Selection for behavior and hemopoiesis in American mink (Neovison vison)

Accepted Manuscript Selection for behavior and hemopoiesis in American mink (Neovison vison) A.G. Kizhina, L.B. Uzenbaeva, V.A. Ilyukha, L.I. Trapezova, N.N. Tyutyunnik, O.V. Trapezov PII:

S1558-7878(16)30149-6

DOI:

10.1016/j.jveb.2016.09.004

Reference:

JVEB 998

To appear in:

Journal of Veterinary Behavior

Received Date: 5 May 2016 Revised Date:

7 September 2016

Accepted Date: 7 September 2016

Please cite this article as: Kizhina, A.G., Uzenbaeva, L.B., Ilyukha, V.A., Trapezova, L.I., Tyutyunnik, N.N., Trapezov, O.V., Selection for behavior and hemopoiesis in American mink (Neovison vison), Journal of Veterinary Behavior (2016), doi: 10.1016/j.jveb.2016.09.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Selection for behavior and hemopoiesis in American mink (Neovison vison)

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Kizhina A.G. 1*, Uzenbaeva L.B. 1, Ilyukha V.A. 1, Trapezova L. I. 2, Tyutyunnik N.N. 1,

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Trapezov O.V. 2

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(11 Pushkinskaya St., 185910 Petrozavodsk, Karelia, Russia; tel. +7 (8142) 573107, Fax: (8142)

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573107)

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Russia (10 Prospekt Lavrentyeva, 630090, Novosibirsk, Russia; tel. +7 (383) 363-49-63*1230, Fax:

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(3833) 33 12 78)

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*Corresponding author, E-mail: [email protected]

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Institute of Cytology and Genetics, Siberian Department, Russian Academy of Sciences, Novosibirsk,

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Institute of Biology, Karelian Research Centre, Russian Academy of Sciences, Petrozavodsk, Russia

Abstract

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It has been known that selective breeding of animals for behavior associated with

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domestication changes a variety of physiological traits including response to stress and hypothalamo–

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pituitary–adrenocortical system (HPAS) activity. The aim of our research is to find association

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between behavioral type and differential leukocyte counts as an indicator of homeostasis activity. We

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used farm-raised 5-mo-old minks (Neovison vison) that had been selective bred for 17 generations on

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the basis of behavior towards human. After selection, minks were divided into the following groups:

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aggressive (from -4 to -1), ‘domestic’ (from +1 to +5). Blood leukocytes were analyzed in 5-mo-old

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minks (n=105) of different behavior lines: aggressive (-1, -2), ‘domestic’ (+1 and from +3 to +5),

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fearful, and minks never subjected to selective breeding (control). We found strong effects of selection

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for behavior of minks on differential blood leukocyte count. There were differences between behavior

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types in differential leukocyte counts. Neutrophils and eosinophils were the leukocyte types the most

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sensitive to selection for behavior. The counts of eosinophils in peripheral blood in ‘domestic’ (+3,

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+5) minks were higher than in control and aggressive (–2) minks. The percentage of neutrophils was

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higher in the most aggressive minks (–2) in comparison to ‘domestic’ minks. We have concluded that

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differences found in leukocytes profile are the result of neuroendocrine system modification.

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Key words: leukocytes, selection, domestication, aggressiveness, hypothalamo–pituitary– adrenocortical system, American mink

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1. Introduction

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The American mink (Neovison vison), a North American native semi-aquatic generalist

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predator, has expanded its range globally as a result of their breeding in fur farms (Dunstone, 1993;

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Bonesi and Palazon, 2007). First attempts to keep wild individuals under human care, thus starting the

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domestication process, have reportedly taken place around 1866 (Shackelford, 1949, 1984), when

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trappers in Canada began to keep and breed wild mink in enclosures. Farm mink after domestication

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differ from their wild counterparts in fur color, skull dimensions, brain, body size and temperament

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(Belliveau et al., 1999; Kruska, 1996; Kruska and Sidorovich, 2003). Domesticated mink are bred to

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be larger, with higher reproductive success and less aggressive than wild ones. Fearfulness and fear-

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induced aggression in mink may be beneficial in a natural context, but may be detrimental to the

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animal’s welfare in the captive environment. Therefore one of the first selected traits in the history of

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mink domestication was tame behavior to humans (Trapezov, 2000).

