Supplemental Material can be found at: http://jn.nutrition.org/content/suppl/2015/01/07/jn.114.19787 1.DCSupplemental.html
The Journal of Nutrition Nutrition and Disease
Quantitative Feed Restriction Rather Than Caloric Restriction Modulates the Immune Response of Growing Rabbits1–3 Christelle Knudsen,4–6 Sylvie Combes,4–6 Christophe Briens,7 Joe¨l Duperray,8 Gwenae¨l Rebours,9 Jean-Marc Salaun,10 Ang´elique Travel,11 Delphine Weissman,12 Thierry Gidenne,4–6* and Isabelle P Oswald13,14 4
INRA (Institut National de la Recherche Agronomique), GenPhySE (G´en´etique, Physiologie et Syste`mes dÕElevage), Castanet-Tolosan, France; Universit´e de Toulouse, INP (Institut National Polytechnique), ENSAT (Ecole Nationale Sup´erieure Agronomique de Toulouse), GenPhySE (G´en´etique, Physiologie et Syste`mes dÕElevage), Castanet-Tolosan, France; 6Universit´e de Toulouse, INP, ENVT, GenPhySE (G´en´etique, Physiologie et Syste`mes dÕElevage), Toulouse, France; 7CCPA, Janz´e, France; 8Evialis, Vannes, France; 9TECHNA, Coueron, France; 10Sanders, Bruz, France; 11ITAVI, UMT BIRD (Unit´e Mixte Technologique Biologie et Innovation pour la Recherche et le D´eveloppement en aviculture), Centre INRA de Tours, Nouzilly, France; 12 INZO, Chierry, France; 13INRA, UMR 1331, Toxalim, Toulouse, France; and 14Universit´e de Toulouse, INP, UMR 1331, Toxalim, Toulouse, France 5
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Abstract Background: Short-term feed restriction strategies are used in rabbits to reduce postweaning digestive disorders, but little is known about the involvement of the immune system in these beneficial effects. Objective: In the present study, the consequences of feed and energy restriction on immune response were investigated. Methods: At weaning, 320 male and female rabbits were assigned to 4 groups differing in dietary digestible energy (DE) concentrations and intake levels: a low-energy ad libitum–feed (LE100) group, a low-energy restricted-feed (LE75) group, a high-energy ad libitum–feed (HE100) group, and a high-energy restricted-feed (HE75) group. The high-energy groups consumed 10.13 MJ DE/kg of feed, whereas the low-energy groups consumed 9.08 MJ DE/kg (formulated values). Intake amounts for the restricted groups were 75% those of the ad libitum groups. Rabbits consumed these diets until age 63 d, after which they consumed feed ad libitum for 9 d. Ten rabbits per group and per age were killed at ages 42, 50, 63, and 72 d. Spleens and appendixes were weighed; PeyerÕs patch surface area was determined by image analysis; plasma total immunoglobulin (Ig) G and anti-ovalbumin IgG; and fecal and plasma IgA concentrations were determined by ELISA; and ileal expressions of cytokines were measured by quantitative reverse transcriptase-polymerase chain reaction at ages 50 and 63 d. Results: The relative weight and size of the lymphoid organs were not affected by treatments. Concentrations of plasma total IgA (241% at 63 d and 229% at 72 d), IgG (222% at 72 d), and anti-ovalbumin IgG (241% at 63 d) were lower with feed restriction. Fecal IgA concentrations were lower with quantitative restriction (240%, 252%, and 265% at age 42, 50, and 63 d, respectively) and energy restriction (256%, 246%, and 273% at ages 50, 63, and 72 d, respectively). Feed-restricted rabbits tended to have greater expressions of interleukin (IL) 1b and IL-2 and lower expressions of tumor necrosis factor a (P < 0.1). Conclusion: These results demonstrated that, in rabbits, restriction and, to a lesser extent, dietary energy concentration modulate gut immunity. J Nutr 2015;145:483–9. Keywords: rabbit, feed restriction, energy intake, health, immune response
Introduction Severe dietary changes often cause digestive disorders. In mammals, weaning generates important changes as the young animal 1 This project was funded by partners involved in the project, including Sanders, Techna, CCPA, Evialis, Inzo, Itavi (Institut Technique de l’Aviculture), INRA (Institut National de la Recherche Agronomique), and the CLIPP (Comite´ Lapin Interprofessionnel pour la Promotion des Produits). 2 Author disclosures: C Knudsen, S Combes, C Briens, J Duperray, G Rebours, J-M Salaun, A Travel, D Weissman, T Gidenne, and IP Oswald, no conflicts of interest. 3 Supplemental Tables 1–3 and Supplemental Figure 1 are available from the ‘‘Online supporting Material’’ link in the online posting of the article and from the same link in the online table of contents at http://jn.nutrition.org. * To whom correspondence should be addressed. E-mail: thierry.gidenne@ toulouse.inra.fr.
shifts from milk to solid feed, and is often responsible for various digestive disorders (1, 2). These disorders are particularly important in agricultural species because they result in economic losses for the breeder. Moreover, current European legislation and recommendations strongly discourage the use of antibiotics (3); thus, new alternatives are needed to preserve animalsÕ health (4). Short-term feed restriction was proven to reduce postweaning digestive disorders in rabbits and has been used by breeders in France for over 10 y as an efficient method to preserve the digestive health of growing rabbits (5). Indeed, a reduction in feed intake (below 20% of free intake) reduces postweaning mortality and morbidity (6). Previous studies in mice and rats suggested
ã 2015 American Society for Nutrition. Manuscript received June 16, 2014. Initial review completed July 11, 2014. Revision accepted December 2, 2014. First published online January 7, 2015; doi:10.3945/jn.114.197871.
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that long-term feed restriction and the nutritional value of the diet could affect gut immunity (7–9). In growing rabbits, the effect of some nutrients, such as starch and fiber, on the immune response was documented. Indeed, increased intake of fiber could improve mucosal immunity through an increased number of membranous epithelial cells in the appendix of postweaning rabbits (10). However, the involvement of the immune system in the reduction in postweaning digestive troubles through short-term feed restriction is poorly documented. Likewise, to our knowledge, the short-term effect of dietary energy concentration on the immune system has not yet been studied in agricultural species. Thus, the present study was conducted to evaluate the impact of both quantitative feed restriction and dietary digestible energy (DE)15 concentration on gut immunity in growing rabbits.
Methods
15 Abbreviations used: B2m, b2 microglobulin; DE, digestible energy; HE, high energy; HE100, high-energy ad libitum feed; HE75, high-energy restricted feed; Hprt1, hypoxanthine phosphoribosyltransferase 1; LE, low energy; LE100, lowenergy ad libitum feed; LE75, low-energy restricted feed.
