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Long-Distance Transport of Finisher Pigs in the Iberian Peninsula: Effects of

Season on Thermal and Enthalpy Conditions, Welfare Indicators and Meat pH

Article in Animals · August 2021

DOI: 10.3390/ani11082410

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 animals
Article
Long-Distance Transport of Finisher Pigs in the Iberian
Peninsula: Effects of Season on Thermal and Enthalpy
Conditions, Welfare Indicators and Meat pH
Genaro C. Miranda-de la Lama 1, * , Rubén Bermejo-Poza 2 , Nora Formoso-Rafferty 3 , Malcolm Mitchell 4 ,
Pilar Barreiro 5 and Morris Villarroel 3

1 Department of Animal Production & Food Science, Agri-Food Institute of Aragon (IA2), University of
Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain
2 Department of Animal Production, Veterinary School, Complutense University of Madrid,
28036 Madrid, Spain; rbermejop89@gmail.com
3 Department of Animal Science, ETSIAAB Technical University of Madrid, 28036 Madrid, Spain;
nora.formosorafferty@upm.es (N.F.-R.); morris.villarroel@upm.es (M.V.)
4 Animal & Veterinary Sciences, Scotland’s Rural College-SRUC, Roslin Institute, Midlothian EH25 9RG, UK;
malcolm.mitchell5@btinternet.com
5 Department of Agroforestry Engineering, ETSIAAB Technical University of Madrid, 28036 Madrid, Spain;
pilar.barreiro@upm.es
* Correspondence: genaro@unizar.es; Tel.: +34-876554150






Simple Summary: Long-distance transport in the global swine industry is more the rule than the
Citation: Miranda-de la Lama, G.C.; exception. We tested the impact on the rates of temperature change and air enthalpy on the stress
Bermejo-Poza, R.; Formoso-Rafferty, response and muscle pH in pigs subjected to long-distance travel from Spain to Portugal performed
N.; Mitchell, M.; Barreiro, P.; in the summer and winter. We found that winter journeys are more adverse for the animals because
Villarroel, M. Long-Distance
during the journey, abrupt variations in rates of temperature change and air enthalpy caused a
Transport of Finisher Pigs in the
marked physiological stress response and effects on the meat pH after 45 min. These results indicate
Iberian Peninsula: Effects of Season
the need to develop new environmental control strategies that mitigate abrupt temperature changes
on Thermal and Enthalpy Conditions,
during travel to attenuate the biological cost of such long-distance transport on the animals.
Welfare Indicators and Meat pH.
Animals 2021, 11, 2410. https://
doi.org/10.3390/ani11082410
Abstract: Current legislation in the European Union places limits on live pig transport according
to outside temperature, but less is known about the effects of sudden changes in the thermal
Academic Editor: Frank J.C.M. Van microenvironment in trailers, particularly during long-distance transport. In this study, we measured
Eerdenburg the temperature and relative humidity inside livestock vehicles carrying 1920 Spanish finisher pigs
(live weight 100 kg and 240 animals per journey) during eight long-distance (>15 h) commercial
Received: 6 July 2021 journeys to slaughter from northern Spain to Portugal in the summer and winter. Here, we report
Accepted: 11 August 2021 the rate of change in the air temperature (◦ C × min−1 ) and air enthalpies in the transport vehicle
Published: 16 August 2021 (kg water kg dry air-1). At sticking, blood samples were taken for to measure cortisol, glucose, and
creatine kinase (CK) as stress response indicators, and the meat pH after 45 min and the pH after
Publisher’s Note: MDPI stays neutral
24 h were also determined. The rate of change in the air temperature and enthalpy was higher inside
with regard to jurisdictional claims in
the livestock vehicle during the winter months and was positively related with higher cortisol and
published maps and institutional affil-
glucose levels and lower pH after 45 min (p < 0.05). It is proposed that the rate of temperature change
iations.
and air enthalpy represent useful integrated indices of thermal stress for pigs during transport.

Keywords: pig welfare; long-distance transport; enthalpy; thermal stress; meat pH

Copyright: © 2021 by the authors.

