Protective Effect of Taxifolin on Cisplatin-Induced Nephrotoxicity in Rats
ThesisWAppendix
1. F A C U L T Y O F H E A L T H A N D M E D I C A L S C I E N C E S
U N I V E R S I T Y O F C O P E N H A G E N
Non-invasive biomarker measurements in cattle and mink: does
housing have an effect on hair cortisol levels?
Masters thesis
May 2015
Regitze Cecilie Charmac - student ID: lsz630
Academic advisor: Professor Christopher Harold Knight
Department of Veterinary Clinical and Animal Sciences
Faculty of Health and Medical Sciences
University of Copenhagen
2. ! 2
Title sheet
Danish thesis title: ”Non-invasive målinger af biomarkører hos kvæg og mink: har
opstaldningsmetode en indflydelse på hår-cortisol niveauer?”
English thesis title: ”Non-invasive biomarker measurements in cattle and mink: does housing have
an effect on hair cortisol levels?”
Master’s thesis (45 ECTS points) in Physiology, Department of Veterinary Clinical and Animal
Sciences, Faculty of Health and Medical Sciences, University of Copenhagen.
By Animal Science student Regitze Cecilie Charmac, student ID lsz630.
Submitted: May 2015
Academic advisor:
Professor Christopher Harold Knight
Department of Veterinary Clinical and Animal Sciences,
Grønnegårdsvej 7, 1870 Frederiksberg C,
Faculty of Health and Medical Sciences,
University of Copenhagen
________________________________________________________________
Regitze Cecilie Charmac, date
3. ! 3
Preface and acknowledgements 5
List of abbreviations 6
Abstract 7
Resumé 8
1 Introduction 9
1.1 Scope
1.2 Collaborators
2 Natural living environments 11
2.1 The natural habitat of mink
2.2 The natural habitat of cattle
2.3 Adaptations to housing systems
2.3.1 Adaptation by breeding
2.3.2 Adaptation by learning
3 Stressors in housing systems 15
3.1 Indoor housing
3.2 Outdoor housing
3.3 Shelter access
4 Stress physiology 20
5 Anatomy, structure, growth and composition of hair 23
5.1 Anatomy and structure of hair
5.2 Growth of hair
5.3 Composition and growth of mink fur
5.4 Composition and growth of cattle hair
6 Hair cortisol 26
6.1 Transfer of cortisol to hair
6.1.1 Simple Diffusion (Passive transport)
6.1.2 Complex Multi-Compartment Model
6.2 Effect of hair colour on cortisol levels
6.3 Sources of sampling varitations
6.4 Using hair cortisol as a welfare biomarker
7 Aim and experimental design 33
8 Materials and methods 34
5. ! 5
PREFACE
In the accomplishment of my Master of Science degree in Animal Science I am submitting this
master’s thesis on ”Non-invasive biomarker measurements in cattle and mink: does housing have an
effect on hair cortisol levels?” The question of whether hair cortisol can be used as a non-invasive
biomarker for the assessment of animal welfare has been studied through the sampling of hair from
mink and cattle, and the following hair cortisol analysis. The results of the analysis are then studied
by means of statistical tools, and some tentative conclusions are drawn on the basis of the results.
ACKNOWLEDGEMENTS
I would like to thank Professor Christopher Harold Knight of the University of Copenhagen,
Denmark, for providing continuous support and help throughout the project. Furthermore I thank
Professor Alberto Prandi of the University of Udine, Italy for providing me with the opportunity of
hair cortisol analysis, and Antonella Comin and Marta Montillo for performing the analysis. Thanks
to Mariann Chriél of DTU Vet and Morten Vissing of Aqua Akvarium & Dyrepark, for giving me
the opportunity to sample hair from wild- and semi naturally housed mink, and to all contributing
farmers for welcoming me to sample and examine their animals.
6. ! 6
LIST OF ABBREVIATIONS
AA Aberdeen Angus
ACTH Adrenocorticotropic hormone
ANOVA Analysis of variance
C Charolais
CNS Central nervous system
CRH Corticotropin releasing hormone
D Dexter
DHEA Dehydroepiandrosterone
H Hereford
HPA Hypothalamic-pituitary-adrenal
ILH Indoor loose housing
JC Jutland Cattle
L Limousine
LHO Loose housing with outdoor access
MSH Melanocyte stimulating hormone
RDM Red Danish Milking Breed
RIA Radioimmunoassay
S Simmental
SF Slatted floors
SH Scottish Highland
TG Tiroler Grauvieh
TMR Total mix ration
TS Tie stalls
X Crossbreeds
* Dairy breeds
7. ! 7
ABSTRACT
Cortisol is an important component of the hypothalamic-pituitary-adrenal (HPA) ”stress” axis and a
metabolic regulatory hormone. Plasma cortisol is subject to minute-to-minute variation, whereas
cortisol accumulates in hair gradually over prolonged periods. Measurement of hair cortisol may,
therefore, give a better impression of long-term stress. In this study both cattle and mink hair were
sampled to measure concentration of hair cortisol. We used the Danish national cattle registry to
contact 232 beef cattle farmers from 7 postcode areas throughout Denmark. From 35 positive
responses we were able to visit 24 farms to obtain hair samples, comprising approximately 2cm of
forelock hair taken close to the skin. Mink samples were obtained from a mink farm used by the
University of Copenhagen for research, from a wildlife park housing mink in semi-natural
conditions, and from DTU Vet, the National Veterinary Institute who provided samples from dead
wild mink. The samples comprised approximately 5*5cm of mink fur taken close to the skin.
Samples were carefully washed, extracted and analysed for cortisol following a standardized
procedure. A total of 16 breed were represented in the overall sample of 306 cattle, comprising 97
heifers, 142 cows and 71 young bulls. A total of 63 mink were represented in the overall sample,
comprising 48 female, 14 male mink and one mink of unknown gender. Data are reported as mean
± SE pg/mg. Since this was a survey and the data were not balanced, single factor ANOVAR
analyses (Minitab release 11) were used as tests for significance of various effects. The overall
mean hair cortisol value for cattle was 2,98±3,68 pg/mg and for mink it 0,78±0,64 was pg/mg. For
cattle, cortisol concentrations varied between herds (1,36±0,36 pg/mg to 4,68±0,27 pg/mg, p-value
= 0,000), between breeds (Scottish Highland having the lowest value: 1,61±0,19 pg/mg, p-value =
0,000) and with gender (F<M, p-value = 0,002). Surprisingly, pregnant cattle had lower cortisol
(2,36±0,10 pg/mg) than either lactating (2,61±0,14 pg/mg) or young females (2,79±0,12 pg/mg), p-
value = 0,017. Age was a significant factor in the full dataset, p-value = 0,000, and analysis
restricted to males (to remove the effects of physiological state) confirmed that younger animals
had higher cortisol concentration. In a subset of farms where each had some permanently housed
and others allowed access to an outdoor paddock, the latter had lower cortisol concentration (p-
value = 0,012). The majority of cows were healthy and free from clinically evident disease at the
time of sampling. There was no evident effect of recent health status as reported by the farmer (p-
value = 0,296). For mink, cortisol concentrations varied between housing (0,60±0,05 pg/mg to
0,93±0,09 pg/mg, p-value = 0,003) and between gender (0,53±0,07 pg/mg to 0,82±0,05 pg/mg, p-
value = 0,001). Housing does seem to have an effect on hair cortisol levels, but more research is
8. ! 8
needed in order to confirm, that the significant variation found was due to housing, and not other
factors such as breed or management. The data are complex and final interpretation will require
care to exclude compounding factors. Nevertheless, the data suggest that hair cortisol may be of
value in assessing stress and point to several factors that are worthy of more rigorous investigation.
RESUMÉ
Cortisol er en vigtig faktor hos hypothalamus-hypofyse-binyre (HPA) ”stress” aksen, og et
metabolisme regulerende hormon. Plasma cortisol varierer fra minut til minut, mens cortisol lagres i
hår gradvis over en længerevarende periode. Målinger af cortisol i hår kan derfor muligvis give en
bedre analyse for kronisk stress. I dette studie blev hårprøver taget fra både kvæg og mink for at
måle koncentrationen af hår cortisol. Vi brugte det danske kvægregister til at kontakte 232 avlere af
kødkvæg fra 7 postnummerområder i Danmark. Ud af 35 positive svar kunne vi besøge 24 landbrug
for at indsamle hårprøver, som bestod af ca. 2 cm hår fra kvægets forlok klippet tæt ved huden.
Minkprøver blev taget fra en minkfarm brugt af Københavns Universitet til forskning, fra en
vildtpark som holder mink i semi-naturlige omgivelser, og fra DTU Vet, det danske
veterinærinstitut, som fremskaffede prøver fra døde vilde mink. Prøverne bestod af omkring 5*5 cm
minkpels klippet tæt ved huden. Alle prøver blev vasket, ekstraheret og analyseret for cortisol ved
en standardiseret procedure. I alt 16 racer var repræsenteret i den fulde prøve af 306 kvæg, udgjort
af 97 kvier, 142 køer og 71 unge tyre. I alt 63 mink var repræsenteret i den fulde prøve af mink,
udgjort af 48 hunmink, 14 hanmink og 1 mink af ukendt køn. Data bliver præsenteret som
middelværdi ± SE pg/mg. Da dette var en undersøgelse, og data ikke var balanceret, blev single
factor ANOVAR analyser (Minitab udgave 11) brugt som test for signifikans af forskellige effekter.
Den generelle middelværdi af hår cortisol for kvæg var 2,98±3,68 pg/mg og for mink var den
0,78±0,64 pg/mg. Hos kvæg varierede cortisol koncentrationerne mellem besætninger (1,36±0,36
pg/mg to 4,68±0,27 pg/mg, p-værdi = 0,000), mellem racer breeds (Scottish Highland havde den
laveste værdi: 1,61±0,19 pg/mg, p-værdi = 0,000) og mellem racer (F<M, p-værdi = 0,002).
