Behavioural and physiological profiles following exposure to novel environment and social mixing in lambs

Small Ruminant Research 103 (2012) 158–163

Contents lists available at ScienceDirect

Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres

Behavioural and physiological profiles following exposure to novel environment and social mixing in lambs G.C. Miranda-de la Lama a,∗ , M. Villarroel b , G.A. María a a b

Department of Animal Production and Food Science, Faculty of Veterinary Medicine, University of Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain Department of Animal Science, E.T.S.I.A. Polytechnic University of Madrid, Madrid, Spain

a r t i c l e

i n f o

Article history: Received 24 May 2011 Received in revised form 8 August 2011 Accepted 10 August 2011 Available online 4 September 2011 Keywords: Novel environment Social mixing Logistic chain Lamb welfare Aggression Affiliation Stereotypes

a b s t r a c t Most livestock production systems, animals are classified by sex, age or weight at different times in their productive life. In recent years, the pre-slaughter logistics for lamb production has been modified to include an intermediate step between the farm and the abattoir at classification centres (CC), where animals are classified by weight upon arrival and finished to the appropriate slaughter weight. In this study we describe the changes in social behaviour and stereotype profiles of lambs during one month after exposure to novel environment (CC) and social mixing as well as changes in key physiological variables associated with stress. We used a total of 36 male lambs (live weight of 17 ± 0.9 kg, approximately 60 days old and weaned at 45 days). Lambs were allocated to three different pens with 12 lambs each (0.75 m2 per lamb) and took behavioural and physiological measurements at 1, 7, 14 and 28 days after classification and mixing. Behaviour was video recorded continuously for 8 h on each sampling day (08:00–17:00 h) to note aggressive, affiliation and stereotypic behaviours. Blood samples were taken by jugular venipuncture after the behavioural observations to measure plasma cortisol, lactate, glucose, creatine kinase (CK), non-esterified fatty acid (NEFA) and haematological counts. The number of interactions with physical contact and stereotypes decreased with time (P < 0.01), while aggressive and affiliative interactions without physical contact remained the same throughout the trial. The average number of aggressions and affiliative interactions with contact were significantly higher on day 1. Total aggressions peaked on day 1, but decreased to a similar frequency as total stereotypes later on, while total affiliative interactions also peaked on day 1 but were much less frequent. Oral stereotypes peaked on day 1 as did body-rubbing. Plasma cortisol was significantly higher (P < 0.01) in lambs sampled on days 1 and 7, compared with days 14 and 28. Plasma CK (P < 0.05) was higher on day 1 but then decreased. In conclusion, exposure to novel environment (CC) and social mixing can be considered an important stress for lambs, and increased levels of aggression, stereotypes and plasma cortisol levels can be used reliably to indicate welfare problems at the CC. © 2011 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding author at: Departamento de Producción Animal, y Ciencia de los Alimentos, Facultad de Veterinaria, Universidad de Zaragoza, Miguel Servet 177, E-50013 Zaragoza, Spain. Tel.: +34 976 761000x4146; fax: +34 976 761612. E-mail address: [email protected] (G.C. Miranda-de la Lama). 0921-4488/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2011.08.007

A group is defined as a collection of animals in which the animals are of the same species and the composition of the group is relatively stable over time (Kondo and Hurnik, 1990), whereas grouping can be defined as the formation of a group of animals by natural means or by human action (Boe and Farevik, 2003). The term mixing is often

