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Small Ruminant Research 106S (2012) S21–S28

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Extruded linseed supplementation in the diet of dairy sheep: Theeffects on immune response and oxidative stress as affected by thephysiological state�

G. Acuti a, M. Trabalza-Marinuccia,∗, M. Cagiolab, M. Pelab, G. Curinab, L. Moscatib

a Dipartimento di Patologia, Diagnostica e Clinica Veterinaria, Università degli Studi di Perugia, via S. Costanzo, 4, 06126 Perugia, Italyb Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche, via G. Salvemini 1, 06126 Perugia, Italy

a r t i c l e i n f o

Keywords:Transition eweExtruded linseedPolyunsaturated fatty acidsImmune responseOxidative state

a b s t r a c t

Several investigations have studied the influence of polyunsaturated fatty acids on theimmune function of ruminants but many contradictory observations have been reportedin this field. The aim of this study was to evaluate the influence of an extruded linseeddietary supplementation on the immune response and oxidative stress in the transitiondairy ewe. The experiment was performed using 58 Sarda pluriparous ewes, 42 pregnant(T) and 16 non-pregnant and non-lactating (NP). Both groups were fed an isoenergeticand isonitrogenous diet from 60 d before to 15 d after lambing of the T ewes. Within eachphysiological state, one sub-group received a commercial pelleted concentrate (CTR) andthe other an extruded linseed-enriched (10 g/100 g) concentrate (EL). Blood samples werecollected from all animals at −28, −21, −14, −7, +3 and +15 d to parturition, based onexpected date of lambing of the T ewes, to evaluate the innate immunity (serum lysozyme,haemolytic complement and bactericidal activity), the acquired immunity and the oxidativestress. The dietary supplementation with extruded linseed did not markedly influence theinnate immunity response of the animals. The antibody production against Salmonella enter-ica subsp. enterica serovar Abortusovis was shown to be increased by the linseed-enricheddiet in the T ewes (2.37 vs. 2.19 log10 for the T and the NP ewes, respectively). As for thecell-mediated immunity, no differences between dietary treatments were observed. Theextruded linseed-enriched diet was able to induce a transitory overproduction of oxidantsin the NP ewes, whereas no effects were observed in the T ewes. In conclusion, under theadopted experimental conditions, the use of extruded linseed in the diet did not greatlyaffect the oxidative state and the immune response of the transition ewe.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Several investigations have studied the influence ofpolyunsaturated fatty acids (PUFAs) on the immune

� This paper is part of a supplement titled “SIPAOC Congress 2010”Guest Edited by Antonello Carta, Maria Teresa Manfredi, GiuseppeMoniello and Massimo Trabalza-Marinucci.

∗ Corresponding author at: Dipartimento di Patologia, Diagnostica eClinica Veterinaria, via S. Costanzo, 4, 06126 Perugia, Italy.Tel.: +39 0755857707; fax: +39 0755857764.

E-mail address: massimo.trabalza@unipg.it (M. Trabalza-Marinucci).

function of ruminants, monogastric species and experi-mental animals (Agazzi et al., 2004; Calder, 1998; Calderet al., 2002; Caroprese et al., 2009; Harbige, 2003; Healyet al., 2000; Lessard et al., 2003; Parmentier et al., 1997,2002; Savoini et al., 2010).

Many contradictory observations have been reportedin this field. Studies in humans have indicated that anincreased intake of omega-3 PUFA with the diet can inhibitthe immune system and the inflammatory response (Triconet al., 2004). This effect is probably due to a decreased levelof circulating proinflammatory cytokines, such as IL1�, IL6,and TNF�, and a decreased proliferation of peripheral blood

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mononuclear cells (macrophages, T lymphocytes, plasmacells), without inhibition of the T and B cells’ immuneresponse (Kelly, 2001). On the contrary, an improvementin cellular immune reactivity to phytohaemoagglutinin,an increased anti-OVA IgG concentration and a reductionof IL-10 secretion was observed in dairy cows (Caropreseet al., 2009). Lessard et al. (2003) have demonstrated thatthe immunomodulatory effects of omega-3 PUFA in cat-tle are directed towards the response of lymphocytes andmonocytes/macrophages, whereas the antibody responseto ovalbumin and the in vitro production of interferon � donot seem to be affected by the PUFA dietary supplementa-tion.

