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Behavior of lambs at different ages during brief periods of increased sensorial isolation from their mothers
Accepted Manuscript Behavior of lambs at different ages during brief periods of increased sensorial isolation from their mothers Patricia Mora, Daniel Mota, Emilio Arch-Tirado, Patricia Roldán, Carlos VázquezCruz, Angélica M. Terrazas, Marcelino Rosas, Agustín Orihuela PII:
S1558-7878(17)30004-7
DOI:
10.1016/j.jveb.2017.09.004
Reference:
JVEB 1079
To appear in:
Journal of Veterinary Behavior
Received Date: 6 January 2017 Revised Date:
24 August 2017
Accepted Date: 6 September 2017
Please cite this article as: Mora, P., Mota, D., Arch-Tirado, E., Roldán, P., Vázquez-Cruz, C., Terrazas, A.M., Rosas, M., Orihuela, A., Behavior of lambs at different ages during brief periods of increased sensorial isolation from their mothers, Journal of Veterinary Behavior (2017), doi: 10.1016/ j.jveb.2017.09.004. 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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Behavior of lambs at different ages during brief periods of increased sensorial
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isolation from their mothers
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Patricia Moraa,f, Daniel Motab, Emilio Arch-Tiradoc, Patricia Roldánd, Carlos
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Vázquez-Cruze, Angélica M. Terrazasf, Marcelino Rosasg, Agustín Orihuelah*
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a,f
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Metropolitana Xochimilco-Iztapalapa-Cuajimalpa. México D.F. Calzada del Hueso 1100,
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Col. Villa Quietud, Delegación Coyoacán, C.P. 04960.
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b
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Doctorate Program in Biological Sciences and Health at Universidad Autónoma
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Stress Physiology and Farm Animal Welfare, Department of Animal Production and
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Agriculture, Universidad Autónoma Metropolitana, Ciudad de México. Calzada del Hueso
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1100, Col. Villa Quietud, Delegación Coyoacán, C.P. 04960.
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c
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México Xochimilco No. 289 Col. Arenal de Guadalupe, C.P. 14389.
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d
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Calzada de Tlalpan, No. 3016 y 3058, Col. Ex Hacienda Coapa, Coyoacán 04910
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e
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Superiores Cuautitlán, Cuautitlán Izcalli, Estado de México, CP 54714
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f
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Estudios Superiores Cuautitlán, Cuautitlán Izcalli, Estado de México, CP 54714
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g
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Estudios Superiores Cuautitlán, Cuautitlán Izcalli, Estado de México, CP 54714
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h
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Universidad 1001. Col. Chamilpa. Cuernavaca, Morelos. C. P. 62209
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*Corresponding author. E-mail address: [email protected] (A. Orihuela).
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Laboratory of Bioacoustic at National Rehabilitation Institute, Ciudad de México. Calz.
Universidad del Valle de México, Medicina Veterinaria y Zootecnia, Ciudad de México,
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Computer Center, Universidad Nacional Autónoma de México, Facultad de Estudios
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Livestock Sciences Department, Universidad Nacional Autónoma de México, Facultad de
Biological Sciences Department, Universidad Nacional Autónoma de México, Facultad de
Facultad de Ciencias Agropecuarias, Universidad Autónoma del Estado de Morelos. Av.
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ABSTRACT
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To determine the behavior of different-aged lambs during short periods of
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increased sensorial isolation from their mothers, 60 ewe-lamb pairs were randomly
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assigned to one of the following groups: “C”, intact lambs that remained with their
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mothers; “A”, lambs with restricted auditory capacity; “A+V”, lambs with restricted
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auditory and visual capacities; “A+V+W”, lambs with restricted auditory and visual
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capacities plus separation from their mothers by an open wire mesh; and,
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“A+V+S”, lambs with restricted auditory and visual capacities plus separation from
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their mothers by a solid wall. These treatments were repeated in the same animals
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when lambs were 3 (G3), 10 (G10) and 20 (G20) days old. The behavior of lambs
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was recorded during each 15-min separation period, and data were examined
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using covariance analysis to determine differences for repeated measures over
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time. In general, after the physical separation from the ewes (A+V+W and A+V+S
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groups) lambs emitted more vocalizations (3, 10 and 20 days old), displayed more
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frequent urination (3 days old), exploration of objects (20 days old) and
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conspecifics (3 and 20 days old), escape attempts (A+V+W at 10 and 20 days of
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age; A+V+S at 20 days old), or walking (A+V+W at 10 and 20 days of age; A+V+S
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at 3, 10 and 20 days old) and lying down (10 and 20 days old for A+V+W and 3
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days old for A+V+S) in comparison to the other treatments. G10 and G20 lambs of
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the A+V, A+V+W and A+V+S groups also displayed greater values for walking
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activity (P<0.05) in comparison with G3 lambs. In general, it is concluded that the
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exhibition of the examined behavioral parameters was increased with the degree of
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sensorial isolation, and particularly when the physical contact between the lambs
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and their dams was restricted. As lambs became older, they showed an increased
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behavioral reactivity in response to the increased sensorial isolation.
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Keywords: Stress; Attachment; Separation; Welfare; Sheep; Ewe
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Introduction
Lambs stay with their mothers during the first weeks after birth (Morgan et al.,
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1974) when ewes display various signals that trigger specific behavioral patterns in
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their newborns. In turn, young lambs exhibit a variety of signals that induce care-
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giving responses from their mothers (Terrazas et al., 2002; Rödel, et al., 2013).
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Efficient communication between ewe and lamb is crucial for the survival and
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development of the lambs. This communication includes acoustic, olfactory, visual
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and tactile stimuli. The ewe-lamb bond is reinforced after birth through their
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interaction during the first few minutes postpartum and this early period appears to
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be critical for the survival of the lambs.
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communication stimuli, Morgan et al. (1974) concluded that ewes recognize their
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lambs primarily by sight, hearing and smell. Other studies have demonstrated that
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newborn lambs also use acoustic signals to catch the ewes’ attention or alert their
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conspecifics from dangerous situations (Rödel et al., 2013). In addition, ewes tend
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to keep their lambs in close proximity for several days after birth and offer the care
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they require, a behavior that intensifies their bond. At first, this bond seems
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stronger for the ewe, but decreases as the lamb grows, while in the lambs the
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opposite is true (Maldonado et al., 2015), though individual variation exists
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(Galeana et al., 2007).
After studying some of those
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Sheep are, by nature, gregarious animals, so isolation is perceived as a threat
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to their homeostasis (Moberg, 2000), learning (Bouissou and Gaudioso, 1982), and
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emotional reactivity (Doyle et al., 2009), in such an extent that any restriction
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during the early stages of their life may have consequences on several behavioral,
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hormonal and immunological indicators (Orgeur et al., 1998; Orihuela et al., 2004).
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As a result, when separated, both ewes and lambs manifest distress reactions that
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are also followed by adrenal responses. Appeasement of the distress is obtained
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only upon being reunited with the attachment figure, i.e., the ewe (Nowak et al.,
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2011). Alexander (1977) found that when lambs are separated from their mothers
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even temporarily, they show clear signs of stress, including a higher frequency of
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bleating and increased locomotion, two behavioral parameters that demonstrate
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their efforts to maintain contact with the ewes by locating, encountering and
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identifying them. Moreover, Hinch et al. (1990) observed a general increase in the
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spatial distance between family members at older ages, a finding that was
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interpreted as reflecting a gradual decline in the intensity of attachment over a
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period of 2.5 years.
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Despite the conclusions of the former studies, our understanding regarding the
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behavioral consequences of short periods of restriction on the sensorial signals
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displayed by dams and nursing lambs is still relatively poor. This information could
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be of practical importance since in several management practices, lambs are
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separated from their mothers during short periods of time at early ages (Kent et al.,
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1998; Mellor and Stafford, 2000). Thus, our objective was to determine the
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behavior of nursing lambs subjected to different sensorial impairment at different
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ages. In detail, two hypotheses were tested: 1) do lambs manifest greater
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behavioral reactivity as the degree or intensity of sensorial isolation increases? and
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2) are younger lambs less affected by the separation from their dams?
