Research in Veterinary Science 93 (2012) 202–209

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Research in Veterinary Science

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Immune response and milk production of dairy cows fed graded levelsof rumen-protected glutamine

M. Caroprese a,b,⇑, M. Albenzio a,b, R. Marino a,b, A. Santillo a, A. Sevi a,b

a Faculty of Agriculture, Department of Production and Innovation in Mediterranean Agriculture and Food Systems (PRIME), University of Foggia, Foggia, Italyb Food Quality and Health Research Center (BIOAGROMED), University of Foggia, Foggia, Italy

a r t i c l e i n f o

Article history:Received 5 May 2011Accepted 15 July 2011

Keywords:GlutamineImmune functionsAmino acidMilk coagulating properties

0034-5288/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.rvsc.2011.07.015

⇑ Corresponding author at: Faculty of Agriculture, DInnovation in Mediterranean Agriculture and FoodQuality And Health Research Center (BIOAGROMEDNapoli, 25, 71122 Foggia, Italy. Tel.: +39 0881 589332

E-mail address: m.caroprese@unifg.it (M. Caropres

a b s t r a c t

The objective of the study was to determine the effects of dietary supplementation with glutamine on theimmune function and milk production of dairy cows. The experiment involved 24 Friesian cows, dividedinto three groups of eight each, according to the level of rumen-protected glutamine supplementation: adiet with no supplementation (Control), a diet supplemented with 160 g/day/cow (G160) and a diet sup-plemented with 320 g/day/cow (G320). At 0, 30, and 60 days of the experiment, lymphocyte response tophytohemoagglutinin (PHA) was determined in vivo for each animal. Humoral response to chicken eggalbumin (OVA) and interleukin – (IL)-1b, IL-6 and IL-10 plasma levels were measured at 0, 15, 30, 45,and 60 days. Results demonstrate that supplementing 160 g/day/cow of glutamine can modulate immuneresponses of dairy cows and enhance the amino acid profile of cow milk.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Nonessential amino acids (NEAAs) have an important role in thefunction of the immune system. Among NEAAs glutamine (Gln) isone of the most versatile amino acids (AAs) in terms of metabolicuses applied to animal science (Lobley et al., 2001). Gln is also usedby immune cells for energetic needs in the same way as glucose: ithas been observed that the rate of utilization of Gln by both newlyisolated and cultured lymphocytes and macrophages is high (Arda-wi and Newsholme, 1983; Fitzpatrick et al., 1993). A previousstudy demonstrated that duodenal infusion of 300 g/d of Gln todairy cows did not enhance immunocompetence (Doepel et al.,2006). However, cows infused intravenously with 106 or 212 g/dof Gln showed a modulation of acute phase proteins, suggestinga possible role for Gln in the enhancement of the production ofcytokines by cells of the immune system (Jafari et al., 2006). Recentstudies (Peng et al., 2011) on chronic ethanol-fed rats demon-strated that glutamine administration might suppress the inflam-matory response. No studies have evaluated the effects ofglutamine administration in the diet on inflammatory responsein cows.

Though NEAAs can be synthesized by mammals, they maybecome conditionally essential during particular physiological

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epartment of Production andSystems (PRIME) and Food), University of Foggia, via; fax: +39 0881 589331.e).

conditions, such as lactation. Bequette et al. (1996) reported thathigh producing dairy cows produced about 1 kg of milk proteinsduring lactation. In a previous study it was suggested that Glncan be a limiting amino acid for milk protein synthesis becausethe decrease of Gln plasma concentration exceeded that of essen-tial AAs from weeks 2 to 15 after parturition (Meijer et al., 1995).In midlactation the intravenous infusion of Gln in a mixture ofessential AAs (EAAs) and NEAAs did not result in a further milkprotein increase compared with the infusion of EAAs only (Metcalfet al., 1996). Recently, Doepel et al. (2007) suggested that Gln infu-sion during early lactation did not increase milk protein synthesisthrough an increased Gln uptake by the mammary gland, eventhough milk yield increased by 3%. Altogether, these studies dem-onstrate that the administration of Gln by infusion to high produc-ing cows does not result in a consistent improvement of milk yieldand protein. As lactation progresses, milk coagulation ability dete-riorates and rennet coagulation time begins to increase from 120 dof lactation (De Marchi et al., 2007). No studies have investigatedthe effects of Gln supplementation during mid lactation on milkcoagulation properties, which largely depend on milk casein distri-bution. Gln administration during lactation, beside providing theelements for the increase of milk protein synthesis (Meijer et al.,1993), can sustain the synthesis of bovine casein which are richin Gln (Eigel et al., 1984). Gln and glutamic acid, together withproline, which originates from glutamic acid, represent 24–35%of bovine casein (Creamer, 2003).