An appropriate PUFA concentration in the diet, togetherwith a balanced omega-6/omega-3 ratio, is thereforeimportant. This ratio is estimated to be approximately15–20 in the current Western diet, and should decreaseto below 5 or 4 (British Nutrition Foundation, 1992; Woodet al., 2004) to avoid the prothrombotic and proaggregatorystate induced by a high level of n-6 PUFA.

Furthermore, a balanced omega-6/omega-3 ratio in thediet is essential for normal growth and to decrease cardio-vascular disease and other chronic diseases (Simopoulos,1999).

Dietary lipids, such as supplemental oil seeds richin PUFA, can be significant contributors to the load offree radicals in animals (Andrews et al., 2006). Oxidativestress leads to peroxidative damage to lipids and othermacromolecules, with a consequent alteration of cell mem-branes and other cellular components (Toyokuni, 1999). Inaddition, oxidative stress can be related to alterations ofphysiology and pathologies in man and in other animals(Miller et al., 1993).

Most published studies on the effects of dietary PUFAhave focused on the bovine species, while the knowledgeof small ruminants is limited and discordant (Caropreseet al., 2006, 2009; Thanasak et al., 2005). It is not clearwhether the role of PUFA may be influenced by the phys-iological state of the animal and the transient states ofdepressed immune function. The peripartum is consideredthe most critical period for dairy ruminants in consider-ation of the feeding- and management-related diseasesassociated with the depression of the immune system. Theaim of this research was to verify the effects of extrudedlinseed supplementation in the dairy ewe’s diet on theinnate and adaptive immune response and on the oxida-tive stress during two different physiological states, thetransition and the dry period, which are characterised bydifferent immunological conditions.

2. Materials and methods

2.1. Animals and dietary treatments

The experiment was performed using 58 Sarda pluriparous ewes,42 pregnant (T, two months before parturition) and 16 non-pregnant(ultrasonography diagnosis) and non-lactating (NP), homogeneous forage (4–5 years), body condition score (BCS) and milk yield at previ-ous lactation. The ewes had been subjected to oestrus synchronisationand ovulation induction using a combination of commercially available(Cronogest®) progestogen sponges plus PMSg injections (Intervet Srl,Milano, Italy) and then subjected to natural mating. The sheep werehoused in a stable with an outdoor paddock. The animal care procedures

followed the European directives for the protection of experimental ani-mals (Directive 2010/63/EU).

In order to provide the same amount of nutrients, the nutritional planand protocol were the same for the T and NP ewes. The animals weregroup-fed. Both T and NP ewes were randomly allocated into two sub-groups of equal size and fed mixed (pregnancy) or alfalfa (lactation) hay adlibitum and either a commercial pelleted concentrate (CTR) or an extrudedlinseed-enriched (10 g/100 g as fed) concentrate (EL). The experimentaldiets were fed from 60 d before to 15 d after lambing of the T ewes. Thetwo concentrates were formulated to be isoenergetic and isonitrogenousand were administered at a rate of 200, 400 and 800 g/d at the beginningof the trial, during the last 30 d of pregnancy and at early lactation of theT ewes, respectively.

Two months before, 5 d before and 15 d after delivery in the T ewes, thetotal dry matter intake (TDMI) was assessed over a 3-d period by weighingthe residual feed intake. A 0–5 scale (Russel et al., 1969) was used by fourtrained operators to assess the BCS of the sheep (60 and 15 d before and15 d after lambing).