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Materials and methods
The study was conducted at a research station in the National Autonomous
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University of Mexico located at 99°11´42´´ W and 19 °41´35´´ N, with a mean
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temperature of 15.7 °C.
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Animals
Sixty multiparous Columbia ewes aged 2-5 years old, and their single born
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male lambs, were housed permanently under semi-intensive handling conditions at
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a density of 10 m2/animal. Diet consisted of alfalfa hay, commercial concentrate
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mix, and minerals according to the animals’ physiological needs. Water was ad
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libitum offered. In addition, from 10:00 to 16:00 h, ewes were left to pasture in
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alfalfa meadows.
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After birth, the ewes remained with their newborns as a group in a single
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pen. The experiment consisted of 3 phases according to the lambs’ age: 3, 10 and
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20 days. At 48 h of age, lambs were ear marked, sexed and each ewe-lamb pair
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was identified by painting a number with a non-toxic aerosol on their sides. This
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number indicated the treatment group to which they were randomly assigned. In
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case of the 10- and 20-day-old lambs, 24 h before testing, ewe-lamb pairs were re-
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marked.
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Experimental groups All lambs were tested between 9:00 and 10:00 h for 15 min at the ages of 3
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(G3), 10 (G10) and 20 (G20) days. They were randomly allocated into 5 treatment
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groups (n=12 ewe-lamb pairs each). In the Control group, “C”, ewes and lambs
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remained in constant contact in the same pen, free to generate and receive all
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sensorial stimuli. In group “A”, lambs were only subjected to auditory restriction,
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where the handler placed cotton plugs in the lambs’ ears, which were then covered
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with a headband to reduce perception of acoustic stimuli. In group “A+V”, lambs
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were subjected both to auditory + visual restriction, where in addition to the
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previous treatment, lambs were also visually restricted to impede reception of
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visual stimuli. In group “A+V+W”, in addition to the auditory and visual restrictions,
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the lambs were physically separated from their ewes by an open wire mesh that
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divided the pen, while in the “A+V+S” group, lambs were treated as in the previous
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group but the open wire mesh was replaced by a solid wall.
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Behavioral observations
For each test, the 12 pairs of ewe-lambs were moved to a 4 X 4 m pen located
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100 m from their home pen. Preparation of the lambs according to its respective
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restriction regimen lasted less than 3 min. All preparatory work was performed by
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the same person to avoid variations in handling, and after this period, behavioral
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parameters were recorded for a 15 min period. At the end of the observation
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period, the lambs returned to their dams.
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The behavioral responses of the lambs were continuously recorded using 2
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portable SONY Handycam video cameras for later analysis. One camera was
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placed on top of the pen to provide a panoramic view of the entire available area,
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while the second one provided a lateral view of the pen. Videos were analysed
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using Observer TM XT Software, version 10.0 (Noldus IT, The Netherlands). The
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examined behavioral variables were: the number of vocalizations, urinations and
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defecations, exploration of objects, exploration of conspecifics, escape attempts,
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number of walking and lying episodes, defined as in the ethogram illustrated in
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Table 1.
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Statistical analysis
Descriptive statistics were obtained for all the examined variables using the
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SAS statistical package (PROC MEANS) (SAS, 2004). Normality tests were
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performed for all variables in all groups and the 3 ages, using the SAS 9.2 program
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(PROC UNIVARIATE) (SAS, 2004). Since the study analysed the responses of the
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same lambs at three ages, data were assessed using a mixed model appropriate
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for repeated measures (SAS, 2004). The model included the treatment, the time,
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the replication of each treatment, as well as the interaction between treatments and
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time, and the interaction between treatments and replication for each treatment as
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fixed effects. The lamb in each treatment and repetition was considered as a
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random effect. Differences between treatments and time were detected using the
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Tukey-Kramer test. In all cases, the level of significance was set at α=0.01. The
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researchers who conducted the assessments and gathered the study results were
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unaware of the treatments and did not participate in selecting the animals or data
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analysis; the researcher who performed the statistical analyses was also unaware
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of the treatments.
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Results
In general, after the physical separation from the ewes (A+V+W and A+V+S),
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lambs in at least one age group had greater values for vocalizations (3, 10 and 20
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days of age), displayed more frequent urination (3 days of age), exploration of
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objects ( 20 days of age), exploration of conspecifics (3 and 20 days of age),
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escape attempts (A+V+W at 10 and 20 days of age; A+V+S at 20 days of age), or
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walking (A+V+W at 10 and 20 days of age; A+V+S at 3, 10 and 20 days of age)
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and lying down (A+V+W at 10 and 20 days of age; A+V+S at 3 days of age)
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behaviors. In contrast, lambs in treatment groups C and A, showed no significant
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differences in vocal reactivity, urination, exploration of conspecifics, escape
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attempts or lying down. Depending on the degree of the applied restriction, the
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average frequency of the different examined behaviors varied according to the
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lambs’ age (Table 2).
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Vocalizations
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No differences were found in the number of vocalizations (P>0.01) emitted by
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the lambs in groups C and A, while lambs in A+V group had greater values of
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vocalization (P<0.01) compared to that of C and A groups. However, when 8
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physical contact with the ewe was restricted, the number of vocalizations increased
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(P<0.01) even more (Table 2). Regarding the age, the lambs in C and A groups showed no differences in the
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number of vocalizations emitted, but the A+V lambs at the age of 10 and 20 days
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vocalized more than at the age of 3 days (P<0.01). In addition, in A+V+S group,
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lambs emitted on average 19 more vocalizations at 10 than 3 and 20 days of age
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(P<0.01).
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Urination
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Urination was the behavior performed least frequently, but lambs in A+V+W
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and A+V+S groups urinated more often than those in C, A and A+V at the age of 3
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days old (P<0.01; Table 2). No significant differences were found among the
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experimental groups at the age of 10 and 20 days. No defecation was observed
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during the 15 min test.
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Exploration of objects
Values for exploration of objects were only significant higher in A+V+W and A+V+S compared to the other groups at the age of 20 days (Table 2).
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Exploration of conspecifics
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A+V+W lambs explored their conspecifics 2.1-to-4.2 times more than controls.
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A+V+W and A+V+S lambs explored their conspecifics less when separated from
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their ewes at the age of 3 and 10 compared to 20 days of age (P<0.01; Table 2). 9
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Escape attempts Escape attempts were also rarely observed, but significant differences (P<0.01)
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appeared when the lambs were physically separated from their mothers (A+V+W
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and A+V+S) at the age of 20 days, while no differences were observed at the age
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of 3 days among the experimental groups (P>0.01; Table 2).
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Walking
Lambs in groups A+V+W and A+V+S walked more than those in C, A and A+V
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groups at 10 and 20 days old (P<0.01). In addition, the highest value for walking
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was observed in A+V+S group at the age of 10 days (P<0.01; Table 2).
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Lying down
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No significant differences were observed regarding this behavior among C, A
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and A+V groups at all the examined ages (G3, G10 and G20). However, A+V+W
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lambs at the age of 10 and 20 days and A+V+S lambs at 3 days old, had higher
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values for lying down than that of C, A and A+V groups (P<0.01; Table 2).
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Discussion
The present study further contributes in the understanding of the behavior of
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lambs at early ages as a result of a brief separation period from their dams and
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when different sensorial stimuli were restricted. The observed annoyance reactions
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coincide with previous studies of LeDoux (2012) and Panksepp (2005), who noted 10
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that when ewe-lamb pairs are abruptly and prematurely separated, both ewes and
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lambs display alerting behaviors. Results show that the majority of behavioral
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parameters assessed in this study were increased with the degree of sensorial
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isolation, particularly when lambs were physical isolated from their dams. Also our
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observations suggest that these differences are age dependent as older lambs
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showed greater behavioral reactivity in response to a severe sensorial isolation
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and separation from their mothers. Habituation could play an important role in the
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display of stress over time, since constant periods of temporary separation could
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minimize the negative effects of weaning. However, lambs in the present study
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displayed increased signs of stress in subsequent separations, suggesting that the
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perceived stress was so intense that overcame habituation (or there was no
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habituation at all).