Licensee MDPI, Basel, Switzerland.
This article is an open access article
1. Introduction
distributed under the terms and
conditions of the Creative Commons Pig production in the European Union is increasingly industrialized and special-
Attribution (CC BY) license (https:// ized [1], with the EU being the second largest pig producer in the world, with 24.1 million
creativecommons.org/licenses/by/ tons of pork produced in 2019 [2]. Spain is one of the most important pig producers in
4.0/). Europe and almost a third of national production is exported [3]. To maintain this high

Animals 2021, 11, 2410. https://doi.org/10.3390/ani11082410 https://www.mdpi.com/journal/animals

Animals 2021, 11, 2410 2 of 11

level of competitiveness, the pig sector depends on road transport as one of its strategic
components in the European pork supply chain [4]. The high demand for pork meat in
some member countries has stimulated intracommunity trade involving the long-distance
transport of live pigs [5]. Even under favorable conditions, long-haul transport can cause
different degrees of stress to animals, ranging from discomfort and aversion to death [6].
Extreme ambient temperatures during long-distance journeys are considered as one of the
most important risk factors for dead on arrivals, non-ambulatory animals, skin lesions,
and carcass downgrading, especially when microclimate conditions are outside of the
optimal thermal comfort zone for pigs [7]. These thermal conditions may be complex and
result from the interaction of several factors such as external climatic conditions, heat and
water production from the animals, ventilation regimes, distribution and flow rates, and
additional external sources of heat and/or moisture [8].
The temperature humidity index (THI) forms the basis for calculating ventilation
on farms [9]. The Livestock Weather Safety Index (LWSI) is derived from the THI and is
widely applied across species, as described by Eigenberg et al. [10]. The LWSI has been
related to mortalities during pig transport in Denmark [11]. Barbosa-Fihlo et al. [12] have
compared using enthalpy with the Black Globe and Humidity Index (BGHI) proposed by
Curtis [13] in the assessment of thermal comfort in broilers. Enthalpy was deemed the
better index and the same authors [14] later applied an Enthalpy Comfort Index (ECI) to
poultry during transport, reporting that hostile thermal conditions are well predicted by
this index. Enthalpy, a concept that combines temperature and relative humidity, is the
heat energy of the air and is the major determinant of the dry and latent heat loss to the
environment. It can be calculated using simple tools (i.e., a thermometer and hygrometer)
and mathematical models [15]. Advances in this area will underpin improvements in
handling and the transport of livestock, especially under more extreme climate conditions.
Current legislation in Europe: European Union Council Regulation 1/2005 [16] defines
temperature limits during the transport of livestock on journeys over 8 h in duration.
However, the legislation does not specify any corresponding limits for humidity or water
vapor content in the air. Similarly, there is no consideration of the effects upon the animals
regarding the rate of change of the thermal conditions. According to EC 1/2005, the upper
temperature limit for the long-distance transport of livestock on long journeys is 30 ◦ C
with a tolerance of 5 ◦ C, meaning the absolute limit is 35 ◦ C. Although integrated indices
of temperature and humidity are used extensively in other areas of animal production
to predict their impact on production and welfare, this approach has not been applied
to animal transport in the European legislative context, and few studies have considered
the potential application of enthalpy as an integrated index of thermal load. In this study,
the aim was to develop and compare psychrometric charts as well as rates of change in
the temperature and enthalpy from long distance (>8 h) journeys carrying pigs across the
Iberian Peninsula from northern Spain to southern Portugal. In addition, the potential use
of rates of change of temperature and enthalpy as non-invasive indicators and their impact
on blood stress indicators and meat pH in pigs were evaluated.

2. Materials and Methods

A total of eight long-distance commercial journeys from a pig finishing farm located
in the town of Campanas in the autonomous community of Navarra in Spain (42.69 N,
1.65 W, and 575 m.a.s.l.) to a EU licensed abattoir at Vila Franca do Rosario in the Lisbon
region in Portugal (38.97 N, 9.25 W, and 88 m.a.s.l.) were studied. The distance for all
of the journeys was 936.5 km with an average duration of 14 h and 30 min. Campana’s
climate is Cf2b according to Köppen–Geiger’s climate classification; warm and humid with
cold winters and mild summers with rainfall throughout the year, except for two relatively
dry months. The annual average temperature is around 12 ◦ C with an average monthly
temperature of 5 ◦ C in the coldest month (January) and 20.9 ◦ C in the warmest month
(August), attending to the typical climate data [17]. Pigs were carefully handled during the
pre-slaughter period: they were handled at unloading and in lairage using plastic paddles
Animals 2021, 11, 2410 3 of 11

only. They were loaded and unloaded by the workers hired by the farm and the abattoir
and were kept together in familiar groups during transport and lairage. All procedures
were conducted in accordance with the guidelines for the ethical treatment of animals in
applied animal welfare studies [18].