Samples of the feeds were collected weekly during the trial and ana-lysed for chemical composition (Table 1). The dry matter, crude protein,ether extracts and ash were determined according to AOAC procedures(AOAC, 1990, 2000). The methods of Van Soest et al. (1991) were usedfor the analyses of the neutral detergent fibre (NDF), acid detergent fibre(ADF) and lignin. Sodium sulphite was used in the NDF procedure. Boththe NDF and ADF are expressed inclusive of ash. The calcium and phospho-rous concentrations were determined according to Julshamn et al. (1998)and AOAC (1996), respectively.

Fatty acid extractions were performed according to the method ofFolch et al. (1957). Methylation of feed fat was conducted using thesodium methoxide methylation procedure (Christie, 1982). Fatty acidmethyl esters were quantified using a gas-chromatograph Perkin-Elmer-8410 (Perkin-Elmer, Norwalk, CT, USA) fitted with a fused silica capillarycolumn [SP150-2380, 100 m × 0.25 mm (internal diameter) with 0.2 �mfilm thickness; Supelco, Inc., Bellefonte, PA, USA]. Hydrogen was used asthe carrier gas with a pressure of 120 kPa. The injector and detector tem-perature was 250 ◦C. Gas-chromatography conditions were as follows: aninitial oven temperature of 70 ◦C was maintained for 1 min, then rampedup at a rate of 5 ◦C/min to a final temperature of 100 ◦C and maintained for2 min; the temperature was then increased to 175 ◦C at 10 ◦C/min and heldat 175 ◦C for 28 min; the temperature was ramped up to 225 ◦C at a rateof 5 ◦C/min and maintained for 25 min. The split ratio was 40:1. Fatty acidpeaks were identified by comparing the retention times of sample peakswith those of the standard mixture (37-Component FAME Mix, Supelco,Bellefonte, PA, USA).

The metabolisable energy (ME) content of feeds was estimatedaccording to Andrieu et al. (1981) and Sauvant (1981).

2.2. Blood analytical determinations

Blood samples were collected from the T ewes during pregnancy (28,21, 14 and 7 d before delivery) and during early lactation (3 and 15 d afterdelivery), at 8:00 am via jugular venipuncture using vacuum tubes (Vac-uette, Greiner, Austria) containing lithium heparin or no anti-coagulant.At the same time, blood samples were also taken from the NP ewes. Thesamples without anti-coagulant were incubated at room temperaturefor 2 h and then centrifuged at 3520 × g for 16 min at 4 ◦C; the aliquotsof serum were stored at −80 ◦C under sterile conditions until analysis.The serum samples were tested to evaluate the innate immunity [serumlysozyme (SL), haemolytic complement (HC) and serum bactericidal activ-ity (SBA)], the acquired immunity (antibodies against Salmonella entericasubsp. enterica serovar Abortusovis, hereinafter referred as to S. Abortuso-vis) and the oxidative stress [antioxidant power of plasma (AOP) andreactive oxygen metabolites (ROMs)]. The samples collected with lithiumheparin were used to evaluate the cellular immunity (total lymphocytes,CD4+ and CD8+ lymphocytes). For this determination, a follow-up samplewas collected at 90 d after lambing.

To eliminate systematic error arising from inter-assay variability, allthe samples from each animal were analysed within the same assaysession, which included an equal number of animals belonging to theexperimental groups.

The SL was measured using a lyso-plate assay (Osserman and Lawlor,1966), performed at 37 ◦C for 18 h in a humidified incubator. The methodwas based on the lyses of Micrococcus lysodeikticus in 1% (w/v) agarose. Thediameters of the lysed zones were measured with a ruler and compared

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Table 1Ingredients, chemical composition and metabolisable energy content of the concentrates and the hays used in the experimental diets (g/100 g, unlessotherwise indicated).

