El

Ma

b

a

ARRAA

KHSFS

0d

Small Ruminant Research 102 (2012) 177– 185

Contents lists available at ScienceDirect

Small Ruminant Research

jou rn al h om epa ge: www. elsev ier .com/ locate /smal l rumres

ffects of shade and flaxseed supplementation on the welfare ofactating ewes under high ambient temperatures

. Caropresea,∗, M. Albenzioa, A. Brunob, G.Annicchiaricob, R. Marinoa, A. Sevia

Department of Production and Innovation in Mediterranean Agriculture and Food Systems (PriMe), Università di Foggia, Via Napoli 25, 71100 Foggia, ItalyCRA - Istituto Sperimentale per la Zootecnia, Segezia–Foggia, Italy

r t i c l e i n f o

rticle history:eceived 11 April 2011eceived in revised form 8 July 2011ccepted 12 July 2011vailable online 11 August 2011

eywords:igh ambient temperatureolar radiationlaxseedheep welfare

a b s t r a c t

The objectives of this study were to evaluate the effects of protection from solar radiationand whole flaxseed supplementation on the welfare of ewes under high ambient temper-atures. The experiment was carried out during the summer (July and August) of 2007:40 Sarda ewes were divided into four groups of ten each, and either exposed (EXP; notoffered shade) or protected from solar radiation (PRO; offered shade). For each solar radi-ation treatment, ewes were supplemented with whole flaxseed (EXP-F and PRO-F) or not(EXP-C and PRO-C). At the beginning of the experiment and at d 21 and 44 of the trial, thebody weight and the body condition score of the ewes were recorded. Respiration rate andrectal temperature were measured twice weekly. At d 29 of the experiment, blood cortisolconcentrations were measured after an injection of ACTH. Cellular immune response wasevaluated by intradermic injection of phytohemagglutinin at the beginning of the exper-iment, and at d 14, 29, 44 of the trial. Humoral response to ovalbumin was measured at8, 14, 28, and 44 d of the study period. At d 1, 14, 28, and 44 of the experiment, bloodsamples were collected from each ewe for the determination of the blood metabolites andenzymes. Behavioral observations of ewes were recorded by trained observers weekly.Flaxseed supplementation resulted in significantly lower values of respiration rate both inshaded and non-shaded ewes. Small but significant differences were found in ewe rectaltemperatures, which were lower in protected than in exposed ewes, irrespective of flaxseedsupplementation. The exposure to solar radiation resulted also in lower BCS. Supplementedewes displayed higher anti-OVA IgG and cortisol levels than non supplemented ewes. Dietand solar radiation affected plasma concentration of glucose which were higher in theexposed than in the protected ewes and in the supplemented than in no supplementedewes. Plasma levels of Cl and Na were higher in supplemented than in non supplementedewes. Shaded groups had lower plasma concentration of NEFA, gamma-glutamyl transpep-tidase, and K, and higher levels of ALT/GPT and Mg compared with non shaded groups.

Solar radiation affected eating and ruminating activities: greater proportions of ewes ofthe exposed groups were observed eating than ewes in the shaded groups. Whole flaxseedsupplementation enhanced humoral immune and thermoregulatory responses during hotseason. As expected, the provision of shaded areas reduced the mobilization of body lipidresources.

∗ Corresponding author. Tel.: +39 0881 589332; fax: +39 0881 589331.E-mail address: m.caroprese@unifg.it (M. Caroprese).

921-4488/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.smallrumres.2011.07.010

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

During summer, dairy sheep raised in the Mediter-ranean area are exposed to ambient temperatures oftenexceeding their thermal neutral zone. Prolonged expo-

inant R

sured in the morning after the milking and before feeding time. The bodyweight was measured by an electronic scale (METTLER MultiRange ID5,KC120/KC240).

Table 1Chemical and fatty acid composition (g/100 g of total fatty acids) of diets.

Control diet Flaxseed diet

DM% 90.56 90.79Ether extract, % of DM 2.71 4.75CP, % of DM 12.89 13.03ADF, % of DM 25.51 26.06NDF, % of DM 50.14 49.21ADL, % of DM 2.99 3.01

Fatty acids, g/100 g of total fatty acidsC16:0 18.59 17.69

178 M. Caroprese et al. / Small Rum

sure to maximum air temperature over 30 ◦C and totemperature-humidity index (THI) higher than 80 preventlactating ewes from maintaining their thermal balance,thus inducing heat stress conditions (Sevi et al., 2001).When exposed to high ambient temperatures, sheep trig-ger a series of physiological compensatory mechanismsthat allow the maintenance of vital functions by dras-tic changes in the animals’ biological functioning, such asreduction of feed intake, changes in the metabolism ofwater, protein, energy, and mineral balances, enzymaticreactions, and hormonal secretions (Marai et al., 2007). Lac-tating ewes under heat stress display increased respirationrate and rectal temperature; the enhanced mobilization ofbody fat reserve for thermoregulation results in increasednon-esterified fatty acids plasma concentration. In addi-tion, in dairy ewes under heat stress alterations of immuneresponses resulting in a depressed cell-mediated immuneresponse and of behavioral responses with increased inac-tive behaviors have been observed (Sevi et al., 2001).

Management and nutritional practices are consideredthe main strategies to improve welfare and productionperformance of lactating animals under high ambient tem-peratures. The provision of shaded areas during the hotseason can facilitate ewes to the maintenance of their ther-mal balance, and energy and mineral metabolism (Seviet al., 2001).

Fat supplementation has been suggested as a strategyto increase the energy ration of the diet and to reduce theadverse effects of heat stress on dairy cows. Liu et al. (2008)demonstrated a positive effect of fat supplementation onthe regulation of body temperature, plasma enzymes, elec-trolyte and hormones. No studies tested the effects of fatsupplementation on lactating ewes exposed to high ambi-ent temperatures. Caroprese et al. (2009) has providedevidence that feeding whole flaxseed rich in omega-3 fattyacids to cows raised under high ambient temperature alterscytokines production, and enhanced cellular and humoralimmune responses of dairy cows. A better understand-ing of the effects of omega-3 fatty acids on immune andphysiological responses of ewes under high ambient tem-perature may be useful in optimizing nutritional strategiesto improve sheep welfare and health.