Overraskende nok viste det sig, at drægtige køer havde lavere cortisol (2,36±0,10 pg/mg) end både
lakterende (2,61±0,14 pg/mg) og unge hundyr females (2,79±0,12 pg/mg), p-værdi = 0,017. Alder
var en signifikant faktor i hele datasættet, p-værdi = 0,000, og analyse kun restrikteret til handyr
(for at fjerne effekter af fysiologisk status) bekræftede at unge dyr havde højere koncentrationer af
cortisol. I en andel af besætninger, hvor nogle havde dyrene permanent opstaldet, og andre havde
dyrene i løsdrift med adgang til udearealer, havde de sidstnævnte dyr den laveste koncentration af
9. ! 9
cortisol (p-værdi = 0,012). Størstedelen af køerne var raske og frie fra kliniske sygdomme ved
tidspunktet for prøvetagningen. Der var ingen evident effekt af sidst observerede helbredsstatus
reporteret af landmanden (p-værdi = 0,296). Hos mink varierede cortisol koncentrationerne mellem
opstaldning (0,60±0,05 pg/mg to 0,93±0,09 pg/mg, p-værdi = 0,003) og mellem køn (0,53±0,07
pg/mg to 0,82±0,05 pg/mg, p-værdi = 0,001). Opstaldningsmetode ser ud til, at have en effekt på
niveauet af målt hår cortisol, mens yderligere forskning skal bruges, for at bekræfte at den
signifikante forskel er grundet opstaldningsmetode, og ikke andre faktorer såsom race eller
management. Dataene er komplekse og en endelig fortolkning kræver opmærksomhed for at
eliminere forstyrrende faktorer. Under alle omstændigheder ser det ud som om, at hår cortisol kan
være af værdi til at vurdere stress, og flere faktorer der kræver nærmere undersøgelse er
identificeret.
1 INTRODUCTION
The interest in animal welfare in today’s modern society is growing. It is no longer enough that the
animals produce meat, milk, eggs or fur, now they must do so while having an adequate amount of
animal welfare. According to Miele (2011), in the last two decades animal welfare has become an
important issue for the European public. There have been several outbreaks of severe diseases on
animal farms such as Bovine Spongiform Encephalopathy and Avian Influenza, and these outbreaks
have been contributing factors to the growing concern for animal welfare (Miele, 2011). They also
gave rise to the Welfare Quality Project funded by the European Union, aiming to explore how best
to assess and improve animal welfare on both farms as well as slaughter plants (Miele, 2011).
Another study by Loveridge (2013) found that, increasingly, government organizations as well as
animal welfare activists use animal welfare standards as an important argument, when discussing
how to secure overseas markets and when responding to local concerns. Loveridge (2013)
conducted an experiment, in which differences in perceptions of animal welfare on farms were
measured. The general public was asked to complete questionnaires, both in 1994 and in 2008, and
the differences in results were examined. Loveridge (2013) found, that the prior conception that
animal welfare only based on physical wellbeing, is now being challenged as more attention is
given to behavioural restriction. Tail docking of pigs and other standard farm practices, were not
given much thought in 1994, but in 2008 these practices are increasingly thought of as methods
compromising animal welfare (Loveridge, 2013). As such we see a growing tendency of people
giving animal welfare more thought and importance. One of the major factors when considering
10. ! 10
animal welfare is thought to be The Five Freedoms. The Five Freedoms describe five guidelines for
the keeping of animals which should be fulfilled, in order to provide said animals with adequate
welfare. The Five Freedoms are 1) Freedom from hunger and thirst, 2) Freedom from discomfort, 3)
Freedom from pain, injury and disease, 4) Freedom to behave normally and finally 5) Freedom
from fear and distress (RSPCA, 2013). As such, housing systems for production animals need to
take The Five Freedoms into account, since they are designed to provide the animals with the least
stressful environment possible.
One way of assessing whether an animal is suffering from stress, is by monitoring cortisol levels in
the animal. By measuring cortisol, any changes in the activity of the hypothalamic-pituitary-adrenal
(HPA) axis can be detected, which regulates energy balance, reproduction, immune responses and
is activated during stress (Minton, 1994). Cortisol can be measured in many different ways, and a
lot of research has been done on detecting cortisol and its metabolites in blood, faeces, urine and
saliva (Bayazit, 2009; Minton, 1994; Möstl et al., 2002; Wernicki et al., 2006). Many of these
measures require an invase approach in order to secure a sample. The metabolic pathway for
cortisol to blood, faeces, urine and salive is also rather short, and so factors such as handling during
sampling time could influence the measured cortisol levels in the sample.
In this study hair was used as a measure for cortisol in the animals. Using hair for cortisol
measurements is a non-invasive approach, and cortisol accumulates in hair over a much longer
time, than diffusion of cortisol into blood for example. This gives us the advantage, that the
sampling procedure in itself may be less stressful for the animals, as it is non-invasive, and should
the animal experience any stress during sampling, the cortisol produced will not accumulate in the
hair during the time of sampling. As such, measuring cortisol in hair has the potential to provide us
with a method that is non-invasive, and enables us to evaluate whether the animal is suffering from
chronic stress, as one sample of hair will reflect cortisol levels over a long period of time. It is
believed that hair cortisol measurements might be used for dairy cattle herds in the future, as the
automated milking systems could be programmed to take a hair sample during milking, and so the
sample could be used to evaluate the welfare of individual animals and the herd in general.
As measurements of cortisol concentration in hair have benefits in terms of being non-invasive as
well as accumulating over long periods of time, this makes the method especially useful when
11. ! 11
wishing to evaluate housing systems. Since stress during handling and sampling will not have time
to accumulate in hair, before the hair is sampled, the measured concentration of cortisol in hair will
reflect chronic stress in the animals. In this study we will attempt to measure the welfare of the
animals housed in different housing systems, in terms of whether the animals seem to be suffering
from chronic stress or not, based on the hair cortisol concentrations measured.
1.1 SCOPE
This report will address key themes in terms of using non-invasive biomarker measurements of
cortisol as a welfare assessment. It will adress adaptations to housing systems, potential stressors in
housing systems, general principles of stress physiology and the use of biomarkers for welfare
assessments.
1.2 COLLABORATORS
Collaborators to this project are Professor Christopher Harold Knight of the University of
Copenhagen, Denmark, who has acted as project supervisor and mentor. Professor Alberto Prandi
of the University of Udine, Italy, has been in charge of radioimmunoassay sample analysis, and
Antonella Comin and Marta Montillo has performed said analysis. Mariann Chriél of DTU VET
provided me with hair samples from wild mink. Zoologist Michael Vissing of Aqua Akvarium &
Dyrepark supplied the samples from the mink kept in semi natural conditions.
2 NATURAL LIVING ENVIRONMENTS
2.1 THE NATURAL HABITAT OF MINK
The mink is a small mammal from the weasel family Mustelidae. There are various breeds of mink
but the one found in Denmark, both in the wild and on fur farms is the American mink, the
Neovison vison. The American mink originates from North America (British Wildlife Centre, 2012)
but was introduced to Denmark as a production animal for fur farming. There is a wild population
of mink in Denmark due to escapes from fur farms, and thus it is an invasive species.
In nature the habitat of the mink is mostly associated with aquatic areas such as swamps, rivers,
streams, ponds and salt water marshes (Butfiloski & Baker, 2005). Research by Bodey et al., 2010
showed that the preferred habitat was coastal. They are carnivorous animals and up to 40% of their
12. ! 12
diet consists of fish (Baker, 1999), which explains why the habitat of the mink is often found to be
near water. However their diet is much dependent on the availability of prey, and so other types of
food such as small rodents, rabbits, muskrats, squirrels, crabs, crayfish, insects, snails, frogs, snakes
and waterfowl have also been found to be part of the mink diet (Baker, 1999). Mink live in dens
which can be located in rock piles, tree roots, bridge crossings and stream bank holes (Butfiloski &
Baker, 2005), and they may also utilize the den of another type of animal, such as an abandoned
beaver den or muskrat bank dens (Schuh, 1997). Mink are known to use their dens to stockpile food
and thus often kill more prey than needed in order to store the food for later use (Schuh, 1997).
Mink are very territorial animals as both sexes keep hunting territories marked with odour from
their anal glands. They are mostly solitary animals, as they only associate with other mink during
breeding season or rivalry fights over territories. Especially male mink often fight with rival males,
and more mink are killed by other mink than by other predators (Schuh, 1997).
2.2 THE NATURAL HABITAT OF CATTLE
The domesticated cattle as we know it, the Bos Taurus, originates from the aurochs, the Bos
Primigenius. The natural habitat of the aurochs is uncertain, but it seems that the animals preferred
swamps, swamp forests, river valleys, river deltas and bogs (Tikhonov, 2008). However the aurochs
was most likely also found in drier forests and maybe even open parkland (Maas, 2014). In Europe,
the European Bison, Bison Bonasus, is primarily thought to have lived in dry forests, whilst the
aurochs in Europe lived in wetter areas (Maas, 2014). The two subspecies however are thought to
have had overlapping habitats as well (Van Vuure, 2002).
Cattle have adapted to their regional environment for example by the process of spring calving and
mating in the early summer. Behavioural patterns such as seeking shade, panting, sweating and
vasodilation has enabled them to adapt to changes in temperature in their living environment as
well. Their natural diet consists of grasses, shrubs, young trees and other types of vegetation, and
cattle move to another area when they have consumed all feed available in one area. Cattle are
social animals that live in herds, with a social ranking system and one dominant bull (Hindshaw,
1993).
As both mink and cattle have since been domesticated, both species have had to adapt to modern
13. ! 13
production systems. In the following section, some methods deployed to adapt animals to housing
systems are described.
!
2.3 ADAPTATIONS TO HOUSING SYSTEMS
Animals in modern farms have to adapt to housing systems, which may not necessarily reflect their
natural environment. Furthermore, on the farm, animals may have to go through various
environments as they move through different phases of the production system. This could include
introductions to new housing systems, new types of feed, housing with unfamiliar conspecifics and
new human handlers (Wechsler & Lea, 2007). How animals cope with the housing systems is most
likely very important for both animal welfare and animal performance (Wechsler & Lea, 2007).
2.3.1 ADAPTATION BY BREEDING
According to D’Eath et al. (2010), breeding goals for farm animals mostly include goals such as
health and functional traits, which has the potential to improve animal production and welfare.
However, behavioural traits are rarely part of the breeding goals, despite also having the potential to
improve production, product quality, reducing labour costs and improving handler safety (Jones &
Hocking, 1999; Boissy et al., 2005; Grandinson, 2005; Turner & Lawrence, 2007; Macfarlane et al.,
2010). One reason to why behavioural traits are less desired as breeding goals, is that they are more
difficult and time-consuming to select for, as lots of animals must be identified in a consistent and
reliable manner, in order to have a population of animals for the breeding programme (D’Eath et al.,
2010). Behavioural traits are still included in some breeding programmes however. For example,
some beef cattle breeding programmes include the behavioural trait ”ease of handling,” (D’Eath et
al., 2010).
