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Effects of dietary quillaja saponin and curcumin on the performance and immune status of weaned piglets¹

School of Biology, University of Leeds, Leeds LS2 9JT, U.K.

	Abstract

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The objective of this study was to determine whether dietary quillaja saponin and curcumin (extract of turmeric) can modify piglet immune status and performance immediately after weaning. Piglets (n = 192) were weaned at 29 ± 0.1 d and allocated to treatment (six replicates of eight pig per treatment) accounting for weight, litter, and gender, using a 2 x 2 factorial arrangement. Factors were diets with or without (as-fed basis) quillaja saponin (750 mg/kg during wk 1, 300 mg/kg during wk 2 to 3) and with or without dietary curcumin (200 mg/kg). Diets were fed ad libitum for 20 d after weaning. Feed intake was measured daily. Piglets were weighed at weaning, d 7, 14, and 20 after weaning. On each of d 6 and 20 after weaning, eight pigs per treatment were sacrificed for blood and tissue collection. Treatment had no effect on piglet growth. The ADFI and G:F were similar for all treatments between d 0 and 14 of the trial. Between d 15 and 20, ADFI and G:F were lower in quillaja-supplemented piglets (ADFI = 621 vs. 572 g/d; G:F = 0.75 vs. 0.85; P < 0.05). Serum immunoglobulin (Ig) G, IgA, interferon-, and C-reactive protein (CRP) did not differ among treatments on d 6 after weaning. On d 20, IgG and CRP were greater (P < 0.05) in saponin-supplemented pigs (IgG = 17.5 vs. 11.4 mg/mL; CRP = 26.98 vs. 12.5 mg/mL). Small intestine villus and crypt measurements did not differ among treatments on either d 6 or 20. Saponin supplementation during the postweaning period seemed to potentiate an immune response in the weaned piglet but had a detrimental effect on the utilization of feed. Dietary curcumin had no influence on any measured aspect of pig performance or immune status.

Key Words: Curcumin • Growth • Immune response • Piglets • Quillaja

	Introduction

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Saponins are compounds found in a number of plants. Structurally they are an aglycone nucleus with one or more carbohydrate side chains. A source of saponins is the plant Quillaja saponaria. Quillaja saponins (QS) are used as vaccine adjuvants due to their ability to potentiate and modulate immune response. Oral and s.c. administration of QS with an antigen can modulate the immune response, including enhanced antibody and cytokine production (Cainelli Gebera et al., 1995; Hoshi et al., 1999). Turner et al. (2002) investigated feeding a crude Q. saponaria extract to weaned piglets challenged with Salmonella typhimurium. No differences in pig performance or serum antibody concentrations were observed with quillaja supplementation. However, the Turner study did not use QS compounds, but a crude quillaja extract likely to be 10% saponin. Additionally, such a crude extract contains tannin (an antinutritional factor) at approximately 15%; thus, it is possible that this extract was not of suitable composition or dose rate to induce a response. Another compound with immunomodulatory properties is curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), extracted from the plant Curcuma longa. South et al. (1997) found that dietary curcumin at 40 mg/kg, fed for 5 wk, enhanced plasma IgG concentrations in rats. Churchill et al. (2000) found that mice fed 1 g of curcumin/kg of feed had increased mucosal CD4+ T cells and B cells in the intestine, indicating curcumin can modulate lymphocyte mediated immune functions.

The objectives of this study were first to determine whether dietary QS or curcumin can influence the growth and feed intake of weaned piglets, and second to assess the immunomodulatory properties of these compounds. It is proposed that supplementation of piglet diets with these plant extracts will potentiate an immune response to some pathogens commonly encountered on commercial pig units, which should improve piglet health status and performance.