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Vocal communication is a key factor in the mutual recognition of ewes and
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their lambs (Carter et al., 1992; Terrazas et al., 2003). Marchant-Forde et al.
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(2002) demonstrated similar results in cattle by showing that cows respond to their
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offspring’s vocalizations by suspending other activities or react to those calls by
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moving their ears, head and/or body, and then walk in the direction of the
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vocalizations emitted by their calves. Similarly, Pérez-Torres et al. (2006) observed
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that calves vocalized more during separation from their dams than in previous and
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subsequent periods.
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The increased number of vocalizations by the lambs in all age groups when
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were physically separated from the ewe confirm the hypothesis that ewes and
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lambs establish a strong bond immediately after birth (Poindron and Le Neindre, 11
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1980) and that vocalization is the primary mechanism of communication when
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separation occurs, since it represents a mechanism of informing the mother for a
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potential danger of the lamb. Usually ewe-lamb pairs remain in very close contact
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during the first 4 weeks of life (Morgan et al., 1974). As a consequence, any
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separation of the lamb from its mother before the natural weaning age, causes
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vocalizations by the lamb in an attempt to catch the mother’s care and attention
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(Alexander, 1978; Weary et al., 2008). This auditory stimulus may also be
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associated with other emotions, such as anxiety (Ross and Young, 2009).
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The increase in the number of vocalizations emitted by the lambs subjected
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to increased auditory and visual restriction in A+V compared to A and C groups
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suggests that vision might be more important than other senses for the detection of
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the ewes by the lambs, although this conclusion is partially correct, since in the
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present experiment auditory stimuli were only restricted and not totally blocked.
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However, previous findings suggest that most lambs will find their own mothers by
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using vocal cues if visual cues are not available (Shillito, 1975). In totally
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anacoustic lambs, results might be different, as Arch-Tirado and Collado-Corona
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(2002) demonstrated in their study with normal-hearing and deaf new-borns
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Guinea pigs, where total loss of hearing increased vocalizations in an attempt to
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catch mother’s attention, results that are similar to those of Zito et al. (1977) in
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lambs.
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Despite these changes in vocalization frequency caused by impairment of
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auditory and visual stimuli, our experiment showed that as long as there was a
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physical contact between the ewes and the lambs, the frequency of these 12
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behaviors was lower compared to that displayed after their separation, highlighting
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the importance of the physical bond, at least for the age groups used in this study.
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Insel and Winslow (1991) described the presence of the ewe as a “calming” factor
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that mediates the release of oxytocin by modulating the behavioral response of the
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lamb. This could explain the low average number of vocalizations in C, A and A+V
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compared with A+V+W and A+V+S groups, where the physical separation of the
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ewe triggered an increased frequency of vocalizations. Similar results have been
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found in new-borns mice, where separation increased the emission of ultrasonic
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vocalizations in response to isolation from their dams (Ross and Young, 2009).
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In general, frequency of the exhibited behaviors displayed by lambs with
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restricted hearing and vision (A+V) was similar to that of the control lambs for all
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ages (apart from vocalizations at all ages and walking at 10 and 20 days of age).
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These findings seem to confirm the hypothesis that the tactile stimulation provided
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by the close physical contact with the ewe could inhibit many of the cerebral
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changes that occur when the signals that lambs exchange with their dams are no
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longer discernible. Levine (2001) described this phenomenon after a thorough
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review of the factors that are involved in the development of the hypothalamic-
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pituitary-adrenal axis in rats. As a result, the presence of the ewe is considered an
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important factor that reduces the stress that can be experienced by the lambs
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when visual and auditory interaction are restricted or eliminated. Contrary to all
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these observations, including ours in this experiment, Simitzis et al. (2012) found
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no discernible effects of temporary deprivation of the mother on manifestations of
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the stress induced on the behavior of lambs. A possible explanation for these
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discrepancies between the present study and that of Simitzis et al. (2012) is the
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difference in the age of tested lambs and in the applied experimental design. In our
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case, lambs were very young -in the pre-weaning stage- and the direct effects of
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ewe deprivation were tested, while in case of Simitzis et al. (2012), lambs were
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subjected to isolation at an early age (3rd of 4th week) but the long term effects of
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this treatment were examined later at an age of total independence from their
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mothers (2 and 3 months old).
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The behavioral parameters categorised in the present study as walking and
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escape attempts, are similar to that used by Cockram et al. (1993), who examined
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the repeated effect of separating lambs by quantifying stress-induced behavioral
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responses. The above authors suggested that the study of the behavioral
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adjustments is a more reliable estimation of sheep’s response to an aversive
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stimulus, because they allow direct detection of behavioral reactivity (Cockram et
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al., 1993).
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Urination, as a response to stress, was very infrequent to be of meaningful
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significance, even though younger lambs showed greater urination episodes than
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older ones. However, it must be considered that urination might be affected by the
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amount of liquid in the bladder during the stress period, and the observation time
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during this experiment was of too short duration (15 min) for the proper evaluation
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of this parameter.
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Our findings show an increase in the behaviors of exploring objects and
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conspecifics, when lambs were separated from their mother at 3 and 20 days of
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age, respectively. Lambs at an early age depend on familiar surroundings to stay 14
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calm (Brennan and Kendrick, 2006), and when they are isolated from them, they
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initiate activities either for finding their mothers (probably by odour) or for becoming
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familiar with their new surroundings. The average frequency of escape attempts and walking behaviors observed
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in our study tended to be higher in older lambs that were subjected to ewe
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deprivation. These behaviors form part of the lamb’s seeking on their mother
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(Panksepp, 2005) as a result of unexpected isolation which in turn causes an
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anxiety response. This response has been shown to be mediated by the release of
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epinephrine, which triggers the “fight-or-flight” response as an alerting mechanism.
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Any factor that disturbed the homeostasis of 10 and 20 days old lambs, such as
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separation from the ewe in our study, would lead to greater manifestations of
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behaviors that reflect phases of the “fight-or-flight” response involving locomotor
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activity, particularly in animals with a more developed locomotor system.
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Another important characteristic of the ewe-lamb bond is that sheep belongs
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to the “follower” animals (Lent, 1974; Walser, et al. 1981; Pillot et al., 2010). This
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means that under normal conditions they tend to follow their mothers all the time,
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and any movement is within a close range from their mothers. The results
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concerning the frequency of the walking behavior in the C group, are a
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confirmation of this hypothesis. The frequency of walking behaviors increased at
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the age of 10 and 20 days in the groups subjected to restrictions (A+V, A+V+W
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and A+V+S, apart from the age of 3 days in A+V and A+V+W groups). These
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findings are further supported by studies by Greiveldinger et al. (2009) and Vögeli
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et al. (2015), who correlated increased locomotion with anxiety behavior or
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restlessness in the presence of negative stimuli due to the release of epinephrine
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from the hypothalamic-pituitary-adrenal axis for restoring homeostasis.
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Conclusion It is concluded that: a) the frequency of the examined behavioral parameters
5
increases with the degree of the lambs’ sensorial isolation, especially when
6
physical contact with the ewes is restricted; and, b) at older ages, lambs show
7
greater reactivity in their behaviors under conditions of sensorial isolation.
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Conflict of interest
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None of the authors has any financial or personal relationship that could inappropriately influence in the content of the paper.
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Acknowledgements
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This study is part of the PhD by Patricia Mora-Medina was supported by a
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Doctoral scholarship from the CONACyT, México. Daniel Mota-Rojas, Agustín
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Orihuela, Angélica M. Terrazas-García and Emilio Arch-Tirado were supported as
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members of the Mexico´s National System of Researchers (SNI). Finally, we would
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like to thank Jesús Emilio Serrano Martínez for taking care of the animals and
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Miguel García-Winder for his review and comments to the manuscript.