Some ethical considerations however, have also been raised over the topic of including behavioural
traits in breeding programmes. In this process animals are being adapted to the environment or
housing system, instead of the other way around. Discussions have arisen on whether this might
compromise the naturalness of the animals, and maybe even the integrity of the animals. In some
cases behavioural traits included in the breeding programme could enhance naturalness, such as
breeding for good maternal behaviour, but in other cases, a trait which could improve animal
welfare, could at the same time compromise the naturalness (D’Eath et al., 2010). Other issues are
that when selecting against certain behaviours, we are at risk that a situation could arise, in which
14. ! 14
the animals bred are generally unreactive, although this could be controlled and measured. Yet
another potential ethical issue is that if we breed against behavioural measures of welfare, it could
results in animals which do not show signs of low animal welfare, even if they are suffering from
this (D’Eath et al., 2010).
2.3.2 ADAPTATION BY LEARNING
A farm animal needs to learn about its surrounding environment, and there a different factors which
require this, such as learning about sites for feeding, drinking, resting etc. Other factors include
learning about what do eat, housing equipment, characteristics of group members and
characteristics of humans (Wechsler & Lea, 2007). It is generally believed, that animals which can
behave naturally and satisfy their behavioural needs, are animals with high animal welfare. This
natural behaviour is defined as the way evolution has shaped the natural behaviour of the animals,
both in terms of evolutionary adaptation, but also in terms of behaviour changes due to learning
(Fraser et al., 1997, Wechsler & Lea, 2007).
Animals learn from interactions with humans, and most likely also from other cues they come into
contact with on a farm, such as noises, smells and management routines. All these aspects will
influence the behaviour of the animal (Wechsler & Lea, 2007). In a study on laying hens, Dawkins
(1977) found that during a preference test, animals that were used to being housed in battery cages,
preferred the cages over outdoor housing. This gradually changed over time, but hens which were
already familiar with the outdoor housing system would prefer this from the start. This indicates
that animals adapt to their environment over time, by learning about the cues of the environment.
Another aspect of learning is social learning, which occurs in farm animals in a variety of
situations. Nicol (1995) suggested using social learning to make new animals adapt to a new
environment. It was suggested that new animals should be housed with a few animals which had
already habituated to the environment, which would then facilitate habituation by the new group of
animals as well (Nicol, 1995). In general, knowledge of species-specific learning could help in
designing housing systems and mangement routines which allow animals to adapt to the
environment more smoothly (Wechsler & Lea, 2007).
Ultimately, potential stressors will be present in all housing systems, and these could compromise
animal welfare. In the next section, some of these stressors will be explored.
15. ! 15
3 STRESSORS IN HOUSING SYSTEMS
When keeping animals in housing systems, the animals are sometimes kept in an environment that
does not resemble the natural habitat of the species. Production animals have adapted to the various
housing systems, for example by means of selection through breeding. The fact remains that some
factors in the housing systems might still be stressful for the animals; we call these housing system
stressors.
3.1 INDOOR HOUSING
One stressor in indoor housing systems in relation to cattle is lameness, as the pain from being lame
will stress the animal. Lameness has negative effects on both resting, walking and feeding
behaviour (Cook & Nordlund, 2009). Lameness is regarded as a major welfare problem, especially
for dairy cows (von Keyserlingk et al., 2009), but beef cattle housed indoors may suffer from the
same complications. Lameness can be caused by different factors such as dermatitis, foot rot and
other diseases, but can also arise from the type of housing. In housing systems the factors
contributing to lameness in cattle is the use of concrete floors, zero grazing and uncomfortable
stalls, and these factors also explain why there are large differences in lameness problems in
between farms, as different farms have different management plans (Cook & Nordlund, 2009).
Concrete flooring is not ideal for cattle to walk on, and is only made worse if the concrete is
covered by manure (Phillips & Morris, 2000).
Cook & Nordlund (2009) conducted a study in which they examined the influence of the
environment on dairy cow lameness, amongst other things. They found that free stall housing shows
the highest rate of lameness amongst dairy cows, mainly because the animals are much more
exposed to concrete walkways and manure whenever they are not resting. Factors influencing the
time the animals spent resting were poor stall designs with obstructions to normal stall use,
overstocking, behavioural changes during the transition period, heat stress, prolonged milking times
and management tasks that keep cows away from stalls (Cook & Nordlund, 2009). Almost all cattle
sampled in this study were beef cattle, but some of these factors still apply to the housing systems
used for beef cattle. Some animals observed during the sampling period for the present study, were
stabled in housing systems of poor design, such as one barn in which the animals had to climb a
steep step, in order to be able to reach the feeding table. This design could give rise to lameness just
from making the animals climb the step and potentially getting hurt. It could also lower the time
16. ! 16
spent resting, as once the animals have climbed this step, they are less likely to climb back down to
rest, knowing that they would then have to re-climb the step again later. As such, instead of resting,
the animals might spend a lot of time standing by the feeding table, which could result in lameness
if the concrete area is not adequately covered with straw, or contaminated with manure. In general,
the problem with lameness in cattle is directly correlated with the sum of total standing time per
day, and the type of flooring the cattle are standing on when not resting (Cook & Nordlund, 2009).
Overstocking is mentioned by Cook & Nordlund (2009) as also being a cause for lameness in cattle.
However, there are other problems associated with the overstocking of animals, one of these being
individual animals subjected to stress because of hierarchy fights. Huzzey et al. (2006) conducted a
study in which they examined the effect of stocking density and feed barrier design on the
behaviour of dairy cattle. It was found, that when the stocking density of the cattle was increased,
more cows were displaced from the feeding table. A direct association to the hierarchy of the
animals was also found, as lower-ranking cattle were displaced from the feeding table more often
than higher-ranking cattle, especially in situations of overstocking. The use of a headlock feed
barrier, which provides some physical separation between the animals, proved to lessen the
competition at the feeding table. Thus, the conclusion of the study recommended avoiding
overstocking at the feeding table (Huzzey et al., 2006). Overstocking is not only an issue when it
comes to feeding situations however, as it can be a problem in hierarchy fights among the animals
as well. When housing cattle indoors at a limited amount of space, it can prove difficult for lower-
ranking cows to move away, when approached by higher-ranking cows. This can give rise to
conflicts and fights over hierarchy, which in turn can cause injuries to the animals involved. This
problem will be even more pronounced, if the stable in question is also overstocked.
According to Dawkins (1988), one of the reasons why animals are sometimes showing signs of
reduced welfare in captivity, is if the animal is unable to perform a specific behaviour which the
animal is strongly motivated for. This inability can be caused either by physical restraints in the
housing system, or by the lack of suitable stimuli (Dawkins, 1988). Therefore it is common to
provide production animals with enrichments in their housing systems in order to improve their
welfare (Vinke et al., 2004). Enriching the environment of the captive animals have shown to have
positive effects on both behavioural variability and social behaviour, and could also positively
influence physiology, neuroendocrinology and cognition (Torasdotter et al., 1998; van den Berg et
al., 1999; Pham et al., 1999; Varty et al., 2000; Woodcock and Richardson, 2000; Williams et al.,
17. ! 17
2001; Larsson et al., 2002). Larsson et al., (2002) furthermore suggests that environmental
enrichment might result in better abilities to cope with stress.
Farmed mink may also be subject to environmental stressors. Vinke et al. (2004) conducted a study
in which mink were placed in normal and enriched cages. The enrichments that were chosen in the
study made by Vinke et al. (2004) were chosen to allow the mink to perform behaviours they are
seen performing in their natural habitat. These behaviours include running, chewing, swimming,
grooming, climbing, playing, hunting, hiding and sleeping (Dunstone, 1993). The results found that
the juvenile mink housed in the most enriched housing system did have the most variable display of
behaviour, as expected, which suggests better coping with the housing system. However a
significant difference in anticipatory behaviour in the different housing systems was also expected,
as Van der Harst et al., (2003) found that, in rats, there was a lower sensitivity to a sucrose-reward,
when rats were placed in enriched cages. In this study, no significant difference in anticipatory
behaviour was found, and a possible explanation could be that the three housing systems did not
differ enough. As such, no significant long-term effects of the enrichments were found, when
measuring anticipatory behaviour and stereotypic behaviour, and this could indicate that, in terms of
stress, there was no difference in the housing systems.
Cage size is an element of mink welfare which has been explored a lot. Some studies, such as Vinke
et al. (2002), has found that bigger cages result in better welfare. However, other studies, such as
the one made by Hansen & Damgaard (1991) found no difference in animal welfare when studying
different cage sizes. This indicates that merely increasing the cage size slightly does not make a
difference for the animals, which could mean that if cage size is used to improve animal welfare,
the increase in size must be quite extensive, and other factors such as environmental enrichments
must be considered too.
3.2 OUTDOOR HOUSING
As cattle are grazers it is often believed that allowing them outdoor access will lead to higher
welfare standards, as behaviours such as grazing and exploration can be achieved (Hemsworth et
al., 1995). However, we cannot know which type of environment the animals actually prefer, but
one idea is to give the animals the choice and observe which type seems to be preferred. There are
some issues with this method, however. One problem is that this method does not tell us how good
or bad one housing system is, it only tells us which one the animals prefer – both systems could be
18. ! 18
good with one being better than the other and, equally, both systems could be bad. Furthermore the
animals could make bad decisions, meaning that their system of choice might not necessarily be the
best choice on a long-term basis (von Keyserlingk et al., 2009).
Legrand et al. (2009) conducted a study in which cows were given the choice of either a well-
designed and managed free-stall barn or an outdoor pasture area. In this study the cows chose the
pasture, but only during night time, and thus preferred to stay in the free-stall barn during the
daylight hours, especially during hot temperatures. Schütz et al. (2009) found that the use of shade
by cattle is positively correlated with solar radiation, meaning that in the case of the study made by
Legrand et al. (2009), the choices of the animals were complex, as factors such as solar radiation
determined whether one option or the other was chosen. It was also found that the cows returned to
the barn to access the TMR (total mix ration) (Legrand et al., 2009). Trials such as these are useful
when trying to determine which type of housing the animals prefer, however more research is
needed. In this case it would be relevant to explore whether the animals would prefer to stay
outdoors on pasture, if shade was available, and if the TMR could also be accessed outdoors.
In general, when housing animals outside, temperature is the probably the greatest potential
stressor. When housing animals indoors, both cattle and mink, the animals can be protected from
environmental extremes such as heat, cold and wet (von Keyserlingk et al., 2009). In regards to
cattle, research has shown that when the climatic conditions change, cattle will change locations in
response to this (Redbo et al., 2001).