Concentratesa Forages

Item CTR EL Mixed hay Alfalfa hay

IngredientsExtruded linseed – 10.0Maize 33.6 15.7Wheat flour shorts 20.0 20.0Soybean meal 18.8 12.4Wheat middlings 16.3 25.3Wheat bran – 5.0Barley 5.0 5.0Sugar cane molasses 2.0 2.0Vitamin and mineral supplement 4.3 4.6

Chemical compositionDry matter 88.27 88.85 93.35 91.91Crude protein 17.37 16.99 9.44 13.97Ether extracts 3.49 6.43 1.25 1.62Ash 6.67 6.96 7.35 9.43Neutral detergent fibre 19.52 21.61 58.21 44.50Acid detergent fibre 6.03 6.44 40.42 33.12Lignin (sa)b 1.20 1.79 5.67 4.58Calcium 1.47 1.53 1.06 1.59Phosphorus 0.62 0.68 0.23 0.16Vitamin A (UI/kg) 60,000 60,000Vitamin D3 (UI/kg) 4000 4000Vitamin E (mg/kg) 75.00 75.00

Lipid profile, g/100 g of fatty acidsC16:0 15.32 10.43 18.99 28.6C18:0 2.00 3.54 3.30 4.4C18:1 n-9 23.03 19.71 6.64 5.2C18:2 n-6 54.08 26.22 27.06 20.4C18:3 n-3 3.49 38.91 36.75 22.1Other fatty acids 2.08 1.19 7.26 19.3

Metabolisable energy (Mcal/kg) 2.10 3.10 1.35 1.69

a CTR, group had no extruded linseed supplementation; EL, group with extruded linseed supplementation.b Lignin determined by solubilisation of cellulose with sulphuric acid.

with the lysed zones of a standard lysozyme preparation (Sigma–AldrichItaly, Milan, Italy). The HC (Barta and Barta, 1993) was performed usingmicrotitre plates. The complement titre was the reciprocal of the serumdilution that caused 50% lyses in the red blood cells of a ram. The SBAanalyses were performed according to a previously validated method forcattle (Amadori et al., 1997). The test is based on a challenged sera withno pathogenic Escherichia coli.

The humoral immunity was evaluated in vivo by determining the pro-duction of Immunoglobulin G (IgG) antibodies against S. Abortusovis. Theanimals were immunised with S. Abortusovis vaccine (Istituto Zooprofilat-tico dell’Umbria e delle Marche, Perugia, Italy) 35 and 21 d before lambing.The S. Abortusovis was cultivated in a trypticase soy agar for 24 h at 37 ◦C.The collected culture was resuspended at 1.5 × 109 CFU/mL, and the bac-teria were inactivated by 0.05% (v/v) formaldehyde. Sixty millilitres ofaluminium hydroxide (AlOH3) was added to the inactivated 150 mL ofculture as an adjuvant. The serum agglutination test assay was performedusing microtest plates. The sera were first diluted by adding 10 �l of serumto 90 �l of normal saline and then adding 100 �l of a previously standard-ised stained microtest antigen to give a final dilution of 1/20. By titratingthe serum in doubling dilutions and adding an equal volume of standard-ised stained antigen, an end-point (titre) was obtained. The plates weresealed and incubated for 18, 24 or 48 h at 37 ◦C. A positive reaction con-sisted of a fine diffuse precipitation, whereas a negative reaction showeda button-like precipitate, titres of 1/40 were usually considered to be pos-itive (Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 7thedition, 2010).

The cellular immunity was evaluated using fluorescence-activatedcell sorting (FACS). The lymphocytic populations were obtained bythe centrifugation of heparinised blood with Ficoll–Hystopaque 1077(Sigma–Aldrich Italy, Milan, Italy) at 400 × g for 30 min. The buffy coat waswashed three times with PBS-A, and the lymphocitic population (1 × 106)

was incubated for 22 min at 4 ◦C in the dark with PBS-A containing amixture of diluted primary antibodies against anti-sheep CD8 FITC (AbDSerotec, Oxford, UK) and anti-sheep CD4 RPE (AbD Serotec, Oxford, UK),respectively.

