Effect of space allowance during transport and fasting or non-fasting during lairage on welfare indicators in Merino lambs

A total of 72 male lambs of Merina breed were sampled in a 3×2 factorial design, testing three different space allowances treatment (SA) during transport [0.16 m 2 /animal (SAL; n= 24); 0.20 m 2 /animal (SAM; n= 24) and 0.30 m 2 /animal (SAH; n= 24)] and two lairage treatments (TL) during 18 h previous slaughter [fasting (FAST; n= 36) vs feeding (FEED; n= 36)] on welfare physiological indicators. After transport, glucose and lactate dehydrogenase (LDH) were highest in SAM group and lowest in SAH one ( p< 0.05). SAL showed intermediate values for both parameters. SA did not affect the rest of the blood parameters studied. TL-FAST treatment decreased glucose values ( p< 0.001) while increased LDH ( p< 0.001). Fasting caused an increase ( p< 0.05) of Red Blood Cell Count values in SAM group. Feed deprivation did not affect cortisol or adrenaline values. Noradrenaline value was higher ( p< 0.001) in TL-FAST groups than in TL-FEED. In conclusion, under the conditions of this study, a range of space allowance during transport between 0.16 and 0.30 m 2 /lamb could be recommended without showing major changes on welfare physiological indicators; and feeding could be more appropriate than fasting during lairage.


Introduction
One of the main Spanish breeds of sheep, the Merina breed, is raised in the region of Extremadura (SW Spain) and it is the foremost commercial sheep breed in the world (http://www.merino2014.com/conference. html). Among the products obtained from this breed, lamb is a very valuable product, due to its highly appreciated organoleptic qualities (Tejeda et al., 2008). The lambs are weaned at 45 days and then fed with a commercial concentrate and cereal straw ad libitum until slaughter (20-30 kg live weight).
Many factors relate directly to pre-slaughter stress, such as transport, fasting, conditions in the abattoir which may affect meat quality (Sañudo et al., 1998). Animal transport is an important phase in all systems of meat production (Ljungberg et al., 2007). Transportation by road is the most common method used for lambs and causes changes in many blood parameters as a consequence of stress response (Knowles, 1998). According to Broom (2008), space allowed during transport is one of the most important factors influencing animal welfare. Protection of animals during transport is a legal requirement according to European law (EC, 2004) to prevent injurie or undue suffering and to ensure appropriate conditions to meet their biological needs. A considerable amount of research has been conducted on the stressfulness of transportation on 2 handling practices were carried out according to statements of the Directive 2010/63/EU (EC, 2010) with regard to the protection of animals used in research and for scientific purposes.
The study was carried out in spring, in two journeys that were performed in April and May, respectively (both in sunny conditions and without rain). Values of the temperature and relative humidity were registered using a WatchDog 15-Temperature/Humidity Monitor. Animals (n=36 per journey) were loaded (starting at 8 am, 7ºC average temperature during loading) randomly in the middle floor and in both sides of the lorry, according to the three spaces allowances: 0.16 m 2 /animal (SAL; n=24; 12/journey); 0.20 m 2 /animal (SAM; n=24; 12/journey) and 0.30 m 2 /animal (SAH; n=24; 12/journey). The same vehicle was used in both journeys.
Lambs were transported by paved roads (334 km, ~ 5½ h) from the farm (Cabeza del Buey, Badajoz, Spain; 38° 43′ N) to the abattoir (Tarancón, Cuenca, Spain; 40° 0′ N) along with other lambs not included in this study. The mean temperature and relative humidity recorded during transport were similar for both journeys, 11ºC and 56% respectively (min. temp. 10.2ºC and 13ºC at the beginning and max. temp. 14ºC and 16ºC at the end of the journey for the first and second journey, respectively). The lorry had two axles and three floors, natural ventilation along the full length of the truck body sides and on each floor, a hydraulic elevator for loading and unloading. When animals arrived to the slaughterhouse were unloaded and rested in lairage for 18 h previous slaughter.
Animals in the slaughterhouse and in each group of density during transport were split randomly in two groups: one group of animals remained in fasting (FAST; n=36) during lairage, while the other group (FEED; n=36) were fed ad libitum the same commercial concentrate as on the farm). During lairage all animals remained in a waiting area, covered with a roof, at a density of 0.36 m 2 per animal and receiving water ad libitum. The average temperature at lairage was 14.7ºC with a relative humidity of 62% (first journey: max. temp.=18.3ºC, min. temp.=7.3ºC; second journey: max. temp=20.9ºC, min. temp.=12.7ºC). After lairage, lambs were slaughtered using standard commercial procedures.