Mink are not housed outdoors as such, but in cages in either closed or open barns. However, a wild
population of mink is present in Denmark, with many of these animals having escaped from fur
farms, and some being the result of a breeding wild population. A mink living in the wild after
having lived on a fur farm will be subjected to many stressors. The environment inside the cage
system is relatively barren, so a change of environment into the wild will be stressful, as the animals
will be exposed to many novel objects. Malmkvist & Hansen (2002) explored fearful behaviour in
mink from two breeding lines; both lines were selected over 10 generations, one line selected for
confident reaction towards humans, another selected for fearful reaction towards humans. Through
six different tests the fearful reaction was monitored. When being tested with a novel object, the
confident mink approached, made contact with and manipulated the object sooner than the fearful
mink. The same tendency was observed when the mink were subjected to unfamiliar mink and
19. ! 19
unfamiliar food. Both types of mink showed signs of fearful behaviour, but the confident line was
able to overcome this quicker than the fearful genetic line. Depending on whether the mink
escaping into nature are of a confident or fearful nature, being subjected to various novel objects
will have different stressful impacts.
3.3 SHELTER ACCESS
In this section the presence or lack of a shelter in housing systems for both cattle and mink will be
explored. This is done in order to establish whether or not providing the animals with shelter could
potentially lead to stressful living conditions.
Olson & Wallander (2002) conducted a study in which they tried to establish whether access to
windbreak shelters for grazing cattle during winter altered the diurnal patterns of the animals. The
cattle were grazing on an exposed pasture, and were observed during two winters. They found that
the time the cattle spent in the shelter ranged between 0% and 30%, and that the animals did not
start using the shelter until the 16th day of the trials. Only a subtle difference in activity patterns
were observed between cattle with shelter access and cattle without shelter access, which indicates
that similar behaviours were used amongst the animals to minimize energy expenditure and
maximize energy gain (Olson & Wallander, 2002). The time spent grazing and standing differed on
a day-to-day basis, which might be explained by the different types of weather and which
behaviours the cattle chose to cope with it. Interestingly, during extreme cold the cattle were not
seen lying down in order to minimize energy expenditure, but rather they were observed standing
up, in order to maximize heat gain from solar radiation. Olson & Wallander (2002) concluded that
cattle behaviour during winter is a balance between maximising energy gain (thermal and food) and
minimising energy loss (thermal and metabolic). They found no significant change in the diurnal
patterns of animals provided with shelter access.
To a certain extent cattle may be able to adapt to various different climatic conditions, especially
when these change gradually. However, according to Van Laer et al. (2014) the substantial negative
effects of hot as well as cold conditions on cattle, such as high temperatures, high humidity, intense
solar radiation and low temperatures combined with precipitation and wind will lessen the welfare
and performance of the animals. The thermo tolerance of the animals may differ greatly between
individuals, and factors such as breed, age, productivity, body condition and coat condition must be
taken into consideration. As several of these factors can vary within the same herd, all animals in
20. ! 20
one herd may not be affected by the weather in the same way (Van Laer et al., 2014). Van Laer et
al., 2014 state, that currently the knowledge on the effect of adverse weather on pastured cattle in
temperate climates is quite limited, but suggests that allowing cattle access to a shelter will benefit
their welfare.
For mink, a permanent nest box with straw bedding is present in all cages used as housing systems
designed according to the new system (Vinke et al., 2002). Several studies have been done in order
to examine the importance of having a nest box present in the cages; Hansen et al. (1994) found that
nest boxes reduces the occurance of stereotypic behaviour, Møller (1990) stated that nest boxes
equipped with straw bedding increases mink kit survival rates, and de Jonge & Leipoldt (1994)
found that straw stimulates manipulation behaviour which results in improved pelt quality. Cooper
& Mason (2000) conducted a study in which they examined if mink were willing to work for visits
to the hay box (a nest box with straw bedding). They found that mink were indeed willing to work
for this, indicating that straw as bedding is an incentive. A study by Hansen & Damgaard (1991)
examined the effect of cage size and nest box equipment on plasma cortisol, number of eosinophil
leucocytes and on frequency of leucocyte groups. Three different cage sizes were used in the
experiment, as well as cages with and without nest boxes. Immobilization in a mink trap and its
effect on the same biomarkers was also examined. Hansen & Damgaard (1991) found that the mink
kept in cages without nest boxes had an increased level of physiological stress, shown by higher
levels of plasma cortisol. The differences between the physiological parameters of mink kept in
cages with nestboxes, and mink kept in cages without nest boxes were significant. They also found
a positive correlation between keeping mink in cages without nest boxes and immobilization of
mink in a trap; the physiological effect of immobilizing mink 30 minutes a day in a trap was similar
to keeping mink in a cage without a nest box.
The potential stressors in housing systems can give rise to physiological reactions in response to the
stressors. The following section will describe the basics of stress physiology in animals.
4 STRESS PHYSIOLOGY
The hypothalamic-adenal medullary system is made up by the hypothalamus, the pituitary gland,
the sympathetic neural pathways to the adrenal medulla, and the release of epinephrine by the
adrenal gland. This is a short acting stress response, and was originially referred to as the fight-or-
flight syndrome (von Borell, 2001). The longer-term and sustained response to stressors is the
21. ! 21
hypothalamic-pituitary-adrenocortical (HPA) stress response system. Corticosteriods and
aldesterone are the major adrenal cortical hormones (von Borell, 2001).
Three phases of the stress response were described by Selye (1946) as being first alarm, followed
by resistance by the release of corticosteroids, which would then lead to exhaustion of the response
system or even death if unsuccessful. Mason (1971) questioned the theory of nonspecificity of
stressors and concluded, that a stressor may not necessarily activate the HPA system, as it depends
on how the animal responds to the stressor; if the animal does not perceive the stressor as stressful,
then there may be no response at all from the HPA system. In relation to animal welfare, stress
could refer to a state where the animal is unable to adapt to its environment, because it is being
challenged beyond its behavioural and physiological capacities (Terlouw et al., 1997).
In a stressful situation an animal will react with various biological mechanisms in response to the
stressor (von Borell, 2001). As stressful situations can be potentially harmful to the animal, the
body responds by activating neurophysiological mechanisms to resist and prevent major damage
(Ewing et al., 1999). Various sensory detectors receive information about the threat at hand, and
transform the information into neural signals. These signals are then transmitted to cognitive and/or
non-cognitive centers of the nervous system, in order to make a coordinated response to the stressor
(von Borrell, 2001). Interactions between the central nervous system (CNS), endocrine system and
immune system influence how the animal reacts, as they respond to stressful stimuli in a
coordinated manner (see figure 4.1, van Borell, 2001).
Figure 4.1: Interactions between CNS, endocrine system and immune system (van Borell, 2001).
22. ! 22
A hypothalamic response in response to stress is to release corticotropin releasing hormone (CRH).
CRH will then stimulate the anterior pituitary into releasing adrenocorticotropic hormone (ACTH)
and subsequently release glucocorticoids from the adrenal gland. This effect is a result of
communication between the hypothalamus and the pituitary gland (Bale et al., 2000).
Cortisol is the main stress indicator in this project, and is derived from cholesterol as is every other
steroid hormone. The main purpose of cortisol is to control the metabolism of glucose through the
process of gluconeogenesis, but cortisol also facilitates the body’s response to stress as well as
regulating the immune system. Cortisol regulates the immune system by inhibiting the production
of leukotrienes and prostaglandins, thereby having anti-inflammatory effects as both of these
substances are highly involved with inflammation. Cortisol also has anti-immune effects as it
inhibits the growth of some immune cells, and cortisol can thereby stop the immune system from
overreacting to minor infections (Widmaier et al., 2011).
When cortisol is produced by the body due to stress multiple effects will take place. Cortisol has
several effects on the organic metabolism such as stimulation of protein catabolism in bone, lymph
and muscle, stimulation of liver uptake of amino acids as well as gluconeogenesis, maintenance of
plasma glucose levels and stimulation of triglyceride catabolism in adipose tissue, with the purpose
of releasing glycerol and fatty acids into the bloodstream. All these effects on the organic
metabolism are found, because cortisol acts to mobilize the energy sources of the body by
increasing the plasma concentrations of amino acids, glucose, glycerol and free fatty acids. When
an animal faces a situation which is stressful, and more cortisol is produced, these effects are very
useful for the animal, because the increase of plasma concentrations of amino acids, glucose,
glycerol and free fatty acids will enable the animal to stay alive even without eating. Fasting is
usually costomary behaviour in stressful situations, and so these effects are imperative for the
survival of the animal. The amino acids produced from the protein catabolism in bone, lymph and
muscle will not only provide nutritional advantages by supplying glucose via hepatic
gluconeogenesis, but can also help with tissue repair, which could be an issue in stressful situations
(Widmaier et al., 2011).
The adrenal cortex secretes five major hormones; aldosterone, corticosterone,
dehydroepiandrosterone (DHEA), androstenedione and cortisol, which I will be focusing on. The
23. ! 23
adrenal cortex is composed of the zona glomerulosa, which are the cells of the outer layer that
contains enzymes which convert corticosterone to aldosterone. The zona fasciculata and the zona
reticularis in contrast produce cortisol and androgens. The zona fasciculata is found to primarily
produce cortisol in humans whereas the zona reticularis mainly produce androgens, but it is possible
for both zones to produce both steroids (Widmaier et al., 2011).
Cortisol is transported by plasma because of its lipid nature. As such, after formation cortisol will
not be stored in the cytosol, because the lipophilic properties of cortisol will allow it to diffuse over
the lipid bilayer of the cell membrane. Cortisol will diffuse into the interstitial fluid, and then enter
the circulation system, where a carrier protein such as albumin will transport it (Widmaier et al.,
2011).
5 ANATOMY, STRUCTURE, COMPOSITION AND GROWTH OF HAIR
5.1 ANATOMY AND STRUCTURE OF HAIR
When looking at hair with the naked eye each strand looks like a fairly simple structure. However,
this is not the case, as the structure of each hair is quite complex. Each strand grows from a folicle,
which is a sac-like organ. From this follicle compacted cells make up cylindrical shafts, and these
shafts compose the strand of hair. The toughness of hair is caused by the sulfur-rich protein keratin
(Harkey, 1992). The process in which the hair is hardened is called keratinization, in which the hair
is hardened and solidified first by syntheetization of melanin, and then later by enrichment in these
sulfur-rich keratins (Boumba et al., 2006).
Each strand of hair is made up of an outer cuticle and a central cortex, and the central cortex may
also contain a central medulla. These contain three different types of cells; cuticular cells, cortical
cells and medullar cells. The cuticle protects the interior fibers of the hair, and also makes sure the
hair shaft stays attached to the follicle. Both chemicals, heat, light or injury can cause trauma to the
cuticle, which may cause it to fall apart (Harkey, 1992). The central cortex contains the cortical
cells. Cortical cells contain pigment granules which determine the colour of the hair, depending on
the type of pigment and alignment (Harkey, 1992). Every colour of hair is made by different types
of pigments produced in different amounts; the most principal pigment of hair is melanin, which is
synthesized in the hair bulb from the amino acid tyrosine (Harkey, 1992). A central medulla is only
found in certain types of animal hair, and therefore medullar cells are also restricted to only some
24. ! 24
kinds. In fine animal hair only the cuticle and central cortex are present, whilst thick hair – namely
horse hair – also contains a central medulla and medullar cells. Medullar cells increase as the fiber
diameter of the hair strand increases (Harkey, 1992).