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of digestive disorders, such as diarrhea, cecal impaction, and suspicion of epizootic rabbit enteropathy, or other pathologies such as respiratory problems and injuries. Rabbits without clinical signs of illness but showing weight loss or very low growth (3 SD below the mean) were considered morbid and excluded from the study. Ovalbumin immunization. At age 37 d, 32 rabbits per group were immunized with a 1 mL subcutaneous injection of 600 mg ovalbumin (Sigma-Aldrich) in incomplete FreundÕs adjuvant (Sigma) and challenged 9 d later (age 46 d) with 300 mg ovalbumin in incomplete FreundÕs adjuvant. The weights (means and SDs) at age 35 d of the rabbits selected for immunization and those of the remaining rabbits were equivalent in order to have a representative sample of the study rabbits. Killings and samplings. Ten healthy rabbits per treatment were killed at ages 42, 50, 63, and 72 d. All rabbits killed at ages 63 and 72 d had been immunized with ovalbumin, whereas those killed at ages 42 and 50 d had not been immunized. The weights (means and SDs) of the rabbits selected for sampling and those of the remaining rabbits were equivalent in order to have a representative sample of the study rabbits. Rabbits were killed by electrical stunning followed by exsanguination. Blood was collected from the aorta in heparinized tubes (Vacuette 9 mL sodium heparin; Greiner Bio-One) and immediately stored on ice. After centrifugation (1000 3 g for 10 min at 4°C), plasma samples were stored at 220°C until further analysis. Lymphoid organs (spleen and vermiform appendix) were collected and weighed individually. Feces were collected from the rectums of the rabbits killed and transferred to dry tubes on ice until arrival at the laboratory, where the samples were stored at 220°C until further analysis. A 5 cm-long section of the ileum was collected 10 cm from the ileocecal junction, snap frozen in liquid nitrogen, and stored at 280°C until analysis. The first PeyerÕs patch encountered from the ileocecal junction was isolated, the ileum was cut up, and a picture was taken of the patch next to a ruler. Images were analyzed through the use of ImageJ software (14) to evaluate the surface of the patches. Total and anti-ovalbumin IgG ELISA measurements. Concentrations of plasma immunoglobulin G (IgG) were measured by ELISA as described previously (15). Goat anti-rabbit IgG (Fc fragment–specific) was used as a capture antibody (Bethyl Laboratories) and HRP-labeled goat anti-rabbit IgG was used as a detection antibody in conjunction with a tetramethylbenzidine substrate (1:1 H2O2:tetramethylbenzidine) (Thermo Fisher Scientific). The OD of each well was read at 540 nm and subtracted from the readings at 450 nm (Infinite M200, Tecan) to correct for optical imperfections in the plates. The mean OD of each sample was then calculated, and the mean value of the negative control was subtracted from all sample values. IgG concentrations were then obtained from the standard curve constructed with the OD values of an IgG standard solution. Concentrations of specific anti-ovalbumin IgG were also measured by ELISA (16). Briefly, ELISA plates were coated with ovalbumin diluted
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Study design, rabbits, housing, and feed. The study was conducted at the INRA (Institut National de la Recherche Agronomique) UE (Unit´e Exp´erimentale) PECTOUL (Poˆle Exp´erimental Cunicole de Toulouse) breeding unit in Castanet-Tolosan, France, on 320 healthy male and female hybrid rabbits (Oryctolagus cuniculus) at a 50:50 male-to-female ratio. A bifactorial design was used with 2 levels of feed intake—ad libitum feed consumption vs. restricted feeding at 75% of ad libitum— and 2 diets differing in DE concentration—a low-energy (LE) diet, formulated with 9.08 MJ DE/kg, and a high-energy (HE) diet, formulated with 10.13 MJ DE/kg according to the European Group on Rabbit Nutrition tables from Maertens et al. (11). The rabbits were assigned to 4 treatment groups: a low-energy ad libitum–feed (LE100) group, a lowenergy restricted-feed (LE75) group, a high-energy ad libitum–feed (HE100) group, and a high-energy restricted-feed (HE75) group. The diets were formulated to meet the nutritional requirements of growing rabbits (12), without drug supplementation (antibiotics or coccidiostatics). Special attention was given to obtaining a theoretic deviation of 1 MJ/kg of DE between the diets, with the constraint of obtaining similar ratios of digestible fibers, starch, and fat to DE, starch to digestible fibers, and digestible proteins to DE. Likewise, both diets were formulated with equivalent concentrations of acid detergent fiber and neutral detergent fiber (Supplemental Table 1). The feeds were manufactured and pelleted with the use of one batch of raw materials by Euronutrition. The rabbits were housed in collective cages of 5 rabbits with a density of 15 rabbits/m2 in a closed unit in which the environment (temperature, lighting, and ventilation) was monitored and controlled. A total of 320 male and female rabbits were randomly assigned at weaning (age 35 d) to 1 of the 4 treatments. Thus, mean weights were similar between treatments and litters were split to avoid any maternal effect. The HE100 and LE100 groups were then fed freely, and the LE75 and HE75 groups were given a restricted diet for 4 wk, until age 63 d. Afterwards, all 4 groups were fed freely until the end of the study at age 72 d. Moreover, 32 rabbits per group were immunized with ovalbumin as described below (Figure 1). The amounts of feed distributed to the restricted groups were calculated on the basis of a theoretic ad libitum ingestion curve and readjusted for each diet according to the real ingestion of the ad libitum–feed groups (LE100 and HE100) for periods of 3–4 d. Feed was given daily in a single distribution between 0800 and 0830, and water was consumed ad libitum. Rabbits were handled according to recommendations for the care of experimental animals in accordance with French national legislation (13). Rabbits were weighed individually at weaning (age 35 d), after 1 wk of feed restriction (age 42 d), during the restriction period (age 50 d), at the end of the restriction period (age 63 d), and at the end of the study, after 9 d of ad libitum feed consumption by all 4 groups (age 72 d). The health status of the rabbits was assessed by monitoring for clinical signs
FIGURE 1 Study design. AL, ad libitum feeding; BW, body weight; FR, feed restriction; HE100, high-energy ad libitum–feed group; HE75, highenergy restricted-feed group; LE100, low-energy ad libitum–feed group; LE75, low-energy restricted-feed group; OVA, ovalbumin immunization.
in carbonate buffer (4 g/L NaHCO3 0.1 mol/L; pH 9.6) and incubated overnight at 4°C. Diluted plasma samples were then added to the plates, and the anti-ovalbumin antibodies were detected with HRP-labeled antirabbit IgG (Bethyl Laboratories). Plasma samples were quantified by reference to standard curves constructed with hyperimmune rabbit serum. Fecal IgA extractions and IgA ELISA measurements. A small amount of fecal matter (200–600 mg) was diluted to 50 g/L in cold PBS. The samples were dispersed for 10 s on ice (T25 Ultra-Turax, IKA Labortechnik) and centrifuged at 3000 3 g for 10 min at 4°C. The supernatants were then collected and stored at 220°C until analysis. Concentrations of plasma and fecal IgA were quantified by ELISA through the use of a specific polyclonal goat anti-rabbit IgA antibody (Bethyl Laboratories). Sample relative IgA concentrations were obtained from the standard curve constructed with the OD values of a diluted reference serum (Bethyl Laboratories).