This study was conducted with the aim of evaluatingthe effects of protection from solar radiation and of wholeflaxseed supplementation on physiological, immune, andbehavioral responses of lactating ewes under high ambienttemperatures.

2. Materials and methods

2.1. Experimental design and ewe feeding

The experiment was conducted during the summer (July–August)of 2007 at Segezia research station of the Council for Research andExperimentation in Agriculture (CRA-ZOE) and lasted 44 d. Forty late-lactation Sarda ewes (d 202.1 ± 5.3 of lactation, mean ± SD) were dividedinto four groups of ten each, balanced for parity (2.6 ± 0.7), milk yield(740.5 ± 9.43 g/d), body weight (39.11 ± 0.26 kg) and BCS (1.61 ± 0.06).During the study, animals were either exposed (EXP; not offered shade)

or protected from solar radiation (PRO; offered shade). Groups were sep-arately reared in external pens of 5 × 12 m bounded with mesh-fence. Theshade was provided by 3 × 8 m and 3.5 m high brickwork rooms adjacentto the open pens; the trough and the crib were located in the externalareas. Both ewes exposed to and protected from solar radiation bene-

esearch 102 (2012) 177– 185

fited from the same space allowance, which included shaded areas in PROgroups. For each solar radiation treatment, ewes were supplemented withwhole flaxseed (EXP-F and PRO-F) or not (EXP-C and PRO-C). Ewes werefed twice daily; EXP-C and PRO-C ewes were fed 1 kg/ewe/d of pellet-ted concentrate (Pecorlat 19, Raggio di sole mangimi SpA, Potenza, Italy)and 1.5 kg/ewe/d of vetch and oat hay; EXP-F and PRO-F ewes were sup-plemented with 21% whole flaxseed (Lin Tech, Tecnozoo srl, Torreselledi Piombino Dese, Italy) in substitution of an equal amount of concen-trate and were fed 790 g/ewe/d of pelletted concentrate, 1.5 kg/ewe/d ofvetch and oat hay, and 210 g/ewe/d of whole flaxseed. Water was avail-able ad libitum for all groups from automatic drinking troughs at any timeof day. The chemical composition of vetch and oat hay, pelleted concen-trate and whole flaxseed was carried out by standard procedures (AOAC,1990): crude protein (CP), fat (by ether extract), neutral detergent fiber(NDF), acid detergent fiber (ADF), acid detergent lignin (ADL) were ana-lyzed. Net energy for lactation (NEL) of the diet was 1.38 Mcal/kg for EXP-Cand PRO-C ewes and 1.36 Mcal/kg for EXP-F and PRO-F ewes (NRC, 2001).The chemical composition of the ingredients of diets is reported in Table 1.

Lipid extraction for fatty acid analysis of the diet ingredients was car-ried out according to a modified Folch method (Ardvisson et al., 2009).Separation and quantification of the methyl esters were carried out usinga gas chromatograph (Agilent 6890N) equipped with a flame ionizationdetector (FID), autosampler, split injection port and a fused silica capillarycolumn (100 m, internal diameter 0.25 mm, film thickness 0.25 �m) (HP-88 capillary column, Agilent Technologies Spa, Italy). Helium was usedas the gas carrier (0.42 ml/min). Injector temperature was maintained at220 ◦C whereas the detector temperature was of 250 ◦C. Fatty acid pro-file was determined using the following temperature gradient program:initial temperature of 150 ◦C that was increased to 220 ◦C at 1 ◦C/min.

Each peak was identified and quantified using pure methyl ester stan-dards: 37 component FAME mixture and methyl trans vaccenate C18:1trans-11 octadecenoate (Matreya Inc., Pleasant Gap, PA); conjugatedlinoleic acid (CLA) isomers were identified by comparison of retentiontimes with CLA methyl ester standards: C18:2 cis-9 trans-11 octadeca-dienoate and methyl C18:2 trans-10 cis-12 octadecadienoate (MatreyaInc., Pleasant Gap, PA). Results were expressed as g/100 g of total fattyacids analyzed (Table 1).

During the trial, ambient temperature and relative humidity in pro-tected and exposed area were monitored with thermo-hygrographs (LSI,I-20090 Settala Premenugo-Milano, Italy) placed at 1.5 m from the floor.Average of temperature-humidity index (THI) was calculated using theKelly and Bond’s (1971) formula.

2.2. Weight gain and body condition score

At the beginning of the experiment and at d 21 and 44 of the trial,the body weight and the body condition score (BCS, six-point scale 0:thin, 5: fat) of the ewes were recorded. Body weight and BCS were mea-

C18:0 2.65 2.74C18:1 cis-9 16.07 16.13C18:2n6 cis-9, cis-12 33.52 30.48C18:3n3 17.38 21.30Others fatty acids 11.85 11.73

inant R

2

1pa

2

iSsjtii

2

th(Aoeawi

2

aiosMAai2sWp1tadIw(2aw2

((aag

2

latnss

M. Caroprese et al. / Small Rum

.3. Respiration rate and rectal temperature

Respiration rate (RR) was measured in all animals twice weekly at430 h by counting the rate of flank movements. Subsequently, rectal tem-erature (RT) was measured by an electronic thermometer (LSI) with anccuracy of 0.1 ◦C.

.4. Cortisol level determination

At d 29 of the experiment, the ewes were intravenouslynjected with 2 IU porcine ACTH/kg BW0.75 (adrenocorticotropic hormone,igma–Aldrich, Milan, Italy). Blood samples (7 ml) for evaluation of corti-ol concentrations were collected in heparinized vacuum tubes from theugular vein immediately before and 60, 120 and 240 min after ACTH injec-ion. Hormone concentration was determined by a competitive enzymemmunoassay (Radim, Cortisolo EIA WELL KS18EW, Rome, Italy), accord-ng to Caroprese et al. (2010).