2.1. Study Description

The eight long-distance journeys monitored the transport of a total of 1920 Spanish
finisher pigs (240 animals per journey) in the winter (n = 3) and in the summer (n = 5). All of
the pigs were Large White/Landrace × Duroc pigs with a mixed group males and females
who were six-months-old (with an approximate average live weight of 100 kg). Pigs
were off feed for 12 h before transport. Loading was usually conducted at approximately
05:00 a.m. by three farm operators, avoiding mixing between unfamiliar pens. The farm
operators went to one pen at a time to drive the pigs toward the loading platform using
plastic bags and plastic boards. The loading procedures lasted approximately 2 h per
journey. The animals were loaded and transported from Campanas (Spain) to a commercial
abattoir in Vila Franca do Rosario (Portugal). This journey duration is compliant with the
overarching transport regulation, EC 1/2005, which prescribes a maximum journey time
for adult pigs of 24 h when undertaken on higher standard vehicles and with constant
access to water. For each journey from the farm to the abattoir, members of the research
team accompanied the truck to ensure and verify that the journey met the objectives of the
study. All journeys always took the same route and had the same lorry and driver.
The vehicle that was used was an articulated lorry with a tractor unit (MAN, Munich,
Germany) towing a trailer (Carrozzeria Pezzaioli, Montichiari, Italy). The trailer had three
floors with six compartments per floor (each compartment measured 220 cm long × 245 cm
wide and 84 cm high), giving a total surface area of 5.39 m2 per compartment and an
average stocking density of 0.42 m2 /pig). The total loading capacity of the truck was
about 27,000 kg and was equipped with suitable drinking systems with nipples to provide
water during the journey. The trailer had both natural and mechanical ventilation systems,
which consisted of twelve automatic fans per floor or six per side. The fans, nine in each
truck, were 225 mm in diameter with a 11,700 m2 /h flow in compliance with EC 1/2005.
The trailer had a hydraulic controlled elevator for loading and unloading and provided
anti-slip floors with incorporated side guards.

2.2. Enthalpy Assessment

Data on temperature and relative humidity were collected inside the livestock vehicles
during loading, transport, and unloading using Hobo data loggers (Hobo H8 loggers, Onset
Computers, Bourne, MA, USA). Prior to loading, two loggers were placed on the lorry at
the same level as the pigs in the middle floor of the trailer, and the loggers had an inside that
was specifically designed perforated metal tubing to let air in while avoiding contact with
the animals. Sensors were pre-programmed to record temperature and relative humidity
at regular 5 min intervals and were fitted and removed by a member of the research team
before and after each journey. Approximately 20 mm of wood shavings were placed on
each floor of the vehicle as bedding.

2.3. Slaughter
The abattoir operated from Monday to Friday (from 06:00 a.m. to 15:00 p.m.) with a
slaughter capacity of 2000 head/day at a rate of 220 heads/h. On arrival at the abattoir,
pigs were unloaded with an adjustable-slope metal ramp with an anti-skid floor. After
unloading at the abattoir, the pigs were showered for 15 in the winter or for 30 min in the
summer, and the pigs were kept in lairage pens without mixing the groups on arrival and
were given access to water through nipple drinkers. The lairage time for all animals was
at least 12 h from arrival, including overnight rest and slaughter the following morning.
At the end of the lairage period, pigs were stunned using a CO2 chamber with 70% CO2
atmosphere for approximately 60 s in a one-gondola dip-lift system. Following stunning,
Animals 2021, 11, 2410 4 of 11

pigs were horizontally exsanguinated. Carcasses were then eviscerated and split before
being placed in a chiller set at 4 ◦ C for 24 h.