Calculations and statistical analysis. The DE contents of the diets were obtained from digestibility measurements presented in a companion paper (19). Mean DE intakes were then estimated for each treatment at each age as the mean gross intake per rabbit in the sampled cages, from weaning to killing, multiplied by the DE content of the diet for each treatment. Mean fecal IgA concentrations were plotted according to the mean DE intake at ages 42, 50, and 63 d. Because the feed-restricted rabbits weighed less than the rabbits consuming feed ad libitum, the weights of the organs (spleen and appendix) were expressed relative to body weight to correct for body size. All measurements were analyzed with the use of the GLM procedure (SAS). To account for unequal variances, logarithmic transformation of the data was performed when necessary. In each age group, relative organ weights, PeyerÕs patch surface areas, and antibody concentrations were tested for the effects of feed intake level, the diet, and their interactions as fixed effects. Body weight was added as a covariable for the analysis of PeyerÕs patch surface areas. The effect of age was analyzed for each treatment with the use of age as a fixed effect. Finally, the model for cytokine measurements used the feed intake level, diet, age, and all interactions between these factors as fixed effects. Mean comparisons were performed with the use of TukeyÕs HSD (Honestly Significant Difference) procedure. Differences were considered significant at P < 0.05, whereas differences in which 0.05 < P < 0.1 were considered tendencies, and only differences in which P < 0.1 are shown in the figures. Values in the text, tables, and figures are means 6 SEMs. Tendencies were discussed only for cytokine expression.
Total and anti-ovalbumin plasma IgG concentrations were lower with quantitative feed restriction after 4 wk of treatment. The main objective of this study was to assess the effects of feed intake level and dietary energy concentration on the immune response. Therefore, the systemic immune response, assessed by plasma IgG concentrations, was studied. Plasma total IgG concentrations were not affected during feed restriction (Figure 4). However, after returning to ad libitum feed consumption, rabbits that previously were restricted had lower IgG concentrations than rabbits that previously consumed feed ad libitum (222%, P < 0.01). Concerning the effect of dietary energy concentration, after 1 wk of the study, IgG concentrations were higher in rabbits fed the HE diet than in those fed the LE diet (+23%, P < 0.05), but at all subsequent time points (50, 63, and 72 d), the diet energy concentration did not affect plasma IgG concentrations (Figure 4). The ovalbumin immunization protocol allowed us to investigate the effects of feed intake level and dietary energy concentration on
Results Body weight and PeyerÕs patch surface area was lower with quantitative feed restriction without affecting relative spleen and appendix weight. The feed intake of the rabbits consuming feed ad libitum averaged 135 g/d from age 35 to 63 d. The feed intake of the rabbits consuming the restricted diets was
FIGURE 2 Effect of feed intake level and dietary DE concentration on the growth of rabbits from age 35 to 72 d. Values are means 6 SEMs, n = 10. DE, digestible energy; HE100, high-energy ad libitum–feed group; HE75, high-energy restricted-feed group; LE100, low-energy ad libitum–feed group; LE75, low-energy restricted-feed group. Feed restriction modulates rabbit immunity
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Expression of mRNA encoding for cytokines by real-time PCR. Tissue RNA of 50- and 63-d-old rabbits was processed as previously described (17). Concentrations, integrity, and quality of RNA were determined spectrophotometrically (OD 260) through the use of the Nanodrop ND1000 (Labtech International). The sequences of primers used in the PCR for Il1b, Il2, Il8, Il10 and Tnfa are detailed in Supplemental Table 2 and were purchased from Sigma. Real-time qPCR was performed in 384 well plates with the use of SYBR Green as the reporter. qRT-PCR data were expressed as 2(2DDCt) and normalized to a housekeeping gene. b2 microglobulin (B2m) was chosen among 3 other candidate reference genes [Gapdh, hypoxanthine phosphoribosyltransferase 1 (Hprt1), and cyclophilin A (Ppia)] because its expression was not affected by our experimental treatments or age, and it was thus considered to be a valid reference. Amplification efficiency and initial fluorescence were determined by using the same method as for real-time PCR (18). Finally, gene expression was expressed relative to the LE100 group. The specificity of qRT-PCR products was assessed at the end of the reactions by analyzing dissociation curves.
consistent with the level of 75% initially planned (75% and 74% for the LE and the HE diets, respectively, from age 35 to 63 d). As expected, growth rate was lower with feed restriction, leading to lower weight in the rabbits for which feed was restricted starting at age 42 d, whereas diet energy concentration did not affect growth (Figure 2). The relative weights of each organ were not affected by the experimental treatments during the feed restriction period. However, the relative weight of the appendix was greater in rabbits fed the LE diet after 1 wk of ad libitum feed consumption at age 72 d (P < 0.05, 0.32% 6 0.01% compared with 0.28% 6 0.01%, data not shown). The surface area of the PeyerÕs patches was not affected by the experimental treatments at age 42 and 50 d but was lower with feed restriction at age 63 d (P < 0.01, 20.2 cm2). After 1 wk of ad libitum feed consumption, the surface area of the PeyerÕs patches of the rabbits for which feed previously was restricted was smaller than that of rabbits that consumed feed ad libitum throughout the study (P < 0.05, 20.2 cm2) (Figure 3). However, when the body weight of the rabbits was added as a covariable in our model, no effect from our treatments was detectable, regardless of age. In conclusion, body weight was lower with feed restriction, but the growth of secondary lymphoid organs (spleen and appendix) and the surface area of the PeyerÕs patches were not affected by feeding level, whereas dietary energy concentration only had a moderate effect on the growth of the appendix.
antigen-specific immunity. Plasma anti-ovalbumin IgG concentrations were lower with feed restriction at age 63 d (241%, P < 0.05) (Figure 5), but after 1 wk of ad libitum feed consumption, previous feeding levels did not affect specific antibody concentrations. Diet energy concentration did not affect anti-ovalbumin IgG concentrations in any of the age groups. Taken together, these results demonstrate that feed restriction and dietary energy concentration had a limited effect on plasma total IgG concentrations, but feed restriction greatly affected treatment-specific anti-ovalbumin IgG production after 4 wk of treatment. Quantitative feed restriction reduced fecal and plasma IgA concentrations to a greater extent than did energy restriction. Another aim of this study was to assess the effect of feed intake level and dietary energy concentration on the local immune response in the digestive tract through measurements of fecal IgA concentrations. Fecal IgA concentrations were lower during feed restriction (240% at age 42 d, P < 0.05; 252% at age 50 d, P < 0.001; and 265% at age 63 d, P < 0.001) (Figure 6A). When rabbits returned to ad libitum feed consumption, no effect from the previous feeding levels was observed. Fecal IgA concentrations were higher in rabbits fed the HE diet than in those fed the LE diet from age 50 d onward (+56% at age 50 d, P < 0.01; +46% at age 63 d, P < 0.05; and +73% at age 72 d, P < 0.05).