The cells were then sorted using an FACS Calibur flow cytometer(Becton Dickinson, Franklin Lakes, USA) equipped with a BLUE laserat 488 nm. Data analyses were performed using the CellQuest® Prosoftware (Becton Dickinson Immunocytometry Systems, Franklin Lakes,USA) and Kaluza 1.1 (Beckman Coulter Inc., Fullerton, USA). The condi-tions for forward scatter (FSC) and side scatter (SSC, orthogonal lightscatter) were adopted from literature (Thompson et al., 1985). Thegranulocytes and cell doublets were excluded by gating. The sort was con-sidered complete after 500,000 cells from each subpopulation had beenaccumulated.

The ROMs were evaluated using a commercially available kit (DiacronInternational Srl, Grosseto, Italy). The AOP values were measured using acommercially available kit (Diacron International Srl, Grosseto, Italy) thatevaluates the serum’s ability to oppose the massive oxidative action of ahypochlorous acid (HClO) solution.

2.3. Statistical analysis

The obtained data were analysed with the GLM procedure of the SASsoftware (2001), using a model of variance for repeated measurements.Data are reported as least square means ± standard error. Overall dif-ferences between treatment means and their interactions were testedaccording to the Tukey test and considered to be significant whenP ≤ 0.05. The values of the antibody concentration against S. Abortuso-vis were transformed in logarithm form to normalise the frequencydistribution.

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Fig. 1. Metabolisable energy (ME) and crude protein (CP) intake by tran-sition (T) and non pregnant and non lactating (NP) ewes during theexperiment.

3. Results and discussion

3.1. Feed intake and BCS

At the beginning of the experiment, the TDMI was1.70 ± 0.03 kg/d for the T ewes and 1.65 ± 0.03 kg/d for thedry ewes, respectively. Five days before lambing in the Tewes, the TDMI remained fairly constant in both the T andNP (1.73 ± 0.04 kg/d vs. 1.67 ± 0.04 kg/d) ewes, and then atearly lactation, the values recorded were 2.51 ± 0.05 kg/dvs. 1.78 ± 0.03 kg/d for the T and NP ewes, respectively. Theincrease in hay DM intake in the T ewes (1.80 ± 0.03 kg/dvs. 1.07 ± 0.03 kg/d for the T and the NP ewes, respectively)was clearly related to the higher nutritional requirementsof lactation (Bocquier et al., 1988), which also determinedthe large decrease in BCS in the post-partum period asopposed to the non-lactating ewes (2.3 ± 0.1 vs. 3.3 ± 0.2,respectively).

Fig. 1 shows the ME and crude protein intake by the twogroups of animals. To some extent, the NP ewes were ableto regulate their energy and protein intake by reducing theamount of forage eaten when the concentrate was fed at themaximum level (due to an increased forage to concentratereplacement rate).

The TDMI was not influenced by the two experimen-tal concentrates, which were formulated to be isoenergeticand isonitrogenous. Similarly, the BCS values of the eweswere not affected by the presence of the EL in the diet(2.7 ± 0.2 vs. 2.8 ± 0.2 for the EL and the CTR ewes, respec-tively).

3.2. Innate immunity

The SL values were significantly (P = 0.001) affected bythe time of sampling in both the T and NP ewes (Fig. 2).A significant (P = 0.03) interaction between diet and timeof sampling was observed in the NP ewes: the presenceof linseed in the diet produced an increase in SL values at7 d before lambing [average values across sampling times:1.72 ± 0.12 �g/mL vs. 1.26 ± 0.12 �g/mL (P = 0.03) in EL andCTR group, respectively]. The SL values (1–3 �g/mL) were

Fig. 2. Serum lysozime (SL) concentration in transition (A) and in nonpregnant and non lactating (B) ewes during the experiment. EL, extrudedlinseed-enriched diet; CTR, control diet; sampling times with differentletters differ (P < 0.001); *the EL group differs from the CTR group (P = 0.03).

always in the physiological ranges (Kostro et al., 2010;Sotirov et al., 2005).

The HC values were influenced by the time of sam-pling in both T (P < 0.001) and NP (P = 0.01) ewes (Fig. 3).The T group fed the EL-enriched diet showed a decrease(P < 0.001) of complement concentration (reported asCH50%) 15 d after lambing. No differences were observedin NP ewes because of the nutritional plan.