2.4. Physiological Assessment

Blood samples were taken at the time of slaughter to evaluate physiological stress from
20 pigs per journey (one 10 mL tube per animal, with anticoagulant, EDTA-K3), totaling 160
sampled animals. Once all of the samples were collected for each journey, the samples were
refrigerated for 10 h until they were centrifuged at 1300× g for 10 min to obtain plasma.
The parameters measured in the plasma were cortisol, glucose, and creatine kinase. Plasma
cortisol was assessed by ELISA. Calibrators were prepared with vials of cortisol in PBS and
BSA and lyophilized at the concentrations of 0, 10, 30, 100, 300, and 900 ng/mL. For the col-
orimetric reading of samples, the blank and calibrators were performed within 20 min from
the end of the assay using a spectrophotometer (Hitachi 717® ) at 405 (for concentrations
below 30 ng/mL) and 450 nm (for concentrations between 30 and 900 ng/mL). Plasma
glucose was determined by the enzymatic colorimetric method (GOD/PAP). All of the
solutions were pipetted into a cuvette and were incubated for 20 min at room temperature
(15–25 ◦ C). The absorbance of the samples and the standard absorbency was read against
the blank using a spectrophotometer (Hitachi 717® ) at 505 nm. CK levels were measured
using a Roche/Hitachi 717 Chemistry Analyzer (Roche Diagnostics, S.L., Sant Cugat del
Valles, Spain) with Boehringer Mannheim reagents.

2.5. pH Measurements
Muscle pH was measured on the carcasses from the 160 animals sampled during the
slaughter. After slaughter and dressing but before carcass cooling the initial pH of the
loin was measured (pH 45 min) using a portable pH meter (HANNA, mod. HI9125) with
temperature compensation. The electrode was inserted in the Longissimus dorsi muscle (LD)
between the 13th and 14th intercostal space, perpendicular to the midline of the left held
carcass, at an average depth of 2.5 cm. Afterward, the ultimate pH was measured from the
same animals at 24 h post-mortem at the same location on the carcass.

2.6. Enthalpy Models and Statistical Analyzes

Psychrometric graphs were obtained for each of the eight journeys using data collected
by the sensors placed at animal height inside the vehicles. The graphs were obtained based
on the ASBE model, which included temperature, relative humidity, absolute humidity
and enthalpy. The psychrometric data ASAE D271.2, defined in April 1979 and reviewed
in 2005 (ASABE 2006, ST. Joseph, MI, USA), were used to calculate the psychrometric
properties of the air surrounding the pigs. The temperature gradient was calculated
using the Savitzky–Golay algorithm for one dimension, tabulating the data. The numeric
derivatives were calculated using the Savgol routine in Matlab version 7.0 (Mathworks Inc.,
Natick, MA, USA). A polynomial routine was also used to test the neighboring data around
each point. The points were processed by replacing them with the value of the polynomial.
The derivatives were presented after programming the derivatives of the polynomials of
each point. A window of 21 points with a fifth order polynomial was used. After that, we
calculated and graphed the speed of temperature change (i.e., temperature gradient ◦ C/s)
and the apparent temperature in each case [19,20].Enthalpy (h) is a thermal comfort index
that expresses the heat amount in 1 kg dry air in kJ and is determined by the equation as
seen in Barbosa-Fihlo et al. [21].
   
RH 7.5t
H = 6.7 + 0.243t + ·10[ 237.3+t ] 4.18
100

where:
H = enthalpy (kJ/kg dry air);
t = temperature (◦ C);
RH = relative humidity (%).
Animals 2021, 11, 2410 5 of 11

All of the statistical analyses were performed using the statistical program SAS/STAT
(Statistical System Institute Inc. Cary, NC, USA. 2000). The data for temperature and
humidity were analyzed using repeated measures, while the data on cortisol, glucose
levels, and meat pH were analyzed using PROC MIXED. The experimental unit was
each journey. Averages were compared by the least significant distance, with a level of
significance of 5% (p < 0.05).

3. Results
In the eight journeys that were studied, there was no mortality or non-ambulatory ani-
mals. There were five journeys that corresponded to the winter season (January–February),
and the remaining three occurred in the summer season (June–August). Table 1 summa-
rizes the average temperatures and relative humidity for each journey. The average inside
temperature during winter journeys was 13.8 ± 3.9 ◦ C (CV = 28.3%) and was 28.9 ± 4.1 ◦ C
(CV = 14.0%) in the summer. Figure 1 shows the changes in temperature inside of the
truck over time for both the summer and winter journeys. During most journeys, the tem-
perature increased as the transport progressed, with two journeys in summer surpassing
30 ◦ C. Figure 2 shows the psychrometric graph for each journey, with more data points
for the summer journeys in the upper right quadrant (higher temperatures and higher
water content in the air). The average humidity for the winter months was 5.5 g water/kg
dry air and was 9.8 g water/kg dry air in the summer. Figure 3 summarizes the speed
of temperature change or gradient for the summer and winter journeys. The average
speed of change for the winter months was 11.8 H/min−1 and for the summer months,
it was 12.8 H/min−1 . On average, plasma cortisol levels (±SD) were significantly higher
(p < 0.05) in the winter (57.8 ± 11.7 nmol/L) than in the summer (28.8 ± 4.3 nmol/L).
Plasma glucose was also significantly higher (p < 0.05) in the winter (309.7 ± 37.9) than
in the summer (60.2 ± 26.6). CK exhibited no significant differences among the seasons
(p > 0.05) and averaged 4887.3 ± 2649.1 U/L. The pH (±SD) of the LD muscle after 45 min
was significantly lower (p < 0.05) in the winter (6.08 ± 0.24, range 5.29–6.56) compared to in
the summer (6.24 ± 0.24, range 5.33–6.8). However, the pH after 24 h was not significantly
different between the seasons (p > 0.05) and was 5.56 ± 0.13 (range 5.34–5.94).