	Materials and Methods

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Animals and Management
This study and procedures used were carried out according to U.K. Home Office regulations on animal experimentation and were approved by the Ethical Review Committee of the University of Leeds, U.K. One hundred ninety-two piglets (62.5% Large White, 25% Landrace, 12.5% Duroc), derived from 24 litters, were weaned at 29 ± 0.1 (SEM) d of age. Piglets were weighed and ear tagged at weaning and allocated to treatment, balancing for litter, live weight, and gender (101 females and 91 males). Treatments were (as-fed basis) quillaja saponin (Acros Organics, Geel, Belgium) at 0 or 750 mg/kg of the diet (300 mg/kg during Phase 2) and curcumin (Axiss France SAS, Bellegarde-sur-Valserine, France) at 0 or 200 mg/kg of the diet, using a 2 x 2 factorial arrangement. This design aimed to determine individual and potential interactive effects of both QS and curcumin. Dose rates for quillaja were calculated based on findings of increased performance and immune modulation of previous studies (Hoshi et al., 1999; Francis et al., 2001, 2002) using an intake based on grams per kilogram of BW. Curcumin dose rates were recommended by the manufacturer based on unpublished data in other species. Dose rate of the saponin was decreased after the first week to coincide with the increasing feed intake of the piglets, so as to prevent excessive consumption of the saponin due to the known adverse effects of high saponin intake on the intestinal mucosa. Piglets were housed in groups of eight in commercial slatted flat deck pens measuring 1.99 m². Each pen represented one treatment replicate, with six pens per treatment. Piglets were weighed on d 7, 14, and 20 after weaning. For the first 7 d after weaning, piglets were fed the Phase 1 diet, and between d 8 and 20, the Phase 2 diet was offered. Diet composition is shown in Table 1. Diets contained no antibiotic growth promoters. Piglets were fed ad libitum and troughs were weighed back then topped up daily at an allocated time to determine pen feed intake. Water was available ad libitum via two nipple drinkers per pen. Environmental temperature was on a sliding scale, decreasing from 30°C at weaning to 23°C on d 20. Piglet health was monitored. Pen health scores were recorded daily using a subjective scale, and any ill health, including scouring, was noted and treated with appropriate therapeutic agents if necessary. The pig unit is positive for postweaning multisystemic wasting syndrome. Piglets were vaccinated against enzootic pneumonia at weaning. Health status of nursery and weaned piglets on the unit was regarded as low.

In addition, on each of d 6 and 20 after weaning, eight piglets were selected from each treatment (two pigs from Replicates 3 to 6 of each treatment; Replicates 1 and 2 were not sampled from to allow initial assessment of health and performance) and slaughtered for blood and tissue analyses. Piglets were matched for litter of origin and were selected as being most representative of pen performance in terms of weight gain and health.

Slaughter Procedure
Piglets were killed by i.v. administration of 200 mg/mL of pentobarbitone solution (Animalcare Ltd., York, U.K.), with Lignol local anesthetic injection (Arnolds Veterinary Products, Shrops, U.K.). Death was confirmed by exsanguinations, and blood was withdrawn from the renal artery into tubes containing no anticoagulant, and then centrifuged for 20 min at 2,500 x g. The small intestine was immediately removed, and 5-ccm sections were taken from 25, 50, and 75% sites along its length, measured accurately from the pyloric sphincter muscle. Sections were filled with, and then suspended in, 10% formalin solution. Tissue samples were then dehydrated, cut, stained with Toludeine blue (Sigma Chemical, St. Louis, MO), and mounted on slides to make measurements of intestinal morphology.

Laboratory Analyses
All assays and measurements described were carried out in duplicate. Serum immunoglobulins G and A (IgG and IgA), C-reactive protein (CRP), and interferon-(IFN-) were measured as indicators of immune activation in the piglet. The CRP as an acute-phase protein is released rapidly at the onset of infection, IFN- is released by activated T-helper cells, and measurement of the antibodies IgA and IgG indicates activation of the humoral arm of the immune system. Serum was analyzed for IgG and IgA concentrations using a quantitative sandwich ELISA protocol supplied by Bethyl Laboratories (Montgomery, TX). Antibodies were also purchased from Bethyl Laboratories. Goat anti-pig affinity-purified antibody was the coating antibody, goat anti-Pig IgG/IgA peroxidase conjugate was used as detection antibody, pig reference serum (18.2 mg/mL IgG; 0.65 mg/mL IgA) was used as the standard. A standard curve was created for each plate and a sample repeated in each assay as quality control; interassay CV was 9%. Serum CRP was measured using a solid-phase sandwich immunoassay kit purchased from Tridelta Ltd. (Bray, Ireland). The assay was validated by Tridelta Plc. (Toussaint et al., 1999). Intra- and interassay CV were 4.9 and 13.8%, respectively. The assay is specific for porcine CRP. Plates were precoated with anti-porcine CRP antibody. Following incubation with standards and samples, incubation with HRP-labeled anti-porcine CRP antibody was followed by addition of TMB enzyme substrate. Serum IFN- was measured using the Pierce endogen porcine IFN- ELISA kit (Chan and Perlstein, 1987). Plates were precoated with anti-porcine IFN- antibody. Wells were then incubated with biotinylated anti-porcine IFN- antibody reagent and streptavidin-horseradish peroxidase solution before addition of 3,3',5,5'-tetramethylbenzidene enzyme substrate. Limit of sensitivity was 2 pg/mL, with an assay range of 8 to 500 pg/mL. The assay was validated by the manufacturer, is specific for natural and recombinant porcine IFN-, and does not cross-react with other cytokines.