.5. In vivo cell-mediated immunity

At the beginning of the experiment, and at d 14, 29, and 44 of therial, the skin test was performed to induce non-specific delayed-typeypersensitivity by intradermic injection of 1 mg/mL phytohemagglutininPHA) (Sigma–Aldrich, Milan, Italy) dissolved in sterile saline solution.t each sampling time, the injection was administered into the centerf a 2 cm diameter circle marked on shaved skin on the upper side ofach shoulder. The determination of lymphocyte proliferation, measureds skinfold thickness, was calculated from the two measurements madeith calipers as the difference between 24 h post-injection thickness–pre-

njection thickness.

.6. Humoral immune response

To evaluate the humoral response to ovalbumin (OVA, chicken egglbumin) and to measure anti-OVA IgG concentrations at d 1 of the exper-ment the ewes were subjected to subcutaneous injection of 6 mg ofvalbumin (OVA, Sigma–Aldrich, Milan, Italy) dissolved in 1 mL of sterilealine solution and 1 mL of incomplete Freund’s adjuvant (Sigma–Aldrich,ilan, Italy). A second injection without adjuvant was repeated 7 d later.

ntibody titres were determined in blood samples collected in hep-rinized vacuum tubes (Becton Dickinson, Plymouth, United Kingdom)mmediately before the first antigen injection (1 d) and then at 8, 14,8, and 44 d of the study period. An ELISA (Enzyme Linked Immunoas-orbent Assays) was performed in 96-well U-bottomed microtiter plates.

ells were coated with 100 �L of antigen (10 mg of OVA/mL of phos-hate buffered saline—PBS) at 4 ◦C for 12 h, washed and incubated with% skimmed milk at 37 ◦C for 1 h to reduce nonspecific binding, andhen washed. The sheep serum (1:1000 dilution in PBS) was addednd incubated at 37 ◦C for 1 h. The extent of antibody binding wasetected using a horseradish peroxidase-conjugated donkey anti-sheep

gG (Sigma–Aldrich, Milan, Italy) (1:20,000 dilution in PBS). After otherashing, 100 �L of substrate (1 mg of 3,3′ ,5,5′-tetramethylbenzidine

TMB), 1 mLof dimethylsulfoxide (DMSO), 9 mL of phosphor-citrate buffer, �L of H2O2) were added in each well. The reaction was completeddding 50 �L of H2SO4 after 30 min. Optical density was measured at aavelength of 450 nm with titer-ELISA spectrophotometer (Rosys Anthos

020).Anti-Ova IgG evaluation was a quantitative assay: a standard curve

y = 0.010x + 0.136, R2 = 99.030%) was made up by ovine IgG dilutionsA5295, Sigma–Aldrich, Milan, Italy); all plasma samples were readgainst the curve. Data were expressed as mg of anti-OVA IgG/mL. Thessay was optimized in our laboratory for concentrations of coating anti-en, serum, and antibody.

.7. Blood sampling and analyses

At d 1, 14, 28, and 44 of the experiment, blood samples were col-ected from each ewe for the determination of the following metabolites

nd enzymes: aspartate amino-transferase (AST/GOT), alanine amino-ransferase (ALT/GPT), gamma-glutamyl transpeptidase (GGT), glucose,on-esterified fatty acids (NEFA), calcium (Ca), magnesium (Mg), potas-ium (K), sodium (Na), chlorine (Cl), according to Sevi et al. (2001). Bloodamples were centrifuged for 15 min 1400 × g at 20 ◦C. The plasma sam-

esearch 102 (2012) 177– 185 179

ples were subdivided in three equal rates and stored at −20 ◦C untilanalyses.

2.8. Behavioral observations

Weekly behavioral observations of ewes were recorded by trainedobservers. Scan samples were taken every 15 min from 0900 to 1200 hand from 1300 to 1600 h for a total of 6 h-period in each group of ewes.Behavioral recordings were performed on individual animals marked onthe their heads and rumps.

During each observation and for each animal, posture (standing orlying) and the following behavioral activities were recorded: standingidle, idling, walking, eating, and ruminating. In addition, the number ofaggressive interactions were measured by continuous recordings dur-ing the whole period of observation. Postural and behavioral data wereexpressed either as percentage of the total observations or number ofevents observed (i.e. aggressive interactions).

2.9. Statistical analysis

All variables were tested for normality using the Shapiro–Wilk test(Shapiro and Wilk, 1965), and blood cortisol data were transformed intologarithm form to normalize their frequency distribution. Data were pro-cessed using ANOVA for repeated measures (SAS, 1999) having exposureto solar radiation (non repeated factor), diet (non repeated factor), timeof sampling, and their interactions as repeated factors.

The model utilized was:

yijkl = � + ˛i + ˇij + �k + �l + (˛�)ik + (˛�)il + (��)kl + (˛��)ikl + εijk ln

where � is the overall mean; ̨ the diet (i = 1, 2); ̌ the animal effectwithin groups; � the day of sampling effect; � the exposure to solar radi-ation (l = 1, 2); ˛� are the interaction of diet × day of sampling; ˛� are theinteraction of diet × exposure to solar radiation; �� are the interaction ofexposure to solar radiation × day of sampling; ˛�� are the interaction ofdiet × exposure to solar radiation × day of sampling and ε is the error. Dataon BCS, respiration rate, rectal temperature and cell-mediated immuneresponse were covariated on initial values. Blood cortisol levels and antiOVA-IgG concentrations were processed using ANOVA for repeated mea-sures (SAS, 1999) having exposure to solar radiation (non repeated factor),diet (non repeated factor), time of blood collection (0, 10 and 60 min afterisolation test for cortisol test and 1, 8, 14, 28, and 44 d for anti OVA-IgG),and their interactions as repeated factors. For behavioral data the varia-tion due to solar radiation (non repeated factor), diet (non repeated factor),time of day, day of sampling and their interactions were tested. Individualanimals were nested within treatments. Where significant effects werefound (P < 0.05) Student’s t-test was used to locate significant differencesbetween means.

