Veterinary Immunology and Immunopathology 145 (2012) 134– 142
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Veterinary Immunology and Immunopathology
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mprovements of immune status, intestinal integrity and gainerformance in the early-weaned calves parenterally supplementedith l-alanyl-l-glutamine dipeptide
Department of Animal Science, College of Agriculture, Ningxia University, 489 W. Helanshan Road, Xixia District, Yinchuan, Ningxia 750021, PR ChinaCollege of Life Science, Ningxia University, 489 W. Helanshan Road, Xixia District, Yinchuan, Ningxia 750021, PR ChinaInstitute of Animal Nutrition, Inner Mongolian Academy of Agricultural Animal Science, Huhhot, Inner Mongolia 010030, PR China
r t i c l e i n f o
rticle history:eceived 27 July 2011eceived in revised form 23 October 2011ccepted 27 October 2011
Glutamine (Gln) is an important substrate for the innate immune cells including lympho-cytes and macrophages. In this report, the effects of alanyl-glutamine dipeptide (Ala-Gln)on the naïve immune system, intestinal integrity and gain performance of early-weanedcalves were investigated. Early-weaned Chinese Holstein calves were intravenously admin-istered different dosages of Ala-Gln. The effects of Ala-Gln administration were ascertainedby evaluating the blood for naïve T lymphocyte subpopulations, the concentrations of serumIgG, serum IgA and intestinal mucosal secretory IgA (s-IgA), the intestinal integrity, aswell as the gain performance. Results demonstrated that intravenous administration ofAla-Gln dipeptide (1.01 g/kg × d−1) for 7 days had a positive effect on gain performance,intestinal integrity and the immune system. Calves administered doses of Ala-Gln dis-played an improvement in gain performance and health status concurrent with increasesin blood CD2+ and CD4+ lymphocytes, the ratio of CD4+/CD8+, serum IgA and IgG, intesti-nal mucosal s-IgA while decreasing the occurrence of diarrhea. Moreover, we found that
animals given the effective dose (1.01 g/kg × d−1) of Ala-Gln resulted in improved immunestatus and intestinal integrity relative to those given a lower (0.49 g/kg × d−1) or higherdose (1.99 g/kg × d−1) of Ala-Gln. These findings suggest that maintaining a certain con-centration of plasma and/or tissue glutamine in the early stages of weaning is an effectivealternative approach for improvement of growth performance in early-wean calves.
. Introduction
Calves are traditionally weaned from their dams whenhey are between 108 and 220 days of age. However,
eaning often occurs before the suggested traditionalre-weaning age, due to the consideration of economic via-ility, marketing options and/or adverse production (such
as adverse weather condition and market trends) by theproducers. Early weaning is defined as separating calvesfrom their dams at less than 108 days of age (Rasby, 2007).Weaning is one of the most stressful periods of time dur-ing a calf’s life. Successful weaning is important for calvesduring the transition from a pre-ruminant to a function-ing ruminant animal. However, inadequate weaning maystunt ruminal development, consequently often leading tosickness and inferior calf performance. In early-weaned
calves, weaning stress may elevate concentrations of corti-sol in the blood, which may be deleterious to cell-mediatedimmunity (Quigley et al., 1994), and suppress many innateimmune responses thus causing a predisposition to disease,
regardless of weaning age (Carroll et al., 2009; Hulbert et al.,2011). The decreased immune function may impair overallcalf health by increasing susceptibility of calf to diseases,despite immunoglobulins derived from colostrum contin-uing to provide passive protection in the animal. Thesefindings imply that enhancing immune status may increaseweaning success for young calves.
The physiological immaturity of the immune systemand gut barrier function in the early-weaned animalsincrease the susceptibility to infectious diseases and diges-tive disorders. Nutritional strategies could be employedto support host immune system as most nutrients haveproven roles in mounting a viable immune response (Fieldet al., 2002). Glutamine (Gln) is the most abundant freeamino acid in the body (Young and Ajami, 2001), as wellas in maternal milk and the colostrum (Davis et al., 1994).It is a conditionally essential amino acid during certaincatabolic stress states such as weaning, illness or injury(Mondello et al., 2010; van der Hulst et al., 1996; Wuet al., 1996). Gln serves as an essential precursor for syn-thesizing proteins and amino sugars, and a major fuel inrapidly dividing cells—including lymphocyte and intesti-nal epithelial cells. It has also been demonstrated as animportant substrate for lymphocytes, macrophages andintestinal mucosal cells (Abcouwer, 2000; Cynober, 1997;Newsholme et al., 1999; Rogero et al., 2008a, 2008b; Sacksand Kudsk, 2003; Yaqoob and Calder, 1997). Gln supple-mentation has been shown to improve immune function,growth performance and intestinal integrity in prematureinfants, as well as patients undergoing surgery or radiationtherapy (Jiang et al., 2009; Rogero et al., 2008b; van denBerg et al., 2009; Wu et al., 1996).