Glutamine might have a role in the modulation of immuneresponses. In addition, although the postruminal and parenteralinfusion of Gln to lactating cows both in early and in midlactation

M. Caroprese et al. / Research in Veterinary Science 93 (2012) 202–209 203

has been proved not to improve milk yield and protein, there are alack of data on the effects of dietary supplementation of Gln onmilk production (Metcalf et al., 1996; Doepel et al., 2006, 2007).We hypothesized that Gln administration in the diet might (1)have a role in modulating immune responses by acting on the pro-duction of cytokines by immune cells (2) influence AA profile andprotein composition of milk, and (3) improve milk coagulationproperties by affecting protein composition. This study, therefore,was undertaken to evaluate the effects of dietary Gln supplemen-tation on (i) the immune functions and cytokine production (ii)milk production and milk nutritional and processing propertiesof dairy cows. In particular, the changes in proinflammatory andanti-inflammatory cytokines were studied to evaluate the effectsof Gln administration on the regulation of inflammatory responseconnected to chronic diseases.

2. Materials and methods

2.1. Animals and experimental treatments

The experimental site was a commercial farm located approxi-mately 20 kilometres north-east of Foggia, Apulia, Southern Italy(latitude: 41� 270 600 and longitude: 15� 330 500). The experimentlasted 60 days and was performed from February to April 2007,on 24 Italian Friesian cows, divided into three groups of eight each,which were balanced according to days in milk (121.50 ±7.55 days), parity (2.60 ± 0.20), milk yield (22.60 ± 1.73 kg/day),milk fat (3.76 ± 0.43%), and protein content (3.28 ± 0.29%). Priorto, and during the experiment all cows were assessed by veterinar-ians to evaluate the presence of mastitis or intramammary infec-tions, and any with signs of mastitis were excluded from theexperiment.

Diets given to cows (Table 1) were (1) control diet (C) contain-ing corn, oat hay, concentrate (2) control diet supplemented with400 g/day rumen-protected L-Glutamine 40% (Ascor Chimici,Capocolle di Bertinoro, Italy) providing 160 g/day rumen-protectedGln (G160), and (3) control diet supplemented with 800 g/day

Table 1Dry matter intake, ingredients and chemical composition of diets.

Group

Controla G160 G320

DMI (kg/d) 16.6 ± 0.2e 17.0 ± 0.2 17.4 ± 0.2Ingredients (% of DM)Concentrateb 60.87 59.43 58.09Corn 5.37 5.25 5.13Oat hay 33.76 32.96 32.22

L-Glnc 0 2.36 4.55

Chemical composition (% of DM)DM (%) 92.06 92.22 92.37Ether extract (% of DM) 3.20 4.53 5.76CP (% of DM) 15.33 15.9 16.43ADF (% of DM) 23.03 22.48 21.98NDF (% of DM) 41.18 40.21 39.3ADL (% of DM) 4.01 3.92 3.83NeL (Mcal/kg)d 1.67 1.72 1.76

a G160 = group supplemented with 160 g/d of Gln, G320 = group supplementedwith 320 g/d of Gln.

b Contained: Corn Meal, Roasted Soybean Meal, Wheat Germ Meal, Wheat Meal,Roasted Soybean Seeds, Barley Meal, Wheat Fine Bran, Sugarcane Molasses, Par-tially Debarked Sunflower meal, Calcium Carbonate, Calcium Hydrogen Phospate,Sodium Bicarbonate, Sodium Chloride, Magnesium Oxide, 40 IU/g vitamin A, 4 IU/gvitamin D3, 0.004% vitamin E, 0.0005% vitamin B1, 0.0002% vitamin B2, 0.02%vitamin PP, 0.05% Cl, 0.003% Fe, 0.00025% Co, 0.0006% I, 0.009% Mn, 0.001% Cu,0.025% Zn, 0.00003% Se.

cL-Glutamine 40% (Ascor Chimici srl, Capocolle di Bertinoro, Italy).

d Calculated according to NRC (2001).e DMI are expressed as ± SE.