3. Results

3.1. Meteorological data

In the exposed areas, during the day (from 08.00 to20.00) averages of ambient temperatures ranged from 26.5to 34.4 ◦C and peaked during the wks 5 and 6 of the trial(Fig. 1). In addition, in the exposed areas, weekly aver-ages of maximum ambient temperatures were near or over30 ◦C throughout the study period (data not shown). In theprotected areas averages of ambient temperatures rangedfrom 23.5 to 30.9 ◦C; as a consequence, during daytime,in the exposed areas, mean ambient temperatures were2.3–5.6 ◦C higher than in the protected areas. On the con-trary, during the night (from 20.00 to 08.00) mean ambienttemperatures ranged from 15.6 to 21.7 ◦C in the exposed

areas, and from 21 to 27 ◦C in the protected areas; as aresult, during the night, in the protected areas mean ambi-ent temperatures were more than 5 ◦C higher than in theexposed areas. In the protected areas weekly averages

180 M. Caroprese et al. / Small Ruminant Research 102 (2012) 177– 185

eas me

Fig. 1. Means ± SD of ambient temperature in exposed and protected ar0800).

of nocturnal temperatures never falls below the criticalthreshold of 21 ◦C.

In the exposed areas the maximum temperature-humidity index (THI) values were near 79 on average, androse to 83 and 84 during wks 5 and 6 of the trial. In theprotected areas THI never exceeded 80 during daytime and

was about 6 points higher than in the exposed areas duringnight (data not shown).

Table 2Least square means ± SEM of respiration rate, rectal temperature, BCS, and bodyand fed flaxseed (EXP-F, PRO-F) or control diet (EXP-C, PRO-C).

Treatment

Item Day period EXP-C EXP-F

Respiration rate, breath (min) 0–44 d 99.69a 92.76b

Rectal temperature (◦C) 0–44 d 39.62a 39.70a

BCSb 21 d 2.61b 2.69ab

44 d 2.61b 2.64b

Body weight change (kg) 0–21 d 1.56b 2.63ab

22–44 d 1.47 1.41

Means followed by different letters are significantly different at P < 0.05.a SR: solar radiation, TM: time of sampling; NS, not significant.b Body condition score.

*** P < 0.001.

asured during: (a) daytime (0800 to 20.00), and (b) nighttime (20.00 to

3.2. Respiration rate, rectal temperature, body conditionscore, and body weight change

Flaxseed supplementation resulted in significantlylower values (P < 0.001) of RR both in shaded and non-shaded ewes (Table 2). Respiration rate was also affected

by time of sampling (P < 0.001), and the highest values wereobserved during wks 2, 5 and 6 of the experiment in all thegroups when the highest THI values were recorded.

weight change of ewes when exposed or protected from solar radiation

Effects, P

PRO-C PRO-F SEM SRa Diet TM

100.63a 92.68b 0.93 NS *** ***

39.51b 39.47b 0.04 *** NS ***

2.81ab 2.93a2.96a 3.03a 0.09 *** NS ***

3.19a 2.91a1.51 1.41 0.35 NS NS ***

M. Caroprese et al. / Small Ruminant R

Fig. 2. Antibody titres to OVA (least squares means ± SEM) detected inewes when exposed or protected from solar radiation and fed flaxseed(tw

ieR(wahido(0d(

3

wflfT

3

a(iniaFe

3

c((

EXP-F, PRO-F) or control diet (EXP-C, PRO-C), at 1, 14, 28, and 44 d ofhe trial. a,b Values with different superscripts differ between treatmentsithin a sampling day (P < 0.05).

Small but significant differences (P < 0.001) were foundn the RT, which was lower in the protected than in thexposed ewes, irrespective of flaxseed supplementation.ectal temperature was also affected by time of samplingP < 0.001). In all groups the highest RT was observed duringks 2, 5 and 6 of the experiment. Solar radiation (P < 0.001)

nd time of sampling (P < 0.001) affected BCS. At 21 d theighest BCS was observed in PRO-F group and the lowest

n EXP-C group, while at 44 d both the protected groupsisplayed higher BCS than the exposed ones. An effectf time of sampling for body weight changes was foundP < 0.001) due to higher body weight changes during the–21 d period than during the 22–44 d period. EXP-C ewesisplayed the lowest weight gain during the 0–21 d periodP < 0.001).

.3. In vivo cell-mediated immune response

Ewe cell-mediated immune response to PHA injectionas not affected by the exposure to solar radiation or byaxseed supplementation, whereas an effect of time was

ound (P < 0.001) because in all the groups a decrease in-lymphocyte proliferation was observed at 29 d.

.4. Humoral immune response

Flaxseed supplementation, time of blood collectionnd their interaction affected humoral response of sheepP < 0.001, Fig. 2). On average, anti-OVA IgG were highern sheep supplemented with flaxseed than in sheep witho supplementation (41.97 mg/mL vs 28.52 ± 0.8 mg/mL),

rrespective of shading. In particular, significantly highernti OVA-IgG concentrations were found in EXP-F and PRO-

than in EXP-C and PRO-C ewes at d 14, 28 and 44 of thexperiment.

.5. Plasma cortisol levels

No significant effects of solar radiation on cortisol con-entrations were found. On the contrary, an effect of dietP < 0.01), and of the interaction of diet × solar radiationP < 0.05) emerged with higher cortisol levels in the sup-

esearch 102 (2012) 177– 185 181

plemented than in no supplemented ewes (2.50 Log ng/mLvs 2.37 ± 0.03 Log ng/mL). The highest plasma cortisol lev-els were found in EXP-F ewes and the lowest in EXP-C ewes(P < 0.05; Table 3).

