1.1. Animal-Assisted Interactions

The difference between Animal-Assisted Interaction (AAI) and Animal-Assisted Therapy (AAT) must be clarified as these categories are confused in the public consciousness [11]. While the primary goal of AAI is to improve the life quality of the patients through human–animal bonds, in AAT the animals are integral parts of the treatment process. During AAT, the animal and their handler are specially qualified, cooperating with a therapist with credential titles [12]. In contrast, AAI is a less definite human–animal interaction [13].

The first recorded use of animals in therapy was in 1792 in England by William Tuke who began to make efforts to improve the living conditions of mentally ill people [14,15]. To improve the health of people in general, farm animals were used in the earliest decades, while nowadays the most popular therapy animals are dogs [16]. In the last 30 years, other species have also been reported, such as companion horses [17,18], rabbits [19,20,21], cats [22], birds [18], guinea pigs [23] and reptiles [24].

1.2. Rabbits in Animal-Assisted Interactions

Rabbits as therapy animals have been used in several social areas, from kindergartens to residential care homes and nursing homes, just as in schools or veterans’ homes [15,25,26]. Rabbits as partner animals in AAIs can function well as an alternative to a dog or a cat, but they need to go through a careful screening process. There are several criteria that must be met in order to use rabbits in AAI programs, according to Granger and Kogan’s [27] and Mallon and Cow’s [28] suggestions. These animals need to be trained:

• (i) for being transported and for the use of animal transport boxes/cages;
• (ii) to accept a stranger’s hands-on interaction and to cooperate while being held in their lap for 2 min;
• (iii) for being stroked or petted by more people at the same time;
• (iv) to allow being petted in different body parts and areas, including mouth area, teeth, ears and paws, by strangers.

Animals need to be tolerant:

• (i)

  with disabled persons in their wheelchairs, crutches, walkers and walking aids for medical purposes, etc.;

• (ii)

  to sudden noise, sound and possibly shouting as well.

The rabbits satisfying the above requirements could be involved in various ways in AAI, therefore, they can easily become the children’s favorites [28]. Petting the animal is ideal for developing fine motor skills, learning pet care from an early age, sensitizing children and teaching children responsible pet ownership [27].

AAI could not exist without animals. Therefore, it is of high importance to ensure the health and well-being of the animals in all aspects of AAI. As AAI is beneficial to people through the human–animal bond, it is significantly important not to affect the animals negatively. Whereas the benefits of AAI to people are becoming more and more evident, its advantages for animals are not prominent and hardly measurable. In AAI, the well-being of animals can be harmed in many areas, there is a risk of zoonoses, not only from animals to humans but also from humans to animals, and the physical and mental stress that therapy animals suffer from (even for a short time) can have a negative effect on the well-being of the animal [14]. Menna et al. (2019) do not advise the use of exotic and special pet animals—and rabbits are also included in this category—for AAI purposes. Menna et al., justify their opinion by saying that these species are not closely related to humans on an ethological level [29]. Human–rabbit interactions were measured due to questionaries by Dobos et al., in 2023. In the survey, owners were asked about the interactions and keeping conditions of their pet rabbits. Three principal components were established: amicability, aggression with the owner and aggression with a stranger. Dobos et al., found that individually kept rabbits were considered to be more amicable compared to those that had other rabbits as companions. This human-oriented behavior might be caused by the extra care that a singleton rabbit gets compared to a group-kept one. Dobos et al., also found that large traditional meat rabbit breeds—kept as pet rabbits—are less amicable than dwarf pet rabbit breeds. Even the type of feeding showed an association both with amicability and aggression. Pet rabbit owners often offer muesli-type feed that is calorie-rich and soft. These have negative consequences for the rabbits’ body condition and can cause painfully overgrown teeth which could possibly result in more frequent aggressive responses towards their caregivers [30]. In order to involve rabbits in AAI it is essential for kits that right after birth—while they are in the sensitive period—they become accustomed to the touch of the human hand. This early handling—called imprinting—has a positive impact on stress tolerance throughout the lifetime of the rabbit. Consequently, to ensure the rabbits’ well-being, it is beneficial for therapy rabbit breeders to familiarize kits with human hands and to have prior knowledge about the rabbits’ temperament [31].

1.3. Considering Transportation Stress

Transportation of animals, regardless of the species, is a stressful experience [32,33,34], therefore, commercial animals’ transportation processes are regulated by law and must be documented. Specific provisions relating to carrying companion animals are universalized and give ordinary wellbeing instructions [35].

The effects of carriage on dogs are well documented, such as the methodology of observing the dogs’ behavior during transportation [36,37,38]. Specific data, such as stress hormone (cortisol) examinations, are also available [39,40,41,42].