The fast-growing body mass in young calves elevatesmetabolic demands for nutrients thus creating competi-tion for their use. Furthermore, deprivation of maternalmilk from premature early-weaned calves could result ina systemic insufficiency and the inability to meet all ofthe competing demands. The immature immune systemin pre-weaning calf leads to an increasing susceptibil-ity to infections under metabolic stress conditions likeweaning. An increasing body of studies has indicated thatsupplementation of Gln enables maintenance of the normalimmune response and the integrity of intestinal epitheliumin the face of catabolic stress (Jiang et al., 2009; Newsholmeet al., 1999; Wallace and Keast, 1992). For example, dietarysupplements of Gln toadied in the prevention of jejunumatrophy in weaned pigs (Wu et al., 1996), and enhanced theimmunological functions of peritoneal macrophages andhaematopoiesis in mice that were weaned early and inoc-ulated with Mycobacterium bovis bacillus Calmette-Guerin(BCG) (Rogero et al., 2008a, 2008b). Though parenteral orenteral Gln treatment in human patients has extensivelybeen investigated (Cetinbas et al., 2010; Fuentes-Orozcoet al., 2008; Mondello et al., 2010; O’Riordain et al., 1994;Wischmeyer, 2003), the immunological consequencesassociated with the parenteral Gln in early-weaned calveshave yet to be established.
We hypothesized that Gln might have a positiveimpact on the immune status and growth performance inearly-weaned calves based on the fact that Gln adminis-tration was required for proliferation and enhancement of
munopathology 145 (2012) 134– 142 135
T-lymphocyte responsiveness in mice (Kew et al., 1999;Yaqoob and Calder, 1997), and parental supplementationof Gln improved immune function and intestinal integrityin infants, patients undergoing surgery or radiation ther-apy, as well as early-weaned piglets and mice. Therefore,we investigated the effects of intravenous infusion of Ala-Gln, a dipeptide form of Gln, on the gain performance,intestinal epithelial integrity and naïve immune systemascertained by evaluating the blood naïve T lymphocytesubpopulations, and the concentrations of IgG and IgA inserum, secretion of intestinal secretory IgA (s-IgA), in early-weaned calves.
2. Materials and methods
2.1. Animals and management
The Chinese Holstein calves were bred at the exper-imental farms of the Inner Mongolian Academy ofAgricultural Animal Science (Inner Mongolian, China) andthe College of Agriculture at Ningxia University (Yinchuan,China). The calves used in this study were half-sib neonatalbull clinically healthy calves that were weight and age-matched at pre-weaning age in this study. The animalswere individually housed in a pen with wood slatted floorsin a facility with a consistent temperature of 20 ◦C duringthe experimental period. The neonatal calves were directlyfed 4 times daily with colostrum from mother cows duringthe first 7 days after birth. Followed by feeding three timesdaily with 4.2 kg/day of raw milk from dams for each animalon days 8 and 9 postnatally. Then they were fed twice dailywith 4.3 kg/day of raw milk, and provided ad libitum alfalfahay and a basic calf diet (Table 1) during the pre-weaning10–40 days after birth. Calves were allowed free access topre-warmed water (35 ◦C) during the 0–40 days postna-tally. The calves were weaned on day 41 and provided onlyad libitum alfalfa hay, a basic calf diet and water (room tem-perature) from the beginning of weaning to the completionof the experiments. The composition of the basic calf dietis shown in Table 1. Animals that had watery stools wereconsidered to have a diarrheic sign regardless of the sever-ity and duration. The feed efficiency was represented asthe ratio of feed/gain as determined by the consumption offeed (kg) per kg of weight gain.
2.2. Experimental design and treatment
The study was conducted using approved protocols bythe Animal Use and Care of the Institutional Commit-tee at Ningxia University. A total of 36 newborn Holsteinbull calves were used in three trials conducted at twolocations (two trials were performed at the Inner Mongo-lian Academy of Agricultural Animal Science, and anotherat the College of Agriculture at Ningxia University). Thegenetic background of these animals and the feeding con-dition between these two locations were identical. Eachtrial consisted of 12 calves randomly divided into four
groups. The data shown for each group (nine animals)was derived by pooling data collected from the threetrials. The starting Ala-Gln dosage (1.01 g/kg × d−1) wasdetermined by previous studies (Doepel et al., 2007). To
136 Y. Zhou et al. / Veterinary Immunology and Immunopathology 145 (2012) 134– 142
Table 1Composition and nutrient level of the basic diet for calves.