Table 1. Average temperature (T) and relative humidity (RH) inside (pig level) and outside the
livestock vehicle during each pig transport journey from Spain to Portugal.

Journey Season Tint (◦ C) Text (◦ C) RHint (%) RHext (%)

1 Summer 20.6 24.3 66.9 52.3
2 Summer 29.2 29.3 38.2 38.8
3 Summer 29.7 32.9 34.7 37.7
4 Winter 13.7 15.1 76.9 72.5
5 Winter 16.4 15.2 56.9 57.2
6 Winter 11.6 6.8 36.4 57.5
7 Winter 12.6 9.7 52.9 56.9
8 Winter 14.6 19.8 52.6 44.7
Tint : inside temperature; Text : exterior temperature; RHint : inside relative humidity; HRext : exterior relative
humidity.
 Animals 2021, 11, x FOR PEER REVIEW 6 of

Animals
Animals 2021,2021, 11, x FOR PEER REVIEW
11, 2410 6 of611of 11

Figure 1. Evolution of the average temperatures (in °C) inside the livestock vehicle during transp
from Spain
Figure 1. Evolution to average
of the Portugaltemperatures
in (a) summer (inand
◦ C)(b) winter
inside the throughout the day
livestock vehicle beginning
during from loading
transport
Figure 1.6:00
Evolution of the average
until unloading temperatures (in °C) inside the livestock vehicle during transport
at 23:00.
fromfrom
Spain to Portugal
Spain in (a)insummer
to Portugal and and
(a) summer (b) winter throughout
(b) winter the day
throughout beginning
the day fromfrom
beginning loading at at
loading
6:00 6:00
untiluntil
unloading at 23:00.
unloading at 23:00.

Figure 2. Psychrometric graph of the average temperatures (in ◦ C) inside the livestock vehicle during
transport from Spain to Portugal in (a) summer and (b) winter throughout the day with humidity (H
Figure 2. Psychrometric graph of the average temperatures (in °C) inside the livestock vehicle d
kg water/k of dry air) plotted against temperature for each journey.
ing transport from Spain to Portugal in (a) summer and (b) winter throughout the day with hum
Figure 2.ity
Psychrometric
(H kg water/kgraph
of dryofair)
theplotted
averageagainst
temperatures (in °C)for
temperature inside
each the livestock vehicle dur-
journey.
ing transport from Spain to Portugal in (a) summer and (b) winter throughout the day with humid-
ity (H kg water/k of dry air) plotted against temperature for each journey.
Animals 2021, 11, 2410 7 of 11
Animals 2021, 11, x FOR PEER REVIEW 7 of 11

Figure 3. Speed of change or gradient in temperature (°C/s) inside the livestock vehicle during
Figure 3. Speed of change or gradient in temperature (◦ C/s) inside the livestock vehicle during transport from Spain to
transport from Spain to Portugal in (a) summer and (b) winter plotted against the average temper-
Portugal in (a) summer and (b) winter plotted against the average temperatures (◦ C).
atures (°C).