L-Glutamine 40% providing 320 g/day rumen-protected Gln(G320). Rumen by-pass of microencapsulated (145 lm Ø) supple-ment was P85%, and was performed according to Eldem et al.(1991). Cows were housed in tie stalls and water was availablead libitum. The diet was individually administrated twice daily(06.00 and 14.00) as total mixed ration, and feed intake wasrecorded daily. A pooled sample from each experimental diet wastaken weekly and analyzed along the 60-day period of the experi-ment. The chemical composition of the diets was determined bystandard procedures (AOAC, 1990). Briefly, ether extract was deter-mined by the Soxhlet method using petroleum ether, crude proteinwas determined using the Kjeldahl method. Acid Detergent Fibre(ADF) was determined according to Van Soest (1963a), and NeutralDetergent Fibre (NDF), and Acid Detergent Lignin (ADL) accordingto Van Soest (1963b), using chemical extraction followed by gravi-metric determination of the residues.

2.2. Cell-mediated response

At 0, 30, and 60 days of the experiment cell-mediated immuneresponse was determined in vivo on each cow. At each sampling,1 mg of phytohemoagglutinin (PHA) (Sigma Chemical Co., Italia)dissolved in 1 ml of sterile saline solution was injected intrader-mally into the centre of a 2 cm diameter circle marked on theshaved skin on the dorso-lateral side of each shoulder. The deter-mination of cell-mediated immune response, measured as skinfoldthickness using a millimetre graduated caliper, was calculated asthe difference between 24 h post-injection thickness and pre-injec-tion thickness.

2.3. Humoral response

At day 0 of the experiment, 2 mg of chicken egg albumin (OVA)(A5503, Sigma Chemical Co., Italia) dissolved in 1 ml of sterilesaline solution and 1 mL of incomplete Freund’s adjuvant (SigmaChemical Co., Italia) were injected subcutaneously into both shoul-ders of each cow. A second injection without adjuvant wasrepeated 15 days later. At 0, and then at 15, 30, 45, 60 days ofthe experiment blood samples were taken from the caudal veinof each cow for immunological assays. Blood samples werecollected in duplicate in heparinized vacuum tubes (BectonDickinson, Plymouth, United Kingdom) and centrifuged at 1200gfor 15 min at 25 �C. Plasma samples were stored at �80 �C, beforeELISA to evaluate the anti-OVA IgG titer, IL-1b, IL-6 and IL-10concentrations.

The anti-OVA antibody titre in cow plasma samples was evalu-ated by an ELISA test performed in 96-well U-bottomed microtiterplates. Wells were coated with 100 ll of antigen (10 mg of OVA/mlof phosphate-buffered saline – PBS) at 4 �C for 12 h, washed andincubated with 1% skimmed milk (200 ll) at 37 �C for 1 h to reducenon specific binding. After washing, the plasma (1:5000 dilution inPBS; 100 ll per well) was added and incubated at 37 �C for 1 h. Theextent of antibody binding was detected using a horseradish per-oxidase (HRP)-conjugated rabbit anti-bovine IgG (A5295, SigmaChemical Co., Italia) (1:20,000 dilution in PBS; 100 ll per well).After a further washing, 100 ll of substrate consisting of tetramethyl benzidine free base tablets (TMB, Sigma Aldrich, Italy), di-methyl sulphoxide (DMSO, Sigma Aldrich, Italy), dissolved in0.05 M phosphate-citrate buffer (pH 5.0), and H2O2 was added toeach well and incubated for 30 min at 37 �C. Finally, 2 M H2SO4

was added to terminate reactions. Optical density was measuredat a wavelength of 450 nm. Plasma samples were read against astandard curve obtained using scalar dilution of IgG from bovineserum (I5506, Sigma Chemical Co., Italia). Data were expressed asmg of anti-OVA IgG/ml. The inter- and intra-assay CV were 3.6%and 4.5%, respectively.

a

b

2

4

6

8

10

12

IgG

(m

g/m

l)