FIGURE 5 Effect of feed intake level and dietary DE concentration on plasma anti-OVA IgG concentrations at ages 63 and 72 d in growing rabbits. Values are means 6 SEMs, n = 10. DE, digestible energy; HE100, high-energy ad libitum–feed group; HE75, high-energy restricted-feed group; LE100, low-energy ad libitum–feed group; LE75, low-energy restricted-feed group; OVA, ovalbumin.
When we plotted the mean fecal IgA concentrations against the mean DE intake per treatment, a linear relation between these variables was observed at ages 50 and 63 d but not at age 42 d (Figure 7). To compliment the fecal analyses, plasma measurements of IgA concentrations were performed. Plasma IgA concentrations were lower with feed restriction after 4 wk of treatment (age 63 d, 241%, P < 0.001) and after 1 wk of ad libitum feed consumption (age 72 d, 229%, P < 0.05). Diet energy concentration did not affect plasma IgA concentrations (Figure 6B). These results indicate that fecal IgA concentrations were lower with limited feed intake and reduced dietary energy concentration, and plasma IgA concentrations were also, to a lesser extent, lower with feed restriction. Quantitative feed restriction tended to modify cytokine expression in the ileum. Cytokines play a key role in regulating immunity, and more specifically, inflammation. Expressions of IL-1b and IL-2 were greater with feed restriction (+30% and +77%, respectively, 0.05 # P < 0.1), whereas those of TNF-a were lower with feed restriction (215%, 0.05#P < 0.1). Feed restriction did not affect the expression of IL-8 and IL-10 (Table 1). Immunologic values were affected by age. The relative weight of the spleen decreased with age (P < 0.01) for all groups, whereas the relative weight of the appendix increased with age until age 63 d and decreased afterwards for all groups except LE75, for which age did not affect the relative weight of the appendix (Supplemental Table 3). The surface area of the PeyerÕs patches moderately increased with age for all groups (P < 0.01) (Supplemental Table 3). Fecal IgA concentrations increased with age (P < 0.01) for the HE100 and HE75 groups but were unaffected by age for the LE100 and LE75 groups (Supplemental Figure 1A). Plasma total IgG and IgA concentrations increased with age regardless of study group (P < 0.001) (Supplemental Figure 1B and C).
FIGURE 4 Effect of feed intake level and dietary DE concentration on plasma total IgG concentrations from age 42 to 72 d in growing rabbits. Values are means 6 SEMs, n = 10 per treatment and per age. For an age, labeled means without a common letter differ, P , 0.05. DE, digestible energy; HE100, high-energy ad libitum–feed group; HE75, high-energy restricted-feed group; LE100, low-energy ad libitum–feed group; LE75, low-energy restricted-feed group. 486
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Discussion Dietary strategies influence animal health. The present study aimed to analyze the effects of quantitative feed restriction and dietary energy concentration on the immune response of
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FIGURE 3 Effect of feed intake level and dietary DE concentration on the development of PeyerÕs patches from age 42 to 72 d in growing rabbits. Values are means 6 SEMs, n = 10. For an age, labeled means without a common letter differ, P , 0.05. DE, digestible energy; HE100, high-energy ad libitum–feed group; HE75, high-energy restricted-feed group; LE100, low-energy ad libitum–feed group; LE75, low-energy restricted-feed group.
growing rabbits. We demonstrated that feed restriction rather than dietary energy concentration is responsible. The spleen, the appendix, and PeyerÕs patches are major secondary lymphoid organs. As previously demonstrated in mice (20), feed restriction did not affect the relative weight of the spleen or the appendix. Dietary energy concentration had only a very moderate effect on the relative weight of the appendix at the
FIGURE 7 Fecal IgA concentrations per treatment according to the mean digestible energy intake at ages 50 and 63 d in growing rabbits. Values are means 6 SEMs, n = 10. HE100, high-energy ad libitum– feed group; HE75, high-energy restricted-feed group; LE100, lowenergy ad libitum–feed group; LE75, low-energy restricted-feed group.
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FIGURE 6 Effect of feed intake level and dietary DE concentration on fecal (A) and plasma (B) IgA concentrations from age 42 to 72 d in growing rabbits. Values are means 6 SEMs, n = 10. For an age, labeled means without a common letter differ, P , 0.05. DE, digestible energy; HE100, high-energy ad libitum–feed group; HE75, high-energy restricted-feed group; LE100, low-energy ad libitum–feed group; LE75, low-energy restricted-feed group.
end of this study. Similarly, the smaller surface area of the PeyerÕs patches in the rabbits consuming a restricted diet may be correlated to the lower weight of these rabbits. Feed restriction and energy restriction therefore did not impair growth of the secondary lymphoid organs. Similar results were reported by Rogers et al. (20), who showed that a 30% reduction in feed intake did not affect the number of PeyerÕs patches or their number of cells. However, Kubo et al. (21) demonstrated that 50% feed restriction reduced the number of cells in the spleens of growing mice. Thus, immune activity might be compromised by feed restriction despite the lack of effect on the physiologic variables of the secondary lymphoid organs measured. Concerning the adaptive systemic immune response, feed restriction affected total IgG concentrations only after its application. A similar delayed dietary effect was reported by Zhu et al. (10) in growing rabbits fed diets differing in starch and fiber concentrations. As for the effects of feed restriction on the immune response to a specific antigen, the response was much more prompt: specific anti-ovalbumin concentrations were considerably lower after 4 wk of feed restriction. Similar results were obtained by Martin et al. (22) in 30% feed–restricted deer mice submitted to a keyhole limpet hemocyanin challenge. These findings could indicate that immunologic memory is an energetically costly process, which is negatively affected by feed restriction. Another hypothesis could be that antibody production is indirectly lowered through variations in nutrient or metabolite signals caused by reduced feed intake. Surprisingly, previous feeding level had no effect on the specific response to ovalbumin after 1 wk of ad libitum feed consumption. This could indicate that the specific immune response is readjusted very quickly after a change in the amount consumed. The present study evaluated the effect of feed intake level and dietary energy concentration on the local immune response through fecal and plasma IgA concentrations and ileal cytokine expressions. Fecal IgA concentrations were lower with feed restriction in growing rabbits, confirming the results obtained in intestinal fluids and submandibular gland cultures in mice subjected to long-term feed restriction (8, 23). However, to our knowledge, this is the first study to find effects of short-term feed restriction on fecal IgA concentrations. As we published previously (19), DE intake was greater with the HE diet and ranked in the following order: LE75 < HE75 < LE100 < HE100. In this study a linear relation between DE intake and fecal IgA concentrations was found. We could therefore hypothesize that fecal IgA concentration might be regulated by DE intake rather than the quantity of feed ingested. However, the regression analyses were based on the mean energy intake of a sample of rabbits and not on individual measurements. Thus, additional studies would be necessary to confirm our hypothesis. Muthukumar et al. (23) showed that a 40% reduction in feed intake resulted in lower submandibular gland IgA concentrations and attenuated the onset of diseases in autoimmune-prone mice, indicating a possible protective effect from a reduction in IgA concentrations. The mycotoxin deoxynivalenol also induces an increase in IgA levels in mice (24) and is associated with intestinal lesions in pigs (25). Thus, the lower fecal IgA concentrations could be related to the lower incidence of digestive disorders observed in rabbits consuming the restricted diet and detailed in our companion paper (19). In addition, our results showed that plasma IgA concentrations were lower with feed restriction after 4 wk of treatment. The change in plasma IgA concentrations lagged behind the one occurring at the fecal level (age 63 compared with 42 d), suggesting that the immune response in peripheral blood occurs subsequent to the response in the digestive tract. This hypothesis is consistent with the results obtained in plasma IgG concentrations in our study and the study by Zhu et al. (10).