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The investigation of the effects of domestication provides insight into evolution problems and

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is of practical interest. One of the first selection experiments for domestic behavior was conducted

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with silver fox (Vulpes vulpes) at the Institute of Cytology and Genetics, Novosibirsk, Russia

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(Belyaev, 1979). Belyaev (1979) supposed that selection of silver fox for behavior in response to

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human interference can be a pivotal factor of domestication. Long-term selection of farm-bred mink

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(Neovison vison) for tame and aggressive defensive reaction towards man has been carried out at the

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Institute’s experimental farm since the 1980s. Tame and aggressive lines of standard (+/+) minks

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differing in their defensive response towards humans were developed using a breeding protocol

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designed to study the effects of destabilizing selection (Klotchkov et al., 1998). As revealed in studies

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using silver foxes and minks, behavior-controlling genes have a broad pleiotropic effect. Selection of

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silver foxes and minks for low aggression and tameness modifies numerous regulatory processes and

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leads to a variety of morphological, behavioral and physiological traits. Trapezov (2013) and Wilkins

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et al. (2014) note certain traits of domestication in mammals: depigmentation, floppy ears, shorter

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muzzles, smaller teeth, docility, smaller brain or cranial capacity, more frequent estrous cycles,

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neotenous (juvenile) behavior, curly tails.

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The influence of selection on the activity of regulatory systems controlling homeostasis has

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been demonstrated for numerous animal models. Selection for low aggressiveness to humans leads to

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a change in behavioral manner and is accompanied by genetically–determined changes of the

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neuroendocrine system (Klotchkov et al., 1998). A result of selection for domestic behavior in minks

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is alteration of the hypothalamo–pituitary–adrenocortical system (HPAS) activity (Trapezov, 2000)

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and levels of serotonin and other monoamines in the mink brain (Nikulina et al., 1985). Animals

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selected over several generations for tameness showed lower basal and stress-induced hypothalamo–

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pituitary–adrenocortical activity, reached sexual maturity earlier and were easier to mate than more

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fearful animals (Jeppesen and Pedersen, 1998; Nikula et al., 2000; Malmkvist, 2001).

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There is a need to study the association of behavioral type and differential leukocyte counts

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as the indicator of homeostasis activity. According to some authors, the effects of HPAS under stress

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include elevated plasma corticosteroids, reduced T and B lymphocyte proliferation and neutrophil

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phagocytosis, increased neutrophil/lymphocyte ratio, as well as leukocyte adhesiveness/aggregation

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(Irwin et al., 1990; Song et al., 1994). Since domestication changes neuroendocrine system activity we

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can expect animals within distinct behavior categories to show discrepancy in differential leukocyte

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count. As revealed in previous works by Davis et al. (2008), the counting of white blood cells from

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blood smears (leukocyte profiles) can be used to assess stress and welfare in animals. They concluded

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that this given method can provide a reliable assessment for stress in all vertebrate taxa.

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with different behavior types under prolonged experimental domestication. 2. Materials and methods

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The aim of the present study is to research differential leukocyte counts in American mink

2.1. Experimental animals

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Experiments were performed with standard dark brown (+/+) minks at the experimental

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farm facility of the Institute of Cytology and Genetics (Novosibirsk, Russia). The animals were tested

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for defensive reaction to man only once a year at 5 months age (September) by one person (Trapezov)

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throughout the selection period (17 generations). The “hand catch test” was applied for this purpose.

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Both males and females were tested for defensive reaction towards man during an entire day, with

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breaks for the morning and evening feeding. The experimenter opened the cage where the tested

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animal was kept, slowly reached for the animal, and tried to catch it with a hand protected with a

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special mitten.

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The technique allowed discrimination among three types of behavior towards man:

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aggressive, fearful, and ‘domestic’ (tame). To study minks of different behavioral categories we

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followed the classification of Trapezov (1987). Mink with fearful behavior tried to avoid contact with

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man as much as possible and when the experimenter tried to catch them, they ran about the cage in

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panic, shrieking. This behavior was assigned score “0”.