Control

G160

G320

204 M. Caroprese et al. / Research in Veterinary Science 93 (2012) 202–209

2.4. Determination of interleukin IL-1b, IL-6 and IL-10 by indirectsandwich ELISA-test

The level of IL-6 and IL-1b in plasma was determined by captureELISA performed on 96-well microtiter plates, according to Carop-rese et al. (2006). Mouse monoclonal antibodies specific for bovineIL-6 and for sheep IL-1b (Serotec Ltd., UK) (100 ll, 5 lg/ml) dis-solved in 50 mM carbonate buffer (pH 9.6) were used to coat wellsand incubated overnight at 4 �C. After washing with PBS (pH 7.2)and 0.05% Tween 20 (PBST) plates were incubated with 100 ll of3% BSA diluted in PBST at room temperature for 1 h to block nonspecific binding. Plates were then washed twice with PBST andthe plasma (50 ll per well) added and incubated at room temper-ature for 90 min. Buffer alone or 3% BSA in PBST provided a nega-tive control. Rabbit polyclonal antibody anti-sheep IL-6 and anti-bovine IL-1b conjugated to biotin (Serotec Ltd., UK) were used asdetecting antibodies (1:500 diluted in 3% BSA in PBST) to deter-mine captured IL-6 and IL-1b and incubated at room temperaturefor 90 min. The presence of binding for IL-6 was detected usinggoat anti-rabbit IgG conjugated to HRP (Sigma Aldrich, Italy). IL-1b concentrations were detected using HRP conjugated streptavi-din. The following steps were carried out as described above forthe anti-OVA IgG detection. Plasma samples were read against astandard curve obtained using scalar dilution of bovine IL-1b (Sero-tec Ltd., UK). Data were expressed as ng of IL-1b/ml. IL-6 data wereexpressed as optical density.

IL-10 determination in cow plasma samples was carried outby an ELISA test according to Kwong et al. (2002). The ELISAwas standardized using biologically-active recombinant ovineIL-10 expressed in Chinese hamster ovary (CHO) cells using theGln synthetase expression vector™ (Lonza, UK). Production ofrecombinant ruminant cytokines using this system has beendescribed in detail elsewhere (Graham et al., 1995; Entricanet al., 1996). Recombinant ovine IL-10 was provided by theBBSRC/RERAD Immunological Toolbox. ELISA standards wereprepared by doubling dilutions of IL-10 from 3.645 to 0.043bU/ml to generate a standard curve (y = �0.007271 + 0.0994x +0.0065x2, R2 = 99.09%).

0

15 30 45 60Days

Fig. 1. Antibody titres to OVA (Least Squares means ± SEM) detected in blood ofcows fed control diet, 160 g/d Gln (G160), or 320 g/d Gln supplemented diets(G320), at 15, 30, 45, and 60 days of the trial. a,b Values with different superscriptsdiffer between feeding treatments within a sampling day (P < 0.05).

a

b

a

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

0 30 60

Days

Skin

fold

thic

knes

s (m

m)

Control

G160

G320

Fig. 2. Skinfold thickness (Least Squares means ± SEM) after PHA injection detectedin cows fed control diet, 160 g/d Gln (G160), or 320 g/d Gln supplemented diets(G320), at the beginning, 30 and 60 days of the trial. a,b Values with differentsuperscripts differ between feeding treatments within a sampling day (P < 0.05).

2.5. Milk sampling and analysis

Milk samples from morning and afternoon milking (07.00 and17.00) were collected every 14 days from each cow for the durationof the experiment. One aliquot was stored at �20 �C for amino acidanalysis. Fresh samples were used for the following analysis: pH(GLP 21 Crison, Spain), total protein, casein, fat, and lactose contentusing an infra red spectrophotometer (Milko Scan 133B; Foss Elec-tric, Hillerød, Denmark) according to the International Dairy Feder-ation standard (IDF, 1990); somatic cell count (SCC) using a FossElectric Fossomatic 90 cell counter (IDF, 1995); evaluation of thecoagulation ability (clotting time, rate of clot formation, and clotfirmness after 30 min) using a Foss Electric formagraph (Foss Elec-tric, Hillerød, Denmark).

Milk samples for AA determination were hydrolyzed with 6 Nhydrochloric acid at 160 �C for 60 min. Amino acid compositionwas determined using an HPLC system (Agilent Technologies1100 Series, Waldbronn, Germany). The separation was carriedout by ZORBAX Eclipse AAA column (150 � 4.6 mm ID, prepackedwith 3.5 lm particles). Individual amino acid peaks were identifiedby comparing their retention times with those of standards (SigmaChemical Co., Italia). Results are expressed as mg amino acid/g to-tal amino acid. During the hydrolysis Gln was converted to gluta-mate and asparagine was converted to aspartate, thus, the valuesreported as glutamate include both glutamate and Gln and thosefor aspartate include both aspartate and asparagine.