TABLE 1 Effect of feed intake level and dietary DE concentration on cytokine relative expressions in the ilea of growing rabbits between ages 50 and 63 d1 50 d
IL-1b TNF-a IL-8 IL-2 IL-10
P values2
63 d
LE100
LE75
HE100
HE75
LE100
LE75
HE100
HE75
Pooled SEM
Age
Diet
Intake level
1.05 1.09 1.18 1.26 0.89
1.77 0.73 0.88 3.79 1.57
1.18 0.95 1.01 1.67 0.95
1.47 0.84 1.06 2.41 0.93
1.02 1.03 1.04 1.11 1.24
1.21 0.86 1.24 1.96 1.31
1.10 0.93 1.13 1.56 1.35
1.24 0.96 1.56 1.80 1.29
0.08 0.04 0.07 0.30 0.10
0.38 0.59 0.16 0.87 0.30
0.97 0.91 0.49 0.57 0.10
0.053 0.07 0.52 0.07 0.65
Values are means and pooled SEMs, n = 10. DE, digestible energy; HE100, high-energy ad libitum–feed group; HE75, high-energy restricted-feed group; LE100, low-energy ad libitum–feed group; LE75, low-energy restricted-feed group. 2 All interactions between factors were nonsignificant. 1
Acknowledgments We thank P Aymard, E Balmisse, and J-M Bonnemere from the INRA (Institut National de la Recherche Agronomique) UE (Unit´e Exp´erimentale) PECTOUL (Poˆle Exp´erimental Cunicole de Toulouse) for rabbit handling and care during the study. We also thank J Laffitte, AM Cossalter, and A Pierron from the TOXALIM (Toxicologie Alimentaire) unit and Y Lippi from the GeT-TRIX (G´enome et Transcriptome-Impact Transcriptomique des X´enobiotiques) platform for their technical support in ELISA protocols and qRT-PCR analyses. CK, CB, JD, GR, J-MS, AT, DW, and TG designed the research. CK, SC, and TG conducted the research. IPO provided the immunologic reagents and materials. CK performed the statistical analysis. CK, SC, TG, and IPO wrote the paper. TG had primary responsibility for final content. All authors read and approved the final manuscript. 488
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Lalles JP, Boudry G, Favier C, Le Floc’h N, Lurona I, Montagne L, Oswald IP, Pie S, Piel C, Seve B. Gut function and dysfunction in young pigs: physiology. Anim Res 2004;53:301–16. Fortun-Lamothe L, Boullier S. A review on the interactions between gut microflora and digestive mucosal immunity. Possible ways to improve the health of rabbits. Livest Sci 2007;107:1–18. Landers TF, Cohen B, Wittum TE, Larson EL. A review of antibiotic use in food animals: perspective, policy, and potential. Public Health Rep 2012;127:4–22. Gallois M, Rothkotter HJ, Bailey M, Stokes CR, Oswald IP. Natural alternatives to in-feed antibiotics in pig production: can immunomodulators play a role? Animal 2009;3:1644–61. Gidenne T, Combes S, Fortun-Lamothe L. Feed intake limitation strategies for the growing rabbit: effect on feeding behaviour, welfare, performance, digestive physiology and health: a review. Animal 2012;6:1407–19. Gidenne T, Combes S, Feugier A, Jehl N, Arveux P, Boisot P, Briens C, Corrent E, Fortune H, Montessuy S, et al. Feed restriction strategy in the growing rabbit. 2. Impact on digestive health, growth and carcass characteristics. Animal 2009;3:509–15. McGee DW, McMurray DN. Protein malnutrition reduces the IgA immuno response to oral antigen by altering B-cell and suppressor T-cell functions. Immunology 1988;64:697–702. Lara-Padilla E, Campos-Rodriguez R, Jarillo-Luna A, Reyna-Garfias H, Rivera-Aguilar V, Miliar A, de la Rosa FJB, Navas P, Lopez-Lluch G. Caloric restriction reduces IgA levels and modifies cytokine mRNA expression in mouse small intestine. J Nutr Biochem 2011;22:560–6. P´erez-Berezo T, Franch A, Ramos-Romero S, Castellote C, Perez-Cano FJ, Castell M. Cocoa-enriched diets modulate intestinal and systemic humoral immune response in young adult rats. Mol Nutr Food Res 2011;55:S56–66. Zhu YL, Wang CY, Wang XP, Li B, Sun LZ, Li FC. Effects of dietary fiber and starch levels on the non-specific immune response of growing rabbits. Livest Sci 2013;155:285–93. Maertens L, Perez JM, Villamide M, Cervera C, Gidenne T, Xiccato G. Nutritive value of raw materials for rabbits: EGRAN tables 2002. World Rabbit Sci 2002;10:157–66. De Blas C, Mateos GG. Feed formulation. In: De Blas C, Wiseman J, editors. Nutrition of the rabbit. Wallingford (United Kingdom): CABI, 2010:222–32. Journal Officiel de la R´epublique Franc¸aise 0278. Arreˆt´e du 30 novembre 2009 modifiant l’arreˆt´e du 19 avril 1988 fixant les conditions d’attribution de l’autorisation de pratiquer des exp´eriences sur les animaux. 2009:20652. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods 2012;9:671–5. Accensi F, Pinton P, Callu P, Abella-Bourges N, Guelfi JF, Grosjean F, Oswald IP. Ingestion of low doses of deoxynivalenol does not affect hematological, biochemical, or immune responses of piglets. J Anim Sci 2006;84:1935–42. Meissonnier GM, Laffitte J, Raymond I, Benoit E, Cossalter AM, Pinton P, Bertin G, Oswald IP, Galtier P. Subclinical doses of T-2 toxin impair acquired immune response and liver cytochrome P450 in pigs. Toxicology 2008;247:46–54. Grenier B, Loureiro-Bracarense AP, Lucioli J, Pacheco GD, Cossalter AM, Moll WD, Schatzmayr G, Oswald IP. Individual and combined effects of subclinical doses of deoxynivalenol and fumonisins in piglets. Mol Nutr Food Res 2011;55:761–71.