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Ethical statement
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The animals were handled humanely throughout the study, and their care and
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all experimental treatments complied with norm NOM-062-ZOO-1999 of Mexico’s 16
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Department of Agriculture, Ranching, Rural Development, Fishing and Alimentation
2
for animal-based experimentation. The study was approved by the Internal
3
Committee for the Care and Use of Experimental Animals at the FES-Cuautitlán-
4
Universidad Nacional Autónoma de México (UNAM). No phase of the study
5
entailed a permanent interruption of the ewe-lamb bond, and no ewe or lamb was
6
injured during the experiment.
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Authorship statement
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The idea for the paper was conceived by Daniel Mota. The experiment was
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designed by: Daniel Mota, Emilio Arch-Tirado, Angélica M. Terrazas and Agustín
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Orihuela. The experiment was performed by: Patricia Mora, Patricia Roldán and
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Marcelino Rosas.The data were analyzed by Carlos Vázquez-Cruz. The paper was
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written by: Patricia Mora and Agustín Orihuela.
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stimuli in sheep living in different housing conditions. Front. Vet. Sci. 2, 9. Walser, E. S., Willadsen, S., Hague, P., 1981. Pair association between lambs of
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Table 1.
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Ethogram of the examined behavioural parameters during the 15 min period of sensory
Description
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Behaviour
Bleats (Centoducati et al., 2015).
Urination
Elimination of urine (Centoducati et al., 2015).
Defecation
Elimination of faeces (Centoducati et al., 2015).
Exploring
The snout is in contact with the wall, the ground of the pen, or any
objects
other object in the pen (Anderson et al., 2015).
Exploring
The snout is in contact with other lambs or ewes present in the
conspecifics
pen (Anderson et al., 2015).
Escape
The lamb is observed to press on the dividing mesh or to jump
attempts
over the walls of the pen. The lamb exhibits jumping and part of its
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Vocalization
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body contacts the ground, or it runs a fast race to no apparent destination (Anderson et al., 2015). Elevating at least two feet simultaneously and moving the body
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Walking
rapidly or slowly in any direction; leaving the assigned area
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(Anderson et al., 2015). Each step was counted.
Lying down
Lambs lying down with their body resting on the sternum with the abdomen slightly inclined to one side and the forefeet and at least one hind foot flexed under its trunk (Centoducati et al., 2015). Each change in position from standing to lying was counted.
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restriction
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Table 2.
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Frequency of the behavioural parameters displayed by 3, 10 and 20 days-old
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lambs during a 15-min separation from their dams, under 5 different sensory
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isolation conditions
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AGE (days) Behaviour.
Group
1.8 ± 2.8 a,1
0.1 ± 3.1 a,1
14.5 ± 2.8 a,2
24.6 ± 3.3 b,2
24.8 ± 3.6 b,2
59.1 ± 2.8 b,3
50.7 ± 3.3 a,3
69.2± 3.6 c,3
54.6 ± 2.8 a,3
74.0 ± 3.3 b,4
54.2 ± 3.6 a,4
0.0 ± 0.1 a,1
0.0 ± 0.0 a,1
0.0 ± 0.0 a,1
A
0.1 ± 0.1 a,1
0.0 ± 0.0 a,1
0.1 ± 0.1 a,1
A+V
0.0 ± 0.1 a,1
0.0 ± 0.0 a,1
0.1 ± 0.1 a,1
A+V+W
0.3 ± 0.1 b,2
0.1 ± 0.0 a,1
0.1 ± 0.1 a,1
A+V+S
0.3 ± 0.1 b,2
0.0 ± 0.0 a,1
0.0 ± 0.1 a,1
C
3.1 ± 1.9 a,2
2.4 ± 1.6 a,2
2.0 ± 1.7 a,1
A
2.7 ± 1.5 a,2
3.1 ± 1.3 b,2
1.6 ± 1.3 a,1
A+V
0.2 ± 1.7 a,1
2.3 ± 1.5 a,2
1.8 ± 1.5 a,1
A+V+W
5.0 ± 1.7 c,2
4.3 ± 1.5 b,2
3.0 ± 1.5 a,2
A+V+S
4.2 ± 1.7,b,2
0.9 ± 1.5 a,1
4.8 ± 1.5 b,2
C
0.7 ± 1.1 a,1
1.1 ± 1.7 a,1
1.4 ± 1.8 a,1
A
1.0 ± 0.9 a,1
2.3 ± 1.3 a,1
1.1 ± 1.4 a,1
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1.3 ± 2.4 a,1
C
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1.2 ± 4.0 a,1
A
A+V+S
Exploration of objects
Exploration of conspecifics
0.6 ± 3.6 a,1
1.4 ± 3.8 a,1
A+V+W
Urinations
20
C
A+V
Vocalizations
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2.3 ± 1.5 b,1
2.6 ± 1.7 b,1
A+V+W
3.4 ± 1.0 a,2
3.3 ± 1.5 a,1
5.7 ± 1.7 b,2
A+V+S
2.6 ± 1.0 a,2
2.7 ± 1.5 a,1
3.3 ± 1.7 b,2
C
0.0 ± 0.0 a,1
0.0 ± 0.0 a,1
0.0 ± 0.2 a,1
A
0.0 ± 0.0 a,1
0.0 ± 0.0 a,1
0.0 ± 0.1 a,1
A+V
0.0 ± 0.0 a,1
0.0 ± 0.1 a,1
0.0 ± 0.2 a,1
A+V+W
0.0 ± 0.0 a,1
0.3 ± 0.1 b,2
2.4 ± 0.2 c,3
A+V+S
0.1 ± 0.0 a,1
0.0 ± 0.1 a,1
0.6 ± 0.2 b,2
C
9.4 ± 1.4 b,2
7.2 ± 2.7 a,1
5.7 ± 2.5 a,1
A
5.9 ± 1.1 a,1
9.5 ± 2.1 b,1
5.7 ± 2.0 a,1
7.0 ± 1.2 a,1
15.8 ± 2.5 b,2
12.3 ± 2.3 b,2
8.3 ± 1.2 a,1,2
31.4 ± 2.5 b,3
35.5 ± 2.3 b,3
35.0 ± 1.2 a,3
54.5 ± 2.5 b,4
36.0 ± 2.3 a,3
0.2 ± 0.5 a,1
0.1 ± 0.2 a,1
0.0 ± 0.2 a,1
A+V
Walking
A+V+W A+V+S
0.3 ± 0.4 a,1
0.1 ± 0.2 a,1
0.2 ± 0.1 a,1
A+V
0.0 ± 0.4 a,1
0.0 ± 0.2 a,1
0.0 ± 0.2 a,1
A+V+W
0.7 ± 0.4 a,1,2
0.6 ± 0.2 a,2
0.8 ± 0.2 a,2
A+V+S
1.4 ± 0.4 b,2
0.5 ± 0.2 a,1,2
0.3 ± 0.2 a,1
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A
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Lying down
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0.9 ± 1.0 a,1
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Escape attempts
A+V
2
a,b,c
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(P<0.01).
4
1,2,3
5
age (P<0.01).
Letters in the same row indicate differences among ages within the same treatment
Numbers in the same column indicate differences among treatments within the same
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C= Control; A= Auditory restriction*; A+V= Auditory + Visual restrictions*; A+V+W=
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Auditory + Visual restrictions + Wire mesh fence-line; A+V+S= Auditory + Visual
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restrictions + Solid fence-line restriction.
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Highlights:
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1. Behavioural responses increase with the degree of sensorial isolation.
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2. Older lambs show greater behavioural reactivity to sensorial isolation.
3. A short separation period should be promoted in very young lambs during husbandry practices.
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4. Regardless of age, physical separation induced the highest behavioural response.
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allowed.