2.6. Statistical analysis

All variables were tested for normality using the Shapiro–Wilktest (Shapiro and Wilk, 1965) and transformed into logarithm formto normalize their frequency distribution, when necessary. Then,data were processed by ANOVA using the GLM procedure for re-peated measures of SAS (1999), having time as repeated factor.Themodel utilized was: yijkl = l + ai + bij + ck + (ac)ik + eijkl

Where l = the overall mean; a = feeding treatment b = animaleffect within feeding treatment; c = day of sampling effect;ac = interaction of feeding treatment x day of sampling and e = er-ror. For humoral response, antibody titres measured at day 0 wereused as covariates. When significant effects were found (atP < 0.05), the Student t-test was used to locate significant differ-ences between means.

3. Results

3.1. Immunological determinations

Cows of the G160 group had a lower anti-ova IgG concentrationthan cows in the other two groups (P < 0.05) at 30 days of the

b

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40

60

80

100

120

140

160

0 15 30 45 60

Days

IL-1

0 (b

U/m

l)

Control

G160

G320

Fig. 3. Interleukin-10 secretion (Least Squares means ± SEM) detected in blood of cows fed control diet, 160 g/d Gln (G160), or 320 g/d Gln supplemented diets (G320) at 0,15, 30, 45, and 60 days of the trial. a,b Values with different superscripts differ between feeding treatments within a sampling day (P < 0.05).

M. Caroprese et al. / Research in Veterinary Science 93 (2012) 202–209 205

experiment, while no differences across groups emerged at theother sampling times (Fig. 1).

A significant decrease of cell-mediated immune response afterthe injection of PHA was observed in all groups throughout thetrial (P < 0.001). In addition, an interaction of diet supplementationand time of sampling was found as a result of a more marked de-crease of cell-mediated immune response (P < 0.05) in cows fedG120 compared with G320 and control cows at the end of the trial(Fig. 2).

Average IL-10 secretion was affected by diet (P < 0.01), showinghigher values in G160 (89.25 ± 2.94 bU/mL) than in control andG320 cows (77.76 and 75.35 ± 2.94 bU/mL, respectively). Effectsof time of sampling and of diet � time of sampling were found be-cause IL-10 was higher in G160 cows than in G320 cows at 45 daysand in G160 cows than in G320 and control cows at 60 days(P < 0.001) (Fig. 3).

IL-1b concentration was affected by time of sampling(P < 0.001) because at 45 and 60 days IL-1b secretion was higherthan before in all the experimental groups (Fig. 4). An effect of timefor IL-6 concentration was also found (P < 0.001) with a lower IL-6

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 15 30 45 60

Days

IL-1

beta

(lo

g ng

/ml)

Control

G160

G320

Fig. 4. Interleukin-1b secretion (Least Squares means ± SEM) detected in blood ofcows fed control diet, 160 g/d Gln (G160), or 320 g/d Gln supplemented diets(G320) at 0, 15, 30, 45, and 60 days of the trial.

concentration measured at days 15, 45 and 60 than at days 0 and30. At 15 days of the experiment, IL-6 concentration was higherin G160 and G320 than in control cows (P < 0.05) (Fig. 5).

3.2. DMI, milk production, composition, and coagulation properties

Supplemented cows received 0.6–1.1% more CP and 0.05–0.09 Mcal/kg more Net Energy of lactation (NeL) in their diets com-pared with control cows (Table 1). No significant differencesemerged in milk yield and composition of cows fed different ratesof Gln; cows fed 320 g/day/animal of Gln displayed an increase inmilk production of 7% compared with control cows and of 11%compared with cows fed 160 g/day/animal of Gln (Table 2). No dif-ferences were found for fat, protein, and casein content of milkamong groups, although a tendency to an increase of casein con-tent in G160 and G320 milk was observed, as expressed by caseinto protein ratio (P = 0.09). Somatic cell counts were not differentamong groups. As expected, with the advancement of lactation a

b

a

a

0

0.2

0.4

0.6

0.8

0 15 30 45 60

Days

IL-6

(O

.D.)