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Aggressive behavior varied qualitatively, which allowed scoring it on a four-point scale as follows.

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Score – 1. Fearful response towards humans. When attempts were made to catch the mink, it rapidly

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retreated, hid in its nest box, gaped and bared its teeth, cried shrilly or hissed; its posture was tense,

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showing severe emotional stress.

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Score – 2. Attack from the nest box. When attempts were made to catch the animal, it jumped to the

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entrance of the nest box and hid there to attack the experimenter’s gloved hand from the box and bit it

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fiercely.

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Score – 3. Active attack outside the shelter. When attempts were made to catch the mink, it

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immediately attacked the experimenter’s hand instead of hiding.

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Score – 4. Onset of attack in response to a human approaching. Even before the test began, i.e. before

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the experimenter opened the cage, the mink loudly shrieked, ran about the cage, and gnawed the bars.

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Animals with domesticated behavior did not show any signs of fear or aggression on contact with

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man, which allowed the experimenters to work barehanded. On the whole, the domestic behavior was

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defined as absence of fear or defense reaction towards man. The expression of domesticated behavior also varied qualitatively, making it possible to score

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it using a five-point scale as follows.

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Score +1. Exploratory responses. The mink calmly responded to the experimenter’s stretched hand

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and showed an exploratory response, sniffing the hand and quivering the vibrissae.

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Score +2. Calm response to contact with human hand. The mink accepted the forced contact with the

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experimenter’s hand and did not retreat, allowing the hand to touch its face, chest, and paws.

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Score +3. Active contact on the part of the animal. When the experimenter opened the cage, the mink

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got up leaning against the open door and reached out with its snout to the human hand. Within the

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cage, the mink actively sniffed about the hand, examined it, and often leant against the hand with its

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paws.

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Score +4. The mink allowed any part of its body to be touched. The animal displayed an active

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exploratory response, examined the experimenter’s hands, played with them, but resisted all attempts

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to be handled.

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Score +5. The mink allowed humans to handle it. These individuals displayed the most pronounced

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domesticated behavior, allowing the experimenters to handle them without showing any signs of fear

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or aggression.

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Selective breeding specific patterns of handler-directed behavior has been ongoing for 17

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generations based on the results of annual tests. The study reported here was performed on 5-mo-old

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male and female minks (n=105) selected for ‘domestic’, aggressive and fearful behavior. The control

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group included minks of the same age that had never been subjected to selective breeding. Animals

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were chosen at random from the population of minks scoring +1,+3, +4, +5 (‘domestic’ group), and

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from the population scoring -1, -2 (aggressive group). The sample did not include +2 and -3, -4 scored

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minks, since animals within these groups were too few. Тable 1 shows the numbers of animals in each

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group and their sex distribution.

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Table 1. Behavioral categories of minks used in the experiment. Behavioral categories N ‘Domestic’ Females Males +5 9 10 +4 8 9 +3 1 5 +1 2 Avoidant fear (0) 10 9 Aggressive -1 4 -2 9 8 Control (Non selected) 8 13

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All animals were homed in regulation cages on the same farm and given diet and water. All

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the experiments were conducted according to EU guidelines on the use of animals for biochemical

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research (86/609/EU).

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2.2. Blood sampling

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Blood samples were obtained from claw capillaries in the morning after overnight fasting.

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Blood smears were stained with May-Grünwald and Romanowski stains (MiniMed, Russia).

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Differential leukocyte counts were performed by identifying 200 leukocytes in each blood smear using

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light microscope Axioscop 40 (Zeiss, Germany) and computer software for image analysis

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“VideoTest” (VideoTest Ltd, Russia). All investigations were performed in accordance with the “Guide for the Care and Use of

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Laboratory Animals” published by the US National Institute of Health (NIH publication No. 85–23,

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1996). The study conforms to the principles outlined in the Helsinki Declaration (2000).