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The results for cytokine expressions suggested that feed restriction tended to regulate the proinflammatory response (IL-1b and TNF-a) and increase the expression of IL-2. In contrast, LaraPadilla et al. (8) found greater expressions of TNF-a and IL-10 and lower expression of IL-2 in the duodenum of mice feeddeprived on alternate days for 17 wk, whereas Kurki et al. (26) recently found that long-term feed restriction at 70% of ad libitum intake in lean mice increased the protein expression of IL-2, IL-1b, IL-10, and TNF-a in adipose tissues. This suggests that either the cytokines are differentially expressed in the ileum, or the immune response is dependent on the duration and type of feed restriction. Finally, IL-2 and TNF-a regulate the synthesis of IgA (27); thus, the potential differential expression in these 2 cytokines could be related to the reduced IgA concentrations observed with feed restriction. However, this hypothesis remains to be confirmed, because the mechanisms by which cytokines regulate the production of IgA are complex. In conclusion, the present study demonstrated the impact of short-term feed restriction on the systemic and local digestive immunity of growing rabbits. Fecal IgA concentrations rapidly decreased with both feed restriction and energy restriction, whereas the effects on the systemic immune response (plasma IgG and IgA concentrations) lagged behind. Thus, the local immune response was modulated at an earlier stage than the systemic immune response. The innate immune response seemed less affected by our treatments because cytokine expressions in the ileum only tended to be modulated by quantitative feed restriction. Taken together, our results demonstrated that feed restriction and, to a lesser extent, the dietary energy concentration of the feed modulated gut immunity. However, it is not known whether these variables are associated with the beneficial effects of feed restriction on postweaning digestive health in rabbits.
18. Peirson SN, Butler JN, Foster RG. Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis. Nucleic Acids Res 2003;31:e73. 19. Knudsen C, Combes S, Briens C, Coutelet G, Duperray J, Rebours G, Salaun J-M, Travel A, Weissman D, Gidenne T. Increasing the digestible energy intake under a restriction strategy improves the feed conversion ratio of the growing rabbit without negatively impacting the health status. Livest Sci 2014;169:96–105. 20. Rogers CJ, Berrigan D, Zaharoff DA, Hance KW, Patel AC, Perkins SN, Schlom J, Greiner JW, Hursting SD. Energy restriction and exercise differentially enhance components of systemic and mucosal immunity in mice. J Nutr 2008;138:115–22. 21. Kubo C, Day NK, Good RA. Influence of early or late dietary restriction on life-span and immunological parameters in MRL/Mp-lpr/lpr mice. Proc Natl Acad Sci USA 1984;81:5831–5. 22. Martin LB, Navara KJ, Weil ZM, Nelson RJ. Immunological memory is compromised by food restriction in deer mice Peromyscus maniculatus. Am J Physiol Regul Integr Comp Physiol 2007;292:R316–20.
23. Muthukumar AR, Jolly CA, Zaman K, Fernandes G. Calorie restriction decreases proinflammatory cytokines and polymeric Ig receptor expression in the submandibular glands of autoimmune prone (NZB X NZW) F-1 mice. J Clin Immunol 2000;20:354–61. 24. Pestka JJ. Deoxynivalenol-induced IgA production and IgA nephropathyaberrant mucosal immune response with systemic repercussions. Toxicol Lett 2003;140–141:287–95. 25. Bracarense A-PFL, Lucioli J, Grenier B, Pacheco GD, Moll W-D, Schatzmayr G, Oswald IP. Chronic ingestion of deoxynivalenol and fumonisin, alone or in interaction, induces morphological and immunological changes in the intestine of piglets. Br J Nutr 2012;107:1776–86. 26. Kurki E, Jin S, Martonen E, Finckenberg P, Mervaala E. Distinct effects of calorie restriction on adipose tissue cytokine and angiogenesis profiles in obese and lean mice. Nutr Metab (Lond) 2012;9. 27. Lebman DA, Coffman RL. Cytokines in the mucosal immune system. In: Ogra PL, Lamm ME, McGhee J, Mestecky J, Strober W, Bienenstock J, editors. Handbook of mucosal immunology. San Diego: Academic Press, 1994:243–9.