G.C. Miranda-de la Lama et al. / Small Ruminant Research 103 (2012) 158–163

used to describe the same procedure, especially in pigs (Tuchscherer et al., 1998; Boe and Farevik, 2003) and lambs (Ruiz-de la Torre and Manteca, 1999a,b). In most livestock production systems, animals are classified by sex, age or weight at different times in their productive life (Fernandez et al., 2007). When young animals are classified they often have to cope with the stress of weaning, a novel environment, mixing with unfamiliar animals, different feeding regimes and increased susceptibility to multi-factorial diseases, which all affect their behaviour and physiology. Social mixing of unfamiliar animals, which may disturb the social structure of the group (Mounier et al., 2006), and increase agonistic interactions with the establishment of a new social hierarchy, as seen in pigs (Rushen, 1987; Tuchscherer et al., 1998; Andersen et al., 2000), cattle (Mounier et al., 2005), horses (Houpt and Wolski, 1980), goats (Fernandez et al., 2007), and sheep (ewes, Sevi et al., 2001; lambs, Ruiz-de la Torre and Manteca, 1999a,b). Other studies have also found that social mixing increases the amount of time that unfamiliar animals spend fighting which implies higher activity of hypothalamic-pituitaryadrenal (HPA) axis (Rushen, 1987; Tuchscherer et al., 1998; Fernandez et al., 2007). In sheep, plasma cortisol levels increase when they are exposed to adverse conditions (Minton et al., 1992; Rhodes III et al., 1994; Parrott et al., 1998). In lambs, Sevi et al. (2001), also report that mixing increases plasma cortisol and agonistic behaviour. However, there is little data available on the effect of social mixing under commercial conditions on the behaviour and physiology of young lambs, despite the fact that problems during the relatively short finishing process may have a large impact on the final product. In Spain, lambs produced for meat are light and less than 3 months old (24–28 kg live weight). Many are Ternasco de Aragón® , a protected geographical indication for light lambs which are usually kept indoors, and fed a concentrate based diet and milk until weaning (45–50 days old), and ˜ thereafter with concentrate and straw (Sanudo et al., 1998). In recent years, the logistics chain for these lambs has been modified to include an intermediate step between the farm and the abattoir at classification centres (CC) (Miranda-de la Lama et al., 2009). There are several commercial and productive reasons why CC are useful in the lamb meat production chain, including simplification of farm management, scarcity of specialized man power and product standardization (Miranda-de la Lama et al., 2010a). Lambs are classified by weight upon arrival to the CC. Depending on their live weight class, they are finished for several days or up to one month until they reach the appropriate slaughter weight (Miranda-de la Lama et al., 2010b). As a result, young lambs, shortly after weaning, are exposed to a new environment, and mixed with lambs from other farms, but it is unclear how this affects their behaviour and physiology in the following weeks. Having this information will help to recommend improvements in handling that reduce its biological cost and maximize efficiency during the finishing process. In this study we describe the changes in social behaviour and stereotype profiles of lambs during one month after social mixing in novel environment (CC) as well as changes in key physiological variables associated with stress.

159

2. Materials and methods The study was carried out in the Autonomous Community of Aragon (northern Spain) in October 2008, at a cooperative classification centre located in Zaragoza (41◦ 41 N). The area is located in the Ebro river depression, characterized by a dry Mediterranean climate with average annual temperature of 15 ◦ C, and about 317 mm annual rainfall. All protocols were approved by the Animal Experimentation Ethics Committee of the University of Zaragoza. The CC belongs to a cooperative with 10 CC in Aragón with a capacity for more than 180,000 lambs per year. The CC used in our study had three barns. The central barn is used for classification and has a curved race system with a scale. Animals are sorted by weight class and kept in a holding pen until either transported to the abattoir or sent to a fattening pen in the two lateral barns. 2.1. Study description We used 36 male lambs of the Rasa aragonesa breed, clinically healthy, with a live weight of 17 (±0.9) kg and approximately 60 days old (weaned at 45 days). The classification processes involved moving the lambs through a chute in single file. The handling system includes an elevated platform, scales, a loading ramp and a classification mill. Once all the lambs reach the preloading pen, they are sorted by weight using the classification mill and released into temporary holding pens according to their weight class. Lambs were randomly selected from the temporary holding pen from the 16–18 kg category. This category was chosen since most lambs arrive to the CC at this weight and they remain at the abattoir up to one month for finishing. Experimental lambs were allocated to three different pens with 12 lambs (3 m × 3 m pens, stocking density 0.75 m2 per lamb) with straw bedding. Feeding was ad libitum with pellet concentrate in a feeder and straw was provided as well as fresh water. All animals followed the same feeding regime and health program, established by the standards of the cooperative. The commercial concentrate (Ovirum high energy® ) contained barley, corn, wheat, vegetable fat, soya tort, sugar cane molasses, calcium carbonate, sodium chloride and a vitamin mineral corrector. 2.2. Behavioural measurements Lambs were individually identified by coloured numbers and a videorecording device (model VDVR-9, Circontrol S.A., Terrassa, Spain) was set up in a room close to the pens to record animal behaviour. The camera was placed in front of each pen 220 cm above the ground. Each group was recorded continuously for 12 h per day (08:00–20:00 h) on days 1, 7, 14 and 28 (a total of 48 h per pen) after social mixing. Each video was observed twice by the same trained observer, first to note social interactions and again to note stereotypic behaviours. Behaviour sampling (Martin and Bateson, 1986) was used to record all social interactions. Only the total number of aggressions and affiliations initiated by each individual per day were considered for statistical analyses. Because animals were individually marked, we could also note the initiator and receiver of the social interaction. Each interaction was classified as aggressive or affiliative and whether it involved physical contact or not (Miranda-de la Lama et al., 2011). Aggressive interactions with contact included butts (when the lamb used its forehead to hit another lamb on any part of its body), mounting (when a lamb mounted another lamb from behind to move the latter, without an apparent sexual function), and kicking (when a lamb hit another lamb on any part of its body, with its forelegs). Aggressive interactions without contact included threats (when a lamb turned towards or approached another lamb with its head down and then lunged, without making contact), avoidance (when a lamb actively moves away from another individual whether or not previous interaction has occurred between the two individuals), and persecution (when a lamb moved towards another lamb, causing the latter to walk or run away). Affiliative interactions with contact included licking (licking another animal’s body), and nibbling (grooming another lamb’s body using teeth). The affiliative interaction without contact was sniffing (sniffing another animal’s body). Regarding stereotypes, two types were measured, oral (licking or gnawing repeatedly on feeders, walls, fences, or wood or metal objects without feed consumption) and body-rubbing (up and down) against a fence. For statistical analysis, all aggressive and affiliative interactions were subdivided into those that involved physical contact and those without contact. The results are expressed as the average per animal per day.