Ingredient of diet Ratio (g/kg) Nutrient level
Expanded corn 410.0 Net energy for gain (NEG) (MJ/kg) 4.86Cooked bean cake 150.0Soybean meal 110.0 Crude protein (g/kg) 215.2Wheat bran 140.0 Calcium (g/kg) 15.3Corn gluten meal 80.0 Phosphorus (g/kg) 8.6Whey powder 54.0 Acid detergent fiber (ADF) (g/kg) 54.9Dicalcium phosphate 18.0 Neutral detergent fiber (NDF) (g/kg) 170.5Powder 20.0Salt 8.0Premix feeda 10.0
aw(ttodoaae4wTbwicsttaS3fwffm
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a Mixture of multivitamin and multimineral supplement.
scertain any dose-dependent effects of Ala-Gln on early-eaned calves a saline control (no Ala-Gln), a low dosage
0.49 g/kg × d−1, equal to 0.34 g/kg × d−1 of free Gln, half ofhe starting dosage) and a high dose (1.99 g/kg × d−1, equalo 1.34 g/kg × d−1 of free Gln, twice of the starting dosage)f Ala-Gln, were included in these studies. The Ala-Gln waselivered to calves in 500 mL of saline daily using continu-us intravenous infusion (via the external jugular vein) onn average of 5 mL/min for seven consecutive days initi-ted on the day of weaning. Blood (10 mL) was drawn fromach animal at 0, 6, 7 and 14 days post-weaning (41, 47,8 and 55 days after the birth, respectively). The calvesere slaughtered 18 days post weaning (post-natal day 59).
he intestine was harvested and the fecal mater removedy flushing 3 times with cold saline before the length andeight were recorded. The intestine was then segmented
nto the duodenal, jenunal, and ileal regions and a 10-m piece was removed from the middle portion of eachegment for analysis of the mucosal tissues. The mucosalissue from individual sample was harvested by scrapinghe mucosal layer 10 times up and down the segment using
tissue scraper (TPP, Techno Plastic Products, Trasadingen,witzerland). The resulting samples were then vortexed for0 s and centrifuged at 3000 × g (Beckman JA25.50 rotor)or 10 min to remove insoluble material. The supernatantas then passed through a 0.45-�m filter (Millipore, Bed-
ord, MA), aliquoted and stored at −20 ◦C or directly usedor Bradford analysis of protein concentration and s-IgA
Plasma samples for glutamine concentration mea-urements were deproteinized with perchloric acid2%), neutralized with potassium hydroxide (KOH)/Tris2 mol/L/0.5 mol/L), and centrifuged 3000 × g for 15 min at4 ◦C. The concentration of glutamine in plasma and in
he tracer infusate was determined using a Waters 600igh-performance liquid chromatography (HPLC) systemWaters Corporation, Milford, MA, USA), according to areviously described protocol with modifications (van Eijk
t al., 1993). The parameters used in HPLC were as follows:olvent (mobile phase), methanol–10 mmol/L phosphateuffer (pH 6.85) (85:15); stationary phase, Waters Symme-ry (R) C18 chromatography column; column temperature,
White blood cells (WBC) from individual peripheralblood samples were used to analyze T lymphocyte sub-populations by flow cytometry methods. Mouse primaryantibodies against the surface antigens of bovine totalperipheral T cells (CD2), T helper cells (CD4), cytotoxicT cells (CD8), and normal mouse IgGl were obtainedfrom GenWay Biotech Inc. (San Diego, CA, USA). The sec-ondary fluorescein isothiocyanate (FITC)-conjugated goatanti-mouse IgG secondary antibody was a product ofBD Bioscience Inc. (Heidelberg, Germany). The stainedcells sorted by flow cytometry using a FACScan appa-ratus (BD Bioscience) equipped for double fluorescencemeasurements and analyzed using FlowJo software. Nor-mally, lymphocyte gates were set to selectively analyze500–10,000 cells from each sample. The gating was setto excluding red blood cells and other debris, focus-ing on the population of lymphocytes. Cells stainedwith FITC-conjugated normal mouse IgGl (product of BDbioscience) were used as controls for background fluores-cence.