Statistical Analyses
Data were analyzed as a randomized complete block design using analysis for a 2 x 2 factorial arrangement of treatments, in the GLM procedure of Minitab 12.2 (Minitab Inc., Philadelphia, PA). Time was also included in the model to examine the effect of time on measured variables. Pen represented block and was included in the model. For performance data, piglet weaning weight and weaning age were used as covariates. All means presented are least squares means. Pen served as the experimental unit for statistical analyses of all data. Significance was determined at P < 0.05, and a trend was assumed at 0.05 < P >0.10.

	Results

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No interactions between QS and curcumin supplementation were observed for any measurement; therefore, only the main effects of each supplement are presented.

Piglet Performance
Dietary treatment had no effect on piglet growth (Table 2). Piglet ADFI was did not different across treatments for the first 2 wk after weaning; however, between d 15 and 20, piglets receiving a quillaja-supplemented diet consumed more feed than their counterparts (P < 0.05; Table 3). This resulted in a tendency for feed intake across the 20 d experimental period to be higher for quillaja-supplemented animals (P < 0.10). Curcumin supplementation had no effect on feed intake at any point during the trial. Additionally, G:F was influenced by quillaja supplementation during the third week after weaning, with supplemented animals tending to have a lower G:F during this period (P < 0.10; Table 3). Again, this affected overall G:F values for the 20-d period, with G:F for pigs receiving QS tending to be lower at 0.84 compared with 0.89 (P < 0.10). Curcumin had no effect on G:F at any point.

View this table: [in this window] [in a new window]   Table 4. Main effects of dietary quillaja saponin and curcumin on serum immunoglobulin G, immunoglobulin A, interferon-, and C-reactive protein on d 6 and 20 after weaning

View larger version (26K): [in this window] [in a new window]   Figure 1. Effect of dietary treatment on villus height (top panel) and crypt depth (bottom panel) in small intestine of piglets on d 6 after weaning. Measurements were taken at sites 25, 50, and 75% along the length of the small intestine, measured from the pyloric sphincter muscle. There were no treatment effects on either variable. Quillaja +/–= with or without quillaja supplementation; curcumin +/–= with or without curcumin supplementation.

View larger version (27K): [in this window] [in a new window]   Figure 2. Effect of dietary treatment on villus height (top panel) and crypt depth (bottom panel) in small intestine of piglets on d 20 after weaning. Measurements were taken at sites 25, 50, and 75% along the length of the small intestine, measured from the pyloric sphincter muscle. There were no treatment effects on either variable. Quillaja +/–= with or without quillaja supplementation; curcumin +/–= with or without curcumin supplementation.

	Discussion

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The effects of curcumin on pig performance have not been published and the current study found no effect on growth. Work by Platel and Srinivasan (1996) showed that when curcumin was fed to rats at 5 g/kg of the diet for 8 wk, pancreatic enzyme activity was significantly enhanced. This indicates that curcumin could have properties as a digestive enhancer, which potentially would improve performance, although growth was unaffected in the rat study. However, increased enzyme production may also be due to an increase in feed intake or upregulation caused by decreased digestive capacity, potentially a result of anti-nutritional factors in the diet. Thus, it is possible that curcumin may have antinutritional properties and this would be a route for further investigation. The level of curcumin fed in the present study was much lower than that used in the Platel and Srinivasan (1996) trial, and curcumin was fed for a shorter period of time. It is possible that a higher dose or a longer period of supplementation is required to elicit an effect, which may also be true for effects on immune status. South et al. (1997) found dietary curcumin at 40 mg/kg enhanced nonspecific serum IgG, but this was after 5 wk of supplementation. Additionally, the diets used in the current study were formulated to be highly digestible (although not quantified). Immune stimulation and effects on performance may therefore have been observed with a poorer-quality diet.

Supplementation with QS increased ADFI, but not until the third week of the study. Due to the lack of effect on growth, however, G:F was poorer in these animals. It is unlikely that the increase in ADFI was due to increased diet palatability, as saponins are reputed to be bitter tasting. The QS-supplemented animals may have had improved vigor (although this was not quantified) than their counterparts resulting in increased appetite and feeding activity. Increases in feed intake are desirable in weanling pigs due to the suboptimal growth rates normally observed during this period. Nonetheless, due to the changes in immune function seen in quillaja-supplemented animals on d 20 after weaning, it is most likely that these extra nutrients are being partitioned away from growth and towards the immune system to support an immune response, resulting in lower G:F (Klasing and Barnes, 1988).

