1. Introduction
Animals should be slaughtered as near to the point of production as possible. The journey time for slaughter animals should never exceed the physiological needs of the animal for food, water, or rest [
1]. In China, more than 95% of the live animals slaughtered for the Hong Kong market are transported by truck and special cargo trains from inland provinces. This can include provinces in northeast China and Inner Mongolia, which involves travelling distances of over 3500 km [
2].
The welfare of sheep during transport on the road can be influenced by many factors: the duration of the journey [
3,
4], stocking density [
5], driving behaviour, vibration and noise, change in acceleration and cornering, road type [
6,
7], the design of the vehicle [
1,
8], loading and unloading [
9,
10], mixing with unfamiliar sheep or breeds, the novel environment, hunger, thirst and fatigue [
1],temperature, humidity, excess ventilation and cold or heat stress [
11,
12,
13].
Cold exposure is potentially one of the major stressors of livestock in winter. In a cold environment, homeothermic animals, including mammals, have to generate additional heat from their diet and body reserves [
14]. In the fields, temperatures below the lower critical temperature and chilling winds can cause cold stress in livestock, as well as increasing their energy requirements, leading to decreases in productivity. For adult sheep, the lower critical temperature is –3 °C [
2], below which daily metabolizable energy requirements of a 60 kg sheep increase by 0.14–0.64 MJ for each 1 °C decrease in ambient temperature from January to December in New Zealand [
15]. A previous study has recommended that the appropriate ambient temperature for adult ewes in northwest pastoral regions of China should be above 2 °C [
16]. Sheep are transported at temperatures far below this in the winter throughout the northern hemisphere, and in the region of this study, Inner Mongolia, winter temperatures can commonly be as low as −20 °C. The problems sheep may experience during transport at these temperatures, especially the effects of different journey lengths, vehicle design and level of feeding, have not been reported in the literature.
While sheep are considered more tolerant of transportation than other livestock, it can be stressful for both adult sheep and lambs, which show physiological responses such as increased heart rate, elevated plasma cortisol concentration and hyperthermia [
17,
18,
19]. It has been noted that sheep do not experience changes in plasma osmolality even after 48 h without food and water, and after transport for 14 h, their immediate priority is for food; and only after 1–3 h do they start to drink [
20]. It may therefore be more important to provide feed than water for sheep before their transportation.
Vehicle design is critical for the transport of animals [
21]. It is recommended that transport vehicles should never be completely enclosed, as a lack of ventilation will cause excessive stress and even suffocation [
21], but this could be different in cold temperatures. Additionally, it might also affect profitability—in pigs, cold stress can lead to weight loss [
22,
23] and mortality during transport [
24]. In winter, the thermal properties of the trailer can be modified to reduce heat loss, including polystyrene insulation [
25] and an insulated floor [
26].
The duration of transport has also been investigated. Transport of sheep for 12, 30, and 48 h has resulted in reduced body weight and increased haemoconcentration on arrival [
4]; however, after 9 h [
27] or longer [
28], sheep appear able to adapt to the stressors of the journey. However, this might not remain true in the context of very cold conditions.
The objectives of this research were to evaluate the effects of cold conditions on welfare during transportation of sheep and to establish what the perceived animal welfare issues in sheep transport are at cold temperatures under different transport conditions (an enclosed and open truck, with different durations of transport, and with or without feeding before transport). This will inform our understanding of the welfare issues associated with the transport of sheep in cold temperatures.
2. Materials and Methods
Sheep were cared for in accordance with the guidelines for animal experiments of Inner Mongolia Agricultural University. The experimental protocol (No: NND20212037) was approved by the Institutional Ethics Committee of Inner Mongolia Agricultural University.
2.1. Experimental Facilities
At the start and the endof the transportation, the location we tested was the Hailiutu experimental farm (Location: 40°51′~41°8′ North Latitude, 110°46′~112°10′ East Longitude) of the Inner Mongolia Agricultural University, Hohhot, Inner Mongolia, China. This sheep farm comprises an indoor barn area covered with straw and hay bedding and an outdoor area of yards, which, in winter, is covered with snow. Sheep were free to choose whether to be in the outdoor area or in the barn. Sixty 4-month-old Dorper × Mongolian female sheep, with average body weight of 18.02 ± 2.60 kg and fleece length of 40 mm, were randomly selected from a flock of eighty on the farm. They were divided into 20 groups of three similar sheep, each with its own identical house and outdoor area.
The sheep were fed in groups of three with the same ration: alfalfa hay (1.5 kg/head/d) first and then a pelleted feed (0.5 kg/head/d) one hour later, for thirty days. The diet contained 90.7% dry matter, 11.1 MJ/kg digestible energy and 13.6% crude protein. This was split into two feeds daily at 08:00/09:00 h and 15:00/16:00 h and was offered in the research facility. The physical composition of the diet included corn, soybeanmeal, cottonseedmeal, and a vitamin and mineral premix. Water was provided ad libitum both in the barn and in the outdoor area.
The transport journeys took place on 15 and 16 January 2020. Mean maximum and mean minimum temperatures on these two days were −13 °C/−21 °C and −11 °C/−18 °C; the humidity was 69% and 77%, respectively [
29]. Three experiments were conducted with two treatments in each and 10 sheep in each treatment. The factors investigated in the three experiments were: Experiment 1—enclosing the vehicle (truck with or without a plastic cover and 1 h trip); Experiment 2—transport duration (1 or 2 h); and Experiment 3—pre-feeding (feeding the sheep before loading or not, 2 h trip). The 60 sheep were allocated to the six treatments so that the ten animals in each treatment had a similar body weight. Each sheep was used for one experiment only. Apart from Experiment 3, all sheep were held in an outdoor paddock and provided with access to alfalfa hay and fresh water for 2 h before each experiment.