2.5. Determination of the immunoglobulinconcentrations
The concentrations of IgA and IgG from serum, ands-IgAs from duodenal, jejunum and ileal mucus were deter-mined using a sandwich ELISA detection kit for bovineImmunoglobulin A and G against standards, according tothe manufacturer’s suggested protocol (CUSAbio BiotechInc., Wuhan, China). Standard curves for the concentra-tions of IgG or IgA were generated using known standards.The unknown IgA or IgG samples were assayed in tripli-cate using the following protocol. The serum and intestinalmucosal samples were diluted 1:2000 and 1:5000 usingELISA wash buffer. A 96-well plate pre-coated with aspecific primary antibody against bovine IgG or IgA was
washed three times with 200 �L ELISA wash buffer, and100 �L of diluted samples were added to individual wells.Samples were incubated for 2 h with shaking on a horizon-tal mixer at room temperature (RT). Wells were washed
and Immunopathology 145 (2012) 134– 142 137
Fig. 1. Plasma glutamine (Gln) concentration in the early-weaned calves(�mol/L). Plasma Gln concentration was measured from the serum ofearly-weaned calves given varied doses of Gln on the indicated days postweaning. Compared to the group of saline control, **p < 0.01; *p < 0.05.
Y. Zhou et al. / Veterinary Immunology
and 100 �L peroxidase-labeled anti-bovine IgA or IgG anti-body was added to each well and incubated for 1 h at RT.The unbound antibody was washed away by washing for5 min × 3 min prior to incubating with 100 �L tetramethyl-benzidine substrate solution for 10 min at RT. Finally, 50 �Lof stop solution was added to each well and mixed briefly.The absorbance of each well at 450 nm was determinedusing an ELISA reader. The immunoglobulin concentrationswere determined using the standard curves.
2.6. Intestinal wall morphometric analysis
Intestinal wall morphometric analysis was performedon duodenum and jejunum tissues for measurements of thevillus height, mucosal thickness and crypt depth as previ-ously described with modifications (Hermes et al., 2008). Inbrief, a 1.0 cm central portion of the duodenum and proxi-mal, medial and distal jejunum were collected and fixed ina phosphate buffered (pH 7.3) 10% formalin solution. Thetissues were fixed for three days and processed for paraffinembedding in a RMC Paraffin Tissue Processor (Model 1530,Research & Manufacturing Inc.). Longitudinal 5 �m-thicksections were cut and stained with haematoxylin–eosin(H&E). The H&E images were captured using a Leica DMRBoptical microscope equipped with a 10× objectives andLeica DFC 300F digital camera. Three measurements (two ateach end and one at the middle) were carried out over eachlongitudinal section. A total 50 sections with a five-sectionsinterval from each animal were evaluated. All measure-ments were carried out with Motic Images Plus 2.0 (Hermeset al., 2008). Duodenum and jejunum tissues from threeanimals of each group were evaluated in this study.
2.7. Statistical analysis
SAS Software (SAS for Windows, Release 6.12) was usedfor statistical analysis. A one-way ANOVA was used tocompare the means between groups of more than twoand the comparison between two means of the multi-ple groups were based on the Homogeneity-of-Variance. Avalue p < 0.05 was taken to indicate a significant differencebetween groups. Data were presented as mean ± standarderror of the mean (SEM).
3. Results
3.1. Effects on the plasma glutamine concentration
The plasma Gln concentrations of the early-weanedcalves at days 0, 6, 9, 12, 15 and 18 post-weaning weremeasured in this study. The plasma Gln concentration wasdecreased during the first 10 days post-weaning, and theinfusion of various doses of Gln significantly increased theplasma Gln concentrations in these early-weaned calves(p < 0.01 and p < 0.05) (Fig. 1). The increased Gln concen-tration was not only determined on those days given Gln(Day 6 in Fig. 1), but also on the first several days after the
Gln supplementation was withdrawn (Day 9 in Fig. 1). Theplasma Gln concentration returned to the pre-weaning lev-els by 12 days post-weaning with no differences observedbetween the calves given saline control and various doses
Data were derived from an n = 9 for each group from three trials. Resultsrepresent the mean ± SEM from three independent trials with three ani-mals per group per trial.
of Gln after this time point. Increased plasma Gln con-centrations were detected in all experimental groups postday 15 of post-weaning onward in all experiment groups(Fig. 1). The supplementation of Gln did not alter plasmaconcentration of other amino acids determined on day 6of infusion, with the exception of plasma concentrations ofleucine and alanine, which were increased in the calvesgiven a dose of 1.01 g/kg × d−1 of Ala-Gln (p = 0.004 andp < 0.03, respectively, and data not shown). These resultsdemonstrate that the regime of infusion of Gln for 7 con-secutive days was able to maintain serum Gln in theearly-weaned calves at the concentration prior to weaning.