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5. If lamb-ewe separation is necessary, some kind of contact should be
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S1558-7878(17)30004-7
DOI:
10.1016/j.jveb.2017.09.004
Reference:
JVEB 1079
To appear in:
Journal of Veterinary Behavior
Received Date: 6 January 2017 Revised Date:
24 August 2017
Accepted Date: 6 September 2017
Please cite this article as: Mora, P., Mota, D., Arch-Tirado, E., Roldán, P., Vázquez-Cruz, C., Terrazas, A.M., Rosas, M., Orihuela, A., Behavior of lambs at different ages during brief periods of increased sensorial isolation from their mothers, Journal of Veterinary Behavior (2017), doi: 10.1016/ j.jveb.2017.09.004. 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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Behavior of lambs at different ages during brief periods of increased sensorial
2
isolation from their mothers
3
Patricia Moraa,f, Daniel Motab, Emilio Arch-Tiradoc, Patricia Roldánd, Carlos
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Vázquez-Cruze, Angélica M. Terrazasf, Marcelino Rosasg, Agustín Orihuelah*
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a,f
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Metropolitana Xochimilco-Iztapalapa-Cuajimalpa. México D.F. Calzada del Hueso 1100,
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Col. Villa Quietud, Delegación Coyoacán, C.P. 04960.
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b
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Doctorate Program in Biological Sciences and Health at Universidad Autónoma
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Stress Physiology and Farm Animal Welfare, Department of Animal Production and
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Agriculture, Universidad Autónoma Metropolitana, Ciudad de México. Calzada del Hueso
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1100, Col. Villa Quietud, Delegación Coyoacán, C.P. 04960.
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c
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México Xochimilco No. 289 Col. Arenal de Guadalupe, C.P. 14389.
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d
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Calzada de Tlalpan, No. 3016 y 3058, Col. Ex Hacienda Coapa, Coyoacán 04910
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e
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Superiores Cuautitlán, Cuautitlán Izcalli, Estado de México, CP 54714
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f
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Estudios Superiores Cuautitlán, Cuautitlán Izcalli, Estado de México, CP 54714
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g
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Estudios Superiores Cuautitlán, Cuautitlán Izcalli, Estado de México, CP 54714
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h
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Universidad 1001. Col. Chamilpa. Cuernavaca, Morelos. C. P. 62209
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*Corresponding author. E-mail address: [email protected] (A. Orihuela).
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Laboratory of Bioacoustic at National Rehabilitation Institute, Ciudad de México. Calz.
Universidad del Valle de México, Medicina Veterinaria y Zootecnia, Ciudad de México,
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Computer Center, Universidad Nacional Autónoma de México, Facultad de Estudios
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Livestock Sciences Department, Universidad Nacional Autónoma de México, Facultad de
Biological Sciences Department, Universidad Nacional Autónoma de México, Facultad de
Facultad de Ciencias Agropecuarias, Universidad Autónoma del Estado de Morelos. Av.
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ABSTRACT
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To determine the behavior of different-aged lambs during short periods of
3
increased sensorial isolation from their mothers, 60 ewe-lamb pairs were randomly
4
assigned to one of the following groups: “C”, intact lambs that remained with their
5
mothers; “A”, lambs with restricted auditory capacity; “A+V”, lambs with restricted
6
auditory and visual capacities; “A+V+W”, lambs with restricted auditory and visual
7
capacities plus separation from their mothers by an open wire mesh; and,
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“A+V+S”, lambs with restricted auditory and visual capacities plus separation from
9
their mothers by a solid wall. These treatments were repeated in the same animals
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when lambs were 3 (G3), 10 (G10) and 20 (G20) days old. The behavior of lambs
11
was recorded during each 15-min separation period, and data were examined
12
using covariance analysis to determine differences for repeated measures over
13
time. In general, after the physical separation from the ewes (A+V+W and A+V+S
14
groups) lambs emitted more vocalizations (3, 10 and 20 days old), displayed more
15
frequent urination (3 days old), exploration of objects (20 days old) and
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conspecifics (3 and 20 days old), escape attempts (A+V+W at 10 and 20 days of
17
age; A+V+S at 20 days old), or walking (A+V+W at 10 and 20 days of age; A+V+S
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at 3, 10 and 20 days old) and lying down (10 and 20 days old for A+V+W and 3
19
days old for A+V+S) in comparison to the other treatments. G10 and G20 lambs of
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the A+V, A+V+W and A+V+S groups also displayed greater values for walking
21
activity (P<0.05) in comparison with G3 lambs. In general, it is concluded that the
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exhibition of the examined behavioral parameters was increased with the degree of
23
sensorial isolation, and particularly when the physical contact between the lambs
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and their dams was restricted. As lambs became older, they showed an increased
2
behavioral reactivity in response to the increased sensorial isolation.
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Keywords: Stress; Attachment; Separation; Welfare; Sheep; Ewe
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Introduction
Lambs stay with their mothers during the first weeks after birth (Morgan et al.,
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1974) when ewes display various signals that trigger specific behavioral patterns in
8
their newborns. In turn, young lambs exhibit a variety of signals that induce care-
9
giving responses from their mothers (Terrazas et al., 2002; Rödel, et al., 2013).
10
Efficient communication between ewe and lamb is crucial for the survival and
11
development of the lambs. This communication includes acoustic, olfactory, visual
12
and tactile stimuli. The ewe-lamb bond is reinforced after birth through their
13
interaction during the first few minutes postpartum and this early period appears to
14
be critical for the survival of the lambs.
15
communication stimuli, Morgan et al. (1974) concluded that ewes recognize their
16
lambs primarily by sight, hearing and smell. Other studies have demonstrated that
17
newborn lambs also use acoustic signals to catch the ewes’ attention or alert their
18
conspecifics from dangerous situations (Rödel et al., 2013). In addition, ewes tend
19
to keep their lambs in close proximity for several days after birth and offer the care
20
they require, a behavior that intensifies their bond. At first, this bond seems
21
stronger for the ewe, but decreases as the lamb grows, while in the lambs the
22
opposite is true (Maldonado et al., 2015), though individual variation exists
23
(Galeana et al., 2007).
After studying some of those
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Sheep are, by nature, gregarious animals, so isolation is perceived as a threat
2
to their homeostasis (Moberg, 2000), learning (Bouissou and Gaudioso, 1982), and
3
emotional reactivity (Doyle et al., 2009), in such an extent that any restriction
4
during the early stages of their life may have consequences on several behavioral,
5
hormonal and immunological indicators (Orgeur et al., 1998; Orihuela et al., 2004).
6
As a result, when separated, both ewes and lambs manifest distress reactions that
7
are also followed by adrenal responses. Appeasement of the distress is obtained
8
only upon being reunited with the attachment figure, i.e., the ewe (Nowak et al.,
9
2011). Alexander (1977) found that when lambs are separated from their mothers
10
even temporarily, they show clear signs of stress, including a higher frequency of
11
bleating and increased locomotion, two behavioral parameters that demonstrate
12
their efforts to maintain contact with the ewes by locating, encountering and
13
identifying them. Moreover, Hinch et al. (1990) observed a general increase in the
14
spatial distance between family members at older ages, a finding that was
15
interpreted as reflecting a gradual decline in the intensity of attachment over a
16
period of 2.5 years.
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Despite the conclusions of the former studies, our understanding regarding the
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behavioral consequences of short periods of restriction on the sensorial signals
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displayed by dams and nursing lambs is still relatively poor. This information could
20
be of practical importance since in several management practices, lambs are
21
separated from their mothers during short periods of time at early ages (Kent et al.,
22
1998; Mellor and Stafford, 2000). Thus, our objective was to determine the
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behavior of nursing lambs subjected to different sensorial impairment at different
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ages. In detail, two hypotheses were tested: 1) do lambs manifest greater
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behavioral reactivity as the degree or intensity of sensorial isolation increases? and
3
2) are younger lambs less affected by the separation from their dams?