Generally, rabbit transport-related publications are focused on slaughterhouse deliveries in order to improve meat quality [43,44,45]. The European Food Safety Authority (EFSA), panel on Animal Health and Welfare (AHAW) 2022 gave a scientific opinion about rabbits’ container transport’s welfare consequences [46] in order to reduce the animals’ suffering of trauma. As a result of inappropriate animal handling during transportation typical injuries occur, such as hemorrhages, bruises and broken bones. The EFSA and the above publication’s common recommendations are the following:

• (i) careful rabbit handling,
• (ii) to prevent the transfer of urine and feces, solid floors of the crates are recommended,
• (iii) to avoid heat or cold stress, rabbits should travel in their thermal comfort zone. To fulfill this criterion the temperature inside the van should be kept permanent, between 10 °C and 20 °C.
• (iv) Prolonged hunger (more than 12 h feed withdrawal) causes weight loss and is pernicious to their welfare. In the thermal comfort zone, the total time of food and water delimitation should not exceed 12 h.
• (v) the crates’ height should be enough to let the rabbits sit in a natural position.

Buil et al. [44] highlight that the critical point of commercial rabbits’ transport to slaughter is the condition of transport while the time of the journey is a less stressful aspect. Therefore, the details of transportation need to be well planned and executed in advance.

Herbel et al. [47] examined the stress response of beagle dogs where 18 animals attended the study and undertook repeated short-distance road transports. Transport cages were placed on the floor of a minibus that had all its rear seats removed. The route took 60 min included approximately 25% city traffic and 75% four-lane motorways. The driver of the car and a second person accompanying the transports had handled the dogs during the four-week accustomization phase before the study and were, thus, familiar to the dogs. The same two persons were also present during control experiments. Our present study was based on a similar design and carried out parallel methodology in several respects (detailed in Section 2).

2.2. Housing and Feeding

In the rabbit shed each rabbit was housed individually as presented in Figure 1. The Hungarian and EU legislation in force Decree of the Ministry of Agriculture (32/1999./III. 31./ and 178/2009./XII. 29./) in the “General rules for rabbit does and bucks, and suckling and growing rabbits” gives a clear policy: the rabbits must be housed individually after 12 weeks of age, with the exception of fattening rabbits. The individual cages of our rabbits were placed next to each other and the animals were in visual contact. The pet rabbit cages (95 × 57 × 46 cm) have a corrosion-resistant metal-coated wire mesh (on the top) and a dark plastic bottom. The spacious rabbit cage provided all the necessary things rabbits needed in their cage, like a feeder, a hay container, a hanging water bottle, a separate sleeping area and a rabbit litter box filled with wood pellets (compressed wood shavings and sawdust from untreated wood). To be a house-trained animal, rabbits used the litter box to urinate and defecate. All the accessories were made of plastic except the stainless-steel feeder.

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Figure 1

Rabbit shed.

As for feeding, the rabbits received a complete diet (Versele-laga complete cuni adult rabbit) which is an all-in-one pellet to avoid selective feeding behavior and to ensure consumption of all the essential nutrients for optimal health. This feed does not contain a coccidiostat or other additives. In addition to the pellets, the rabbits had free access to the water nipple drinker, were given hay ad libitum and a daily 35–40 g of fresh raw carrot sticks and 30–35 g apple pieces, both from organic farming, as a treat and vitamin complementary. The cages were equipped with gnawing sticks and supplementary mineral blocks as environmental enrichment. Although the animals were housed individually to prevent harm and social stress, the cages were placed side-by-side, enabling them to sniff each other and stay in visual contact. In good weather conditions, the rabbits were grazing for at least two hours in their mobile open-air hutches every second day as presented in Figure 2a,b.

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Figure 2

(a) Rabbit free grazing. (b) Rabbit free grazing.

Veterinary checkups, including physical examinations like dental health condition checks and assessment of the appearance of the Spilopsyllus cuniculi and Psoroptes, were conducted on all rabbits prior to the transportation examination. All rabbits were vaccinated against myxomatosis and RHD, and none of them were infected with parasites or zoonotic diseases.