The time of sampling influenced (P < 0.001) the SBA inboth the T and NP ewes during the trial. As there were nodifferences between the two dietary treatments, data fromEL and CTR groups are pooled together in Fig. 4. The physi-ological SBA level in Holstein-Friesian cattle is reported tobe >90% (Amadori et al., 1997).

The modifications reported in this work for the SL andHC values, related to the dietary treatment, did not show aclear pattern. At present, no valid reasons for this responsecan be found. As for the SBA response, a series of envi-ronmental influences other than diet and physiologicalstate might have played a role in the experimental con-ditions of the present study. These results suggest that anumber of different factors can affect the non-antigen spe-cific response of the organisms to the external pathogens(Caroprese et al., 2009; Savoini et al., 2010). Studies inhumans reported that the innate immune response inyoung subjects is not affected by the PUFA intake, whereasolder subjects are more sensitive to the immunologiceffects of the PUFA intakes (Dinka et al., 2006).

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Fig. 3. Haemolytic complement (HC) concentration in transition (A) andin non pregnant and non lactating (B) ewes during the experiment. EL,extruded linseed-enriched diet; CTR, control diet; sampling times withdifferent letters differ (P < 0.001); *the EL group differs from the CTR group(P < 0.001).

The last fifteen days of pregnancy and the days aroundlambing negatively affected the innate immune response.This condition is probably due to the effects of physio-logical immunosuppressive products originating from thefoetus (e.g. fetoprotein) or progesterone, a steroid prod-uct deriving from the placenta (Bonizzi et al., 2003). Inaddition, it must be stressed that the BCS recorded dur-ing the experiment followed opposite trends for NP and

Fig. 4. Serum bactericidal activity (SBA) concentration in transition (T)and in non pregnant and non lactating (NP) ewes during the experiment.Sampling times with different letters differ (P < 0.001).

Fig. 5. Antibodies against Salmonella Abortusovis (AB) concentration intransition (A) and in non pregnant and non lactating (B) ewes duringthe experiment (vaccine inoculation: first dose, 35 d before lambing andbooster shoot 21 d before lambing). EL, extruded linseed-enriched diet;CTR, control diet; sampling times with different letters differ (P < 0.001);*the EL group differs from the CTR group (P = 0.04).

T groups. The possible interactions between physiologicalstate, metabolic conditions, and innate immune responseof the animals must be taken into account.

Finally, triplet- and twin-bearing ewes tended tohave a lower SBA and a higher SL concentrationthan animals bearing a single foetus (SBA: 70.63 ± 9.70,73.33 ± 3.70 and 82.94 ± 6.67%; SL: 1.91 ± 0.25, 1.86 ± 0.10and 1.66 ± 0.17 �g/mL for triplet-, twin-, and single-bearing ewes, respectively).

3.3. Adaptive immunity

In our research and in agreement with other stud-ies (Caroprese et al., 2009; Parmentier et al., 2002), thehumoral response was influenced by the dietary treatment.Antibody production against S. Abortusovis was affectedin the T ewes (Fig. 5) by both the diet (P = 0.04) and thetime of sampling (P < 0.001). The time of sampling influ-enced (P < 0.001) the humoral immune response in the NPewes as well (Fig. 5). Caroprese et al. (2009) observed thatsupplementing food with omega-3 sources was able toenhance the immune response of dairy cows immunisedwith chicken egg albumin.

In this study, the T ewes experienced considerable stressspecific to the peripartum period. In addition, the BCS lossobserved in the T ewes after parturition (−0.45 vs. +0.56

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Table 2Changes in total, CD4+ and CD8+ lymphocytes in transition (T) and in non pregnant and non lactating (NP) ewes during the experiment.