3.6. Plasma metabolites and enzymes

Plasma concentration of glucose was affected bysolar radiation, diet, and time of sampling (P < 0.001,P < 0.001, and P < 0.05, respectively) (Table 3). Blood glucoseconcentrations were higher in the exposed than in the pro-tected ewes (5.01 mmol/L vs 4.26 ± 0.06 mmol/L). Flaxseedsupplementation also resulted in higher plasma glu-cose (4.85 mmol/L vs 4.42 ± 0.06 mmol/L). Solar radiationaffected plasma concentration of NEFA, which were higherin the exposed than in the protected ewes (P < 0.001). In allthe groups plasma NEFA increased from the first samplingto d 28 of the experiment (P < 0.001). Higher ALT/GPT lev-els were found in the protected than in the exposed ewes(17.87 U/L vs 16.21 ± 0.4 U/L, P < 0.01). Plasma concentra-tion of both ALT/GPT and aspartate amino-transferase(AST/GOT) decreased from the beginning to the end of theexperiment (P < 0.05) in all the groups. The ewes exposedto solar radiation showed a higher plasma concentration ofgamma-glutamyl transpeptidase (GGT) concentration thanthe shaded ewes (58.52 U/L vs 51.65 ± 1.21 U/L, P < 0.001).In addition, GGT was affected by the interaction of solarradiation × diet and higher GGT levels were measured inEXP-F and PRO-F than in EXP-C and PRO-C groups (P < 0.05).Cl and Na plasma levels were affected by diet (P < 0.05 andP < 0.01, respectively); the highest levels of both Cl and Nawere found in EXP-F ewes. The ewes exposed to solar radia-tion showed higher K and lower Mg plasma levels (P < 0.05)than the protected ewes (4.62 vs 4.48 ± 0.04 for K and0.93 mmol/L vs 0.97 ± 0.01 mmol/L for Mg). In addition aneffect of solar radiation × diet for K plasma levels emerged,with the highest plasma K in EXP-F ewes (P < 0.01). CABdid not change across treatments, but and an increase inCAB levels was observed in all the groups at d 14 and 28(P < 0.001).

3.7. Behavioral observations

In Table 4 some of the main behavioral traits observedwere reported. During the trial no effects of solar radia-tion on behavioral traits of ewes were found except foreating and ruminating activities (P < 0.01). Indeed, greaterproportions of ewes of the exposed groups (EXP-C and EXP-F) were observed eating than ewes in PRO-C and PRO-Fgroups. On the contrary, greater proportions of ewes ofthe protected groups (PRO-C and PRO-F) were observedruminating than ewes in EXP-C and EXP-F groups. In themorning, higher proportions of ewes were observed lyingand idling (P < 0.001) than during afternoon hours in EXP-C,PRO-C and PRO-F groups. Greater proportions (P < 0.001) of

ewes from EXP-C, EXP-F and PRO-F groups were observedeating in the afternoon. In the protected groups greaterproportions of ewes stayed out during the afternoon hoursthan during the morning hours (31.38 vs 10.00 ± 4.13).

182 M. Caroprese et al. / Small Ruminant Research 102 (2012) 177– 185

Table 3Least square means ± SEM of cortisol, metabolites and enzymes in plasma of ewes when exposed or protected from solar radiation and fed flaxseed (EXP-F,PRO-F) or control diet (EXP-C, PRO-C).

Treatment Effects, P

Item EXP-C EXP-F PRO-C PRO-F SEM SRa Diet TM

Cortisol (Log ng/mL) 2.31b 2.55a 2.42ab 2.44ab 0.05 NS ** ***

Glucosio (mmol/L) 4.85b 5.17a 3.99d 4.53c 0.08 *** *** *

NEFAb (mmol/L) 0.52a 0.52a 0.49b 0.49b 0.01 *** NS ***

AST/GOTc (U/L) 105.25c 127.3a 120.18ab 114.48bc 4.15 NS NS *

ALT/GPTd (U/L) 14.43c 18.00a 19.13a 16.63b 0.60 ** NS *

GGTe (U/L) 61.40a 55.65b 50.48c 52.83ab 1.71 *** NS NSNa (mmol/L) 148.23b 150.68a 148.70b 149.20ab 0.64 NS * ***

K (mmol/L) 4.54ab 4.70a 4.53ab 4.43b 0.06 * NS ***

Cl (mmol/L) 107.80b 110.03a 108.25b 109.33ab 0.55 NS ** NSMg (mmol/L) 0.92b 0.94b 0.99a 0.95ab 0.02 * NS NSCABf (mmol/L) 44.97 45.34 44.98 44.3 0.44 NS NS ***

Means followed by different letters are significantly different at P < 0.05.a SR: solar radiation, TM: time of sampling; NS, not significant.b Nonesterified fatty acids.c Aspartate amino-transferase.d Alanine amino-transferase.e Gamma-glutamyl transpeptidase.f Catione-anione balance (CAB) = sodium + potassium − chloride.* P < 0.05.

** P < 0.01.*** P < 0.001.

Table 4Least square means ± SEM of behavioral activities recorded in ewes when exposed or protected from solar radiation and fed flaxseed (EXP-F, PRO-F) orcontrol diet (EXP-C, PRO-C). Values are expressed as either percentage (p) or mean number of events.