Blood sampling
Blood samples were taken by trained personnel by external jugular venipuncture according to Linares et al. (2008) at three different times. The first sample used to determine the basal blood parameter concentrations several sheep breeds [Tadich et al. (2009) in the Corriedale breed; Miranda-de la Lama et al. (2010aLama et al. ( , 2012 in the Rasa Aragonesa breed; De la Fuente et al. (2010, 2012 in the Assaf breed]. Lairage of animals at the slaughterhouse is a common commercial practice that helps livestock recover from transportation stress prior to slaughter (Kannan et al., 2000). Fasting during this period is important for reducing gut contents prior slaughter resulting in a reduced risk of carcass contamination (Gregory & Grandin, 1998). However, it is suggested that provision of feed during lairage could reduce the stress in the animals (Gonyou, 2012). Some studies have considered the effect of lairage on lamb welfare, including time of lairage (Díaz et al., 2014), with or without lairage (Liste et al., 2011).
A variety of welfare indicators can be used to assess the welfare of animals (Broom, 2000). Many physiological parameters have been proposed to evaluate the effects of stress previous slaughter, such as hematological parameters (Liotta et al., 2007); hormones (Mellor et al., 2002), glucose (Tadich et al., 2009) or tissular damage quantified by the change in creatinine kinase and lactate dehydrogenase (Ekiz et al., 2012).
Stressors immediately prior to slaughter, including transport and lairage conditions can have a cumulative effect. To date, no studies have examined the effects of space allowance during transport and fasting or feeding during the lairage prior to slaughter on lamb welfare in this breed. This information would help to recommend appropriate management during the stay of lambs at the abattoir before slaughter. Therefore, the aims of this study were to investigate: (1) the effect of space allowance (SA: low, medium or high) during transport; (2) the management during lairage (TL: fasting vs feeding) on the haematological, hormonal and biochemical blood parameter values for Merina breed lambs; (3) to determine the best combination (space allowance during transport and management during lairage at the slaughterhouse) to obtain the highest animal welfare before slaughter.

Study description
Seventy two Merina breed male lambs (28.03 ± ± 0.05 kg, 90 days old) were used in this trial. Lambs were weaned at 45 days after birth and then fed a commercial concentrate (17% crude protein), containing cereals, soya, calcium carbonate, sodium chloride, mineral-vitamin mix and cereal straw ad libitum. All 3 Lamb welfare during transport and lairage ANOVA was carried out to determine the effects of space allowance treatment during transport on haematological, hormonal and biochemical blood parameters and the differences with the basal values on farm. A Tukey's test at a significance level of p<0.05 was carried out to check the differences between pairs of groups.
A General Linear Model (GLM) was used to examine the effects of space allowance treatment during transport (low, medium, high) and treatment during lairage [Fasting (TL-FAST) or Non Fasting (TL-FEED)] and their interactions on haematological, hormonal and biochemical blood parameters after lairage. In addition a Tukey's test at a significance level of p<0.05 was carried out to check the differences between pairs of groups [space allowance treatment (SA) -treatment during lairage (TL)].
A stepwise discriminant function analysis was carried out to select a linear combination of the independent parameters that best allowed differentiating among the SA groups during transport. A second discriminant analysis was carried out to differentiate among treatment groups during lairage. This last analysis was defined by two canonical discriminant functions which were illustrated by means of a dispersion diagram.