160

G.C. Miranda-de la Lama et al. / Small Ruminant Research 103 (2012) 158–163

2.3. Physiological measurements Blood samples were taken by jugular venipuncture after behavioural observations on days 1, 7, 14 and 28 (two 10 ml tubes were collected per animal, with and without anticoagulant, EDTA-K3). Animal handling and venipuncture required less than 1 min per lamb. Samples were kept on ice for <1 h and taken to the laboratory for routine haematological measurements. The EDTA plasma and serum were centrifuged at 3000 rpm for 10 min and aliquots were frozen and kept at −30 ◦ C until analysed. An automatic particle counter (Vet-ABC, Divasa Farmavid S.A.) was used to count red blood cells (RBC) and white blood cells (WBC) (number per mm3 ), haemoglobin (g/dl) and haematocrit (%). The leukocyte formula was estimated from blood swabs on clean slides. Staining was performed by the rapid panoptic method using dyes from Química Clinica Aplicada Inc. (QCA). With an optic immersion microscope we counted and identified 100 leucocytes per sample (neutrophils, lymphocytes, eosinophils, basophils and monocytes). The neutrophil/lymphocyte ratio (N/L) was used as a chronic stress indicator. In the serum samples, with an Multichannel Technicon Analyser (RA-500) and using reagents from Bayer diagnostics (SA) for RA technicon systems, we determined the concentration of glucose (mg/dl) (Ref. T01-1492-56), and the activity of creatine kinase (CI<), EC 2.7.3.2 (UI/L) (Ref. T01-1885-01). The concentration of cortisol was determined from plasma (EDTA k3) by enzyme inmmunoassay using an “in home-kit” (validated by Chacon et al., 2004). Each sample was determined in duplicate from 50 ␮l of plasma and the results were expressed in ng nl−1 , with the corresponding controls. Variation coefficients of the analysis, inter- and intra-assay, were 7 and 8%, respectively. The concentration of lactate was determined using a Sigma Diagnostic kit (lactate no 735-10) and spectrophotometer (Lambda 5, Perkin Elmer). Serum concentration of non-esterified fatty acid (NEFA) levels were analysed by a multianalyzer ACE® (Clinical Chemistry System of the Alfa Wasserman), with commercial kits (NEFAC Ref. 994-75409 of the Wako). 2.4. Statistical analysis Physiological, haematological and behavioural variables were analysed using SAS/STAT (9.1 SAS Inst. Inc., Cary, NC, USA) using the repeated measures procedure in PROC MIXED to detect differences between sampling day. Least squares means were calculated using the LSMEANS option of the procedure, and differences between means were determined using the PDIFF option. The study was repeated three times and no effect of repetition factor was found. The final model included the animal as a random effect, and the day of sampling (repeated measure), while residuals were considered as random. The estimated covariance structure is used to obtain generalized least square estimates of time differences (Littell et al., 1998). The general representation of the model is: yijk =  + tj + ık + εijk where yijk is N × 1 vector of records,  is the overall mean, tj is the effect of period k, ık is the random effect of the animal and εijk denotes the residual term. Least squares means are reported throughout, and probability values of P < 0.05 were considered to be statistically significant.