Control

G160

G320

Fig. 5. Interleukin-6 secretion (Least Squares means ± SEM) detected in blood ofcows fed control diet, 160 g/d Gln (G160), or 320 g/d Gln supplemented diets(G320) at 0, 15, 30, 45, and 60 days of the trial. a,b Values with different superscriptsdiffer between feeding treatments within a sampling day (P < 0.05).

10121416182022242628

Days

Milk

yie

ld, k

g/d

C G160 G320

2.5

2.7

2.9

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ein,

%

22.12.22.32.42.52.62.72.82.9

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ein,

%

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, x10

3cel

ls/m

l

0.720.730.740.750.760.770.780.790.8

0.81

1 30 60 1 30 60

1 30 60 1 30 60

Days

Cas

ein/

Prot

ein

Fig. 6. Milk yield, protein, casein, casein to protein ratio, and SCC (Least Squares means ± SEM) detected in milk of cows fed control diet, 160 g/d Gln (G160), or 320 g/d Glnsupplemented diets (G320) at 0, 30, and 60 days of the trial.

Table 2Least Square Means ± SEM of milk production and composition, and of efficiency of milk production of cows fed control diet (C), 400 g/d of Gln (G400), or 800 g/d of Gln (G800).

Treatment Effects, P

Control G160 G320 SEM Gln Time Time � Supplementation

Milk yield (kg/d) 19.01 18.38 20.42 1.74 NS ⁄⁄⁄ NS

Milk composition (%)Fat 3.69 3.90 3.76 0.15 NS NS NSProtein 3.15 3.26 3.22 0.12 NS ⁄⁄⁄ NSCasein 2.41 2.54 2.52 0.09 NS ⁄⁄⁄ NSCasein to Protein Ratio 0.76 0.77 0.78 0.01 NS ⁄⁄⁄ NSLactose 4.71 4.78 4.80 0.03 NS ⁄⁄⁄ NS

pH 6.7 6.66 6.63 0.02 NS ⁄⁄⁄ ⁄

SCC (�103 cells/ml) 432.39 532.20 287.03 96.07 NS NS NS

Milk net energya (Mcal/kg) 0.70 0.73 0.71 0.02 NS ⁄ NSEnergy efficiency for milk productionb 0.48 0.45 0.47 0.03 NS ⁄⁄⁄ NSNitrogen efficiency for milk productionc 0.21 0.20 0.21 0.01 NS ⁄⁄⁄ NS

Significance: NS, not significant; ⁄, P < 0.05; ⁄⁄⁄, P < 0.001.a Calculated according to NRC (2001): 0.0929 �milk fat% + 0.0563 �milk protein% + 0.0395 �milk lactose%.b Energy efficiency for milk production = Milk net energy �Milk yield/NeL � DMI.c Nitrogen efficiency for milk production = Nitrogen milk production/Dietary nitrogen intake.

206 M. Caroprese et al. / Research in Veterinary Science 93 (2012) 202–209

change of milk yield and composition was observed, but no effectsof dietary treatment among groups were observed (Fig. 6).

The AA profile of milk is reported in Table 3; glutamate, prolineand total essential amino acids were higher in G160 and G320 milk

Table 3Least Square Means ± SEM of amino acid composition (mg/g total amino acids) of milkfrom cows fed control diet (C), 160 g/d of Gln (G160), or 320 g/d of Gln supplemen-tation (G320).

Treatment Effects, P

Control G160 G320 SEM Gln

Ala 34.70 31.87 31.50 1.25 NSArg 36.85 34.51 34.55 0.90 NSAsp 75.64 b 77.85 a 76.04 b 0.61 ⁄

Cys 5.86 5.65 6.08 0.35 NSGlu 207.59 b 211.55 a 211.00 a 1.05 ⁄

Gly 19.36 17.22 17.42 0.80 NSHis 36.05 33.90 34.20 0.85 NSIle 49.86 50.76 50.98 0.78 NSLeu 91.80 90.51 91.66 0.95 NSLys 68.11 67.91 67.36 1.25 NSMet 27.02 28.77 28.55 0.88 NSPhe 37.80 38.24 39.36 0.76 NSPro 114.25 b 119.05 a 119.27 a 1.10 ⁄