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The research was performed using the facilities of the Equipment Sharing Center of the

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Institute of Biology, Karelian Research Centre, Russian Academy of Sciences. 2.3. Statistical analysis

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All the collected and calculated numerical data were transformed into SI units and

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processed statistically as mean ± standard error of the mean (SEM). Differential leukocyte counts were

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evaluated by nonparametric Mann-Whitney U-test for comparison between behavioral categories. We

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conducted a multivariate analysis of variance (MANOVA) with differential leukocyte counts as

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response variables, and sex and behavior type as factors. Linear regression analyses were employed to

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evaluate the relationships between percentage values of eosinophil, neutrophil counts, neutrophil

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versus lymphocyte (N:L) ratio and expression of domestication behavior.

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The statistical analyses were performed using MS-Excel® (Microsoft Corp., Inc., USA), Sigma-Stat 2.03 (Systat Software Inc., Point Richmond, California, USA). 3. Results

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Figure 1 demonstrates two lines of selection for ‘domestic’ and aggressive behavior in

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standard (+/+) minks covering 17 generations. Selection for domestication showed a more complex

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pattern. The selection effect declined in the 5th and 6th generations: a substantial part of the progeny

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demonstrated aggressive reaction towards humans.

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Fig 1. Changes in the average behavior scores in 17 generations of minks selected for

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domestic and aggressive behavior. The patterns of selection for ‘domestic’ and aggressive behavior

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types differed from one another. The series of aggressive mink generations followed a uniform and

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smooth trend for increase of aggressiveness. The progeny of the minks selected for ‘domestic’

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behavior however included many individuals with aggressive reaction to humans in generations F5

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and F6.

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There were clear-cut distinctions in differential leukocyte counts between behavior types

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(see Table 2). Neutrophil and eosinophil counts significantly varied between groups. The eosinophil

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level in the blood of the minks that had not been specially selected was the lowest as compared to

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other behavioral types. The eosinophil counts in the peripheral blood of ‘domestic’ (+3, +5) minks

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were higher than in control and aggressive (–2) minks. In addition to ‘domestic’ minks, animals from

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group –1 also demonstrated elevated eosinophil level compared to control and aggressive (–2) minks.

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Animals showing cowardice or avoidance reactions (0) yielded increased eosinophil counts compared

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to the control. The neutrophil level was lower in the blood of ‘domestic’ minks and minks from group

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–1 as compared to aggressive (–2) minks. Animals from group +4 delivered the highest monocyte

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counts, whereas individuals from group +5 had the lowest monocyte counts. The highest basophil

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level was detected in the blood of the most aggressive minks (–2). No significant differences in the

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lymphocyte and band neutrophil counts were found among minks of different behavior categories. Therefore, we performed multivariate analysis of variance (MANOVA) with differential

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leukocyte counts as response variables, and sex and expression of domesticated behavior as factors

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(Table 3). MANOVA revealed influence of sex on percentage values of monocytes and basophils. The

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percentage values of neutrophils, basophils and values of neutrophil/lymphocyte ratio were influenced

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by the expression of domesticated behavior, while there was no sex-related effect on lymphocytes and

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eosinophils.

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Linear regression analysis revealed significant positive correlation between expression of

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domesticated behavior and percentage values of eosinophils (Fig. 2C and 2D). Negative correlation

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between expression of domesticated behavior and neutrophils number was observed (Fig. 2A and 2B).

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The expression of domesticated behavior and values of N:L ratio were also negatively correlated with

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each other (Fig. 2E and 2F). No significant correlations were observed between expression of

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‘domestic’ behavior and percentage values of eosinophils in females and expression of domesticated

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behavior scores and values of N:L ratio in males (Fig. 2D and 2F).

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Fig. 2. Correlations between expression of ‘domesticated’ behavior scores and percentage of neutrophils (A, B), eosinophils (C, D) and neutrophil/lymphocyte ratio (E, F).

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The Graphs A, C, E show average data for males and females (♦

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B shows linear regression for females (●

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excluded females (no significant correlation, p>0.05), Graph F excluded males (no significant

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correlation, p>0.05).

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4. Discussion

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Our study has demonstrated that leukocyte profiles vary in minks from different behavioral

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categories. Selection for domestication significantly increased eosinophil content in ‘domestic’ minks.