2.3. The Transportation by Car

For two weeks, every second day the rabbits were taken for a 30 min transport by car at 8:30 AM, imitating the duty of an assisted animal that is working three days per week in AAI and needs to be transported to the intervention’s location. During the examined period, the rabbits were transported on Monday, Wednesday and Friday, while on all the other days they remained at their housing. They were transported in rabbit-sized cages, made of strong plastic and consisting of two parts that are held together with a special fastening system made up of a clasp on the back and two hooks in front. It comes with a plasticized metal door with a safety clip. The upper part has an ergonomic handle to ease carrying and ensure a sturdy grip. Side grids make sure there is air circulating inside. According to the carrier’s direction of use, it is suitable for pets weighing up to 3 kg. The approximate size of the box is 24.5 × 28 × 41.5 cm. The transportation dates were the following: 24 October; 26 October; 28 October; 31 October; 2 November and 4 November 2022. The transportation was carried out by an air-conditioned minibus (Renault Trafic Combi, 2021 model, France) at a temperature of 19 degrees. The placement of the carrier boxes are presented in Figure 3. Each carrier was stabilized on the floor to avoid side slips. Twelve rabbits were transported in the first week and six in the second. While traveling in the car, rabbits had limited access to see each other and the outside environment during the experimental stages. Each trip was preceded by a 10 min familiarization experience when rabbits were permitted to get used to their cages in the non-moving and shaded car. The 30 min (25 km) public road drive consisted of, approximately, 10 min of city traffic and 20 min of a two-line main road. The same route was driven on each occasion. The owner of the rabbits and a second person—the car driver–were present during the transportation.

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Figure 3

Rabbits in their individual transportation boxes placed in the car.

The methodology was based on the study by Herbel et al. [47] which examined dogs’ short-term transportation. The 18 rabbits were divided into three groups:

• Six of them were transported three times to examine intermediate long-period stress effects on the animals;

• Six of them were transported six times to examine prolonged period stress effects on the animals;

• Six animals formed the control group that were not transported at any time and remained at their housing during the whole examination period.

In order to obtain information on whether the experienced stress is reducible by feeds or treats, three subgroups were formed from the transported animals (four rabbits in each group): no feed, hay and hay+ carrot groups, detailed in Table 1.

2.4. Collecting, Storing and Transporting the Feces Samples to Laboratory

Samples were collected seven times from 18 rabbits, so the total sample number of this research was 126. The first samples were collected two days before the examination period in order to obtain information on the individuals starting cortisol levels. The second time sampling was exactly 24 h after the transportation had been carried out as the hormone cortisol’s metabolites appear in feces with a delay [51,53]. The sampling dates were the following: 22 October; 25 October; 27 October; 29 October; 1 November; 3 November and 5 November 2022. The samples were collected as follows:

• The plastic bottom of the animal’s individual cage and the litter box were cleaned and sterilized with a biocidal product with a spectrum of bactericidal, fungicidal and virucidal effects (SteriClean Farm, active ingredient: sodium hypochlorite solution (0.05%)) daily at 7:30 AM.

• After restoring all equipment in the cage, the litter box was filled with wood pellets again.

• At 8:30 AM, if feces appeared in the litter box, they were removed immediately.

• After 8.30 AM, from the first feces of the animals, samples were collected using sterile gloves for each individual. Every 15 min the conductor of the examination (who is the owner of the rabbits—first author) checked the litter boxes and collected the new samples.

• All the samples were labeled (with the rabbit’s number and sampling date) and immediately frozen.

• The samples were kept at −21 °C until they were transported to the laboratory.

The samples were transported in a cold box, that is suitable for vaccine carries, in a frozen condition, on 9 January 2023 to the Department of Diagnostic Laboratory, University of Veterinary Medicine, Budapest, Hungary, where the laboratory examinations were carried out. Cortisol levels were measured from the feces based on their breakdown products according to Benedek et al.’s [53] method. Cortisol was extracted from the feces samples by adding 4 mL of ethanol for 500 mg of feces, followed by vortexing for 3 min. Then, 10 min centrifugation at 2000 rpm was applied. Cortisol-containing supernatant was collected and used as samples.

The concentration of metabolites was determined with a cortisol ELISA kit (DEH3388, Demeditec GmBH, Kiel, Germany), according to the manufacturer’s protocol. Analytical sensitivity 0.38 ng/mL; range 10–800 ng/mL; intra-assay CV < 5%; inter-assay CVs were 5.2 and 7.8% for control 1 and control 2, respectively. Raw cortisol concentration data were translated and expressed as μg/g.

In the groups formed by the number of transportations, the appearance of cortisol concentration, defined by the metabolites in the feces samples (μg/g), was interpreted per day and presented using the following scheme:

• PRE—collected 2 days before the transport began (22 October),

• 1—collected 24 h after the first transport (25 October),

• 2—collected 24 h after the second transport (27 October),

• 3—collected 24 h after the third transport (29 October),

• 4—collected 24 h after the fourth transport (1 November),

• 5—collected 24 h after the fifth transport (3 November),

• 6—collected 24 h after the sixth transport (5 November).