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Materials and methods
The study was conducted at a research station in the National Autonomous
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University of Mexico located at 99°11´42´´ W and 19 °41´35´´ N, with a mean
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temperature of 15.7 °C.
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Animals
Sixty multiparous Columbia ewes aged 2-5 years old, and their single born
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male lambs, were housed permanently under semi-intensive handling conditions at
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a density of 10 m2/animal. Diet consisted of alfalfa hay, commercial concentrate
14
mix, and minerals according to the animals’ physiological needs. Water was ad
15
libitum offered. In addition, from 10:00 to 16:00 h, ewes were left to pasture in
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alfalfa meadows.
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After birth, the ewes remained with their newborns as a group in a single
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pen. The experiment consisted of 3 phases according to the lambs’ age: 3, 10 and
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20 days. At 48 h of age, lambs were ear marked, sexed and each ewe-lamb pair
20
was identified by painting a number with a non-toxic aerosol on their sides. This
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number indicated the treatment group to which they were randomly assigned. In
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case of the 10- and 20-day-old lambs, 24 h before testing, ewe-lamb pairs were re-
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marked.
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Experimental groups All lambs were tested between 9:00 and 10:00 h for 15 min at the ages of 3
4
(G3), 10 (G10) and 20 (G20) days. They were randomly allocated into 5 treatment
5
groups (n=12 ewe-lamb pairs each). In the Control group, “C”, ewes and lambs
6
remained in constant contact in the same pen, free to generate and receive all
7
sensorial stimuli. In group “A”, lambs were only subjected to auditory restriction,
8
where the handler placed cotton plugs in the lambs’ ears, which were then covered
9
with a headband to reduce perception of acoustic stimuli. In group “A+V”, lambs
10
were subjected both to auditory + visual restriction, where in addition to the
11
previous treatment, lambs were also visually restricted to impede reception of
12
visual stimuli. In group “A+V+W”, in addition to the auditory and visual restrictions,
13
the lambs were physically separated from their ewes by an open wire mesh that
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divided the pen, while in the “A+V+S” group, lambs were treated as in the previous
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group but the open wire mesh was replaced by a solid wall.
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Behavioral observations
For each test, the 12 pairs of ewe-lambs were moved to a 4 X 4 m pen located
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100 m from their home pen. Preparation of the lambs according to its respective
20
restriction regimen lasted less than 3 min. All preparatory work was performed by
21
the same person to avoid variations in handling, and after this period, behavioral
22
parameters were recorded for a 15 min period. At the end of the observation
23
period, the lambs returned to their dams.
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.
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The behavioral responses of the lambs were continuously recorded using 2
3
portable SONY Handycam video cameras for later analysis. One camera was
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placed on top of the pen to provide a panoramic view of the entire available area,
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while the second one provided a lateral view of the pen. Videos were analysed
6
using Observer TM XT Software, version 10.0 (Noldus IT, The Netherlands). The
7
examined behavioral variables were: the number of vocalizations, urinations and
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defecations, exploration of objects, exploration of conspecifics, escape attempts,
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number of walking and lying episodes, defined as in the ethogram illustrated in
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Table 1.
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Statistical analysis
Descriptive statistics were obtained for all the examined variables using the
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SAS statistical package (PROC MEANS) (SAS, 2004). Normality tests were
15
performed for all variables in all groups and the 3 ages, using the SAS 9.2 program
16
(PROC UNIVARIATE) (SAS, 2004). Since the study analysed the responses of the
17
same lambs at three ages, data were assessed using a mixed model appropriate
18
for repeated measures (SAS, 2004). The model included the treatment, the time,
19
the replication of each treatment, as well as the interaction between treatments and
20
time, and the interaction between treatments and replication for each treatment as
21
fixed effects. The lamb in each treatment and repetition was considered as a
22
random effect. Differences between treatments and time were detected using the
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Tukey-Kramer test. In all cases, the level of significance was set at α=0.01. The
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researchers who conducted the assessments and gathered the study results were
2
unaware of the treatments and did not participate in selecting the animals or data
3
analysis; the researcher who performed the statistical analyses was also unaware
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of the treatments.
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Results
In general, after the physical separation from the ewes (A+V+W and A+V+S),
8
lambs in at least one age group had greater values for vocalizations (3, 10 and 20
9
days of age), displayed more frequent urination (3 days of age), exploration of
10
objects ( 20 days of age), exploration of conspecifics (3 and 20 days of age),
11
escape attempts (A+V+W at 10 and 20 days of age; A+V+S at 20 days of age), or
12
walking (A+V+W at 10 and 20 days of age; A+V+S at 3, 10 and 20 days of age)
13
and lying down (A+V+W at 10 and 20 days of age; A+V+S at 3 days of age)
14
behaviors. In contrast, lambs in treatment groups C and A, showed no significant
15
differences in vocal reactivity, urination, exploration of conspecifics, escape
16
attempts or lying down. Depending on the degree of the applied restriction, the
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average frequency of the different examined behaviors varied according to the
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lambs’ age (Table 2).
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Vocalizations
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No differences were found in the number of vocalizations (P>0.01) emitted by
22
the lambs in groups C and A, while lambs in A+V group had greater values of
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vocalization (P<0.01) compared to that of C and A groups. However, when 8
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physical contact with the ewe was restricted, the number of vocalizations increased
2
(P<0.01) even more (Table 2). Regarding the age, the lambs in C and A groups showed no differences in the
4
number of vocalizations emitted, but the A+V lambs at the age of 10 and 20 days
5
vocalized more than at the age of 3 days (P<0.01). In addition, in A+V+S group,
6
lambs emitted on average 19 more vocalizations at 10 than 3 and 20 days of age
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(P<0.01).
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Urination
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Urination was the behavior performed least frequently, but lambs in A+V+W
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and A+V+S groups urinated more often than those in C, A and A+V at the age of 3
12
days old (P<0.01; Table 2). No significant differences were found among the
13
experimental groups at the age of 10 and 20 days. No defecation was observed
14
during the 15 min test.
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Exploration of objects
Values for exploration of objects were only significant higher in A+V+W and A+V+S compared to the other groups at the age of 20 days (Table 2).
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Exploration of conspecifics
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A+V+W lambs explored their conspecifics 2.1-to-4.2 times more than controls.
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A+V+W and A+V+S lambs explored their conspecifics less when separated from
23
their ewes at the age of 3 and 10 compared to 20 days of age (P<0.01; Table 2). 9
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Escape attempts Escape attempts were also rarely observed, but significant differences (P<0.01)
4
appeared when the lambs were physically separated from their mothers (A+V+W
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and A+V+S) at the age of 20 days, while no differences were observed at the age
6
of 3 days among the experimental groups (P>0.01; Table 2).
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Walking
Lambs in groups A+V+W and A+V+S walked more than those in C, A and A+V
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groups at 10 and 20 days old (P<0.01). In addition, the highest value for walking
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was observed in A+V+S group at the age of 10 days (P<0.01; Table 2).
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Lying down
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No significant differences were observed regarding this behavior among C, A
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and A+V groups at all the examined ages (G3, G10 and G20). However, A+V+W
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lambs at the age of 10 and 20 days and A+V+S lambs at 3 days old, had higher
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values for lying down than that of C, A and A+V groups (P<0.01; Table 2).
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Discussion
The present study further contributes in the understanding of the behavior of
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lambs at early ages as a result of a brief separation period from their dams and
22
when different sensorial stimuli were restricted. The observed annoyance reactions
23
coincide with previous studies of LeDoux (2012) and Panksepp (2005), who noted 10
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that when ewe-lamb pairs are abruptly and prematurely separated, both ewes and
2
lambs display alerting behaviors. Results show that the majority of behavioral
3
parameters assessed in this study were increased with the degree of sensorial
4
isolation, particularly when lambs were physical isolated from their dams. Also our
5
observations suggest that these differences are age dependent as older lambs
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showed greater behavioral reactivity in response to a severe sensorial isolation
7
and separation from their mothers. Habituation could play an important role in the
8
display of stress over time, since constant periods of temporary separation could
9
minimize the negative effects of weaning. However, lambs in the present study
10
displayed increased signs of stress in subsequent separations, suggesting that the
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perceived stress was so intense that overcame habituation (or there was no
12
habituation at all).