Item Physiological state Days to lambing of the T ewes SEM

−21 −14 −7 +90

Lymphocytes, %* T 23.3 19.6 18.2 20.3 1.4NP 21.3 27.6 26.1 12.8 2.4

CD4+, %* T 14.4 11.8 10.4 12.9 0.8NP 12.1 15.0 13.2 7.7 0.9

CD4+, MFI# T 508.7 660.5 890.0 824.7A 20.5NP 482.4 597.1 903.2 586.1B 35.9

CD8+, %* T 8.7 7.7 7.8A 7.2 0.7NP 9.1 12.2 12.6B 5.0 0.9

CD8+, MFI T 258.9 347.6 497.8 392.0a 15.8NP 279.1 323.7 478.4 246.1b 27.8

Means with different superscripts in a column differ (a, b: P < 0.05; A, B: P < 0.01; A, B: P < 0.001).* Mean percentage of cells in peripheral blood mononuclear cells.# Mean fluorescence intensity.

Table 3Modifications of reactive oxygen metabolites values (mmol H2O2) in transition (T) and in non pregnant and non lactating (NP) ewes during the experiment.

Days to lambing of the T ewes T ewes NP ewes

Diet Sampling time Diet Sampling time

EL CTR EL CTR

−28 4.06 3.73 4.24AB 4.19 3.92 4.03AB

−14 4.65 4.50 5.08ABC 4.75 4.42 4.56A

−7 4.63 4.57 4.83A 4.88 4.76 4.81A

+3 5.63 5.61 5.78C 5.30 3.80 4.55A

+15 3.60 4.02 3.96B 3.68 3.57 3.58B

SEM 0.22 0.21 0.25** 0.18

EL, extruded linseed-enriched diet; CTR, control diet. Means with different superscripts in a column differ (A, B, C: P < 0.001).** EL group differs from CTR group for P = 0.02.

in T and NP ewes, respectively) was likely to make themmore sensitive to the oxidative stress (Bernabucci et al.,2005). The efficiency of the adaptive immune response islimited under stress conditions (Amadori et al., 2009), andT ewes might have taken greater metabolic advantage fromthe EL-enriched diet.

A large number of studies have demonstrated the poten-tial benefits of dietary unsaturated fatty acids on theimmune system in humans and in experimental infectedanimals. It is generally accepted that many of the importantinfections of human populations have been associated withnutritional deficiency (de Pablo and Álvarez De Cienfuegos,2000). Nevertheless, the influence of omega-6 and omega-3

PUFA and their ratio on immune response of ruminants isnot very clear (Lessard et al., 2003, 2004). For instance, inan experiment conducted with 30 lactating Holstein cowsfed whole linseed, calcium salts of palm oil or micronizedsoybeans, no effects on the antibody response to ovalbu-min due to dietary treatments were reported (Lessard et al.,2003).

The litter size tended to influence the antibody pro-duction. Triplet- and twin-bearing ewes showed a lowerresponse than animals bearing a single foetus (2.14 ± 0.13and 2.27 ± 0.05 vs. 2.44 ± 0.09 log10, respectively). Thisresult is in agreement with Berggren-Thomas et al. (1987)that found, in ewes, a tendency towards a decreased

Table 4Modifications of the antioxidant power of plasma values (�mol HClO) in transition (T) and in non pregnant and non lactating (NP) ewes during theexperiment.

Days to lambing of the T ewes T ewes NP ewes

Diet Sampling time Diet Sampling time

EL CTR EL CTR

−28 311.79 268.40 288.95A 267.15 233.75 250.45A

−14 248.93 235.50 2411.24B 248.17 250.83 249.51A

−7 219.98 206.68 212.28B 220.85 240.40 230.61A

+3 158.38 157.92 157.03C 160.90 149.38 155.14B

+15 240.53 235.77 236.74B 247.23 250.40 248.82A

SEM 15.50 10.41 10.52 7.95

EL, extruded linseed-enriched diet; CTR, control diet. Means with different superscripts in a column differ (A, B, C: P < 0.001).

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anti-ovalbumin titre as litter size increases. The level ofantibody secretion against antigens can provide a mea-sure of the metabolic and physical stress connected tomultiple pregnancies. Twin- and triplet-bearing ewes wereshown to suffer more markedly from the physiologicaladjustment related to pregnancy and parturition than thesingle-lambing ewes (Sevi et al., 2002, 2003).