3. Results In general, oral stereotypes and agonistic interactions with contact were high on the first day after social mixing, while affiliative behaviours were less frequent. Physiological stress indicators corresponded with the behaviours observed, indicating more acute stress directly after mixing, which decreased the normal levels in the following weeks. No health problems or serious wounds or injuries were observed. The mean (±SD) slaughter weight was 27.4 (±1.1 kg), and the carcass weight was 10.9 (±1.07 kg). There were no differences between groups for these traits. 3.1. Behavioural profile The least square means (±SE) per animal per day of aggressive and affiliative interactions as well as stereotypes are presented in Table 1. The number of interactions

Fig. 1. Least square means (±SE) per animal per day of total aggressive and affiliative interactions and stereotypes in terms of days after social mixing.

with physical contact and stereotypes decreased with time (P < 0.01), while aggressive and affiliative interactions without physical contact remained the same throughout the trial. The average number of aggressions with contact was significantly lower on day 7 compared to day 1 (a decrease of 32.4%), and lower still on days 14 and 28 (55% less than on day 1). Similarly, affiliative interactions with contact were significantly higher on day 1 compared to days 7, 14 and 28 (an average decrease of 50%). As seen in Fig. 1, total aggressions peaked on day 1, but decreased to a similar frequency to total stereotypes later on, while total affiliative interactions also peak on day one but were much less frequent. As with aggressions and affiliations, the oral stereotypies also peaked on day 1 compared to the other sampling days (a decrease of 44% on average). Body-rubbing also peaked on day 1 but then decreased significantly again in the last week to very low levels (less than 2 per animal per day) on day 28 (90% less than on day 1). 3.2. Physiological profile Plasma cortisol was significantly higher (P < 0.01) in lambs sampled on days 1 and 7, compared with days 14 and 28 (Table 2). Lactate was high on day 1 but decreased significantly (P < 0.01) by day 7, returning to higher values on days 14 and 28. Plasma CK (P < 0.05) was higher on day 1 but then decreased, as with cortisol. There were no significant differences among glucose or haematological values (white and red blood cells, haematocrit and ratio N/L) among then different sampling times. 4. Discussion For the most part, social mixing is necessary in intensive fattening systems (Boe and Farevik, 2003), although it is unclear what effect it may have on the welfare of young lambs during the finishing process. We found that lambs are more aggressive to one another immediately after mixing and show more stereotypes as well as higher physiological levels of stress. High levels of aggression are one of the most obvious outcomes of disrupting established social groups (Weary et al., 2008). Even though, in our study, the frequency

G.C. Miranda-de la Lama et al. / Small Ruminant Research 103 (2012) 158–163

161

Table 1 Least square means (±SE) per animal per day for aggressive interactions with physical contact (total amount of butting, mounting and kicking), aggressive interactions without physical contact (threats, persecution and avoidance) and affiliative interactions with contact (liking, nibbling) and without (sniffing), as well as stereotypic behaviour profile in terms of days after social mixing. Variables

Aggressive interactions With contact Without contact Affiliative interactions With contact Without contact Stereotypes Oral Body-rubbing

Days after social mixing 1

7

14

28

29.3 ± 2.0a 8.9 ± 1.0

19.8 ± 2.0b 8.4 ± 1.0

14.2 ± 2.0c 8.3 ± 1.06

11.9 ± 2.0c 7.0 ± 1.0

11.6 ± 1.0a 1.0 ± 0.1

6.3 ± 1.0b 0.9 ± 0.1

5.3 ± 1.0b 0.5 ± 0.1

5.9 ± 1.0b 0.6 ± 0.1

37.7 ± 2.7a 14.5 ± 1.1a

22.0 ± 2.7b 6.0 ± 1.1b

21.8 ± 2.7b 4.5 ± 1.1b

19.7 ± 2.7b 1.5 ± 1.1c

a,b,c: different letters at the same row mean significant difference within treatments (P < 0.05).

of aggression decreased significantly a few weeks after mixing, it still remained quite high (approximately 30 aggressions per lamb day). Ruiz-de la Torre and Manteca (1999b) analysed the effect of mixing young lambs under comparable conditions to those used here. In their control treatment (lambs not mixed), aggressions were quite low compared to our results (3–4 aggressions per lamb per day). There may be several reasons for a high aggression level that are not solely due to mixing, including the being introduced to a novel environment. On the other hand, affiliative behaviours were also higher immediately after mixing. Affiliative behaviour can be used as an indicator of positive experiences in farm animals under commercial conditions (Boissy et al., 2007), so it may appear contradictory that in our study there were high levels of affiliative interactions as well as aggressive interactions immediately after mixing. However, Miranda-de la Lama and Mattiello (2010) described affiliative interactions (i.e. licking) as behaviours that help reduce aggression. In addition, affiliations were always much less frequent than aggressions (an average of 11 affiliative contacts per animal per day compared to almost 30 aggressions per animal per day). Recent results also suggest a function of social licking in conflict resolution to reduce social tension (Waiblinger et al., 2003). Along those lines, affiliation with contact (licking and nibbling) was higher on the first day of mixing, while sniffing (no contact) was much less frequent. Thus, lambs preferentially use affiliative contact on the first day of mixing but they may not be as obvious an indicator of social stress as