5.2 GROWTH OF HAIR
Hair grows in three phases; the anagen phase, the catagen phase and the telogen phase. Hair also
does not grow continually, but has both periods of growth and quiescence (Harkey, 1992). During
the anagen phase cells divide to form the new strand of hair as they form a filament which pushes
through the follicle. Once the hair has pushed through, the cells transform into cuticle, central
cortex and a central medulla, if the new hair is of a thick diameter (Harkey, 1992). This also onsets
the process of keratinization in which the hair is hardened and thoughened. Trials with laboratory
animals have found, that if radioactive compounds are given to the animals, when the anagen phase
is happening, radioactivity is found in the keratogenous zone, but almost nothing is found in the
follicle. On the other hand radiolabeled glucose also adminstered to the animal at this time is not
found in the keratogenous zone, but in the follicle. This indicates that nutrients from the
surrounding vessels are incorporated into the follicle, whereas chemicals such as radioactive
compounds are absorbed by the keratogenous zone, which in turn means that all substances present
in capillaries and the surrounding tissues, lymph and intracellular fluids might be found in the
completed hair strand (Harkey, 1992). During the anagen phase, human hair will grow about 1 cm
every 28 days. In the catagen phase the growth of hair stops temporarily whilst the hair shaft is
keratinized and the outer root attaches to the root of the hair. In this process the so-called ”club
hair” is made (Harkey, 1992). The telogen phase is a period in which the growth stops completely
and the club hair formation is finalized. The hair can be easily removed when pulling during this
phase, which will reveal a solid white material at the root. The length of the telogen phase increases
with age, and differs for different types of body hair (Harkey, 1992).
5.3 COMPOSITION AND GROWTH PATTERN OF MINK FUR
The moulting pattern of the mink is decribed by Bassett & Llewellyn (1949) in twenty different
stages. The mink has two different types of coats – the summer coat and the winter coat. The
summer coat colour is lighter and less intense, whereas the fur density is less great than in the
winter coat (Bassett & Llewellyn, 1949). The spring molt begins around mid-April, and the growth
of the summer coat, which begins at the nasal area of the animal, is almost completed in July. This
25. ! 25
coat is only kept for about three weeks until the winter coat starts developing, with the onset of a
dusty appearance to the entire coat, and the summer coat shedding begins. The winter coat starts
developing from the tail region of the mink, and then gradually spreads to the rest of the body, and
the winter coat is fully developed around November (Bassett & Llewellyn, 1949).
Figure 5.3: Stages in the moulting pattern of the adult mink (Bassett & Llewellyn, 1949).
Mink fur is composed by underfur and guard hair (Bassett & Llewellyn, 1949). The underfur is the
hairs found closest to the skin of the animal, at the base of the fur. The underfur plays a great role in
thermoregulation, as its principal function is to maintain normal body temperature. The guard hairs
are the longer hairs, which cover the underfur, and also protect the underfur and skin of the animal.
When winter progresses the pigment in the guard hairs, melanin, loses its depth and the entire coat
will fade in colour (Bassett & Llewellyn, 1949). This is why mink pelts are taken just after the
winter coat has developed, as high-quality pelts should have lush, thick underfur and equal, lustrous
guard hair (Kopenhagen Fur, 2014).
5.4 COMPOSITION AND GROWTH PATTERN OF CATTLE HAIR
Cattle hair differs from breed to breed. Scottish Highland cattle as well as Belted Galloway cattle
have double coats, with underfur helping to maintain good isolation in cold weather, and guard
hairs protecting the underfur and skin of the animals. All types of cattle undergo seasonal moulting
of their coats which is regulated photoperiodically (Yeates, 1958).
A study found that the cattle coat reached maximum weight in January, and then decreased to 50%
of the maximum weight during April and May. During May and June the coats increased in weight
26. ! 26
again, reaching 75% of the maximum weight. Then growth continued on from October until
reaching its maximum weight in January (Berman & Volcani, 1961). The hair fibre diameter was at
its lowest from December until March, and then increased drastically until June (Berman &
Volcani, 1961). This indicates that the winter coat in cattle is shed during April and May, where the
summer coat will then be complete. The summer coat will not remain stagnant for long however,
before the growth of the winter coat sets in, which is then complete in January. Day lenght is not
necessarily the only factor influencing the growth cycle of cattle hair however, as Berman &
Volcani (1961) found that the air temperature also has an influence, although hair diameter seemed
to be only influenced be variations in day lenght. Yeates (1958) also found that nutrition has an
influence on coat shedding in cattle, as low nutrition impeded seasonal shedding, however some
individuals among a breed seemed less affected than others.
6 HAIR CORTISOL
6.1 TRANSFER OF CORTISOL TO HAIR
The way in which cortisol is transferred into hair is widely discussed with many different theories
presented (Russell et al., 2012). Figure 3.2 shows several of the processes which might be involved
with the transfer of cortisol to hair. In this figure both transfer via blood, sebum and sweat is
proposed. Various glands empty their ducts into hair follicles, such as sebaceous and apocrine
glands, whilst the eccrine sweat glands do not empty their ducts into hair follicles despite of being
located near the follicle (Boumba et al., 2006). Figure 3.2 shows how cortisol present in blood is
expected to accumulate in hair by entering the hair shaft at the medulla via passive diffusion, whilst
cortisol accumulated in sebum or sweat from sebaceous and eccrine secretions might coat the outer
cuticle of the hair (Pragst & Balikova, 2006; Raul et al., 2004). According to Russell et al. (2012)
however, there have been no studies confirming the presence of cortisol in neither sebum nor sweat.
Brown (1985) found concentrations of alcohol in sweat, Vree et al. (1972) found concentrations of
amphetamine in sweat, Smith and Liu (1986) found cocaine in sweat, Perez-Reyes et al. (1982)
found phencyclidine in sweat and Henderson and Wilson (1973) found methadone, also in sweat.
These drugs were found present in sweat in higher concentrations than in blood, so even though no
studies have confirmed the presence of cortisol in neither sebum nor sweat, it is not unthinkable that
cortisol could be found in both.
27. ! 27
Figure 6.1: Mechanisms for transfer of cortisol to hair via blood, sebum, sweat etc. (Pragst &
Balikova, 2006).
Two models have been proposed for the transport of cortisol into hair; simple diffusion and the
complex multi-compartment model. These models are described in the next section.
6.1.1 SIMPLE DIFFUSION (PASSIVE TRANSPORT)
Boumba et al. (2006) explains how the simplest model proposed to explain cortisol (or any drug)
transfer into hair is by passive transfer via blood. When cortisol is transferred into the hair by
passive transfer via blood, it is moved from the blood and into the hair follicle by means of passive
diffusion (Boumba et al., 2006). The process of keratogenesis, which is where the hair is hardened
and solidified first by synthetization of melanin and then by enrichment in keratins, then binds the
cortisol in the medulla of the hair shaft (Boumba et al., 2006).
Passive transport is a process in which no energy is expended. The simplest model used to explain
how cortisol is transferred to hair is a type of passive transfer named simple diffusion (Boumba et
al., 2006). Simple diffusion can only occur with substances which are lipid soluble such as gases,
cholesterol and some hormones (Mulroney & Myers, 2009). Cortisol is lipid soluble, and so it can
move down its concentration gradient through the cell membrane by means of simple diffusion. The
28. ! 28
movement which the substance makes in order to pass through the cell membrane follows Fick’s
Law (Mulroney & Myers, 2009). This means that when a molecule undergoes passive diffusion
through a membrane, the diffusion will be proportional to the surface area of the membrane and the
concentration difference of the molecule (Mulroney & Myers, 2009).
The simple diffusion model has been criticised however, as Chittleborough and Steel (1980)
reported that there was a poor correlation between drug intake and the levels of the drug found in
hair. Henderson (1993), amongst others, also found this poor correlation between dose adminstered
and the resulting concentration found in hair. In light of several publications showing experimental
data which reported issues with the simple diffusion model, a new model was created; the complex
multi-compartment model.
6.1.2 COMPLEX MULTI-COMPARTMENT MODEL
In the complex multi-compartment model drugs, hormones etc. are thought to be incorporated into
hair via several different pathways. These pathways include the blood circulation during formation
(1), sweat and sebum after formation (2) and the external environment after both hair formation and
the hair has penetrated the skin (3) (Boumba et al., 2006).
This means, that during formation of the new hair, drugs, hormones etc. present in the body will
enter the hair shaft through the blood circulation. After the formation of the hair, drugs, hormones
etc. are believed to be able to enter the hair via sweat and sebum (Boumba et al., 2006). This theory
is based upon several studies in which alcohol (Brown, 1985), amphetamine (Vree et al., 1972),
cocaine (Smith & Liu, 1986), phencyclidine (Perez-Reyes et al., 1982) and methadone (Henderson
& Wilson, 1973) were found in sweat, in higher concentrations that in blood, in test persons
subjected to either of the drugs. A concentration of either drug was also found present in the hair of
each person, though a variability was observed which may be explained by individual variations in
secretions (Henderson, 1993). During washing and extraction in the laboratory a variation is also
found in relation to drug concentration in hair after formation. One theory explaining this, is that
drugs and hormones entering the hair shaft after formation, does not undergo the process of
keratinization, meaning that the drugs and hormones are less tightly bound to the hair shaft, and
may be washed off more easily (Henderson, 1993). After the hair formation is finished and the hair
has penetrated the skin, an external contamination of the hair is thought to be able to occur, in
which substances found in air, water or products applied to the hair are deposited on the surface of
29. ! 29
the keratinized surface of the hair (Boumba et al., 2006). Henderson (1993) also suggests that the
multiple body compartments that surround the hair follicles may transfer substances into the hair.
During hair formation the hair in the follicle is constructed by matrix cells, and the hair follicle
itself is nourished with blood from capillary networks in the skin dermis. The matrix cells are found
3-5 milimeters into the dermis under the scalp surface on humans. In order to produce a new hair
the matrix cells undergo mitosis. The process of transferring drugs to the hair during formation via
blood, is thus thought to be possible by a diffusion of the drug from blood into the matrix cells via
the capillary network, and then deposition of the drug into the hair whilst the matrix cells undergo
mitosis and develop new hair (Joseph et al., 1999). After hair formation, drugs are thought to be
transferred to hair via sweat and sebum. The epidermis layer of the skin, located above the dermis,
has a metabolically active layer which undergoes mitosis and produces new cells in order to renew
the stratum corneum, which is the outermost layer of the skin. The stratum corneum is continously
exposed to sweat and sebum secretions. Thus, the theory is that drugs present in the skin could be
leached by sweat and/or sebum and then deposited in hair (Joseph et al., 1999).