Effect of space allowance during transport (SA)
Physiological parameters values for on farm and after transport per SA are showed in Table 1. Journey did not affect any blood parameter values on farm or after transport in each SA. In the present work haematological parameters after transport did not differ significantly from values detected at the farm. SA during transport did not affect these parameters.
After transport, the SAM group showed the highest cortisol value (142.10 nmol/L) while the lowest was found at the SAH (58.48 nmol/L), while SAL group showed intermediate values (101.53 nmol/L) for this parameter. However, there were not significant differences among SA treatments and values were similar to the values found on farm.
In general, adrenaline concentration was lower on farm than after transport but only significant differences (p<0.05) were found between SAL groups (469.51 nmol/L on farm vs 662.59 nmol/L after transport). SA treatment during transport did not affect adrenaline concentration. Noradrenaline values on farm were similar to the values after transport p> 0.05).
was taken on the farm in resting conditions; a second sample was taken immediately after unloading the animals to evaluate the effect of space allowance during transport. The final sample was collected after 18 h lairage, to determine the effect of the lairage treatment (feeding or fasting) before slaughter. Approximately 20 seconds were necessary for each extraction, and in all samples a 0.8×25 mm bevelled needle (Terumo Neolus, Belgium), 5 mL syringe (BD Discardit TM , Spain) were used; 5 mL of blood was extracted to fill 1 mL tubes containing EDTA (ethylene diaminotetraacetic acid) for haematology and catecholamine determination and 4 mL tubes without additive for cortisol and biochemical parameter concentrations measurements. Blood samples were maintained at 2ºC in a portable refrigerator until they arrived at the clinical analysis laboratory. The tubes were centrifuged at 4000 rpm for 5 min and frozen and kept at −18ºC until processed.
The determination of total cortisol concentration was carried out through a competitive enzyme assay (EIA, RADIM, Pomezia, Italy). The assay sensitivity was 5 ng/mL. The inter-and intra-assay coefficients of variation were 6% and 5%, respectively. The catecholamines were analysed using a competitive enzyme immunoassay kit (Cat Combi-Adrenaline-Noradrenaline ELISA, EIA-4309, DRG Instruments GmbH, Germany). The sensitivity in the analyses was 11 pg/mL for adrenaline and 44 pg/mL for noradrenaline. The inter-and intra-assay coefficients of variation were 14% and 11% for adrenaline and 12% and 13% for noradrenaline. -Biochemical: Glucose, total protein, creatinine, lactate dehydrogenase (LDH), creatine kinase (CK) and lactate, were determined using a clinical system autoanalyzer (Synchron CX4 delta, Beckman Coulter Inc, USA).

Statistical analysis
Data were analysed with the Statistical Package SPSS 19.0 (IBM, 2010). First a Shapiro-Wilk test was carried out to check the normality and homogeneity of variance of all parameters. Then the repeated measured ANOVA test showed significant differences on glucose (p<0.001), total proteins (p<0.001) and LDH activity (p<0.01). In general the lowest values for these serum indicators were found on farm and the highest in SAM group after transport. Tukey test showed significant differences (p<0.05) among SA groups in glucose concentration and in the LDH activity: SAH one had the lowest values (4.99 mmol/L; 296.52 U/L respectively) with significant differences with SAM (5.60 mmol/L, 398.96 U/L respectively). The SAL group (0.16 m 2 /lamb) showed intermediate values for these biochemical parameters (5.34 mmol/L, 350.91 U/L respectively). However, total proteins, creatinine, CK and lactate values were not affected by the space allowance during transport.
The discriminant analysis showed that only the LDH activity marked the difference among SA groups. The centroid values were -0.124, 0.430 and -0.378 for SAL, SAM and SAH respectively; eigen value: 0.125; canonical correlation: 0.334. Table 2 shows the values of blood parameters in each group of animals after lairage. Respect to haemato-logical parameters, a significant effect (p<0.05) of treatment during lairage was found on RBC, with significant differences (p<0.05) between SAM groups. Cortisol after 18 h ranged between >65 nmol/L (TL-FAST-SAH) and <111 nmol/L (TL-FEED-SAM). The highest value of adrenaline (763.45 nmol/L) was found in TL-FAST-SAM group. There was a strong effect (p<0.001) of lairage treatment on noradrenaline, with highest values in groups TL-FAST-SAL (2816.46 nmol/L) and TL-FAST-SAM (3171.40 nmol/L).