aggressions, and were also much lower than stereotypes. However, aggression and affiliative interactions may arise from the global effect of mixing and novelty, whose effects are difficult to isolate under commercial conditions. We observed stereotypes after social mixing, which, to our knowledge, is one of the first times they have been described in lambs in relation to classification and mixing. The occurrence of stereotypic behaviour by farm animals kept in intensive housing systems has long been thought to provide information about the level of welfare of the animals (Mason, 1991; Duncan et al., 1993). When lambs are unable to control a novel situation, negative emotions increase, such as frustration, and are associated with more persistent disorders (Greiveldinger et al., 2009), such as stereotypic behaviour. The tendency towards a higher incidence of stereotypies in the period just after classification and mixing suggests that it can be used as non-invasive criteria to assess animal welfare. In our study, oral stereotypies were very frequent, more than aggressive interactions with contact and more than twice as frequent as body-rubbing (remaining quite frequent throughout the trial). Several previous studies have shown that stereotyped oral behaviours typical in cattle (Redbo and Nordblad, 1997) and lambs (Yurtman et al., 2002), are strongly affected by feeding regime. However, other studies in lambs have reported more factors associated with stereotype oral activities, such as indoor group housing (Savas et al., 2001), isolation (Cooper et al., 1996) and weaning (Karaagac et al., 2005). The high levels of

Table 2 Least square means (±SE) of indicators of physiological profile in terms of days after social mixing. Variables

Days after social mixing 1

Cortisol (ng/ml) Lactate (mg/dl) Glucose (mg/dl) NEFA (nmol/L) CK (UI/L) White blood cells (103 mm−3 ) Red blood cells (106 mm−3 ) Haematocrit (%) Ratio N/L

14.26 19.33 104.42 0.11 335 8.92 10.02 27.86 0.49

7 ± ± ± ± ± ± ± ± ±

1.06a 1.19a 1.88 0.01 122a 0.42 0.14 0.42 0.04

12.2 13.27 107.28 0.05 184 8.23 10.42 28.78 0.55

14 ± ± ± ± ± ± ± ± ±

1.06a 1.19b 1.88 0.01 122b 0.42 0.14 0.42 0.04

9.43 16.45 110.81 0.06 172 8.65 10.41 28.56 0.56

28 ± ± ± ± ± ± ± ± ±

1.06b 1.19a 1.88 0.01 122b 0.42 0.13 0.42 0.04

10.02 16.30 104.94 0.09 126 8.42 10.42 28.54 0.51

± ± ± ± ± ± ± ± ±

1.06b 1.19a 1.88 0.01 122b 0.42 0.14 0.42 0.04

a,b,c: different letters at the same row mean significant difference within treatments (P < 0.05). NEFA: non-esterified fatty acid, CK: creatinine kinase, ratio N/L: neutrophil/lymphocyte ratio.