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The lowest eosinophil counts were revealed in control and aggressive “-2” animals, whereas elevated

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eosinophil level was noted in ‘domestic’ and slightly aggressive “-1” minks. Experiments of Hansen

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and Damgaard (1993) showed that initial eosinophil level in beech martens before an acute stress

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event was higher in aggressive animals than in slightly aggressive ones. However, after an acute stress

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event, eosinophil levels were significantly lower in aggressive animals compared to slightly aggressive

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ones. Hansen and Damgaard (1993) explained this difference by the fact that in slightly aggressive

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beech martens eosinophils returned to the initial normal level sooner than in aggressive animals. There

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are contradictory findings concerning the effect of chronic stress on eosinophil counts. Denial of

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access to a nest box in silver foxes (Jeppesen and Pedersen, 1991) and repeated immobility stress in

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mink (Heller and Jeppesen, 1985) are reported to be accompanied by increased eosinophil counts, after

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initial decreases. Eosinophil counts were however reduced when minks were deprived of the use of

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nest box and immobilized in a mink trap for 30 min daily over 10 days (Hansen and Damgaard, 1991).

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The different leukocyte counts in ‘domestic’ and aggressive minks are promoted via HPAS. It

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has been shown in numerous experiments that HPAS controls physiological reaction to acute stress.

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The blood eosinophil level is a hormone-dependent parameter, and taken together with

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neutrophil/lymphocyte ratios it may help distinguish between leukocyte responses to stress. Both

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emotional (Kerr, 1956) and physiological stress, such as long lasting cold (Denison and Zarrow, 1954)

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or immobilization (Heller and Jeppesen, 1985), have been proved to reduce the eosinophil level.

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Dhabhar et al. (1995, 1996) and López-Olvera et al. (2007) demonstrated in studies on humans and

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mammals that glucocorticoid-induced stress leads to a reduction in eosinophil numbers. Sabag et al.

), p<0.05. Graph D

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) and males (■

both sexes), the Graph

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(1978) reported cortisol-induced migration of eosinophils from the blood to spleen, lymph nodes, and

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thymus, and experimental steroid eosinopenia. As revealed in our study neutrophil counts were also dependent on animal behavioral types.

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The most aggressive minks were characterized by elevated neutrophil levels contrary to ‘domestic’

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ones. Both stress and glucocorticoid treatment caused the number of neutrophils to increase and the

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number of lymphocytes to decrease (Davis et al., 2008). In studies devoted to the effect of treatment

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with cortisol or synthetic glucocorticoids, such as hydrocortisone, dexamethasone or prednisolone,

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these agents produced neutrophilia or lymphopenia, or both, in rats (Cox and Ford 1982), mice (Van

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Dijk et al., 1979), guinea pigs (Fauci, 1975), humans (Fauci and Dale, 1974; Dale et al., 1975), etc.

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Similar research has been conducted using adrenocorticotrophic hormone (ACTH), which stimulates

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glucocorticoids release from adrenal glands. ACTH treatment raised neutrophil/lymphocyte ratio in

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boars (Bilandzic et al., 2006) and horses (Rossdale et al., 1982).

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Some researchers demonstrated that leukocyte response to stress may be distinct between wild

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and domestic mammals. Burguez et al. (1983) showed that injections of cortisol did not significantly

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elevate neutrophil/lymphocyte ratios until 2 h after injection in foals and 4 h later in adult horses. In

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contrast to these studies on domestic mammals, more recent work shows that the leukocyte response

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may occur on a more rapid time scale in wild mammals. In a study with wild ungulates, neutrophil

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numbers were found to double, and lymphocytes reduced by half, within 1 h of capture (López-Olvera

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et al., 2007).

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Studies concerning different leukocyte counts, response of leukocytes to stress, HPAS activity

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in wild and domestic mammals are not so numerous. It is likely that leukocyte profiles should be

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different in wild and domestic mammals; however information regarding this is limited and difficult to

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analyze. Schulte-Hostedde et al. (2012) found that differences in spleen mass between wild and

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domestic mink, and between feral domestic mink and domestic mink on farms are the result of

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discrepancy in the prevalence and abundance of parasites and pathogens. Wild animals are widely

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exposed to many parasites and pathogens that are less prevalent in the domestic environment because

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of veterinary care.

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We found no differences in lymphocyte levels in minks of different behavioral categories.