Haematological, hormonal and biochemical parameters after lairage
A significant effect of treatment during lairage was found on glucose (p<0.001), total proteins (p<0.01), creatinine (p<0.01) and LDH activity (p<0.001). In general, fasting during lairage decreased glucose values, while increased total proteins (in SAM group), creatinine and LDH activity (in SAH groups).
The discriminant analysis (Table 3) obtained two discriminant functions. The first explained the maximum of the existing differences among fasting groups (1: FAST-SAL, 3: FAST-SAM and 5: FAST-SAH) and no fasting groups (2: FEED-SAL, 4: FEED-SAM and 6: FEED-SAH), while the second function (only 10.3% of variance) discriminated between groups 2 and 3 from all other groups (1, 5, 4, 6). This analysis found that the parameters that truly marked the difference among groups were noradrenaline and glucose (the standard-5 Lamb welfare during transport and lairage Table 2. Blood parameters in Merina breed lambs after lairage treatment (TL), fasting or feeding (means ± SE).   Our findings showed that plasma glucose activity was influenced by space allowance. According to the results of Bórnez et al. (2009) in Manchega lambs and Tadich et al. (2009) in Corriedale breed, the highest values found in glucose at medium-SA could be associated with a high stress level. However, De la Fuente et al. (2012) did not find any effect of space allowed in this indicator in Assaf breed lambs. A different reaction to SA could be due to both lamb ages and sheep breeds.

Blood parameters Fasting (TL-FAST) Feeding (TL-FEED) GLM
SA had not effect on total protein values, results that are consistent with the findings by Cockram et al. (1996). Increased creatinine levels have been associated to dehydration (Montané, 2002). Pollard et al. (2002) in red deer subdued to different treatments previous to slaughter, indicated that the creatinine concentration was not a useful parameter to measure animal welfare, which is in agreement with the results found in the present study.
On the other hand, no differences for lactate values were detected among SA treatments, although other authors such as Pollard et al. (2002) or Miranda-de la  found an increase for this welfare indicator related to stress. According to Grandin (1997) genetic factors, such as temperament, interact in complex ways with an animal's previous handling experiences and learning determining future reactions during a particular handling procedure.
High levels of serum LDH or/and CK have been correlated with muscle tissue damage as well as with vigorous exercise (Knowless, 1998;Miranda-de la Lama et al., 2010a). Broom & Fraser (2007) indicated that CK activity could be considered as an indicator that permits determining appropriate minimum acceptable space allowances for transported animals. Although, in our study, no significant effects of space allowance during transport were detected on CK activity, an increase in LDH activity after transport, with highest values for the SAM group, were detected. Ibáñez et al. (2002) found a higher LDH activity in lambs transported at low (4 lambs/m 2 ) than at high stocking density (8 lambs/m 2 ) which is in agreement with our results. On the other hand, Grandin (2000) indicated that sheep may not need to lie down during short journeys. This could explain the absence of differences among SAH and SAL groups on LDH activity.
SA is an economical important factor, because reducing space can reduce costs of transport. In general, smaller space allowances lead to lower unit costs of transport since more animals can be carried in the vehicle (SCAHAW, 2002). A range of 0.16 to 0.30 m 2 / lamb could be acceptable during transport but investigation on behaviour will be required. in graphical form, the results obtained in the statistical procedure: Groups 1, 3 and 5 (FASTING) are located in the area that corresponds to the positive value of the first function (with noradrenaline and glucose as main parameters). In regard to the second function (with the same main parameters), G2 (FEED-SAL) and G3 (FAST-SAM) are located in the area that corresponds to the positive values of this function while the rest of the groups (G1, G4, G5 and G6) are located in the negative part.

Discussion
Many critical points previous slaughter (such as transport or lairage conditions) may compromise lambs welfare (Miranda-de la Lama et al., 2010b). However, sheep reaction to transport is affected by factors such as breed (Hall et al., 1998) or age/weight (Bórnez et al., 2009). Research on space allowances has been published for sheep (Cockram et al., 1996;De la Fuente et al., 2010, 2012Knowless et al., 1998; on Suffolk × Greyface, suckling Assaf lambs, and shorn and unshorn lambs, respectively). There is little information about the effects of space allowance during transport and the lairage conditions before slaughter on welfare for Merino lambs used in the present study.