4. Discussion
4. Discussion
Since the
Since the beginning
beginning of of the
the 20th
20th century,
century, pig pig production
production has has continued
continuedtotoundergo
undergo
massive intensification and specialization in most industrialized countries,
massive intensification and specialization in most industrialized countries,leading
leadingtoto
larger and
larger and fewer
fewer farms
farms andand abattoirs
abattoirs with
with increased
increaseddistances
distancesbetween
betweenthem them[22].
[22].Long-
Long-
distance transport has been reported to be physically, metabolically, and emotionallyvery
distance transport has been reported to be physically, metabolically, and emotionally very
demandingfor
demanding foranimals
animals[23].[23].Thermal
Thermalstress
stressisisoneoneofofthethekeykey factors
factors that
that cancan exacerbate
exacerbate the
the effects of long-distance transport on pig health and welfare [24].
effects of long-distance transport on pig health and welfare [24]. In this context, the current In this context, the
current
study studythat
shows shows
winter thattransport
winter transport
is thermally is thermally
more unstable, more with
unstable,
abrupt with abruptin
changes
changes in temperature and enthalpy compared to during the
temperature and enthalpy compared to during the summer. These microclimatic conditions summer. These microcli-
maticaconditions
have clear impact have onaanimal-based
clear impact on animal-based
welfare indicators welfare
such indicators
as cortisolsuch and as cortisolas
glucose
and glucose as well as on pH after 45 min, but this difference
well as on pH after 45 min, but this difference disappears between the seasons after 24 disappears between theh.
seasons after 24 h. Overall, the results suggest that enthalpy
Overall, the results suggest that enthalpy during transport can be a useful non-invasive during transport can be a
useful non-invasive
indicator indicator of animal welfare.
of animal welfare.
Thermal stress is defined by
Thermal stress is defined by the
the inability
inability to tomaintain
maintainaaconstant
constantbodybodytemperature
temperatureby by
behavioral and physiological adaptation alone. This inability can result inheat
behavioral and physiological adaptation alone. This inability can result in heatstress
stressoror
cold stress
cold stress and,
and, in
in extreme
extreme or or prolonged
prolonged cases, cases, welfare
welfareconsequences
consequencescan canleadleadtotomulti-
multi-
organ failure and death [25]. The European Commission [16], has establishedaamaximum
organ failure and death [25]. The European Commission [16], has established maximum
temperature at
temperature at which
whichlivestock,
livestock,including
including pigs, must
pigs, be transported
must be transported on long
on journeys (i.e.,
long journeys
35 °C), but
◦ there are no accompanying limits for relative or absolute
(i.e., 35 C), but there are no accompanying limits for relative or absolute humidity. The humidity. The ther-
moneutral zonezone
thermoneutral of 100of kg
100pigs is centered
kg pigs around
is centered 20 °C20
around [26],
◦ C but
[26],the
butresults from the
the results from cur-
the
rent study
current study show
showthat temperature
that temperature variations
variations during
during transport
transport vary
varymuch
muchmore morewidely,
widely,
from about
from about 55 toto 35
35 ◦°C.
C. The
The coefficients
coefficients of of variation
variation and andgradients
gradientsin inthe
therelative
relativehumidity
humidity
and enthalpy were also high, especially in the winter, suggesting sudden shiftsininthe
and enthalpy were also high, especially in the winter, suggesting sudden shifts thether-
ther-
mal environment around the animals. The results from the physiological parametersand
mal environment around the animals. The results from the physiological parameters and
the meat
the meat quality
quality measurements
measurements suggest suggest thatthat pigs
pigs subjected
subjectedto tosharper
sharperenthalpy
enthalpygradients
gradients
had poorer
had poorer welfare.
welfare.
Animals 2021, 11, 2410 8 of 11