3.2. Effects on the performance of weight gain
The body weights of the calves were measured dailyfrom the day of weaning to the day that the animals wereslaughtered. As shown in Table 2, parenterally delivereda dose of 1.01 g/kg × d−1 of Ala-Gln significantly improvedthe daily weight gain performance (p < 0.05), increased theefficacy of feed (p < 0.05), and decreased the number ofdiarrheic animals. This dosage was more beneficial to theperformance of weight gain with less occurrence of diar-rhea in early-weaned calves relative to the saline, a lower(0.49 g/kg × d−1), and a higher (1.99 g/kg × d−1) dose of Ala-Gln treated animals, respectively (Table 2).
3.3. Effects on blood lymphocyte frequency
The frequency of blood lymphocyte in these calveswas evaluated by accessing the percentages of bloodT lymphocyte subpopulations for total T lymphoctyes(CD2+), T helper lymphocytes (TH cells, CD4+), and cyto-toxic/suppressor T lymphocytes (CTLs, CD8+) (Fig. 2A–C,respectively). The fraction of CD2+ (Total T lymphocytes)cells significantly increased in the blood of calves given adose of 1.01 g/kg × d−1 of Ala-Gln at day 14, relative to othergroups (p < 0.05) (Fig. 2A). Notably, calves parenterally sup-
plemented with a higher dose of Ala-Gln (1.99 g/kg × d−1)displayed a significant decline in the CD2+ cell popula-tion at 7 days post weaning (p < 0.01), which was back tonormal at 14 days after weaning (Fig. 2A). A significant
138 Y. Zhou et al. / Veterinary Immunology and Immunopathology 145 (2012) 134– 142
Table 2Effects of jugular vein infusion of Ala-Gln dipeptide on early-weaned calves (n = 9).a
Results represent the mean ± SEM for three independent trails for each condition.a The calves were treated and/or evaluated once a day from the day of weaning to the day of slaughter (total 19 days). Data represented from three
independent trials of nine total animals for each group (n = 9), and three animals were used in each group for every trail.b The body weight on the day of weaning (day 0 post-weaning).c Body weight on the day of slaughter (on day 18 post-weaning).
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d The consumption of feed (kg) against the weight gain (kg).e Diarrheic animal was defined by the occurrence of watery stool regar* Relative to the saline treated animals, p < 0.05.
ncrease in CD4+ cells was observed at 7 days (p < 0.01)nd 14 days (p < 0.05) following weaning in animals givenhe 1.01 g/kg × d−1 dose (Fig. 2B). Statistical significanthanges in the CD8+ T cell subpopulation was only observedmong the experimental groups at 7 days after the weaningp < 0.05 or 0.01) (Fig. 2C). The ratio of CD4+/CD8+ cells wasignificantly different among the groups of animals given
he dose of 1.01 g/kg × d−1 of Ala-Gln at 7 and 14 days posteaning (p < 0.01 and p < 0.05, respectively) relative to that
een in the other groups (Fig. 2D). This data demonstrateshat parenteral supplementation of certain amounts of
ig. 2. Effects of intravenous perfusion of Ala-Gln dipeptide on lymphocyte frequeaned on the forty-first day after the birth. The frequencies of peripheral CD2+ (A
arly-weaned calves were evaluated on the indicated days post weaning. Relativef day 0, ††p < 0.01; †p < 0.05. Data were derived from an n = 9 for each group fromrials with three animals per group per trial.
the severity and duration.
Ala-Gln was in favor of increasing the frequency of CD2+
and CD4+ T cells in early-weaned calves.
3.4. Effect on the concentrations of serum IgA and IgG
Similar to the effect on naïve T lymphocyte subpopu-lations, parenteral supplementation of 1.01 g/kg × d−1 of
Ala-Gln significantly increased concentrations of serum IgAin the early-weaned calves (p < 0.01) at 14 days, but not at7 days post-weaning (Fig. 3). Interestingly, no significantchanges in serum IgG concentrations were found (p > 0.05).