Ser 45.19 42.05 42.35 1.12 NSThr 40.51 40.86 41.01 0.85 NSTrp 0.89 0.95 0.98 0.55 NSTyr 43.45 41.85 42.05 0.62 NSVal 64.73 b 68.50 a 66.47 ab 1.05 ⁄

Total EAAa 380.72 b 386.49 a 386.37 a 1.78 ⁄

Significance: NS, not significant; ⁄, P < 0.05.a EAA = Essential amino acids: Phe; Ile; Leu; Lys; Met; Thr; Trp; Val.

aa

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60

Days

Cur

d fi

rmne

ss (

mm

)

Control G160 G320

Fig. 7. Rennet coagulation time (a) and curd firmness (b) (Least Squares mean-s ± SEM) detected in milk of cows fed control diet, 160 g/d Gln (G160), or 320 g/dGln supplemented diets (G320) at 0, 30, and 60 days of the trial. a, b, c Values withdifferent superscripts differ between feeding treatments within a sampling day(P < 0.05).

M. Caroprese et al. / Research in Veterinary Science 93 (2012) 202–209 207

than in control milk (P < 0.05). Furthermore, milk from G160 cowshad higher aspartate and valine content (P < 0.05) than milk fromcontrol and G320 cows.

At 30 days G320 milk showed the highest curd firmness(P < 0.05), whereas G160 and G320 milk showed shorter rennetcoagulation time than control milk (Fig. 7, P < 0.001). These resultsmatched milk pH values, which were lower in G160 and G320 milkthan in C milk (6.64 and 6.60 vs. 6.67, respectively).

4. Discussion

Few reports are available on the influence of Gln on humoral re-sponse: in humans, dietary Gln does not induce an increase of Type1-related antibody response (IgG1 and IgG3) against Keyhole Lim-pet Hemocyanin (KLH), and trauma patients generated a similar denovo humoral response to KLH immunization after 14 days ashealthy volunteers (Boelens et al., 2004). In preruminant calves,Fligger et al. (1997) found that L-Arg supplementation depressedanti-KLH IgG and total IgG concentrations, probably as a result ofoverproduction of nitric oxide by macrophages involved in theantigen processing for IgG producing cells, and using L-Arg to pro-duce nitric oxide. Arginine, proline, and Gln metabolism are bidi-rectional (Cynober et al., 1995) and endogenously synthesizedarginine derives from citrulline in the kidney. Citrulline derivesfrom Gln and glutamate metabolism in the intestine. The adminis-tration of a Gln-enriched diet to rats increased arginine productionby 38% by increasing both the plasma levels of citrulline and thekidney uptake by 30% and 40%, respectively (Houdijk et al.,1994). As a consequence, the reduced anti-Ova IgG production byG160 cows might be an indirect mechanism mediated by Gln con-version to arginine, leading to an overproduction of nitric oxide, assuggested by Yaqoob and Calder (1998). The indirect mechanism ofaction of Gln hypothesized could also help to explain the absenceof differences in IgG production throughout the experiment.

An optimal Gln concentration is required for lymphocyte func-tions and in vitro lymphocyte proliferation (Chang et al., 1999). Rat,mouse and human in vitro proliferative response of T-lymphocyteto mitogens is subject to Gln availability; in addition, Gln utiliza-tion increases with lymphocyte activation (Yaqoob and Calder,1998). Chang et al. (1999) demonstrated that the proliferation ofPHA-stimulated lymphocytes was about threefold higher in thepresence of 0.6 mM Gln than without the addition of Gln. Basedon previous considerations, the reduced T-lymphocyte responseobserved in G160 cows and the lack of differences between controland G320 cows is not easy to explain. There are no data on theoptimal dose of Gln for ruminants, though Jafari et al. (2006) foundthat the administration of 212 g/day to transition dairy cows hadimmunomodulatory effects by increasing the concentrations ofserum amyloid A and LPS-binding protein and reducing the pro-duction of haptoglobin. Based on results from the present experi-ment it can be argued that the dietary administration of 160 g/day of rumen-protected Gln to dairy cows may have suppressiveeffects on cell-mediated immune response; but an increase in thedose of dietary Gln administrated failed to enhance the suppressiveeffect on PHA-stimulated lymphocytes. Chang et al. (1999) re-ported a dose-dependent action of Gln in humans, with an inhibi-tion of lymphocyte proliferation at 2 mM of Gln concentration.