All in all the complex multi-compartment model suggests that the incorporation of substances into
hair is not the result of one process alone, but of several different processes happening during
various stages of the hair growth cycle.
6.2 EFFECT OF HAIR COLOUR ON CORTISOL LEVELS
In relation to glucocorticoid and pigment production, both developmental, physiological and
biochemical similarities are found in mammals. The melanocyte stimulating hormone (MSH) as
well as melanocortin receptors are involved in the biochemistry of pigment and, similarly,
adrenocorticotropic hormone (ACTH) and melanocortin receptors are involved in the biochemistry
of cortisol control. As such, the same families of hormones as well as receptors are involved with
the control of both pigment and cortisol (Bennett & Hayssen, 2010).
Bennett & Hayssen (2010) conducted a study in which they investigated the relationship between
cortisol and coat colour in dogs. Agouti (sable or black and tan) coloured animals were compared
with nonagouti (black) German Shepherds. The content of pheomelanin and eumelanin in
individual hairs of agouti German Shepherds vary a lot, as some hairs are completely black and
thereby all eumelanin, others are all yellow and thereby all pheomelanin, and some are agouti hairs
30. ! 30
banded with both types of pigment in different proportions (Bennet & Hayssen, 2010). Agouti hairs
were defined as hairs having a ratio of eumelanin to pheomelanin that was no higher than 70:30 and
no lower than 30:70 (Bennet & Hayssen, 2010). The results of the study conducted by Bennet &
Hayssen (2010) concluded, that there was a consistently lower concentration of cortisol in black-
haired dogs, when compared to the concentration of hair cortisol in yellow-haired dogs. Agouti hair
cortisol concentration was shown to be intermediate. The same results were found in dogs of the
agouti phenotype; in individual dogs, the black eumelanin hairs had lower cortisol concentrations
than the yellow pheomelanin hairs, and agouti were again found to be intermediate (Bennet &
Hayssen, 2010).
There could be several reasons for the differences in cortisol concentration amongst the various
colours of hair. Stress-associated hair growth inhibition (Botchkarev, 2003) and melanocyte
development and differentiation (Slominski et al., 2004; Roulin et al., 2008) are both control
mechanisms involving glucocorticoids, and as such the reason for the difference in cortisol
concentration between the various coat-colours could possibly be related to these mechanisms
(Bennet & Hayssen, 2010). A different reason could be that hair may be a storage vehicle for
cortisol. As yellow hair contains less pigment than black hair (Russell, 1948; Kaliss, 1942), it may
also have more room to store glucocorticoids than black hair (Bennet & Hayssen, 2010).
Essentially, the differences found in cortisol levels in the various types of pigment, were found
within the same animal, not across breeds or colours (Bennet & Hayssen, 2010). As such, although
differences were found between black, yellow and agouti hairs within individual animals, this
cannot be interpreted as differences in cortisol concentration between breeds or coat colours within
a breed (Bennet & Hayssen, 2010).
6.3 SOURCES OF SAMPLING VARIATIONS
Kobelt et al. (2003) conducted a study in which the aim was to determine the main causes on
sampling variation in saliva cortisol in dogs over time. It was found, that the variation in average
cortisol concentrations between different days was very little, which could be seen as there was a
zero variation for the week and day within week variance components. This indicates that
environmental factors such as weather, did not play a role in varying cortisol concentrations, as the
weather might change from day to day (Kobelt et al., 2003). During the time of day from 14:00 to
16:00 hours, the cortisol concentrations are less pulsatile (Kirschbaum & Hellhammer, 1989). Other
reasons for sample variations could be caused by social interactions between dogs, which could
31. ! 31
change the average cortisol concentration of the group of dogs. If social interactions can affect
cortisol levels, then this factor might be less important for single housed animals (Kobelt et al.,
2003).
Another experiment conducted by Clark et al. (1997), also with dogs, found that plasma cortisol
concentrations changed significantly over time, as dogs had higher cortisol on day zero of the
experiments and on day seven, which indicates that cortisol increased during the first week of the
study and then stabilised. Kirschbaum & Hellhammer (1989) found that environmental and
psychological stressors can give rise to salivary cortisol in humans and other animals, and Beerda et
al. (1999) found that dogs can have variations in cortisol concentrations because of a response to
social and spatial restriction. Tuber et al. (1996) and Hennesy et al. (1998) found that human
contact can change cortisol levels in animals, and, conversely, Beerda et al. (1998) found that
sudden non-social stimuli can have the same effect.
One of the most used ways of measuring cortisol in animals, is by measuring cortisol content in
plasma after having taken a blood sample (Reburn & Wynne-Edwards, 2000). Great variation can
be found in plasma cortisol, if care is not taken with sampling procedure. This can be due to stress
itself, depending on the care taken during the blood sampling, but also variation can be found, if the
sampling procedure is not exactly the same during each sampling. Identifying a method giving rise
to less sample variation could be of great benefit to future research.
Area of sampled hair as well as method used for sampling hair, can also have an effect on the
measured level hair cortisol. This will be described in the next section.
6.4 USING HAIR CORTISOL AS A WELFARE BIOMARKER
At present, cortisol measurements are being used to monitor the hypothalamic-pituitary-adrenal
(HPA) axis activity (Accorsi et al., 2008) and the methods used include faecal, urinary, salivary and
hair corticoid measurements (Cook et al., 2000). The method of using hair cortisol was investigated
by several authors such as Davenport et al. (2006), who conducted a study using rhesus macaques
and validated a procedure for measurement cortisol accumulated in hair. Hair cortisol can be used
to trace pollutants, drugs, anabolic steroids, sex steroids as well as glucocorticoids (Koren et al.,
2002, Yang et al., 1998). In this study we are interested in the glucocorticoids present in hair
sampled from mink and cattle, and we wish to use the hair cortisol as a non-invasive welfare
32. ! 32
biomarker. According to Accorsi et al. (2008), cortisol measurements in hair can be useful when
wanting to study chronic stress and welfare. This is the case because cortisol accumulates slowly in
hair, and so it provides us with a long-term endocrine profile of the animal (Accorsi et al., 2008).
When hair grows at a constant rate, any cortisol found accumulated in the hair can be correlated
with a time this cortisol was present in the blood of the animal (Boumba et al., 2006). This allows
for a specific welfare assessment, as it can be evaluated when the animals had higher levels of
blood cortisol and then one can attempt to identify the reason(s) for this cortisol response.
According to Accorsi et al. (2008), as it provides us with a long-term measure of hormonal activity,
the stress the animals are subjected to during the sampling will not have impact on the results, and
the measurements of hair cortisol might be best suited for evaluations of chronic stress.
Faecal cortisol measurements are already being used as a non-invasive technique to determine HPA
axis activity (Accorsi et al., 2008). A study conducted by Svendsen et al. (2007) examined two
groups of mink, one high stereotyping line and one low stereotyping line. On basis of adrenocortical
activity measured by faecal cortisol metabolites, the welfare of the mink was assessed. Svendsen et
al. (2007) found that the high sterotyping line had higher concentrations of faecal cortisol
metabolites, indicating lower welfare. Other studies by Zanella et al. (1998) found that faecal
cortisol metabolites were lower in high stereotyping lines of mink, but this might be explained by
the low stereotyping lines being less sensitive to stressors (Svendsen et al., 2007), or a drop in
cortisol levels hours following a cortisol response to an accute stressor, also known as rebound
effect or coping (Mason & Latham, 2004). In the study conducted by Accorsi et al. (2008) cortisol
was determined in hair and faeces from domestic cats and dogs, in order to determine the reliability
of hair cortisol measurements. On basis of measurements of both faecal and hair cortisol it was
found that there was a significant correlation between the two, which indicates that cortisol
measured in hair and faeces reflect the same HPA axis activity (Accorsi et al., 2008). The same
correlation was found by Davenport et al. (2006) who compared measurements of hair cortisol to
measurements of salivary cortisol in rhesus macaques. Davenport et al. (2006) found a positive
correlation between hair- and salivary cortisol, as well as establishing that both increased when the
animals were subjected to stressful situations.
Moya et al. (2013) wanted to establish wether hair sampled from beef cattle contains enough
cortisol to be measured, and whether the location of the sampled hair had any influence on the
results. In the study samples of hair were taken from the head, neck, shoulder, hip, and switch (tail)
33. ! 33
from twelve Angus cross bulls. They chose to collect the hair using two methods; plucking, which
included the hair follicles in the samples, and clipping with electric razors (Moya et al., 2013). After
running the samples in the lab, it was found that cortisol could be found in all samples in
concentrations between 0.30 – 5.31 pg/mg (Moya et al., 2013). Hair from the tail contained more
cortisol than hair from the head and shoulder region, whilst hair from the neck and hip contained
more cortisol than hair from the shoulder region (Moya et al., 2013). Furthermore it was found, that
the cortisol levels were higher in hair which was collected by clipping with electric razors, in
comparison to plucking (Moya et al., 2013). Data from the cortisol measured in the sampled hair
was compared to both saliva samples as well as fecal glucocorticoid metabolites, and a positive
correlation was found between cortisol concentration in saliva samples and hair sampled from the
hip and tail, as well as between fecal glucocorticoid metabolites and hair sampled from the neck and
tail (Moya et al., 2013).
In the experiment carried out in the present study, hair was sampled from the forelock of beef cattle.
The site of the hair sampling was chosen from a point of view of convenience. The cattle was
fixated during the sampling procedure, and it was determined that the easiest reachable area for
sampling was the forelock. The hair was sampled by means of a pair of scissors, cutting as close to
the skin as possible. As Moya et al. (2013) established that cortisol was determined in each of their
samples taken from the head, neck, shoulder, hip and switch (tail), the hair sampled from the head
in this study should also contain enough amounts of cortisol to be detected. Moya et al. (2013) did
not find a correlation between cortisol from hair sampled from the head and saliva or fecal
glucocorticoid metabolites however, but that could be explained by the fact, that the hair sampled
from the head in Moya et al.’s (2013) study, contained less cortisol than hair from the hip, tail and
neck, which did correlate with both saliva and fecal glucocorticoids. Even though there is a lack of
correlation between cortisol levels in hair sampled from the head, and saliva and fecal
glucocorticoids, in Moya et al.’s (2013) study, the correlations between hair sampled from the neck,
hip and tail, and saliva and fecal glucocorticoids, indicate that hair samples in general can be used
as a means to measure cortisol levels in beef cattle.