ency in blood of the early-weaned Holstein bull calves. The calves were), CD4+ (B), CD8+ (C) lymphocytes and the ratios of CD4+/CD8+ cells (D) in
control saline treated group, **p < 0.01; *p < 0.05. Compared to the series three trials. Results represent the mean ± SEM from three independent
Y. Zhou et al. / Veterinary Immunology and Immunopathology 145 (2012) 134– 142 139
Fig. 3. Effects of intravenous perfusion of Ala-Gln dipeptide on serum IgAand IgG levels (mg/100 mL) in the early-weaned Holstein bull calves. Thecalves were weaned on the forty-first day after the birth. The concentra-tions of immunoglobulin A (IgA, top panel) and immunoglobukin G (IgG,bottom panel) in the serum were evaluated on the indicated days postweaning. Compared to day 0 levels, **p < 0.01; *p < 0.05. Relative to thesaline control treated animals, ††p < 0.01; †p < 0.05. Data were derived from
Fig. 4. Effects of intravenous perfusion of Ala-Gln dipeptide on intesti-nal mucosal s-IgA concentrations in early-weaned Holstein bull calves(mg/mL). The animals were weaned on the forty-first day after the birth.The concentrations of intestinal mucosal secretory Immunoglobulin A(s-IgA) in different parts of intestine were determined at 18 days postweaning. Compared to the group of saline control, **p < 0.01; *p < 0.05.
an n = 9 for each group from three trials. Results represent the mean ± SEMfor three independent trials for each condition with three animals pergroup per trial. aDay(s) post-weaning.
Calves given a higher dose of Ala-Gln (1.99 g/kg × d−1) dis-played a significant decline of serum IgA (p < 0.05) and IgG(p < 0.01) concentrations at 7 and 14 days post weaningrespectively, unlike the 1.01 g/kg × d−1 dipeptide treatedgroup (Fig. 3). This was consistent with observations in theCD2+ and CD4+ T cells (Fig. 2). This result indicated thatparenteral supplementation of an appropriate amount ofAla-Gln might moderately increase the concentration ofserum immunoglobulins in early-weaned calves.
3.5. Effect on intestinal mucosal secretory IgA (s-IgA)concentration
Mucosal s-IgA plays an important role in the innateimmunity of the digestive system (Mantis et al., 2011;Ohland and Macnaughton, 2010). The s-IgA concentrationsin the ileal mucus of animals parenterally supplementedwith doses of 1.01 g/kg × d−1 and 0.49 g/kg × d−1 of Ala-Glnwere higher relative to the saline control group and thosegiven 1.99 g/kg × d−1 of Gln-Ala (p < 0.01) (Fig. 4). The con-centration of jejunal mucosal s-IgA in animals parenterallysupplemented with Ala-Gln was moderately higher in com-parison to the saline control group (p < 0.05). Furthermore,no significant change in s-IgA concentration was observed
in the duodenal mucosa (Fig. 4). This data suggests thatparenteral Ala-Gln dipeptide was capable of enhancingthe secretion of intestinal mucosal s-IgA in early-weanedcalves.
Data represent an n = 9 for each group from three trials. Results representthe mean ± SEM for three independent trials for each condition, and threeanimals were used for each group in every trial.
3.6. Effect on the intestinal integrity in early-weanedcalves
The rumen development and integrity of intestinalepithelium are important for the transition from thepre-ruminant to mature ruminant animal. Gln has beensuggested to play important roles in intestinal growth,integrity, and function (Marc Rhoads and Wu, 2009; Wanget al., 2008). Though no significant changes in intesti-nal weight and length were found between the animalsgiven Gln and saline control (p > 0.05) (Table 3), highervilli, thicker mucosal layer and more shallow crypt depthswere observed in the duodenum and jejunum of calvestreated with Gln relative to saline controls (p < 0.05 and0.01, respectively) (Table 4). The dose of 1.01 g/kg × d−1 ofGln displayed the most pronounced effects on all abovemeasured criteria relative to the other experimentallytreated groups. These data indicated that the maintenanceof required levels of plasma Gln concentrations in the early-weaned calves might be beneficial to gut development andintegrity of the intestinal epithelium.
4. Discussion
In this report, early-weaned calves given a dose of Ala-Gln dipeptide (1.01 g/kg × d−1) displayed positive effectson gain performance and increased blood CD2+ and CD4+
lymphocytes, concentrations of serum IgA and IgG, intesti-nal mucosal s-IgA and intestinal integrity, as well as thedecreasing incidence of diarrhea. To our knowledge, thiswas the first report to evaluate the effects of parenteralpeptide-bound Gln (Ala-Gln dipeptide) in early-weanedcalves. Because glutamine is presently not included incommercially available amino acid solutions, is unsta-ble in aqueous solution and forms toxic products duringheat sterilization, Ala-Gln was utilized in this study. Fur-
thermore, the dipeptide is more soluble and stable thanfree l-glutamine (Furst et al., 1997). Our results revealedthat immunophenotypic differences occurred followingparental supplementation of 1.01 g/kg × d−1 of Ala-Gln
140 Y. Zhou et al. / Veterinary Immunology and Immunopathology 145 (2012) 134– 142
Table 3Length and weight of the intestines from weaned calves infused different doses of Ala-Gln dipeptide (n = 9).