The enthalpy ranges reported here are similar to a previous study by the research
group that analyzed long-distance pig transport from the UK to Spain [15]. The psychro-
metric graphs underline the large differences between winter and summer journeys, with
higher temperatures and lower humidity in the former and the opposite in the winter, as
also seen in Seedorf et al. [27] and Lucas et al. [9]. Psychrometric graphs are not commonly
used to model the microenvironment around livestock during transport although they
can be used to calculate the absolute humidity and thus provide a better idea of the effort
that animals need to make to lose heat to the environment. Indeed, the THI index can be
graphed onto the psychrometric graphs themselves to designate danger zones. Using GPS
data, we could then backtrack and find where along the way the thermal microenvironment
may be more challenging on-board.
Extreme ambient temperatures during live transport are considered to be one of the
most relevant risk factors for injuries (both ambulatory and non-ambulatory) and deaths
on arrival rates, especially as transport generally occurs when microclimate conditions are
outside the ideal thermal comfort zone of the pigs [28]. However, the results presented
here suggest that even though there was no mortality among the 1920 pigs that were
transported, some winter journeys with high variation in temperature may be pushing
the coping abilities of the animals. Other authors [29] have observed marked mortality
and carcass defects in pigs transported in the winter compared to those transported in the
summer in Spain.
During long-distance transport pigs are exposed to stressful events such as a new
environments, new smells and noises, loading and unloading, mixing with unknown
animals, deprivation of food, among others factors [30]. Elevation of plasma cortisol and
glucose concentrations are considered to be more sensitive and reliable indicators to reflect
the intensity of the stress response during transport [31]. The results showed that the
animal-based measurements of the stress response, cortisol and glucose, showed significant
differences between the seasons, with the winter being more stressful than the summer.
These physiological measures validate the results concerning enthalpy rates. However,
for the plasma CK enzyme variable, no significant differences were found between the
summer and winter journeys. Although the average values we found for this enzyme
are three times higher than those reported in pigs of a similar category during journeys
of less than one hour in the Iberian Peninsula reported by Oliván et al. [32], CK is an
important biochemical marker used to measure muscle exhaustion and fatigue during
pig transport [33,34] because of the greater the amount of muscle microtrauma and the
greater passage of this enzyme to the extracellular environment [7]. It is possible that the
high CK levels found are the result of the interaction between thermal and environmental
conditions (sensory stimuli, social interactions, density), the duration of the journey, and
lairage time.
Acute thermal stress immediately before slaughter accelerates muscle glycogenolysis,
increases lactic acid concentration, and produces a rapid decrease in muscle pH early post-
mortem while the carcass is still hot [35]. In pigs, this results in pale, soft, and exudative
(PSE) meat characterized by a lower water holding capacity. In contrast, animals subjected
to chronic heat stress have reduced muscle glycogen reserves, leading to lower production
of lactic acid and dark, firm, and dry (DFD) meat characterized by high ultimate pH and
greater water holding capacity [36]. The results show that the pH after 45 min was affected
in the pigs that were transported in the winter, however the average values remained within
normal ranges. Surprisingly, at 24 h, this effect disappeared, which possibly related to the
good conditions of the vehicles and the logistics implemented for these types of journeys.
This may be due to the fact that European Legislation provides a series of guidelines that
have substantially improved animal transport in the region [18]. It is possible that the
same type of journey, without forced ventilation, could have greater heat stress effects on
product quality [37].
Animals 2021, 11, 2410 9 of 11

5. Conclusions
Our results correspond to journeys under commercial conditions in specialized trucks,
with a modern pre-slaughter logistics chain and following European regulations governing
pork transport. Under these conditions, long-distance journeys during the winter in the
Iberian Peninsula presented abrupt variations in the rates of temperature change and air
enthalpy. These abrupt changes were reflected in higher values of cortisol and glucose,
but not in CK. Muscle pH was affected at 45 min although at 24 h, these effects were not
observed. Our study has shown that mitigation strategies to avoid thermal stress should
be aimed at controlling abrupt changes in rates of temperature change and air enthalpy
during long-distance transport.

Author Contributions: Conceptualization, G.C.M.-d.l.L., M.V. and P.B.; methodology, M.V. and P.B.;
software, P.B.; validation, M.V. and R.B.-P.; formal analysis, P.B.; investigation, M.V. and N.F.-R.;
resources, M.V.; data curation, P.B.; writing—original draft preparation, G.C.M.-d.l.L.; writing—
review and editing, G.C.M.-d.l.L., M.V., R.B.-P., M.M. and N.F.-R.; visualization, G.C.M.-d.l.L.;
supervision, G.C.M.-d.l.L. and M.V.; project administration, M.V. and M.M.; funding acquisition, M.V.
and M.M. All authors have read and agreed to the published version of the manuscript.
Funding: This work was funded in part by the Madrid Community as part of the projects NEWGAN
(P2009/AGR-1704) and MEDGAN (P2013/ABI2913) and by the UK Government Department for
Environment, Food and Rural Affairs (DEFRA) (project AW0940).
Institutional Review Board Statement: Ethical review and approval were waived for this study
because this study was conducted under commercial conditions that did not involve additional
manipulations to those required by the European Union regulations for the transport and slaughter
of pigs, which are mandatory.
Informed Consent Statement: Not applicable.
Data Availability Statement: The raw data have not been published or stored elsewhere but are
available upon request.
Acknowledgments: We thank Biurrun SL for their help in completing this work as well as the
abattoir Sicasal in Portugal.
Conflicts of Interest: The funders had no role in the design of the study; in the collection, analyses,
or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
The authors declare no conflict of interest.

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