7 AIM AND EXPERIMENTAL DESIGN
The aim of the experiment was to take hair samples from a broad section of cattle farms, and a
selection of mink housed in different environments. As mostly hobby breeders agreed to participate
34. ! 34
in the experiment, the range of cattle farms was not as varied as wished. The experiment was
designed to make hair sampling easily performed, and also to ensure that the hair was cut as close to
the skin as possible.
8 MATERIALS AND METHODS
8.1 TESTING VENUES
The mink testing was conducted at Rørrendegård Pelsdyrfarm located in Taastrup, at the DTU
Veterinary Institute at Frederiksberg and at Aqua Akvarium & Dyrepark at Silkeborg. Cattle testing
was conducted on all contributing cattle farms placed on both Sealand, Funen and Jutland.
8.2 MINK TESTING
The farmed mink involved with this project were all housed at Rørrendegård Pelsdyrfarm. Besides
these, mink caught in the wild and mink housed in Aqua Akvarium & Dyrepark in Silkeborg were
sampled. Materials used in the mink testing were: electric clippers, gloves, envelopes and a
sampling database. The sampling database had information on the individual mink’s breeding
number, the number of the cage they are housed in, the type of barn the cage is placed in, as well as
a comments box for noting behaviour during sampling, health- and physiological status.
8.2.1 ANIMALS
When sampling hair from the mink, at first 12 mink were selected by the daily keeper of
Rørrendegård Pelsdyrfarm, Boye Pedersen. The mink selected are neither part of the best fraction of
mink at the farm, or part of the worst fraction of mink at the farm, but are placed somewhere in the
middle. The 12 mink are all bitches used for breeding. The behaviour during sampling was noted, as
well as the health and physiological status of the animal.
The experimental mink are mink housed in standard cages in closed barns at Rørrendegård
Pelsdyrfarm. These mink took part in a study conducted by the University of Copenhagen, in which
the mink were fed different diets, containing different amounts of various essential amino acids. At
least one mink was found dead in a cage during the experiments, so we can assume that at least
some of the experimental mink were stressed from a metabolic point of view. During the feeding
trials one mink was selected from each trial cage, euthanized and autopsied in order to see which
35. ! 35
effects the trial diet had on the mink. During the autopsy a patch of hair above the tail area was
shaved from each mink, in order for the experimental mink to also be part of this study.
Another 30 mink were sampled with help from Mariann Chriél of the DTU Veterinary Institute.
These mink were all caught in traps or found dead in the wild, in order to examine whether diseases
found on fur farms have spread to wild populations of mink in Denmark. The mink are of both
sexes and of various different colours ranging from the traditional brown to pearl white and grey.
For all mink the weight at capture was noted, which may give indication as to how long the mink
have been living in the wild. Furthermore any diseases found during DTU’s autopsy of the mink
were recorded as well.
Finally two mink living in a wildlife park were sampled, with help from Zoologist Morten Vissing
of Aqua Akvarium & Dyrepark. These mink, one male and one female, were originally housed at a
fur farm, but were moved to animal park in Silkeborg around the year 2010, as part of a project
studying whether farmed mink can readapt to their natural environment and behaviour. The
behaviour during sampling was observed, and the health and physiological status of the animals was
noted.
8.2.2 HAIR SAMPLING
The area chosen to be shaved for the hair sampling of mink was chosen on the basis of two reasons.
The chosen area was a patch just above the tail of the mink. This area was chosen because it is
easily seen and accessible, so monitoring the mink with the purpose of discovering any regrowth
was made easier by choosing a spot which is easily seen. Another motive for chosing this spot was
the moulting and fur growth pattern of the adult mink. The fur which we sample is the winter coat
of the mink. At the time of the first sampling, we did not know whether the mink fur would regrow
during the one month pause between the first and the second hair sampling or not. The growth of
the winter coat in mink starts with a bluish tint developing at the tip of the tail of the mink (Bassett
& Llewellyn, 1949). The growth of the winter coat in mink thereby starts at the area of the tail, with
new guard hair slowly developing on the posteriour third of half of the tail (Bassett & Llewellyn,
1949). The theory was, that if any regrowth did develop in the month between the first and second
sampling, shaving an area close to the tail would be the best strategic option, as we are dealing with
winter fur which, as described, develops from the tail area of the mink.
36. ! 36
Three people were present during the sampling, with two people performing the actual sampling
process, and one person handling and marking the fur samples.
Picture 8.2.2: Left, mink being restrained by two people and shaved. Right, shaved area.
The mink were extracted from their separate holding traps one-by-one. The mink were then held
down on a table, with one person grabbing and fixating the neck and back, and the other person
stretching the mink by tugging on its tail, and holding the electric clippers in the other hand. An
area just above the tail was then shaved off, leaving a hairless space. The area had to be shaved
several times, in order to shave off all fur present. The fur shaved off by the electric clippers was
then placed in envelopes marked with the puppy number of the mink, as well as the number of the
cage it is housed in, and date stamped.
One month following the first hair sampling, a second sampling of mink hair was done. The
hypothesis supposed that the mink hair which was forcibly removed by the electric clippers might
grow back out again in time for the second sampling. However, no regrowth of hair was visible one
month after the first sampling. As such, at the second sampling, instead of collecting the regrowth
of hair, a new sample was taken from each mink. These samples were collected again by use of
fixation and electric clippers, and a small patch above the original shaved area was removed. The
hair removed from this area, in the second sampling, will be analysed in the laboratory, in order to
see whether any difference can be found, in the accumulation of cortisol during the one month of
different housing.
37. ! 37
The experimental mink were sampled following the autopsy performed to examine the effects of the
various diets they were fed. The hair samples were taken with electric clippers from an area above
the tail of each animal.
The wild mink caught or found dead were delivered to the DTU Veterinary Institute which placed
them in freezers until autopsy. During autopsy a patch of skin with fur was separated from hind
quarters of the body, using a picture of a shaved farmed mink as a reference. The patch of skin with
fur taken from the wild mink should therefore be from the same area of the body as the farmed
mink. The skin samples were placed in individual bags wrapped in tissue to protect them, and
labelled with individual numbers, the sex of the mink as well as the area from which it was caught.
The skin samples were kept in a freezer until final sampling of hair. The final sampling was done by
means of electric clippers, shaving the fur off the skin samples and making a database including the
number of the animal, sex, area of capture, weight and any diseases found during autopsy. The hair
samples were placed in envelopes marked with individual samples numbers.
The mink housed in Aqua Akvarium & Dyrepark were sampled by Zoologist Morten Vissing who
works at the park and routinely handles the mink when necessary. The sampled was done by luring
the mink into their nest box by means of food, and then fixating the mink with one hand whilst
using electronic clippers to cut a hair sample from the area just above the tail. After sampling the
hair was placed in envelopes marked with individual sample numbers, and a database was made
with records of the sex of the animals, behaviour during sampling as well as health as physiological
status.
8.2.3 HOUSING
After the shaving had taken place, the farmed mink were placed into cages. 6 of the 12 mink bitches
were chosen to be housed in cages placed in closed barns. These cages are all inside a barn, and
offers the mink protection from wind and weather. The remaining 6 mink were chosen to be housed
in cages placed in open barns. These cages are also inside a barn, but the open barn consists of a
roof and supporting constructions and has no walls. This means that only part of the cages are
protected from wind and weather – the part which consists of the nest box. The shelf and far end of
the cage are placed directly beneath the roof of the barn, and so both wind and weather may affect
this part of the cage. Although some mink were housed in the same types of cages before and after
the shaving, all mink were moved to a new cage after the shaving. This means, that all mink were
38. ! 38
exposed to an unknown cage with new scents and smells, and new neighbouring animals.
The experimental mink were all housed in standard cages in closed barns. The mink who were
sampled for this study were euthanized prior to sampling, and so they were not moved to another
cage afterwards. However the potential stressor for the experimental mink is that they were part of a
feeding trial, and the animals may have been stressed from a metabolic point of view.
The wild mink sampled by the DTU Veterinary Deparment are from different parts of the country.
All the samples in this study are from mink caught in traps or found dead in Northern or Eastern
areas of Denmark. The areas are created by The Danish Veterinary and Food Administration, which
keep track of standards concerning food, feed, animal welfare and animal health. In relation to mink
the Danish Veterinary and Food Administration keep track of wild mink populations, and examine
caught or dead mink to establish whether diseases found on fur farms have spread to wild mink
populations. The Northern areas include the middle and north of Jutland, whilst the Eastern areas
include Sealand and Bornholm. The wild mink were living in the wild in these areas before being
caught in traps or found dead. The weight of the individual animals can give indication as to how
long they have been living in the wild, as a well nourished animal is more likely to have recently
been living on a fur farm, whereas skinny animals are more likely to have spent longer periods
searching for their own food.
The park mink are housed in Aqua Akvarium & Dyrepark in Silkeborg, which is a park housing
animals with special focus on marine and freshwater animals. The two mink housed at Aqua
Akvarium & Dyrepark were originally bred and living on a fur farm in standard cages as the ones
on Rørrendegård Pelsdyrfarm. These mink however where re-housed at Aqua Akvarium &
Dyrepark as part of a project studying mink behaviour. Mink born on fur farms have been bred and
adapted to living in the cages, and the project wanted to examine how long it would take for the
mink to readapt to a natural environment, and to use their natural instincts to hunt for food, swim,
mate etc. The two mink at the Aqua Akvarium & Dyrepark live in a 150 square metre enclosure
designed to resemble a natural mink environment with a fresh water stream, trees, rocks and a
nesting box. They have been housed in this environment for the past four years. They are provided
with food daily, but are also encouraged to find food for themselves in the fresh water stream, such
as live fish.
39. ! 39
8.3 CATTLE TESTING
The cattle involved with this project were all housed on various contributing farms on Sealand,
Funen, Jutland and Bornholm. We used the Danish national cattle registry to contact 232 beef cattle
farmers from 7 postcode areas. From 35 positive responses we were able to visit 24 farms to obtain
hair samples. Materials used in the cattle testing were: cattle (calves, bulls, heifers, cows), pair of
scissors, two types of envelopes and a sampling database. The sampling database has information
on the individual animals’ passport number, the colour of the animal, the age of the animal, the
physiological state of the animal, the animal’s relationship to other cows, the behaviour during
sampling, the cleanliness of the animal on both body and legs, the current health status of the
animal, the social rank, body condition scoring as well as the presense of horns, and a comments
box for other informations given.