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Feed restriction modulates rabbit immunity
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The Journal of Nutrition Nutrition and Disease
Quantitative Feed Restriction Rather Than Caloric Restriction Modulates the Immune Response of Growing Rabbits1–3 Christelle Knudsen,4–6 Sylvie Combes,4–6 Christophe Briens,7 Joe¨l Duperray,8 Gwenae¨l Rebours,9 Jean-Marc Salaun,10 Ang´elique Travel,11 Delphine Weissman,12 Thierry Gidenne,4–6* and Isabelle P Oswald13,14 4
INRA (Institut National de la Recherche Agronomique), GenPhySE (G´en´etique, Physiologie et Syste`mes dÕElevage), Castanet-Tolosan, France; Universit´e de Toulouse, INP (Institut National Polytechnique), ENSAT (Ecole Nationale Sup´erieure Agronomique de Toulouse), GenPhySE (G´en´etique, Physiologie et Syste`mes dÕElevage), Castanet-Tolosan, France; 6Universit´e de Toulouse, INP, ENVT, GenPhySE (G´en´etique, Physiologie et Syste`mes dÕElevage), Toulouse, France; 7CCPA, Janz´e, France; 8Evialis, Vannes, France; 9TECHNA, Coueron, France; 10Sanders, Bruz, France; 11ITAVI, UMT BIRD (Unit´e Mixte Technologique Biologie et Innovation pour la Recherche et le D´eveloppement en aviculture), Centre INRA de Tours, Nouzilly, France; 12 INZO, Chierry, France; 13INRA, UMR 1331, Toxalim, Toulouse, France; and 14Universit´e de Toulouse, INP, UMR 1331, Toxalim, Toulouse, France 5
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Abstract Background: Short-term feed restriction strategies are used in rabbits to reduce postweaning digestive disorders, but little is known about the involvement of the immune system in these beneficial effects. Objective: In the present study, the consequences of feed and energy restriction on immune response were investigated. Methods: At weaning, 320 male and female rabbits were assigned to 4 groups differing in dietary digestible energy (DE) concentrations and intake levels: a low-energy ad libitum–feed (LE100) group, a low-energy restricted-feed (LE75) group, a high-energy ad libitum–feed (HE100) group, and a high-energy restricted-feed (HE75) group. The high-energy groups consumed 10.13 MJ DE/kg of feed, whereas the low-energy groups consumed 9.08 MJ DE/kg (formulated values). Intake amounts for the restricted groups were 75% those of the ad libitum groups. Rabbits consumed these diets until age 63 d, after which they consumed feed ad libitum for 9 d. Ten rabbits per group and per age were killed at ages 42, 50, 63, and 72 d. Spleens and appendixes were weighed; PeyerÕs patch surface area was determined by image analysis; plasma total immunoglobulin (Ig) G and anti-ovalbumin IgG; and fecal and plasma IgA concentrations were determined by ELISA; and ileal expressions of cytokines were measured by quantitative reverse transcriptase-polymerase chain reaction at ages 50 and 63 d. Results: The relative weight and size of the lymphoid organs were not affected by treatments. Concentrations of plasma total IgA (241% at 63 d and 229% at 72 d), IgG (222% at 72 d), and anti-ovalbumin IgG (241% at 63 d) were lower with feed restriction. Fecal IgA concentrations were lower with quantitative restriction (240%, 252%, and 265% at age 42, 50, and 63 d, respectively) and energy restriction (256%, 246%, and 273% at ages 50, 63, and 72 d, respectively). Feed-restricted rabbits tended to have greater expressions of interleukin (IL) 1b and IL-2 and lower expressions of tumor necrosis factor a (P < 0.1). Conclusion: These results demonstrated that, in rabbits, restriction and, to a lesser extent, dietary energy concentration modulate gut immunity. J Nutr 2015;145:483–9. Keywords: rabbit, feed restriction, energy intake, health, immune response
Introduction Severe dietary changes often cause digestive disorders. In mammals, weaning generates important changes as the young animal 1 This project was funded by partners involved in the project, including Sanders, Techna, CCPA, Evialis, Inzo, Itavi (Institut Technique de l’Aviculture), INRA (Institut National de la Recherche Agronomique), and the CLIPP (Comite´ Lapin Interprofessionnel pour la Promotion des Produits). 2 Author disclosures: C Knudsen, S Combes, C Briens, J Duperray, G Rebours, J-M Salaun, A Travel, D Weissman, T Gidenne, and IP Oswald, no conflicts of interest. 3 Supplemental Tables 1–3 and Supplemental Figure 1 are available from the ‘‘Online supporting Material’’ link in the online posting of the article and from the same link in the online table of contents at http://jn.nutrition.org. * To whom correspondence should be addressed. E-mail: thierry.gidenne@ toulouse.inra.fr.
shifts from milk to solid feed, and is often responsible for various digestive disorders (1, 2). These disorders are particularly important in agricultural species because they result in economic losses for the breeder. Moreover, current European legislation and recommendations strongly discourage the use of antibiotics (3); thus, new alternatives are needed to preserve animalsÕ health (4). Short-term feed restriction was proven to reduce postweaning digestive disorders in rabbits and has been used by breeders in France for over 10 y as an efficient method to preserve the digestive health of growing rabbits (5). Indeed, a reduction in feed intake (below 20% of free intake) reduces postweaning mortality and morbidity (6). Previous studies in mice and rats suggested
ã 2015 American Society for Nutrition. Manuscript received June 16, 2014. Initial review completed July 11, 2014. Revision accepted December 2, 2014. First published online January 7, 2015; doi:10.3945/jn.114.197871.
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that long-term feed restriction and the nutritional value of the diet could affect gut immunity (7–9). In growing rabbits, the effect of some nutrients, such as starch and fiber, on the immune response was documented. Indeed, increased intake of fiber could improve mucosal immunity through an increased number of membranous epithelial cells in the appendix of postweaning rabbits (10). However, the involvement of the immune system in the reduction in postweaning digestive troubles through short-term feed restriction is poorly documented. Likewise, to our knowledge, the short-term effect of dietary energy concentration on the immune system has not yet been studied in agricultural species. Thus, the present study was conducted to evaluate the impact of both quantitative feed restriction and dietary digestible energy (DE)15 concentration on gut immunity in growing rabbits.
Methods
15 Abbreviations used: B2m, b2 microglobulin; DE, digestible energy; HE, high energy; HE100, high-energy ad libitum feed; HE75, high-energy restricted feed; Hprt1, hypoxanthine phosphoribosyltransferase 1; LE, low energy; LE100, lowenergy ad libitum feed; LE75, low-energy restricted feed.