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Similarly, Delehantry and Boonstra (2009) found no statistically significant change in lymphocytes in

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squirrels under capture-induced stress. Song et al. (1994) showed that in contrast to neutrophil

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phagocytosis and WBC count, lymphocyte proliferation did not appear to be as sensitive to acute

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behavioral stress. They indicate that there is no close relationship between corticosterone and

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lymphocyte proliferation.

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Others (Miller, 1998) have reported a close relationship between neuroendocrinal and immune

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systems. Selection for domestication and aggressiveness affects differential blood leukocytes content

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and appears to be related to modification of HPAS activity, as reported by others (Veenema et al.,

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2005). Handled animals had lower levels of cortisol than the unhandled control group in experiments

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with silver fox (Pedersen, 1994). Aggressive rat males were much more sensitive even to a soft

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stressor, since the concentration of corticosterone increased more significantly and changes of

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differential leukocyte count appeared earlier and were more expressed than in tame animals. Low-

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aggression mice responded to stress with a higher and longer increase in the plasma corticosterone

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level in comparison to aggressive mice (Kolesnikova and Oskina, 2003).

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Kudryavtseva (2006) o showed that in male mice with long experience with aggression, the

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dopaminergic system was activated and the serotoninergic system inhibited. These alterations in the

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monoaminergic system activity in the brain are accompanied by changes in the activity of certain

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genes in brain regions. Selection for low aggressiveness to humans is known to promote the activity of

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the serotonin system and perhaps of other mediator systems of the mink brain, which decreases the

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activity of the animals. Modification and subsequent hereditary reorganization of this and other

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mediator systems of the brain lead to various functional changes (Naumenko et al., 1987; Nikulina et

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al., 1985).

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We found selection for behavior in minks to strongly influence differential blood leukocyte

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counts. Among the leukocyte types most sensitive to selection for behavior are neutrophils and

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eosinophils. ‘Domestic’ and aggressive minks had significant differences in differential leukocyte

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count: the greatest changes were observed in ‘domestic’ mink. The results of this study suggest that

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differential leukocyte counts differ in aggressive and ‘domestic’ minks, and might be related to HPAS

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activity. We have concluded that differences in differential leukocyte counts in mink selected for

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behavioral traits reflect alterations of the neuroendocrinal regulation system.

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Conflict of interest

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None.

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Acknowledgments

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The study was carried out under state orders (project no. 0221-2014-001 and project no. 0324-

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2015-0004) and was partially funded by Russian Presidential grant for Leading Scientific Schools #

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1410.2014.4. This work was carried out in the Genetic Pools of Fur Animals and Livestock Center for

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Collective Use of the Institute of Cytology and Genetics of the Siberian Branch of the Russian

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Academy of Sciences and was supported by state contract VI.53.2.1 and the Russian Foundation for

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Basic Research (grant no. 13-04-00968-a).

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Table 2. Differential leukocyte counts (Mean±SEM) in Standard mink of different behavioral categories. Leukocyte types, % Monocytes Lymphocytes Band neutrophils Neutrophils Eosinophils Basophils

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+5 (n=19) 5.9±0.4**

‘Domestic’ line +4 (n=17) +3 (n=6) 7.9±0.6♦ 6.9±1.2

+1 (n=2) 6.3±0.7

0 7.6±0.6

Aggressive line -1 (n=4) -2 (n=17) 6.9±0.4 6.8±0.5

7.5±0.4

48.7±1.9

51.4±2.5

53.9±3.54

32.8±13.2

49.2±2.7

50.3±5.6

43.8±2.7

47.1±3.9

0.7±0.1 32.6±2.3◊ 12.1±1.9**◊ 0.2±0.1

2.1±0.9 29.7±1.7◊◊ 8.9±1.4* 0.1±0.5

0.4±0.3 24.6±4.5◊ 13.9±3.1*◊ 0.3±0.1

0.8±0.5 52.5±12.2 7.5±1.2 0.0±0.0

0.9±0.2 34.1±2.6 8.1±1.1* 0.2±0.1

0.8±0.2 28.5±2.3◊ 13.3±3.5*◊ 0.1±0.1

0.8±0.2 41.8±2.7 6.4±0.9 0.4±0.0*

1.1±0.2 38.6±3.9 5.5±0.4 0.1±0.0

(n=19)

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from “+5” group: ♦ p < 0.05 (Mann–Whitney U-test)

p < 0.01, significant differences

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Table 3. Multivariate analysis of variance (MANOVA) of differential leukocyte counts and neutrophil/lymphocyte ratio (N/L ratio).