Treatment Effects, P

Item EXP-C EXP-F PRO-C PRO-F SEM Hour SRa Diet Hour × SR × diet

Lying, pMorning 30.55a 25.00ab 35.55a 41.66a 6.17 *** NS NS NSAfternoon 12.22b 25.00ab 15.00b 10.55bStanding idle, pMorning 41.67 53.89 34.44 32.22 7.17 NS NS NS NSAfternoon 40.00 35.00 50.00 43.89

Idling, pMorning 30.56a 23.33ab 32.22a 40.56a 6.05 *** NS NS NSAfternoon 11.67b 23.89ab 14.44b 9.44b

Walking, pMorning 10.00 7.78 7.78 10.00 4.49 NS NS NS NSAfternoon 3.89 2.22 15.00 5.56

Eating, pMorning 13.33bcd 8.33 cd 7.78 cd 1.11d 5.56 *** ** NS NSAfternoon 31.67a 24.44ab 7.22 cd 20.00abc

Ruminating, pMorning 3.89 7.78 16.67 13.88 3.98 NS ** NS NSAfternoon 8.33 14.44 13.33 20.56

Aggressive interactions, nMorning 16.67 61.11 11.11 33.33 15.95 NS NS NS NSAfternoon 11.00 11.00 38.89 0.00

Means followed by different letters are significantly different at P < 0.05.

a SR: solar radiation, TM: time of sampling; NS, not significant.

** P < 0.01.*** P < 0.001.

4. Discussion

A good indicator of heat stress in animals is theTemperature-humidity index (THI). Sheep production per-formance can be reduced by 20% with THI passing from60–65 to 72–75 (Peana et al., 2007). Also diurnal fluctua-

tions of the ambient temperatures can affect the animals’ability to cope with heat stress: it is well known that the

drops of the minimum temperatures during the night canhelp the animals to dissipate heat load (Silanikove, 2000;Sevi et al., 2001). In the present experiment, both maximumdiurnal temperatures in the exposed areas and minimum

inant R

nhedt(ema1eaitrgistft

dhspvrijdspctheCti

bcdten2to(gttt

sirc1

M. Caroprese et al. / Small Rum

octurnal temperatures in the protected areas were alwaysigher than the suggested limits for the maintenance of thewes physiological and productive responses. It has beenemonstrated that prolonged exposure to maximum airemperature over 30 ◦C can induce heat stress conditionsSevi et al., 2001). Peana et al. (2007) found that in Sardawes a reduction in milk yield can be observed if the maxi-um ambient temperatures are higher than 21–24 ◦C, but

lso if the minimum temperatures changed from 9–12 ◦C to8–21 ◦C. The meteorological conditions registered in thexposed areas during daytime, and in particular a meanmbient temperature of about 30 ◦C throughout the exper-ment, and a mean THI of about 80 over three of the sevenrial weeks, may account for the higher rectal temperaturesegistered in the exposed than in the shaded groups. Thereater energy demand for thermoregulation resulted alson higher mobilization of body lipid resources as demon-trated by the lower BCS values recorded in the exposedhan in the shaded ewes. Accordingly, Sevi et al. (2001)ound that THI values of about 80 for only few hours duringhe day represent a stressful condition to lactating ewes.

Animals exposed to high environmental temperaturesisplay an increase in respiration rate, in order to dissipateeat load by respiratory evaporation (Silanikove, 2000); inheep, heat loss by the increase in respiratory rate is therincipal way of heat dissipation because sweating is pre-ented by the presence of wool coat (Marai et al., 2007). Theespiration rate displayed by the ewes in the present study,rrespective of the shade offered, indicated they were sub-ected to heat stress, according to Silanikove (2000), whoefined the panting rate of 80–120 as indicative of high heattress in farm animals extensively managed. The lower res-iration rate measured in flaxseed supplemented than inontrol groups may be ascribed to the nicotinic acid con-ained in flaxseed. Flaxseeds are known not only for theirigh content in �-linolenic acid, but also for their consid-rable amount of nicotinic acid (Bassett et al., 2009). Diostanzo et al. (1997) reported that dietary supplementa-ion with nicotinic acid has a peripheral vasodilator effectn cows, thus improving heat dispersion by evaporation.

Plasma cortisol can increase during acute heat stressecause of its hyperglycaemic action, which increases glu-ose availability during stress to meet the high energyemand (Matteri et al., 2000). The role of flaxseed on cor-isol response of ewes to ACTH challenge is not easy toxplain. Feeding can affect responses to stress in rumi-ants, via the HPA-axis activation (Munksgaard et al.,006). Evidence suggests that the diet can interfere withhe responses to ACTH challenge in sheep by the regulationf gene expression pathways involved in stress responseStefanon et al., 2009). Results from the present study sug-est further investigations on the possible regulation ofhe gene expression involved in the biological responseso high ambient temperature by flaxseed administration inhe diet.

Hyperthermia and the consequent increase in corti-ol secretion can suppress cell-mediated immunity by

nfluencing the T-helper (Th) 1/Th2 balance with a downegulation of Th1 cytokines in favor of the secretion of Th2ytokines (Elenkov and Chrousos, 1999; Murzenok et al.,997; Webster et al., 2002). In our study the reduction of

esearch 102 (2012) 177– 185 183

cellular immune response coincided with ACTH adminis-tration and the subsequent increase of cortisol secretion.As a consequence, the reduction in cell-mediated immuneresponse could be attributed to the high levels of corti-sol secretion induced in all the groups, thus confirmingprevious findings on cows (Lacetera et al., 2005). Dur-ing heat stress, Caroprese et al. (2009) found in flaxseedsupplemented cows an enhancement of cell-mediatedresponse, probably as a result of reduced IL-10 produc-tion, which helped cows to contrast the lymphocyte shiftfrom Th1 to Th2 type. In the present experiment, flaxseedsupplemented ewes showed higher plasma cortisol con-centration than no supplemented ewes; as a consequence,the high levels of cortisol in flaxseed supplemented ewescan be claimed to explain the absence of the expectedenhancement in lymphocyte functions. In order to preventinfluences of ACTH challenge on immune response, a pos-sible alternative to cortisol evaluation to detect heat stressin sheep could be the evaluation of growth hormone; someauthors (Igono et al., 1988) suggested a suppressed pro-duction of growth hormone in cows subject to heat stressin order to lower metabolic heat production. In line withthe inhibition of lymphocyte responses, we observed anincrease of the humoral responses in supplemented ewes,which can be attributed to the ability of glucocorticoidsto stimulate IL-10 and inhibit IL-12 (Visser et al., 1998).Our results confirm Caroprese et al.’s finding on cows(2009), suggesting that flaxseed supplementation can sus-tain immune responses of lactating ewes during summer,as well. Mechanisms involved in both cellular and humoralresponses observed could be attributed to the ability ofboth glucocorticoids and n−3 fatty acids to influence geneexpression of cytokines (Calder and Field, 2002). In par-ticular, our results suggest that flaxseed supplementationduring heat stress can be a suitable strategy to sustainhumoral immune responses in sheep.