162

G.C. Miranda-de la Lama et al. / Small Ruminant Research 103 (2012) 158–163

oral stereotypes maintained throughout our study could be related to a lack of stimuli in the novel environment, as has also been mentioned as a causing factor for stereotypes in other studies (Cooper et al., 1996). Another explanation for oral stereotypies may relate to a replacement for the lack of contact with the mother, and help to reduce individual stress levels (Latham and Mason, 2008). Early maternal separation can have lasting effects on brain function and on tendencies to stereotype (Bergeron et al., 2006). Rubbing stereotypes have been reported in pigs (Rushen, 1985), horses (McGreevy et al., 1995) and cattle (Fraser and Broom, 1990) with low levels of social contact. In our study, body-rubbing, as opposed to oral stereotypes, decreased to near zero levels by day 28, suggesting that it was more related with an initial frustration. This indicates that rubbing stereotype may be a reliable indicator to assess the effect of the initial social disruption due to mixing. Regarding the physiological profile, it is well accepted that increased aggression after classification and mixing increases the levels of plasma cortisol (Fernandez et al., 2007). In lambs, Sevi et al. (2001), also report that mixing increases plasma cortisol and agonistic behaviour. However, in our study, cortisol levels decreased substantially after the second week, suggesting that the lambs physiological adjusted to the new conditions. Even though the values of cortisol were within normal ranges for lambs (Kaneko et al., 1997), cortisol levels on day 1 were 40% higher than values on day 28. That is a higher increase than reported for the stress response observed in castration (11%, Mellor et al., 2002), increase in stocking density ˜ et al., 2002) and long transport time (9%, De la (5%, Ibánez Fuente et al., 2010). Short term stress or physical exercise increases circulating adrenaline levels, degrading muscle glycogen that is used for gluconeogenesis in the liver, thereby increasing lactate levels (Apple et al., 1995). In our study, lactate concentration was significantly lower on day 7, probably due to the ongoing adaptation process to the housing system. However lactate rose again on days 14 and 28, suggesting that other stressors may affect the process. As seen above, aggressive interactions were higher on day 1, which coincides with higher plasma CK activity. Plasma CK is generally considered a chemical index of physical stress and an indicator of muscular damage and fatigue. It can also be released into the blood under other circumstances, such as vigorous exercise (Van de Water et al., 2003). Higher CK indicates more intense physical stress due to intense activity as social rank was being established. Finally, in our study, the RBC, haematocrit, WBC, and N/L ratio were within normal reference ranges for weaning lambs of the same age (Ullrey et al., 1965; Lepherd et al., 2009). These results indicate that those indicators are not as sensitive to measure the effects of mixing, which may have more acute effects and less of an effect on the overall health of the animals. It should be noted that animal welfare is complex and thus difficult to measure objectively using simple techniques. It is often hard to separately interpret standard physiological, productive and behavioural indicators of stress (Barnett and Hemsworth, 1990; Rushen, 1991; Mendl, 1991). However, our results provide

information about physiological indicators to help understand the biological cost of adaptation to CC. 5. Conclusions Social mixing in novel environments of finishing lambs produced high levels of aggression, stereotypies and elevated plasma cortisol levels, suggesting that they are useful to indicate welfare problems at the CC. Considering that the current tendency in Spain is to increase the number of CC in lamb production, it is necessary to develop protocols to minimize the biological cost of their adaptation after classification and mixing. Out of the 15 behavioural and physiological variables analysed, aggressive interactions, stereotypes and plasma cortisol were most sensitive to social mixing. Acknowledgements This study was funded by the Spanish Ministry of Science (project AGL-2008-02088/GAN). The authors would like to thank the cooperative Oviaragon Grupo Pastores and the laboratory personal in special to Jose Antonio Ruiz, Isabel Vazquez and Gemma Taylor for their help in carrying out the field work. Many thanks to the Santander Central Hispano Bank and the University of Zaragoza for the Latin American Scholarship PhD Program of Genaro C. Miranda-de la Lama from México. References Andersen, I.L., Andenaes, H., Boe, K.E., Jensen, P., Bakken, M., 2000. The effects of weight asymmetry and resource distribution on aggression in groups of unacquainted pigs. Appl. Anim. Behav. Sci. 68, 107–120. Apple, J.K., Dikeman, M.E., Minton, J.E., McMurphy, R.M., Fedde, M.R., Leith, D.E., Unruch, J.A., 1995. Effects of restraint and isolation stress and epidural blockade on endocrine and blood metabolite status, muscle glycogen metabolism and incidence of dark-cutting Longissimus muscle of sheep. J. Anim. Sci. 73, 2295–2307. Barnett, J.L., Hemsworth, P.H., 1990. The validity of physiological and behavioural measures of animal welfare. Appl. Anim. Behav. Sci. 25, 177–187. Bergeron, R., Badnell-Waters, A.J., Lambton, S., Mason, G., 2006. Stereotypic behaviour in captive ungulates: foraging, diet and gastrointestinal function. In: Mason, G., Rushen, J. (Eds.), Stereotypic Animal Behaviour – Fundamentals and Applications for Welfare. , 2nd ed. CABI, Wallingford, UK, pp. 19–49. Boe, K.E., Farevik, G., 2003. Grouping and social preferences in calves, heifers and cows. Appl. Anim. Behav. Sci. 80, 175–190. Boissy, A., Manteuffel, G., Jensen, M.B., Moe, R.O., Spruijt, B., Keeling, L.J., Winckler, C., Forkman, B., Dimitrov, I., Langbein, J., Bakken, M., Veissier, I., Aubert, A., 2007. Assessment of positive emotions in animals to improve their welfare. Physiol. Behav. 92, 375–397. Chacon, G., Garcia-Belenguer, S., Illera del Portal, J.C., Palacio, J., 2004. Validation of an EIA technique for the determination of salivary cortisol in cattle. Span. J. Agric. Res. 2, 45–51. Cooper, J., Haskell, M., Lewis, R.M., 1996. The development of stereotypies in experimental sheep. Anim. Sci. 62, 674. De la Fuente, J., Sánchez, M., Pérez, C., Lauzurica, S., Vieira, C., González de Chavarri, E., Díaz, M.T., 2010. Physiological response and carcass and meat quality of suckling lambs in relation to transport time and stocking density during transport by road. Animal 4, 250–258. Duncan, I.J.H., Rushen, Lawrence, A.B., 1993. Conclusions and implications for animal welfare. In: Lawrence, A.B., Rushen, J. (Eds.), Stereotypic Animal Behaviour: Fundamentals and Applications to Welfare, vol. 21. CAB International, Wallingford, UK, pp. 193–206. Fernandez, M.A., Alvarez, L., Zarco, L., 2007. Regrouping in lactating goats increases aggression and decreases milk production. Small Rumin. Res. 70, 228–232.