IL-10 is considered to be an anti-inflammatory cytokine and soa Gln-induced increase in IL-10 production would be beneficial inanimals with inflammatory complications. Yaqoob and Calder(1998) found that Gln availability induces IL-10 production by hu-man peripheral blood mononuclear cells, even though a growingGln availability did not result in a further increase of IL-10 produc-tion. In agreement with these authors, in the present trial IL-10production increased in G160 cows, but the doubling of Gln

208 M. Caroprese et al. / Research in Veterinary Science 93 (2012) 202–209

supplementation did not result in increased IL-10 secretions. IL-10has an important immunomodulating role by downregulating theexpression of several proinflammatory cytokines, and particularlyof IL12, which is required for Th1 differentiation (Robertsonet al., 2007; Amsen et al., 2009). The IL-10 production results fol-lowing in vivo administration of Gln might explain the reductionin cell-mediated immune response and anti-OVA IgG productionobserved in G160 cows. IL-10 can downregulate the expressionof MHC class II molecules and co-stimulatory molecules, thusreducing the Th1 responses (Wattegedera et al., 2004). In addition,administration of IL-10 to mice can inhibit antigen specific im-mune response in vivo (Rohrer and Coggin, 1995).

The relationship between Gln availability and production ofproinflammatory cytokines is not well established. Human studiesstated that Gln does not influence the in vitro production of IL-1and IL-6 by peripheral blood mononuclear cells (Yaqoob and Cal-der, 1998) and reduces IL-6 production by gut cells (Coëffieret al., 2001). Animal studies showed that administration of Gln inthe diet can enhance the production of IL-1b and IL-6 by macro-phages (Wells et al., 1999). In the present in vivo study cows onGln supplementation had higher IL-6 production at 15 days ofthe experiment, thus matching the results of Wells et al. (1999)in mice. The reduction in IL-6 production in Gln supplementedcows during the subsequent days of the trial might be explainedby the concomitant increase of IL-10. Wattegedera et al. (2004)suggested that IL-10, which can be considered a regulatory cyto-kine, might be produced to control inflammatory processes whenother inflammatory cytokines are produced. Accordingly, Penget al. (2011) found that glutamine administration might suppressthe inflammatory response in rats with chronic ethanol feeding.

The efficiency of utilization of dietary energy and nitrogen wasvery similar across groups. In addition, Gln administration had noeffects on DMI, which was not different among groups, accordingto previous findings on postruminal infusion of Gln (Doepelet al., 2006; Jafari et al., 2006). Gln, and its derivates Glu and Pro,account for 35% of AA residues of b-casein, for about 28% of as1-casein and j-casein, and 24% for as2-casein (Creamer, 2003). Inthis study, though Gln administration did not influence milk yield,the tendency to an increase of casein content in G160 and G320milk observed, together with the AA profile of milk, suggest thatGln supplementation was able to improve AA profile and, probably,the casein composition of milk. These results could help to explainthe improvement of milk coagulation properties observed in Glnsupplemented groups. Milk coagulation properties were monitoredto evaluate the effect of Gln supplementation on milk quality. Ingeneral, a moderate improvement of rennet coagulation time andcurd firmness was found in the groups subject to supplementation.In addition, milk from G320 cows showed the lowest average SCC(287 � 103 cells/ml), which is below the threshold for the defini-tion of high quality milk as reported by Italian DM 91/185.

In conclusion, results from the present study suggest a dose-dependent effect of Gln supplementation on immune functions ofcows. In particular, Gln supplementation of 160 g/day, but not of320 g/day, affected humoral and cell-mediated immune responses,and modulated cytokine concentration of cows. Gln supplementa-tion did not affect milk yield and composition, but improved theAA profile of milk. Milk from cows supplemented with 320 g/dayof Gln displayed ameliorated cheese making quality.

Acknowledgements

The authors would like to thank Concetta Perilli and GiovanniGliatta for expert technical assistance. This work was conductedusing recombinant ovine IL-10 generated within the BBSRC/RERADImmunological Toolbox (grant numbers BBS/B/00255, MRI/094/04), provided by Gary Entrican (G.E.) and Sean Wattegedera

(S.W.) at the Moredun Research Institute (Edinburgh, UK) andfunded by the Scottish Government Rural and Environment Re-search and Analysis Directorate.

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