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Vocal communication is a key factor in the mutual recognition of ewes and
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their lambs (Carter et al., 1992; Terrazas et al., 2003). Marchant-Forde et al.
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(2002) demonstrated similar results in cattle by showing that cows respond to their
16
offspring’s vocalizations by suspending other activities or react to those calls by
17
moving their ears, head and/or body, and then walk in the direction of the
18
vocalizations emitted by their calves. Similarly, Pérez-Torres et al. (2006) observed
19
that calves vocalized more during separation from their dams than in previous and
20
subsequent periods.
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The increased number of vocalizations by the lambs in all age groups when
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were physically separated from the ewe confirm the hypothesis that ewes and
23
lambs establish a strong bond immediately after birth (Poindron and Le Neindre, 11
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1980) and that vocalization is the primary mechanism of communication when
2
separation occurs, since it represents a mechanism of informing the mother for a
3
potential danger of the lamb. Usually ewe-lamb pairs remain in very close contact
4
during the first 4 weeks of life (Morgan et al., 1974). As a consequence, any
5
separation of the lamb from its mother before the natural weaning age, causes
6
vocalizations by the lamb in an attempt to catch the mother’s care and attention
7
(Alexander, 1978; Weary et al., 2008). This auditory stimulus may also be
8
associated with other emotions, such as anxiety (Ross and Young, 2009).
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The increase in the number of vocalizations emitted by the lambs subjected
10
to increased auditory and visual restriction in A+V compared to A and C groups
11
suggests that vision might be more important than other senses for the detection of
12
the ewes by the lambs, although this conclusion is partially correct, since in the
13
present experiment auditory stimuli were only restricted and not totally blocked.
14
However, previous findings suggest that most lambs will find their own mothers by
15
using vocal cues if visual cues are not available (Shillito, 1975). In totally
16
anacoustic lambs, results might be different, as Arch-Tirado and Collado-Corona
17
(2002) demonstrated in their study with normal-hearing and deaf new-borns
18
Guinea pigs, where total loss of hearing increased vocalizations in an attempt to
19
catch mother’s attention, results that are similar to those of Zito et al. (1977) in
20
lambs.
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Despite these changes in vocalization frequency caused by impairment of
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auditory and visual stimuli, our experiment showed that as long as there was a
23
physical contact between the ewes and the lambs, the frequency of these 12
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behaviors was lower compared to that displayed after their separation, highlighting
2
the importance of the physical bond, at least for the age groups used in this study.
3
Insel and Winslow (1991) described the presence of the ewe as a “calming” factor
4
that mediates the release of oxytocin by modulating the behavioral response of the
5
lamb. This could explain the low average number of vocalizations in C, A and A+V
6
compared with A+V+W and A+V+S groups, where the physical separation of the
7
ewe triggered an increased frequency of vocalizations. Similar results have been
8
found in new-borns mice, where separation increased the emission of ultrasonic
9
vocalizations in response to isolation from their dams (Ross and Young, 2009).
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In general, frequency of the exhibited behaviors displayed by lambs with
11
restricted hearing and vision (A+V) was similar to that of the control lambs for all
12
ages (apart from vocalizations at all ages and walking at 10 and 20 days of age).
13
These findings seem to confirm the hypothesis that the tactile stimulation provided
14
by the close physical contact with the ewe could inhibit many of the cerebral
15
changes that occur when the signals that lambs exchange with their dams are no
16
longer discernible. Levine (2001) described this phenomenon after a thorough
17
review of the factors that are involved in the development of the hypothalamic-
18
pituitary-adrenal axis in rats. As a result, the presence of the ewe is considered an
19
important factor that reduces the stress that can be experienced by the lambs
20
when visual and auditory interaction are restricted or eliminated. Contrary to all
21
these observations, including ours in this experiment, Simitzis et al. (2012) found
22
no discernible effects of temporary deprivation of the mother on manifestations of
23
the stress induced on the behavior of lambs. A possible explanation for these
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discrepancies between the present study and that of Simitzis et al. (2012) is the
2
difference in the age of tested lambs and in the applied experimental design. In our
3
case, lambs were very young -in the pre-weaning stage- and the direct effects of
4
ewe deprivation were tested, while in case of Simitzis et al. (2012), lambs were
5
subjected to isolation at an early age (3rd of 4th week) but the long term effects of
6
this treatment were examined later at an age of total independence from their
7
mothers (2 and 3 months old).
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The behavioral parameters categorised in the present study as walking and
9
escape attempts, are similar to that used by Cockram et al. (1993), who examined
10
the repeated effect of separating lambs by quantifying stress-induced behavioral
11
responses. The above authors suggested that the study of the behavioral
12
adjustments is a more reliable estimation of sheep’s response to an aversive
13
stimulus, because they allow direct detection of behavioral reactivity (Cockram et
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al., 1993).
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Urination, as a response to stress, was very infrequent to be of meaningful
16
significance, even though younger lambs showed greater urination episodes than
17
older ones. However, it must be considered that urination might be affected by the
18
amount of liquid in the bladder during the stress period, and the observation time
19
during this experiment was of too short duration (15 min) for the proper evaluation
20
of this parameter.
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Our findings show an increase in the behaviors of exploring objects and
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conspecifics, when lambs were separated from their mother at 3 and 20 days of
23
age, respectively. Lambs at an early age depend on familiar surroundings to stay 14
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calm (Brennan and Kendrick, 2006), and when they are isolated from them, they
2
initiate activities either for finding their mothers (probably by odour) or for becoming
3
familiar with their new surroundings. The average frequency of escape attempts and walking behaviors observed
5
in our study tended to be higher in older lambs that were subjected to ewe
6
deprivation. These behaviors form part of the lamb’s seeking on their mother
7
(Panksepp, 2005) as a result of unexpected isolation which in turn causes an
8
anxiety response. This response has been shown to be mediated by the release of
9
epinephrine, which triggers the “fight-or-flight” response as an alerting mechanism.
10
Any factor that disturbed the homeostasis of 10 and 20 days old lambs, such as
11
separation from the ewe in our study, would lead to greater manifestations of
12
behaviors that reflect phases of the “fight-or-flight” response involving locomotor
13
activity, particularly in animals with a more developed locomotor system.
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Another important characteristic of the ewe-lamb bond is that sheep belongs
15
to the “follower” animals (Lent, 1974; Walser, et al. 1981; Pillot et al., 2010). This
16
means that under normal conditions they tend to follow their mothers all the time,
17
and any movement is within a close range from their mothers. The results
18
concerning the frequency of the walking behavior in the C group, are a
19
confirmation of this hypothesis. The frequency of walking behaviors increased at
20
the age of 10 and 20 days in the groups subjected to restrictions (A+V, A+V+W
21
and A+V+S, apart from the age of 3 days in A+V and A+V+W groups). These
22
findings are further supported by studies by Greiveldinger et al. (2009) and Vögeli
23
et al. (2015), who correlated increased locomotion with anxiety behavior or
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restlessness in the presence of negative stimuli due to the release of epinephrine
2
from the hypothalamic-pituitary-adrenal axis for restoring homeostasis.
3
Conclusion It is concluded that: a) the frequency of the examined behavioral parameters
5
increases with the degree of the lambs’ sensorial isolation, especially when
6
physical contact with the ewes is restricted; and, b) at older ages, lambs show
7
greater reactivity in their behaviors under conditions of sensorial isolation.
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Conflict of interest
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None of the authors has any financial or personal relationship that could inappropriately influence in the content of the paper.