Dose (g/kg × d−1) p value
Saline 0.49 1.01 1.99
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Length (cm) 2143.3 ± 104.7 2266.0 ± 74.7
Weight (g) 1247.7 ± 51.6 1497.3 ± 83.5
ipeptide in early-weaned calves under a feeding regi-en described in this study. These differences included
n increase in levels of CD2+ and CD4+ T lymphocytes, theatio of CD4+/CD8+ fractions, the concentrations of serumgA and IgG and intestinal mucosal s-IgA. These observa-ions were consistent with that found in piglets (Jiang et al.,009; Wu et al., 1996; Yoo et al., 1997), and infants (vanen Berg et al., 2009). These findings suggest that Ala-Glnay be capable of improving naïve immune status in early-eaned calves. Nevertheless, the influence of Ala in thisla-Gln dipeptide form could not be ruled out at this time.-Alanine supplementation has been used for studies tonhance the capacity of high-intensity exercises (Hill et al.,007; Sale et al., 2011). Furthermore, when high doses ofla were used, no toxicity to liver or kidneys, or other obvi-us side effects have been reported (Lochs and Hubl, 1990),uggesting Ala-Gln may be a safe Gln supplement for these in both of humans and animals.
The major function of the innate immune response pro-ides not only a first line of defense, but also is necessaryor the development of the adaptive immune system (Tnd B cells) against infectious agents. Additionally, impair-ents to T-cell function will influence the efficiency of
ell-mediated immunity. Many amino acids including glu-amine and methionine have been reported to be requiredor lymphocyte proliferation (Chuang et al., 1990). Previoustudies have demonstrated that s-IgA was a principal medi-tor of mucosal immunity. It plays important roles in the
mmunity of the digestive and respiratory systems (Kudsk,002; Schroder et al., 1998). Low plasma and tissue Glnoncentrations are correlated with decreases in mucosal-IgA levels and s-IgA producing cells in animals resulting
able 4illus height, mucosal thickness and crypt depth of the duodenum and jejunum frm).
Intestine Dose (g/kg × d
Saline
Duode-num Villus height 72.3 ± 5.9
Mucosal thickness 80.0 ± 3.3
Crypt depth 41.1 ± 4.2
Proximal jejunum Villus height 69.4 ± 3.2
Mucosal thickness 76.7 ± 3.1
Crypt depth 32.8 ± 0.9
Middle jejunum Villus height 79.8 ± 3.7
Mucosal thickness 87.5 ± 4.5
Crypt depth 48.0 ± 1.8
Distal jejunum Villus height 77.8 ± 1.0
Mucosal thickness 84.9 ± 0.9
Crypt depth 39.6 ± 1.6
esults represent the mean ± SEM) for 150 sections from three animals per group* Compared to the group of saline, p < 0.05.
in increased bacterial translocation in the mucosal surface(Kuru et al., 2004; Newsholme and Calder, 1997). In thisstudy, we found that intestinal s-IgA, serum IgA and IgGlevels increased significantly in the animals administeredthe optimal dose of Ala-Gln.
Gln is a preferred fuel source for immune cells knownas lymphocytes and macrophages (Abcouwer, 2000; Kewet al., 1999; Yaqoob and Calder, 1997). Gln-treated ani-mals may have improved gastrointestinal defense andimmune functions against infectious agents as suggestedby reports in other species (Rogero et al., 2008a, 2008b; Wuet al., 1996). In this study, Gln demonstrated its ability toincrease secretion of intestinal mucosal s-IgA and reducethe incidence of diarrhea in the early-weaned calves.Unfortunately, the current study was not designed to testthe effects of Gln on functional immune responses againstinfectious agents. Previous study in cows parenterallygiven Gln demonstrated reduced haptoglobin (indicativeof reduced translocation of bacteria to blood) and anincreased lipopolysacharide binding protein and amyloidA (both indicatives of enhanced endotoxin removal) inthe plasma (Jafari et al., 2006). Together these findingswith our results may imply the potential usefulness ofGln for early-weaning success in calves, particularly inthe transportation of animals, significant changes of feed-ing or weather condition, and the occurrence of infectiousdisease. Although parenteral supplementation of dipep-tide forms of glutamine has broadly been used in human
healthcare, there are several limitations existing in theapplication of Gln supplementation in veterinary practice,especially for large animals like cows. (1) The dipeptideforms of Gln used in human clinics are currently expensive
om weaned calves infused various doses of Ala-Gln dipeptide (n = 3; unit:
and not practical for use in cows. (2) There is no com-mercially available form of rumen-protected glutamine.Thus it requires a parenteral delivery route for Gln sup-plementation in ruminants, and the intravenous delivery ismore time consuming and labour intensive relative to oraladministration. (3) The effect of Gln is largely dependedupon the regiment and dose given. Regardless of the abovelimitations, Gln supplementation would appear to havepotential usefulness in the nutrition of young calves atweaning.