G.C. Miranda-de la Lama et al. / Small Ruminant Research 103 (2012) 158–163 Fraser, A.F., Broom, D.M., 1990. Farm Animal Behaviour and Welfare. Bailliere, Tindell, London, UK. Greiveldinger, L., Veissier, I., Boissy, A., 2009. Behavioural and physiological responses of lambs to controllable vs. uncontrollable aversive events. Psychoneuroendocrinology 34, 805–814. Houpt, K., Wolski, T.R., 1980. Stability of equine hierarchies and the prevention of dominance related aggression. Equine Vet. J. 12, 15–18. ˜ Ibánez, M., De la Fuente, J., Thos, J., Chavarri, González de Chávarri, E., 2002. Behavioural and physiological responses of suckling lambs to transport and lairage. Anim. Welf. 11, 223–230. Kaneko, J.J., Harvey, J.W., Bruss, M.L., 1997. Clinical Biochemistry of Domestic Animals. Academic Press, San Diego, USA, 563 pp. Karaagac, F., Ozcan, M., Savas, T., 2005. Some behaviour traits observed on the Kivircik and crossbred lambs raised in intensive conditions. Turk. J. Vet. Anim. Sci. 29, 803–809. Kondo, S., Hurnik, J.F., 1990. Stabilization of social hierarchy in dairy cows. Appl. Anim. Behav. Sci. 27, 287–297. Latham, N.R., Mason, G.J., 2008. Maternal deprivation and the development of stereotypic behaviour. Appl. Anim. Behav. Sci. 110, 84–108. Lepherd, M.L., Canfield, P.J., Hunt, G.B., Bosward, K.L., 2009. Haematological, biochemical and selected acute phase protein reference intervals for weaned female Merino lambs. Aust. Vet. J. 87, 5–11. Littell, R.C., Henry, P.R., Ammermann, C.B., 1998. Statistical analysis of repeated measures data using SAS procedures. J. Anim. Sci. 76, 1216–1231. Martin, P., Bateson, P., 1986. Measuring Behaviour, An Introductory Guide. Cambridge University Press, Cambridge, U.K. Mason, G.J., 1991. Stereotypies: a critical review. Anim. Behav. 41, 1015–1037. McGreevy, P.D., Nicol, C.J., Cripps, P., Green, L., French, N., 1995. Management factors associated with stereotypic and redirected behaviour in the thoroughbred horse. Equine Vet. J. 27, 86–91. Mendl, M., 1991. Some problems with the concept of a cut-off point for determining when an animal’s welfare is at risk. Appl. Anim. Behav. Sci. 31, 139–146. Mellor, D.J., Stafford, K.J., Todd, S.E., Lowe, T.E., Gregory, N.G., Bruce, R.A., Ward, R.N., 2002. A comparison of catecholamine and cortisol responses of young lambs and calves to painful husbandry procedures. Aust. Vet. J. 80, 228–233. Minton, J.E., Coppinger, T.R., Reddyt, P.G., Davis, W.C., Blechat, F., 1992. Repeated restraint and isolation stress alters adrenal and lymphocyte functions and some leukocyte differentiation antigens in lambs. J. Anim. Sci. 70, 1126–1132. Miranda-de la Lama, G.C., Villarroel, M., Olleta, J.L., Alierta, S., Sanudo, C., Maria, G.A., 2009. Effect of the pre-slaughter logistic chain on meat quality of lambs. Meat Sci. 83, 604–609. Miranda-de la Lama, G.C., Rivero, L., Chacon, G., Garcia-Belenguer, S., Villarroel, M., María, G.A., 2010a. Effect of the pre-slaughter logistic chain on some indicators of welfare in lambs. Livest. Sci. 128, 52–59. Miranda-de la Lama, G.C., Villarroel, M., Liste, G., Escós, J., María, G.A., 2010b. Critical points in the pre-slaughter logistic chain of lambs in Spain that may compromise the animal’s welfare. Small Rumin. Res. 90, 174–178. Miranda-de la Lama, G.C., Mattiello, S., 2010. The importance of social behaviour for goat welfare in livestock farming. Small Rumin. Res. 90, 1–10.