8.3.1 ANIMALS
When sampling the cattle hair, at first animals were chosen on each farm. In general between 10 –
15 animals were sampled at each farm, depending on the size of the herd. The animals were chosen
based on having a variety in the samples, so both cows, bulls, heifers and calves were sampled
when possible. Some animals were specifically chosen if they were known to behave differently
than the rest of the herd, or if they had/had had any health problems recently. All cattle were of
various different breeds depending on the farm visited. Breeds represented in the samples are:
Aberdeen Angus, Danish Belgian Blue, Danish Charolais, Danish Jersey, Danish Shorthorn, Dexter,
Galloway, Hereford, Holstein, Jutland Cattle, Limousine, Red Danish Milking Breed (RDM),
Scottish Highland, Simmental, Tiroler Grauvieh and various cross-breeds. During the sampling the
behaviour of the animal was noted, as well as other factors mentioned in section 6.3.
8.3.2 QUESTIONNAIRES
Prior to the sampling questionnaires were sent out to all participating farms. Questions such as the
breed of the cattle on the farm, the herd size, cattle group sizes, farm size, size of grazing areas and
vet visit frequency were asked. The purpose of the cattle testing was to get samples broad variation
of farms, however, the farmers who expressed an interest of participating in this study were
generally small scale farms and hobby breeders, as opposed to industrial scale farms.
40. ! 40
8.3.3 HAIR SAMPLING
The area chosen for hair sampling for the cattle was the forelock. This was chosen for management
issues because the forelock was deemed fairly easily accessible, especially when the cattle are
fixated. The cattle chosen for sampling was fixated either by headlocks fitted in the barn or by other
means such as using a rope halter. Once fixated a lock of hair from the forelock was chosen and cut
with a pair of scissors, taking care to cut as close to the skin as possible. As the various cattle breeds
have varying lenghts of forelock hair, for long haired breeds the hair sample was cut into two pieces
using scissors. The two centimeters closest to the skin was placed in a small envelope, and the rest
of the hair sample was placed in a big envelopes. The small envelope was then placed inside the big
envelope, and the small envelope holds the sample which will be analysed at the laboratory.
8.3.4 HOUSING
The cattle sampled were housing in different ways according which production method the
individual farmer had chosen. The housing methods observed were 5 different methods; indoor
loose housing system, indoor loose housing system with outdoor access, indoor tie-stalls, outdoor
housing with shelter access and outdoor housing without shelter access. The indoor loose housing
system consists of a barn typically separated into several units of cows, heifers, bulls and calves.
The animals are mostly housed in deep litter bedding though sometimes just on concrete flooring.
In some barns the part of the holding pens facing the feeding table were elevated from the rest of
the pen, with the animals needing to climb a step in order to get to the feeding table. This proved
difficult for some animals, which was explained in section 4.1. The other type of indoor housing
system observed was designed also with indoor barns and deep litter bedding, but in this case the
animals also had the choice to go outside. The outside area was in some cases a field or a dirt
enclosure, and in other cases a smaller enclosed area sometimes partly equipped with concrete
flooring. Some farmers had chosen to have their animals housed indoor in tie-stalls. All tie-stalls
observed were fitted with devices which tied around the animal’s neck, making the individual
animals face the feeding table of the barn at all times. Some farms had all animals housed in tie-
stalls, whereas others had some animals in loose housing systems and others in tie-stalls. Outdoor
housing was also observed, especially in relation to the Scottish Highland breed. Outdoor housing
came both with and without access to a shelter. On farms with shelter access, the shelter was
sometimes a barn with deep litter bedding, and at other times the shelter consisted of a three-wall
41. ! 41
building in the field/pasture. Some outdoor housing systems also came with natural shelters in terms
of trees and forest areas inside the pasture.
8.4 SAMPLE ANALYSIS
All samples were stored in envelopes under room temperature in the time between sampling and
analysis. This was done to protect the samples from contamination for example by condensation
forming on the inside of testing tubes. At the laboratory at the University of Udine, Italy, the strands
of hair were washed with isopropanol and extracted using methanol. Hair cortisol levels were then
determined using a solid-phase microtitre radioimmunoassay (RIA) procedure (Comin et al. 2014).
A commercial kit with human genes was not used, but rather a specially created kit with animal
genes.
9 RESULTS AND STATISTICS
9.1 MINK RESULTS AND STATISTICS
A total of 75 samples of mink hair were analysed at the laboratory in Udine, Italy. The mink
samples were all in the lower range of the normal distribution of all mink-and-cattle samples (see
figure 9.1).
Figure 9.1.1: Distribution of all measured cortisol for the entire dataset (mink and cattle).
42. ! 42
In order to remove any outliers in terms of cortisol concentration, a restricted cortisol limit was
calculated, and all samples scoring higher levels of cortisol than the calculated limit were then
eliminated from the following statistics;
2 ∗ SD!of!cortisol + mean!of!cortisol = restricted!cortisol!limit! ⇔
2 ∗ 0,64 + 0,78 = 2,06
In order to statistically analyze whether the various factors had any influence on the cortisol levels
measured, an Analysis of Variance (ANOVA) was calculated for significance between cortisol and
all of the factors. The model used was the General Linear Model, as most of the data are
unbalanced. Data are reported as mean ± SE pg/mg. In some cases a two-sample t-test was also
performed, in order to examine if there was a signifcant variance between two specific factors. The
results will be analysed more thoroughly in the discussion.
For the farmed mink housed in standard cages there were two datasets, as all animals were sampled
twice. The datasets were analysed using a general linear model, in order to see if there were any
significant variation in cortisol measurements between the two sample dates.
Cortisol and sample date
Figure 9.1.2: Cortisol variation in relation to sampling dates.
There is no significant (p - value = 0,703) variation between hair cortisol concentrations and
43. ! 43
sampling dates (0,92±0,06 to 0,96±0,06). Seeing as there is no significant difference between the
two datasets, the rest of the statistical analysis will be performed using the dataset for farmed mink
in standard cages sampled on January 28th
2014.
Cortisol and housing – all types of housing
Figure 9.1.3: Cortisol variation in relation to housing types.
As can be seen from the p – value = 0,003, there is a significant variation between hair cortisol
concentrations measured in animals housed in different environments. Mink housed in standard
cages in closed barns had the highest concentration of hair cortisol (0,93±0,09), closely followed
by mink housed in standard cages in open barns with cortisol levels of 0,92±0,09. Surprisingly,
mink housed in standard cages in closed barns undergoing feeding trials, had lower hair cortisol
than mink housed in a semi-natural enclosure in a wildlife park (0,61±0,05 to 0,66±0,16) although
it must be remembered that there were only two such animals. The wild mink scored lowest on hair
cortisol concentration with 0,60±0,05).
A two-sample t-test was performed using Minitab, in order to examine whether a significant
variation in cortisol levels could be found between caged (farm mink) and wild mink. The results of
the t-test provided a p-value of p=0,34, which means that no significant variation was found. As
such, the wild mink had slightly lower mean hair cortisol values, but not significantly lower values.
44. ! 44
Cortisol and housing – indoor (farm- and experimental mink) versus outdoor (park- and wild
mink)
Figure 9.1.4: Cortisol variation in relation to indoor- and outdoor- housing.
There is no significant variation in hair cortisol concentrations of animals housed indoors and
outdoors (p – value = 0,074). Indoors housed animals had only slightly higher hair cortisol
concentrations than outdoors housed animals (0,74±0,04 to 0,61±0,05). In order to examine
whether standard caged mink had significant differences in hair cortisol levels when housed in open
versus closed barns, a general linear model was used to analyse these two housing types.
Cortisol and housing – open barn versus closed barn
Figure 9.1.5: Cortisol variation in relation to open- and closed barn systems.
45. ! 45
There is no significant variation (p – value = 0,921) in hair cortisol levels in mink housed in open
versus closed barn systems (0,92±0,07 to 0,93±0,07).
Cortisol and weight (in grams) – all animals
Figure 9.1.6: Cortisol variation in relation to body weight.
There was no significant variation (p – value = 0,517) between hair cortisol concentrations and
different body weights (0,61±0,08 to 0,46±0,11).
Cortisol and colour – categories with too few animals eliminated
Figure 9.1.7: Cortisol variation in relation to coat colour.
46. ! 46
Data were available from mink of 5 different colours (brown, black, pearl, grey and black/white),
but the grey and black/white category comprised only few animals, so these data were excluded
from analysis. There was no significant variation (p – value = 0,498) between measured hair
cortisol levels and the colour of the hair sample (0,71±0,04 to 0,59±0,11).
Cortisol and colour – colours divided into dark and light nuances
Figure 9.1.8: Cortisol variation in relation to coat colour sorted by light or dark colours.
There was no significant variation (p-value = 0,181) between measured hair cortisol levels and light
or dark sampled hair (0,69±0,03 to 0,57±0,08).
Cortisol and gender
Figure 9.1.9: Cortisol variation in relation to gender.
47. ! 47
There was a significant variation (p – value = 0,001) between hair cortisol concentration and the
gender of the sampled animals, with the female animals scoring the highest levels of hair cortisol
(0,82±0,05 to 0,53±0,07).
Cortisol and area of sampling
Figure 9.1.10: Cortisol variation in relation to area of sampling
There was no significant variation (p - value = 0,658) between measured hair cortisol levels and the
area of sampling. Animals sampled in area east only had slightly higher cortisol levels than animals
sampled in area north (0,74±0,05 to 0,66±0,18).
Cortisol and experimental feeding
Figure 9.1.11: Cortisol variation in relation to experimental feeding.
48. ! 48
There was no significant variation (p-value = 0,160) between measured hair cortisol concentrations
and experimental feed compositions. It should be noted that sample size was low for each
individual treatment. Animals fed with feed enriched with methionine had the highest mean levels
of cortisol (0,94±0,14), whereas the two animals fed diets enriched with dextrose had the lowest
levels of mean hair cortisol (0,45±0,14).
9.2 CATTLE RESULTS AND STATISTICS
A total of 311 samples of cattle hair were analysed at the laboratory in Udine, Italy. In order to
remove any outliers in terms of cortisol concentration, a restricted cortisol limit was calculated,
using the formula shown, and all samples scoring higher levels of cortisol than the calculated limit
were then eliminated from the following statistics;
2 ∗ SD!of!cortisol + mean!of!cortisol = restricted!cortisol!limit! ⇔!
2 ∗ 3,68 + 2,98 = 10,34
In order to statistically analyze whether the various factors had any influence on the cortisol levels
measured, an Analysis of Variance (ANOVA) was calculated for the individual factors of interest.
The model used was the General Linear Model, as most of the data are unbalanced. Data are
reported as mean ± SE pg/mg. The results will be analysed more thoroughly in the discussion.
Cortisol and farm number
!
!
!
!
!
!
!
!
!
!
Figure 9.2.1: Cortisol variation in relation to farm number.