As for the cell-mediated immunity, no differencesbetween the sheep fed the EL or CTR concentrate wereobserved. A general tendential increase in the total lym-phocytes and in the subpopulations of CD4+ and CD8+during the period of observation (in particular from −21to −7 d before lambing) and a decrease to basal values 90 dpost-partum were observed (Table 2). At 90 d post-partum,the phenotypic analysis of the lymphocytic populationrevealed that the immune response in the ewes was influ-enced by the physiological status: an increase in CD8+(P = 0.04) and CD4+ (P = 0.01) lymphocytes was recorded inthe T ewes compared to the NP ewes (Table 2). Entrican andWheelhouse (2006) demonstrated that the pregnant sheeputerus has an increased number of intraepithelial CD8+T cells compared to the non-pregnant uterus. These cellsseem to play a significant role in limiting bacteria invasionin pre-lambing and in lambing time.

3.4. Oxidative state

As for the oxidative stress indicators, the ROMs levelsincreased 2 d after delivery in the T ewes (Table 3), probablybecause of the oxidative stress due to metabolic and hor-mone changes, but no differences were observed betweenthe dietary treatments (Table 3). This result is in agree-ment with Rizzo et al. (2008), who found an increase inROS concentration in ewes during the peri-partum period.

On the contrary, the NP ewes (Table 3) showed anincrease (P = 0.02) in the ROMs concentration related to theadministration of EL in the diet (average values: 4.54 ± 0.13vs. 4.07 ± 0.13 mmol H2O2 in the EL and CTR ewes,respectively). The NP ewes, characterised by a maintenancemetabolic rate, might have experienced a greater difficultyin coping with the overload of dietary lipids.

The AOP values were influenced by the time of sam-pling (P < 0.001) in both the T and NP ewes. The AOP levelwas neither influenced by the dietary treatment nor bythe physiological state (Table 4). Blood samples collectedat 90 d post-partum, that is 2 months after the end of theexperimental treatments, revealed that both ROMs andAOP levels were within the normal range for the species(data not shown). Overall, supplementation of PUFA did notresult in an increased lipid peroxidation or overproductionof oxidants (Petit, 2009).

Triplet-bearing produced an increase (P = 0.01) in theROMs concentration compared to twin- and single-delivery (5.34 ± 0.27 vs. 4.59 ± 0.10 and 4.40 ± 0.18 mmolH2O2, respectively). Dairy ruminants experience oxidativestress around lambing (Bouwstra et al., 2010). It can beassumed that the further increase in the ROMs concentra-tion can be associated with the cardiovascular inadequacyto properly oxygenate more than one foetus, which maylead to oxidative stress.

4. Conclusions

The results obtained in this study demonstrated that adietary supplementation with linseed does not markedlyinfluence the innate immunity response of the tran-sition ewe. On the contrary, the antibody productionagainst S. Abortusovis was shown to be increased by thelinseed-enriched diet. This can be of particular impor-tance in the transition period, considering the feeding- andmanagement-related diseases associated with the depres-sion of the immune system in both dairy cattle and sheep.

Under our experimental conditions, the EL concentrate-enriched diet was able to induce a transitory overproduc-tion of oxidants in the NP ewes, whereas no effects wereobserved in the transition ewes.

The effects of litter size on the immune response and theoxidative status need to be confirmed by large-scale exper-iments. Further in vivo investigations using experimentalinfections might be necessary to determine the biologicaleffects of PUFA-enriched diets on the immune response inthe ruminant species.

Conflict of interest

None of the authors have any financial or personalinterest that would inappropriately influence or bias thecontents of this paper.

Acknowledgments

The authors would like to thank E. Cassetta, C. Antonini,and L. Mughetti for help in laboratory analyses and L. Buraniand G. Tenerini for technical assistance and care of the ani-mals. This research was sponsored by the project IZSUM11/08 RC, funded by the Italian Ministry of Health (MinSal).

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