163

Miranda-de la Lama, G.C., Sepúlveda, W.S., Montaldo, H.H., María, G.A., Galindo, F., 2011. Social strategies associated with identity profiles in dairy goats. Appl. Anim. Behav. Sci., doi:10.1016/j.applanim.2011.06.004. Mounier, L., Veissier, I., Boissy, A., 2005. Behavior, physiology and performance of bulls mixed at the onset of finishing to form uniform body weight groups. J. Anim. Sci. 83, 1696–1704. Mounier, L., Veissier, I., Andanson, S., Delval, E., Boissy, A., 2006. Mixing at the beginning of fattening moderates social buffering in beef bulls. Appl. Anim. Behav. Sci. 96, 185–200. Parrott, R.F., Hall, S.J., Lloyd, D.M., 1998. Heart rate and stress hormone responses of sheep to road transport following two different loading procedures. Anim. Welf. 7, 257–267. Redbo, I., Nordblad, A., 1997. Stereotypies in heifers are affected by feeding regime. Appl. Anim. Behav. Sci. 53, 193–202. Rhodes III, R.C., Nippo, M.M., Gross, W.A., 1994. Stress in lambs (Ovis aries) during a routine management procedure: evaluation of acute and chronic responses. Comp. Biochem. Physiol. A 107, 181–185. Ruiz-de la Torre, J.L., Manteca, X., 1999a. Effects of testosterone on aggressive behaviour after social mixing in male lambs. Physiol. Behav. 68, 109–113. Ruiz-de la Torre, J.L., Manteca, X., 1999b. Behavioural effects of social mixing at different stocking densities in prepubertal lambs. Anim. Welf. 8, 117–126. Rushen, J., 1985. Stereotypies, aggression and the feeding schedules of tethered sows. Appl. Anim. Behav. Sci. 14, 137–147. Rushen, J., 1987. A difference in weight reduces fighting when unacquainted newly weaned pigs first meet. Can. J. Anim. Sci. 67, 951–960. Rushen, J., 1991. Problems associated with the interpretation of physiological data in the assessment of animal welfare. Appl. Anim. Behav. Sci. 28, 381–386. ˜ C., Sanchez, A., Alfonso, M., 1998. Small ruminant production sysSanudo, tems and factors affecting lamb meat quality. Meat Sci. 49, S29–S64. Savas, T., Yurtman, I.Y., Karaagac, F., Koycu, E., 2001. Einfluss der intensive gruppen haltung und geschlecht auf die stereotypien und einige verhaltensmerkmale bei mastlammern. Arch. Tierz. 44, 313–322. Sevi, A., Taibi, L., Albenzio, L.M., Muscio, A., Dell’Aquila, S., Napolitano, F., 2001. Behavioral, adrenal, immune, and productive responses of lactating ewes to regrouping and relocation. J. Anim. Sci. 79, 1457–1465. Tuchscherer, M., Puppe, B., Tuchscherer, A., Kanitz, E., 1998. Effects of social status after mixing on immune, metabolic, and endocrine responses in pigs. Physiol. Behav. 64, 353–360. Ullrey, D.E., Miller, E.R., Long, C.H., Vincent, B.H., 1965. Sheep haematology from birth to maturity I. Erythrocyte population, size and hemoglobin concentration. J. Anim. Sci. 24, 135–140. Van de Water, G., Verjans, F., Geers, R., 2003. The eVect of short distance transport under commercial conditions on the physiology of slaughter calves; pH and colour profiles of veal. Livest. Product. Sci. 82, 171–179. Waiblinger, S., Menke, C., Fölsch, D.W., 2003. Influences on the avoidance and approach behavior of dairy cows towards humans on 35 farms. Appl. Anim. Behav. Sci. 84, 23–39. Weary, D., Jasper, J., Hotzel, M.J., 2008. Understanding weaning distress. Appl. Anim. Behav. Sci. 110, 24–41. Yurtman, I.Y., Savas, T., Karaagac, F., Coskuntuna, L., 2002. Effects of daily protein intake levels on the oral stereotypic behaviours in energy restricted lambs. Appl. Anim. Behav. Sci. 77, 77–88.