High plasma glucose levels recorded in the exposedgroups may be caused by an increase of glycogenolysis andgluconeogenesis caused by heat stress conditions. How-ever, the increase in plasma glucose of the ewes receivinglipid supplement is not easy to explain; previous studieson heat stressed cows did not find any effect of supple-mental fat, carbohydrate, or conjugated linoleic acid onglucose concentration (Liu et al., 2008; Drackley et al.,2003). On the contrary, our results suggest that in heatstressed ewes, flaxseed supplementation can play a role inglucose metabolism: plasma glucose concentration foundin supplemented ewes can be the result of flaxseed inducedcortisol increase resulting in an increased availability ofplasma glucose. In addition, also the nicotinic acid con-tained in flaxseed can interfere with glucose metabolism,thus causing an increase of blood glucose levels. Thorntonand Schultz (1980) reported that the administration ofnicotinic acid can be responsible for elevated levels of glu-cose in ruminants.

The increased levels of NEFA observed in ewes exposedto solar radiation is connected to increased mobilization

of body fat reserves necessary to meet the energy require-ments for thermoregulation, according to previous findingson sheep (Sevi et al., 2001). The levels of glucose and NEFAobserved in the exposed ewes compared with the pro-

inant R

184 M. Caroprese et al. / Small Rum

tected ewes, associated to their rectal temperature andBCS, indicated a stress condition caused by the high ambi-ent temperature, and an attempt to cope with heat loadby an enhanced mobilization of body reserves. In addition,exposed ewes showed lower levels of ALT/GPT, probablyas a result of the reduction of thyroid hormone secretionaimed at decreasing the production of endogenous bodyheat (West, 1999). In animals reared under hot climate,alterations in mineral metabolism occur (West, 1999). Inprevious studies carried out on sheep and cows underheat stress, a reduction in plasma levels of magnesiumwas observed, in line with our results (Sevi et al., 2001;Calamari et al., 2007). A reduced Mg intake, together withincreased utilization of Mg for lipolytic enzymes and areduction of Mg transport across the rumen are the mech-anisms claimed to explain the reduction of plasma Mgduring heat stress (Sevi et al., 2001; Calamari et al., 2007).Heat stress can induce an impairment of rumen function-ality in sheep with a reduction in bacterial activity anda dilution of rumen fluid (Bernabucci et al., 2009). Rumi-nant sweat has a high content of K (Maltz et al., 1994), butsweating only slightly contributes to dissipate heat loadin sheep. In the present experiment plasma K levels weredifferent in exposed and shaded ewes but within physio-logical plasma K range in both groups (Srikandakumar et al.,2003). It is well known that the absorption of Na and Clfrom the rumen is connected to rumen metabolism and toconcentration of VFA (Maltz et al., 1994). Our results showthat flaxseed in the diet can contribute to enhance Na andCl levels in sheep under heat stress, probably improvingrumen fermentation.

Generally, sheep are diurnal animals that under heatstress condition tend to change their habits for reducingbody heat load (Silanikove, 2000). The increase of lying andidling activity during the morning hours can be considereda behavioral thermoregulatory strategy aimed at reducingbody heat load by both conductive heat dissipation mecha-nisms and by the reduction of energy demand required forphysical activity. However, apart from the exposure to solarradiation or the provision of shaded areas, the presence offeed in the manger caused an increase in the proportion ofewes observed eating as previously observed by Sevi et al.(2001). Heat load can also interfere with the alternationof periods of ingestive activity and rumination, leading to areduction or cessation of ruminating (Sevi et al., 2001). Thismay account for the ewes exposed to direct solar radiationdisplaying reduced ruminating behavior.

5. Conclusions

Flaxseed supplementation enhanced humoralresponses of ewes under high ambient temperatureand contributed to sustain their thermal balance. At thesame time the provision of shaded areas helped ewes toreduce heat load, as demonstrated by a reduced mobi-

lization of body lipid resources. Further investigations arerequired to investigate the role of flaxseed administrationin the regulation of the gene expression pathways involvedin sheep responses to high ambient temperature.

esearch 102 (2012) 177– 185

References

Ardvisson, K., Gustavsson, A.M., Martinsson, K., 2009. Fatty acids in for-ages: a comparison of different pre-treatments prior to analysis. Anim.Feed Sci. Technol. 151, 143–152.

AOAC, 1990. Official Methods of Analysis, 15th ed. Association of OfficialAnalytical Chemists, Arlington, VA.

Bassett, C.M.C., Rodriguez-Leyva, D., Pierce, G.N., 2009. Experimen-tal and clinical research findings on the cardiovascular ben-efits of consuming flaxseed. Appl. Physiol. Nutr. Metab. 34,965–974.

Bernabucci, U., Lacetera, N., Danieli, P.P., Bani, P., Nardone, A., Ronchi, B.,2009. Influence of different periods of exposure to hot environmenton rumen function and diet digestibility in sheep. Int. J. Biometeorol.53, 387–395.

Calamari, L., Abeni, F., Calegari, F., Stefanini, L., 2007. Metabolic condi-tions of lactating Friesian cows during the hot season in the Po valley.2. Blood minerals and acid–base chemistry. Int. J. Biometeorol. 52,97–197.

Calder, P.C., Field, C.J., 2002. Fatty acids lammation immunity. In: Calder,P.C., Field, C.J. (Eds.), Nutrition and Immune Function. CABI Publishing,Wallingford, UK, pp. 57–92.