Monocytes Lymphocytes Band neutrophils Neutrophils Eosinophils Basophils N/L ratio

Factor of influence

Behavior Type F 1.25 ns

2

η ,% 9.63 0.80 0.50 28.29 14.91 16.29 29.93

0.72 0.42 4.38 1.90 2.19 4.68

η

p

ns

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ns 0.04 ns 0.04 0.002

Sex F

2

p

6.72

6.09

0.02

15.11

1.94

ns

8.81 2.11 2.9×10-5 4.88 0.51

1.05 2.28 0.00 4.58 0.58

ns ns ns 0.04 ns

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◊◊

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0.01, significant differences from “-2” group: ◊ p < 0.05;

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(n=21)

Notes: Significant differences from control group (non selected): *p < 0.05; **p <

408

410

Control

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Table 1. Behavioral categories of minks used in the experiment.

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Behavioral categories N ‘Domestic’ Females Males +5 9 10 +4 8 9 +3 1 5 +1 2 Avoidant fear (0) 10 9 Aggressive -1 4 -2 9 8 Control (Non selected) 8 13

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Table 2. Differential leukocyte counts (Mean±SEM) in Standard mink of different behavioral categories.

+5 (n=19) 5.9±0.4**

‘Domestic’ line +4 (n=17) +3 (n=6) 7.9±0.6♦ 6.9±1.2

+1 (n=2) 6.3±0.7

0 7.6±0.6

Aggressive line -1 (n=4) -2 (n=17) 6.9±0.4 6.8±0.5

7.5±0.4

48.7±1.9

51.4±2.5

53.9±3.54

32.8±13.2

49.2±2.7

50.3±5.6

43.8±2.7

47.1±3.9

0.7±0.1 32.6±2.3◊ 12.1±1.9**◊ 0.2±0.1

2.1±0.9 29.7±1.7◊◊ 8.9±1.4* 0.1±0.5

0.4±0.3 24.6±4.5◊ 13.9±3.1*◊ 0.3±0.1

0.8±0.5 52.5±12.2 7.5±1.2 0.0±0.0

0.9±0.2 34.1±2.6 8.1±1.1* 0.2±0.1

0.8±0.2 28.5±2.3◊ 13.3±3.5*◊ 0.1±0.1

0.8±0.2 41.8±2.7 6.4±0.9 0.4±0.0*

1.1±0.2 38.6±3.9 5.5±0.4 0.1±0.0

(n=19)

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Leukocyte types, % Monocytes Lymphocytes Band neutrophils Neutrophils Eosinophils Basophils

Control (n=21)

Notes: Significant differences from control group (non selected): *p < 0.05; **p < 0.01,

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group: ♦ p < 0.05 (Mann–Whitney U-test)

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Table 3. Multivariate analysis of variance (MANOVA) of differential leukocyte counts and neutrophil/lymphocyte ratio (N/L ratio).

Monocytes Lymphocytes Band neutrophils Neutrophils Eosinophils Basophils N/L ratio

Factor of influence Behavior Type F 1.25 ns

η2, % 9.63

p

6.72

6.09

0.02

1.94

ns

0.80

0.72

ns

15.11

0.50 28.29 14.91 16.29 29.93

0.42 4.38 1.90 2.19 4.68

ns 0.04 ns 0.04 0.002

8.81 2.11 2.9×10-5 4.88 0.51

1.05 2.28 0.00 4.58 0.58

ns ns ns 0.04 ns

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Notes: ns=not significant (p > 0.05)

Sex F

η2

p

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Dependent variable

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ACCEPTED MANUSCRIPT Highlights •

Long-term selection for tame and aggressive behaviour changes differential blood leukocyte

counts. •

Both neutrophils and eosinophils among examined parameters are sensitive and dependent on

•

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behavior type. Differential blood leukocyte counts in mink selected for behavioural traits reflect alterations of

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the neuroendocrinal regulation system.