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Acknowledgements
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This study is part of the PhD by Patricia Mora-Medina was supported by a
15
Doctoral scholarship from the CONACyT, México. Daniel Mota-Rojas, Agustín
16
Orihuela, Angélica M. Terrazas-García and Emilio Arch-Tirado were supported as
17
members of the Mexico´s National System of Researchers (SNI). Finally, we would
18
like to thank Jesús Emilio Serrano Martínez for taking care of the animals and
19
Miguel García-Winder for his review and comments to the manuscript.
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Ethical statement
22
The animals were handled humanely throughout the study, and their care and
23
all experimental treatments complied with norm NOM-062-ZOO-1999 of Mexico’s 16
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Department of Agriculture, Ranching, Rural Development, Fishing and Alimentation
2
for animal-based experimentation. The study was approved by the Internal
3
Committee for the Care and Use of Experimental Animals at the FES-Cuautitlán-
4
Universidad Nacional Autónoma de México (UNAM). No phase of the study
5
entailed a permanent interruption of the ewe-lamb bond, and no ewe or lamb was
6
injured during the experiment.
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Authorship statement
9
The idea for the paper was conceived by Daniel Mota. The experiment was
10
designed by: Daniel Mota, Emilio Arch-Tirado, Angélica M. Terrazas and Agustín
11
Orihuela. The experiment was performed by: Patricia Mora, Patricia Roldán and
12
Marcelino Rosas.The data were analyzed by Carlos Vázquez-Cruz. The paper was
13
written by: Patricia Mora and Agustín Orihuela.
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Table 1.
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Ethogram of the examined behavioural parameters during the 15 min period of sensory
Description
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Behaviour
Bleats (Centoducati et al., 2015).
Urination
Elimination of urine (Centoducati et al., 2015).
Defecation
Elimination of faeces (Centoducati et al., 2015).
Exploring
The snout is in contact with the wall, the ground of the pen, or any
objects
other object in the pen (Anderson et al., 2015).
Exploring
The snout is in contact with other lambs or ewes present in the
conspecifics
pen (Anderson et al., 2015).
Escape
The lamb is observed to press on the dividing mesh or to jump
attempts
over the walls of the pen. The lamb exhibits jumping and part of its
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Vocalization
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body contacts the ground, or it runs a fast race to no apparent destination (Anderson et al., 2015). Elevating at least two feet simultaneously and moving the body
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Walking
rapidly or slowly in any direction; leaving the assigned area
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(Anderson et al., 2015). Each step was counted.
Lying down
Lambs lying down with their body resting on the sternum with the abdomen slightly inclined to one side and the forefeet and at least one hind foot flexed under its trunk (Centoducati et al., 2015). Each change in position from standing to lying was counted.
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restriction
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Table 2.
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Frequency of the behavioural parameters displayed by 3, 10 and 20 days-old
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lambs during a 15-min separation from their dams, under 5 different sensory
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isolation conditions
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AGE (days) Behaviour.
Group
1.8 ± 2.8 a,1
0.1 ± 3.1 a,1
14.5 ± 2.8 a,2
24.6 ± 3.3 b,2
24.8 ± 3.6 b,2
59.1 ± 2.8 b,3
50.7 ± 3.3 a,3
69.2± 3.6 c,3
54.6 ± 2.8 a,3
74.0 ± 3.3 b,4
54.2 ± 3.6 a,4
0.0 ± 0.1 a,1
0.0 ± 0.0 a,1
0.0 ± 0.0 a,1
A
0.1 ± 0.1 a,1
0.0 ± 0.0 a,1
0.1 ± 0.1 a,1
A+V
0.0 ± 0.1 a,1
0.0 ± 0.0 a,1
0.1 ± 0.1 a,1
A+V+W
0.3 ± 0.1 b,2
0.1 ± 0.0 a,1
0.1 ± 0.1 a,1
A+V+S
0.3 ± 0.1 b,2
0.0 ± 0.0 a,1
0.0 ± 0.1 a,1
C
3.1 ± 1.9 a,2
2.4 ± 1.6 a,2
2.0 ± 1.7 a,1
A
2.7 ± 1.5 a,2
3.1 ± 1.3 b,2
1.6 ± 1.3 a,1
A+V
0.2 ± 1.7 a,1
2.3 ± 1.5 a,2
1.8 ± 1.5 a,1
A+V+W
5.0 ± 1.7 c,2
4.3 ± 1.5 b,2
3.0 ± 1.5 a,2
A+V+S
4.2 ± 1.7,b,2
0.9 ± 1.5 a,1
4.8 ± 1.5 b,2
C
0.7 ± 1.1 a,1
1.1 ± 1.7 a,1
1.4 ± 1.8 a,1
A
1.0 ± 0.9 a,1
2.3 ± 1.3 a,1
1.1 ± 1.4 a,1
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1.3 ± 2.4 a,1
C
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1.2 ± 4.0 a,1
A
A+V+S
Exploration of objects
Exploration of conspecifics
0.6 ± 3.6 a,1
1.4 ± 3.8 a,1
A+V+W
Urinations
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C
A+V
Vocalizations
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2.3 ± 1.5 b,1
2.6 ± 1.7 b,1
A+V+W
3.4 ± 1.0 a,2
3.3 ± 1.5 a,1
5.7 ± 1.7 b,2
A+V+S
2.6 ± 1.0 a,2
2.7 ± 1.5 a,1
3.3 ± 1.7 b,2
C
0.0 ± 0.0 a,1
0.0 ± 0.0 a,1
0.0 ± 0.2 a,1
A
0.0 ± 0.0 a,1
0.0 ± 0.0 a,1
0.0 ± 0.1 a,1
A+V
0.0 ± 0.0 a,1
0.0 ± 0.1 a,1
0.0 ± 0.2 a,1
A+V+W
0.0 ± 0.0 a,1
0.3 ± 0.1 b,2
2.4 ± 0.2 c,3
A+V+S
0.1 ± 0.0 a,1
0.0 ± 0.1 a,1
0.6 ± 0.2 b,2
C
9.4 ± 1.4 b,2
7.2 ± 2.7 a,1
5.7 ± 2.5 a,1
A
5.9 ± 1.1 a,1
9.5 ± 2.1 b,1
5.7 ± 2.0 a,1
7.0 ± 1.2 a,1
15.8 ± 2.5 b,2
12.3 ± 2.3 b,2
8.3 ± 1.2 a,1,2
31.4 ± 2.5 b,3
35.5 ± 2.3 b,3
35.0 ± 1.2 a,3
54.5 ± 2.5 b,4
36.0 ± 2.3 a,3
0.2 ± 0.5 a,1
0.1 ± 0.2 a,1
0.0 ± 0.2 a,1
A+V
Walking
A+V+W A+V+S
0.3 ± 0.4 a,1
0.1 ± 0.2 a,1
0.2 ± 0.1 a,1
A+V
0.0 ± 0.4 a,1
0.0 ± 0.2 a,1
0.0 ± 0.2 a,1
A+V+W
0.7 ± 0.4 a,1,2
0.6 ± 0.2 a,2
0.8 ± 0.2 a,2
A+V+S
1.4 ± 0.4 b,2
0.5 ± 0.2 a,1,2
0.3 ± 0.2 a,1
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A
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Lying down
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0.9 ± 1.0 a,1
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Escape attempts
A+V
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a,b,c
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(P<0.01).
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1,2,3
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age (P<0.01).
Letters in the same row indicate differences among ages within the same treatment
Numbers in the same column indicate differences among treatments within the same
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C= Control; A= Auditory restriction*; A+V= Auditory + Visual restrictions*; A+V+W=
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Auditory + Visual restrictions + Wire mesh fence-line; A+V+S= Auditory + Visual
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restrictions + Solid fence-line restriction.
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Highlights:
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1. Behavioural responses increase with the degree of sensorial isolation.
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2. Older lambs show greater behavioural reactivity to sensorial isolation.
3. A short separation period should be promoted in very young lambs during husbandry practices.
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4. Regardless of age, physical separation induced the highest behavioural response.
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allowed.
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5. If lamb-ewe separation is necessary, some kind of contact should be
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