times between male and female calves. For this statistical test, an average suckling time for each type of teat (odor, taste) was calculated for each calf, and the sum of average suckling times was used to calculate each sex average. All statistical analyses were performed using the software R (version 3.3.1, ran of R Studio environment version 0.99.903).