Caroprese, M., Albenzio, M., Marzano, A., Schena, L., Annicchiarico, G., Sevi,A., 2010. Relationship between cortisol response to stress and behav-ior, immune profile, and production performances of dairy ewes. J.Dairy Sci. 93, 2395–2403.

Caroprese, M., Marzano, A., Entrican, G., Wattegedera, S., Albenzio,M., Sevi, A., 2009. Immune response of cows fed polyunsaturatedfatty acids under high ambient temperatures. J. Dairy Sci. 92,2796–2803.

Di Costanzo, A., Spain, J.N., Spiers, D.E., 1997. Supplementation of nicotinicacid for lactating Holstein cows under heat stress conditions. J. DairySci. 80, 1200–1206.

Drackley, J.K., Cicela, T.M., LaCount, D.W., 2003. Responses ofprimiparous and multiparous Holstein cows to additionalenergy from fat or concentrate during summer. J. Dairy Sci. 86,1306–1314.

Elenkov, I.J., Chrousos, G.P., 1999. Stress hormones, Th1/Th2 patterns,pro/anti-inflammatory cytokines and susceptibility to disease. TrendsEndocrinol. Metab. 10, 359–368.

Igono, M.O., Johnson, H.D., Steevens, B.J., Hainen, W.A., Shan-klin, M.D.,1988. Effect of season on milk temperature, milk growth hormone,prolactin, and somatic cell counts of lactating cattle. Int. J. Biometeorol.32, 194–200.

Kelly, C.F., Bond, T.E., 1971. Bioclimatic Factors and Their Measurement. AGuide to Environmental Research on Animals. Nat. Acad. Sci., Wash-ington, DC.

Lacetera, N., Bernabucci, U., Scalia, D., Ronchi, B., Kuzminsky, G., Nardone,A., 2005. Lymphocyte functions in dairy cows in hot environment. Int.J. Biometeorol. 50, 105–110.

Liu, Z.L., Chen, P., Li, J.M., Lin, S.B., Wang, D.M., Zhu, L.P., Yang, D.P., 2008.Conjugated linoleic acids (CLA) moderate negative responses of heat-stressed cows. Livest. Sci. 118, 255–261.

Maltz, E., Silanikove, N., Shalit, U., Berman, A., 1994. Diurnal fluctuationsin plasma ions and water intake of dairy cows as affected by lactationin warm weather. J. Dairy Sci. 77, 2630–2639.

Marai, I.F.M., El-Darawany, A.A., Fadiel, A., Abdel-Hafez, M.A.M., 2007.Physiological traits as affected by heat stress in sheep—a review. SmallRumin. Res. 71, 1–12.

Matteri, R.L., Carroll, J.A., Dyer, C.J., 2000. Neuroendoc responses to stress.In: Moberg, G.P., Mench, J.A. (Eds.), The Biology of Animal Stress. CABIPublishing, Wallingford, UK, pp. 43–76.

Munksgaard, L., Herskin, M.S., Løvendahl, P., Andersen, J.B., 2006. Effectsof nutrition on stress reactivity. In: Sejrsen, K., Hvelplund, T., Nielsen,M.O. (Eds.), Ruminant Physiology. Wageningen Academic Publishers,Wageningen, The Netherlands, pp. 511–525.

Murzenok, P.P., Netukova, N.I., Zhytkevitch, T.I., 1997. Ultrastructure andfunctional state of rabbit lymphoid cells after repeated exposure tolipopolysaccharide and external heating. Arch. Immunol. Ther. Exp.(Warsz) 45, 301–305.

National Research Council, 2001. Nutrient Requirements of Dairy Cattle,7th ed. Natl. Acad. Sci., Washington, DC.

Peana, I., Fois, G., Cannas, A., 2007. Effects of heat stress and diet on milkproduction and feed and energy intake of Sarda ewes. Ital. J. Anim. Sci.

6, 577–579.

SA.S Institute, 1999. SAS User’s Guide: Statistics. Version 8.1 ed. SAS Inst.Inc., Cary, NC.

Sevi, A., Annicchiarico, G., Albenzio, M., Taibi, L., Muscio, A., Dell’Aquila, S.,2001. Effects of solar radiation and feeding time on behavior, immune

inant R

S

S

S

S

4255–4264.

M. Caroprese et al. / Small Rum

response and production of lactating ewes under high ambient tem-perature. J. Dairy Sci. 84, 629–640.

hapiro, S.S., Wilk, M.B., 1965. An analysis of variance test for normality.Biometrika 52, 591–601.

ilanikove, N., 2000. Effects of heat stress on the welfare of extensivelymanaged domenstic ruminants. Livest. Prod. Sci. 67, 1–18.

rikandakumar, A., Johnson, E.H., Mahgoub, O., 2003. Effect of heat stress

on respiration rate, rectal temperature and blood chemistry in Omaniand Australian Merino sheep. Small Rumin. Res. 49, 193–198.

tefanon, B., Sgorlon, S., De Moro, G., Asquini, E., 2009. Action of larch barkin the regulation of cortisol induced stress in sheep. Ital. J. Anim. Sci.8, 162–164.

esearch 102 (2012) 177– 185 185

Thornton, J.H., Schultz, L.H., 1980. Effects of administration of nicotinicacid on glucose, insulin, and glucose tolerance in ruminants. J. DairySci. 63, 262–268.

Visser, J., van Boxel-Dezaire, A., Methorst, D., Brunt, T., de Kloet,E.R., Nagelkerken, L., 1998. Differential regulation of interleukin-10 (IL-10) and IL-12 by glucocorticoids in vitro. Blood 91,

Webster, J.I., Tonelli, L., Sternberg, E.M., 2002. Neuroendocrine regulationof immunity. Annu. Rev. Immunol. 20, 125–163.

West, J.W., 1999. Nutritional strategies for managing the heat stresseddairy cow. J. Anim. Sci. 77, 21–35.