The intestine as an organ acts in absorption of nutrientsand as the first line defense of food/water-borne infections.The immature development of the immune system andimpaired integrity of the gut barrier are main causes of mal-nutrition, retarded growth and gastrointestinal infectionin inadequately weaned animals (Norouzian et al., 2011;Roth et al., 2009; Wang et al., 2008; Wu et al., 1996). Thevillus height and crypt depth are related to the absorp-tion area, the number of mature intestinal cells and thethickness of intestinal mucosal layer are associated withthe integrity of the gut. Evidence suggests that shortervilli, thinner mucosal layer and deeper crypts within theintestinal epithelium may lead to reduced digestive andabsorptive capacities due in part to decreased number ofabsorption cells and increased number of secretory cells.Similar to previous findings in early-weaned piglets (Wanget al., 2008), early-weaned calves parenterally adminis-trated appropriate doses of Ala-Gln demonstrated highervilli and a thicker intestinal mucosal layer and reducedcrypt depth relative to control animals.
The gut as an organ has been suggested to be criti-cal in response to surgical stress and among the largestimmune organs in the body (Alverdy, 1990). In order toprotect the host from invading pathogens, the gut mustmaintain immunological function including the secretionof s-IgA (Alverdy et al., 1992). In this study, early-weanedcalves given the higher dose (1.00 g/kg × d−1) of Ala-Glndisplayed a decrease in the value of naïve immune indicesand gain performance, and an increase in the feed/gain ratioand occurrence of diarrhea. These observations differedfrom the previous findings in clinical practice that high-dose parenteral glutamine (up to 0.3 g/kg × d−1) exhibitedan advantage over low-dose enteral glutamine for criticallyill patients (Goeters et al., 2002; Wischmeyer, 2003). In vitrostudies using cell lines also suggested that concentration iscritical for immune cells to respond to Gln effect (Yaqooband Calder, 1997). TPN (Total Parenteral Nutrition) witha lower dose of Gln for an extended period of time mayresult in similar clinical outcomes to TPN with a higherdose of Gln for a short period. Furthermore, the differentGln form (l-Alanyl-Gln or Glycyl-Gln) in TPN regimens mayinfluence the effects of Gln supplementation on immunity(Mondello et al., 2010). Taken together, these studies alongwith the findings in this report underscore the importanceof dose, period and form of Gln in the outcome of the Glnsupplement. They also suggested that parenteral Ala-Glnmay be useful to improve the gain performance and health
in early-weaned calves, but the optimal dose needs to bedetermined prior to use.
In summary, intravenous administration of Ala-Glndipeptide at a dose of 1.01 g/kg × d−1 for 7 days significantly
munopathology 145 (2012) 134– 142 141
improved the performance of weight gain and intestinalintegrity, increased frequencies of naïve peripheral CD2+
and CD4+ cells, as well as the concentrations of serumand intestinal immunoglobulin in early-weaned calves.This suggested that parental Ala-Gln might be a potentialapproach to reduce the negative impacts resulting fromweaning stress in early-weaned calves. Further validationof the influence of Ala-Gln on lymphocytes and cytokineproduction following an antigen or ConA stimulation, aswell as the protection from challenge of infectious agents,are needed to justify the potential usefulness of Ala-Gln inimprovement of the immune status in the early-weanedcalves.
Conflicts of interest
The authors declare that there are no conflicts of inter-est.
Acknowledgments
This study was supported by a grant of National NaturalScience Foundation of China (31160645), State Key Lab-oratory of Animal Nutrition (2004DA125184F1014), andNatural Scientific program of Ningxia Autonomous Region(No: NZ0710).
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