Effect of space allowance on blood parameters
Similar values were found on haematological parameters among the different SA groups. Miranda-de la  found a similar number of RBC, leucocytes and hematocrit in Aragonesa lambs at similar age (100 days) and weight (25 kg) transported for 3 h. Our results could indicate that SA during transport is not a factor that affects these blood parameters. According to Mellor et al. (2002) cortisol and adrenaline values increase with emotional anxiety, while noradrenaline seems to be related to physical stress. In addition, the cortisol response to handling and transport depends upon the species and breed studied (Hall et al., 1998). In this study the space allowance had no significant effect on cortisol values, results that are in agreement with Cockram et al. (1996) and De la Fuente et al. (2012). Our results showed a significant increase in adrenaline concentration after transport in SAL group but not corresponding increase in noradrenaline, in agreement with Åkerstedt et al. (1983). These findings imply that a space allowance between 0.16 and 0.30 m 2 /lamb did not cause a significant hormonal variation in lambs transported under the conditions provided in the present study. 7 Lamb welfare during transport and lairage centration and lowest plasma glucose values found after lairage in the FASTING groups could be useful as indicators of the intensity of stress, as found for goats (Kannan et al., 2000).
Lairage allows the animals to recover from the stress endured during transport and consequently to improve meat quality (Rabaste et al., 2007). Feed deprivation during lairage is a common practice to reduce gut contents, therefore minimizing the risk of carcass contamination. However, the results of this study indicate that fasting cause some physiological changes which would be indicative of compromised welfare. In addition, after 18 h lairage with access to feed, some physiologic parameters associated with stress (such as glucose, noradrenaline) or physical fatigue (such as LDH) returned to basal (farm) levels. Therefore, these results suggest that feeding during lairage could be more appropriate than fasting, especially when animals are stressed after transport, since it seems to allow overnight recovery. Nonetheless, it is not clear whether these changes are indicative of adaptation to feed deprivation, a sign of metabolic depletion or a compromised welfare. Though recommended from these results, the decision of feeding or fasting would also depend on other factors such as the economic cost, carcass hygiene or meat quality.
Some recommendations to meat industry could be given on the basis of the information obtained in this paper: From welfare standpoint a range of space allowance during transport of between 0.16 and 0.30 m 2 / lamb could be recommended without showing major changes on welfare physiological indicators. Feeding during lairage could be more appropriate than fasting, especially when animals showed more stress after transport, since it allows recuperation overnight.

Effect of handling during lairage on animal welfare
In agreement with the findings by De Boer et al. (1989), fasting induced a rise in catecholamine levels, particularly of noradrenaline. In addition, catecholamine produced a contraction of the spleen with the subsequent increase in blood red cells values (Marco & Lavín, 1999). For this reason, the highest RBC values in the FAST-SAM group after lairage could be associated to the higher catecholamine value found in this group. However, our results contrast with the lack of variations in haematological values reported by Liste et al. (2011). Perhaps the different values in noradrenaline sampled after lairage could be related to the significant interaction SA × TL that was observed and could explain the results on RBC.
Increase in cortisol concentration has been associated with feed-deprivation stress in Merino lamb (Zimerman et al., 2013), which contrasts with the results of cortisol found in the present paper. However, cortisol levels can vary greatly as it is a time-dependent response with high individual variability and therefore, comparisons among studies must be done with caution.
Glucose metabolism decreases due to the decline of propionate production, the major precursor for gluconeogenesis, in the rumen caused by low feed intake (Bergman, 1975). The lowest plasma glucose values found after lairage at the FASTING groups could be useful as an indicator of the intensity of stress, which is in agreement with the results by Kannan et al. (2000) for goats. The higher levels of glucose found in the present study when animals had access to feed during lairage (similar values to the basal conditions, on farm) could indicate recuperation overnight. Liste et al. (2011) indicated that CK increase could be associated with a feed deprivation during lairage. However, in our study CK levels were similar across all TL groups, as found by Zimerman et al. (2011). Gupta et al. (1999) associated the LDH decrease with feed deprivation in equids, which contrast with our results. Obviously this could respond to the different physiology of these two species. In addition, lairage without feed represented an additional stress, especially in lambs of SAM group. For this reason it is speculated that some physiologic parameters such as LDH increased in this treatment (SAM-TLFAST). This could indicate cumulative effects of the transport and lairage conditions.
With regard to the results of the discriminant analysis, the most notable data showed that this analysis only included the glucose and noradrenaline as the main parameters that allow differentiating among lairage treatments In addition, the highest noradrenaline con-