Vehicles
All journeys were made using two similar vehicles: vehicle 1, a Beijing Futian truck manufactured in 2011 (Foton), and vehicle 2, a Wuling Rongguang truck manufactured in 2011. The two vehicles had the following respective dimensions: length 2.7 and 2.7 m, width 1.5 and 1.5 m and axle height 0.32 and 0.30 m. Both of the vehicle trays had 0.4 m solid walls, with a cage to height 1.1 m (truck 1) and 1.0 m (truck 2). In each experiment, trucks were allocated to treatments at random. The trucks were driven on a return route on a straight, single-lane carriageway (road numbers 038, 025 and 024) with minimal traffic at a consistent speed of 60 km/h. The route was chosen so that there was minimal traffic, no traffic lights and no stops, in order to minimise differences over time and between the journeys of the two vehicles.
2.2. Experimental Details
2.2.1. Experiment 1. Enclosed vs. Open Truck
The first experiment compared sheep in a covered and an open truck, which both left the Hailiutu Experimental Farm at 09:00 h on 15 January. In the enclosed truck treatment, the rear tray of vehicle 1 was covered with polythene on the top and sides of the cage on all four sides, secured with rope to prevent the polythene moving during transport and also to prevent incursion of wind into the tray containing the sheep. The polythene was put in place immediately after loading. Truck 2 was used for the open vehicle treatment. The vehicles proceeded down the prescribed route for 1 h and then returned to the farm for a further 1 h.
2.2.2. Experiment 2. Journey Duration
This experiment compared two journeys, one of 2 h and one of 1 h, made on 15 January on the same route as the first experiment, driven twice in the 2 h journey and once in the 1 h journey. The 2 h journey was from 14:00 h to 16:00 h and the 1 h journey from 14:30 h to 15:30 h, so the time midpoint was the same in both journeys. Both trucks were covered with plastic, as in Experiment 1′s covered vehicle. Vehicle 2 was used for the long duration journey and vehicle 1 was used for the short duration journey.
2.2.3. Experiment 3 Feeding the Sheep before Transport
This experiment started at 11:00 h on 16 January. Sheep were fed 60 min before the journey commencement and at 20:00 h the night before, with 0.5 kg pellets /head and 1.5 kg alfalfa hay/sheep, offered in a trough. Sheep not fed before transport were fasted from 20:00 h on the night before, when orts were removed. Due to a breakdown of truck 2, journeys were made consecutively by vehicle 1. Sheep not fed before transport departed at 11:00 h and arrived back at 13:00 h; those fed departed at 14:00 h and arrived back at 16:00 h. In both groups, water was withheld for 12 h before transportation.
2.3. Measurements
2.3.1. Environmental Parameters
Environmental temperature, humidity and wind speed were recorded by a local weather station at the farm at the time of the sheep transport [
29]. In addition, during the transport, the wind speed was measured with an anemometer (Testo 416 Digital Mini Vane Anemometer, 99 Washington Street Melrose, MA 02176, USA) ten times, by holding the device outside the cab window.
2.3.2. Physical Indices and Sheep Temperatures
Body weights of all sheep were measured to a precision of 0.1 kg 1 h before and after transportation. Starting body temperatures were recorded inside the building 1 h before transport, at the following locations on the sheep: head (midpoint between the ears), one ear (left), abdomen, and the foot/pastern region of the lower front leg, with the coronary band being the central point, using an infrared thermometer (Fluke MT4 max, Fluke Corporation P.O. Box 9090 Everett, WA 98206-90909090, USA).
After these initial measurements, the sheep were individually transferred outside next to the vehicle, for more detailed measurement of frostbite risk. A recording of the minimum temperature of one ear and the foot/pastern region was made to measure frostbite (
Figure 1), using an infrared thermography camera (range −15–550 °C, UNI-T, UTi220A, Uni-trend Technology Co., Ltd., Songshan Lake National Hight-Tech Industrial, Guangdong Province, China), as these are the first areas to suffer from frostbite. After the journey, sheep temperatures were measured again at the same places in reverse order, first beside the vehicle and then in the building. For the outdoor recordings, as ambient temperatures were less than sheep body temperatures, it was necessary for the researcher to cup their ungloved hands around the back of the lower limb and ear to ensure that the recording of minimum temperature was of the sheep body part, not the environment.
2.3.3. Physiological Indices
Two jugular blood samples were collected into heparinized tubes 1 h before and after the transportation for the analysis of stress indices. The blood samples were centrifuged and separated into plasma and serum components at the farm, which were then stored in a container with dry ice at a temperature of around −20 ℃, before analysis. Creatine kinase (CK), adrenocorticotropic hormone (ACTH), heat shock protein (HSP) alanine aminotransferase (ALT), lactic acid dehydrogenase (LDH), free fatty acid levels (NEFA), catecholamine (CA) and cortisol concentration were determined with commercial ELISA kits (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China and Ruixin Biological Technology Co., Ltd. Quanzhou, China), according to the manufacturer’s instructions. Glucose was measured using a HITACHI 7020 automatic biochemistry analyser.
2.4. Statistical Analysis
Experiments were individually analysed for statistical differences between treatments. Based on temperatures and blood parameters measured before departure and after return, the changes during the transport were calculated for each animal. These changes were compared between treatments using a general linear model, including treatment as a fixed effect and value at departure as a covariate. In an attempt to find the common patterns in measured temperatures and blood parameters, and to study the effect of transportation under different conditions on those patterns, a principal component analysis was conducted for each experimental dataset. Results were considered statistically significant at p ≤ 0.05. Statistical analyses were performed, and figures were constructed using R version 3.5.3 (R Foundation for Statistical Computing, Vienna, Austria).