The increased IgG and IgA levels of quillaja-supplemented pigs on d 20, alongside the enhanced CRP concentrations, indicate that these animals had mounted an immune response. Although not significant, it is noted that IFN- concentrations are also higher in these pigs, indicating that alongside increased antibody synthesis, a T-helper cell immune response was potentially elicited. Concentrations of IgG on d 6 were similar to those seen in unweaned pigs of a similar age from the same herd (our unpublished data). The similar blood profiles of pigs across treatments on d 6 suggest that the saponin required time to elicit any physiological effects. Antibodies take approximately 1 wk after exposure to antigen before they can be detected in the serum as a primary immune response, and subsequently remain detectable for up to 14 d; thus, it is conceivable that antibody production in response to a challenge peaked after the d-6 sampling and that the adjuvant action of the saponin prolonged production, thereby resulting in higher concentrations at the d-20 sampling time. It may be that IgM levels were higher in the quillaja group at the d-6 sampling. As IgM peaks more rapidly than IgG, it would therefore have been useful to quantify serum IgM concentrations also.

Clearly, the immune challenge the pigs met was not controlled in this study but simply consisted of piglets encountering typical commercial postweaning mixing and housing situations. This will have resulted in a low-level challenge, which may only have triggered a measurable immune response in the saponin-supplemented pigs due to the immunopotentiating properties of the saponin. These properties have clearly been demonstrated with the use of quillaja saponins as vaccine adjuvants. There was no effect of diet on subjective visual measurements of piglet health, which was generally good for all groups, as evidenced by the incidence of scouring and the numbers of piglets requiring antibiotic treatment. Thus, in this instance, it would seem that although quillaja saponin supplementation boosted piglet immune function during the initial postweaning period, it was not accompanied by improvements in performance or health, and in fact, a negative performance effect was observed.

These effects would be more clearly defined with a similar investigation using pigs weaned at an earlier age when they are more susceptible to disease, or alternatively to repeat the work of Turner et al. (2002), using a controlled pathogen challenge with a purified saponin, not a crude extract, as the dietary supplement. The current study indicates that dietary quillaja saponin has the potential to enhance piglet immune response, although in this experiment, it was at the expense of G:F. However, more work is needed to determine whether this may be of benefit in certain commercial situations. Saponins have surfactant activity and influence membrane potential, thereby increasing permeability (Chao et al., 1998). This effect can allow increased antigen uptake in the small intestine, and the combination of saponins with an antigen forms a complex for antigen presentation, which facilitates transport of the antigen to the cytosol of the antigen presenting cells (Lenarczyk et al., 2004). It seems likely that the saponin is potentiating an immune response to the normal environmental, social, and nutritional challenges the newly weaned pig encounters. The changes in the immune profiles of quillaja pigs on d 20 may, however, have been due to other unrelated factors, such as increased stress or a direct immune response to the saponin, which require further clarification. Furthermore, the exact composition of the quillaja saponins used in this study needs to be fully characterized. Unfortunately, this was not possible in this study due to lack of facilities, but this could be accomplished with the use of HPLC. Quillaja saponin preparations have been shown to contain a number of distinct saponins differing in adjuvant activity (Kensil et al., 1991). For example, one distinct fraction has been shown to display potent immunomodulatory activity by enhancing antibody production specifically toward IgG2a, whereas another enhances antigen-specific proliferation and production of IL-2 and IFN (Johansson and Lovgren-Bengtsson, 1999).

	Implications

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The results of this study indicate that dietary quillaja saponins enhance the immune function of the weanling pig in normal commercial rearing situations in the absence of any controlled pathogen challenge. Dietary saponins also increased piglet feed intake; however, no benefit to growth was observed, resulting in lower feed efficiency. The use of controlled enteric disease challenge would allow better assessment of the ability of saponin to potentiate piglet immune function and promote piglet health. Conversely, curcumin offered in the diet did not influence piglet performance or measures of immune function in the immediate postweaning period.

	Footnotes

 
¹ This work was funded by AXISS France SAS.

³ Current address: AXISS France SAS, 2 Rue des Frères Lumières, 01205 Bellegarde-sur-Valserine, France.

² Correspondence: Chance and Hunt Nutrition, Alexander House, Runcorn, Cheshire WA7 2UP, U.K. (e-mail: sian{at}ilsley50.freeserve.co.uk).

Received for publication February 25, 2004. Accepted for publication September 29, 2004.

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