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of digestive disorders, such as diarrhea, cecal impaction, and suspicion of epizootic rabbit enteropathy, or other pathologies such as respiratory problems and injuries. Rabbits without clinical signs of illness but showing weight loss or very low growth (3 SD below the mean) were considered morbid and excluded from the study. Ovalbumin immunization. At age 37 d, 32 rabbits per group were immunized with a 1 mL subcutaneous injection of 600 mg ovalbumin (Sigma-Aldrich) in incomplete FreundÕs adjuvant (Sigma) and challenged 9 d later (age 46 d) with 300 mg ovalbumin in incomplete FreundÕs adjuvant. The weights (means and SDs) at age 35 d of the rabbits selected for immunization and those of the remaining rabbits were equivalent in order to have a representative sample of the study rabbits. Killings and samplings. Ten healthy rabbits per treatment were killed at ages 42, 50, 63, and 72 d. All rabbits killed at ages 63 and 72 d had been immunized with ovalbumin, whereas those killed at ages 42 and 50 d had not been immunized. The weights (means and SDs) of the rabbits selected for sampling and those of the remaining rabbits were equivalent in order to have a representative sample of the study rabbits. Rabbits were killed by electrical stunning followed by exsanguination. Blood was collected from the aorta in heparinized tubes (Vacuette 9 mL sodium heparin; Greiner Bio-One) and immediately stored on ice. After centrifugation (1000 3 g for 10 min at 4°C), plasma samples were stored at 220°C until further analysis. Lymphoid organs (spleen and vermiform appendix) were collected and weighed individually. Feces were collected from the rectums of the rabbits killed and transferred to dry tubes on ice until arrival at the laboratory, where the samples were stored at 220°C until further analysis. A 5 cm-long section of the ileum was collected 10 cm from the ileocecal junction, snap frozen in liquid nitrogen, and stored at 280°C until analysis. The first PeyerÕs patch encountered from the ileocecal junction was isolated, the ileum was cut up, and a picture was taken of the patch next to a ruler. Images were analyzed through the use of ImageJ software (14) to evaluate the surface of the patches. Total and anti-ovalbumin IgG ELISA measurements. Concentrations of plasma immunoglobulin G (IgG) were measured by ELISA as described previously (15). Goat anti-rabbit IgG (Fc fragment–specific) was used as a capture antibody (Bethyl Laboratories) and HRP-labeled goat anti-rabbit IgG was used as a detection antibody in conjunction with a tetramethylbenzidine substrate (1:1 H2O2:tetramethylbenzidine) (Thermo Fisher Scientific). The OD of each well was read at 540 nm and subtracted from the readings at 450 nm (Infinite M200, Tecan) to correct for optical imperfections in the plates. The mean OD of each sample was then calculated, and the mean value of the negative control was subtracted from all sample values. IgG concentrations were then obtained from the standard curve constructed with the OD values of an IgG standard solution. Concentrations of specific anti-ovalbumin IgG were also measured by ELISA (16). Briefly, ELISA plates were coated with ovalbumin diluted
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Study design, rabbits, housing, and feed. The study was conducted at the INRA (Institut National de la Recherche Agronomique) UE (Unit´e Exp´erimentale) PECTOUL (Poˆle Exp´erimental Cunicole de Toulouse) breeding unit in Castanet-Tolosan, France, on 320 healthy male and female hybrid rabbits (Oryctolagus cuniculus) at a 50:50 male-to-female ratio. A bifactorial design was used with 2 levels of feed intake—ad libitum feed consumption vs. restricted feeding at 75% of ad libitum— and 2 diets differing in DE concentration—a low-energy (LE) diet, formulated with 9.08 MJ DE/kg, and a high-energy (HE) diet, formulated with 10.13 MJ DE/kg according to the European Group on Rabbit Nutrition tables from Maertens et al. (11). The rabbits were assigned to 4 treatment groups: a low-energy ad libitum–feed (LE100) group, a lowenergy restricted-feed (LE75) group, a high-energy ad libitum–feed (HE100) group, and a high-energy restricted-feed (HE75) group. The diets were formulated to meet the nutritional requirements of growing rabbits (12), without drug supplementation (antibiotics or coccidiostatics). Special attention was given to obtaining a theoretic deviation of 1 MJ/kg of DE between the diets, with the constraint of obtaining similar ratios of digestible fibers, starch, and fat to DE, starch to digestible fibers, and digestible proteins to DE. Likewise, both diets were formulated with equivalent concentrations of acid detergent fiber and neutral detergent fiber (Supplemental Table 1). The feeds were manufactured and pelleted with the use of one batch of raw materials by Euronutrition. The rabbits were housed in collective cages of 5 rabbits with a density of 15 rabbits/m2 in a closed unit in which the environment (temperature, lighting, and ventilation) was monitored and controlled. A total of 320 male and female rabbits were randomly assigned at weaning (age 35 d) to 1 of the 4 treatments. Thus, mean weights were similar between treatments and litters were split to avoid any maternal effect. The HE100 and LE100 groups were then fed freely, and the LE75 and HE75 groups were given a restricted diet for 4 wk, until age 63 d. Afterwards, all 4 groups were fed freely until the end of the study at age 72 d. Moreover, 32 rabbits per group were immunized with ovalbumin as described below (Figure 1). The amounts of feed distributed to the restricted groups were calculated on the basis of a theoretic ad libitum ingestion curve and readjusted for each diet according to the real ingestion of the ad libitum–feed groups (LE100 and HE100) for periods of 3–4 d. Feed was given daily in a single distribution between 0800 and 0830, and water was consumed ad libitum. Rabbits were handled according to recommendations for the care of experimental animals in accordance with French national legislation (13). Rabbits were weighed individually at weaning (age 35 d), after 1 wk of feed restriction (age 42 d), during the restriction period (age 50 d), at the end of the restriction period (age 63 d), and at the end of the study, after 9 d of ad libitum feed consumption by all 4 groups (age 72 d). The health status of the rabbits was assessed by monitoring for clinical signs
FIGURE 1 Study design. AL, ad libitum feeding; BW, body weight; FR, feed restriction; HE100, high-energy ad libitum–feed group; HE75, highenergy restricted-feed group; LE100, low-energy ad libitum–feed group; LE75, low-energy restricted-feed group; OVA, ovalbumin immunization.
in carbonate buffer (4 g/L NaHCO3 0.1 mol/L; pH 9.6) and incubated overnight at 4°C. Diluted plasma samples were then added to the plates, and the anti-ovalbumin antibodies were detected with HRP-labeled antirabbit IgG (Bethyl Laboratories). Plasma samples were quantified by reference to standard curves constructed with hyperimmune rabbit serum. Fecal IgA extractions and IgA ELISA measurements. A small amount of fecal matter (200–600 mg) was diluted to 50 g/L in cold PBS. The samples were dispersed for 10 s on ice (T25 Ultra-Turax, IKA Labortechnik) and centrifuged at 3000 3 g for 10 min at 4°C. The supernatants were then collected and stored at 220°C until analysis. Concentrations of plasma and fecal IgA were quantified by ELISA through the use of a specific polyclonal goat anti-rabbit IgA antibody (Bethyl Laboratories). Sample relative IgA concentrations were obtained from the standard curve constructed with the OD values of a diluted reference serum (Bethyl Laboratories).
Calculations and statistical analysis. The DE contents of the diets were obtained from digestibility measurements presented in a companion paper (19). Mean DE intakes were then estimated for each treatment at each age as the mean gross intake per rabbit in the sampled cages, from weaning to killing, multiplied by the DE content of the diet for each treatment. Mean fecal IgA concentrations were plotted according to the mean DE intake at ages 42, 50, and 63 d. Because the feed-restricted rabbits weighed less than the rabbits consuming feed ad libitum, the weights of the organs (spleen and appendix) were expressed relative to body weight to correct for body size. All measurements were analyzed with the use of the GLM procedure (SAS). To account for unequal variances, logarithmic transformation of the data was performed when necessary. In each age group, relative organ weights, PeyerÕs patch surface areas, and antibody concentrations were tested for the effects of feed intake level, the diet, and their interactions as fixed effects. Body weight was added as a covariable for the analysis of PeyerÕs patch surface areas. The effect of age was analyzed for each treatment with the use of age as a fixed effect. Finally, the model for cytokine measurements used the feed intake level, diet, age, and all interactions between these factors as fixed effects. Mean comparisons were performed with the use of TukeyÕs HSD (Honestly Significant Difference) procedure. Differences were considered significant at P < 0.05, whereas differences in which 0.05 < P < 0.1 were considered tendencies, and only differences in which P < 0.1 are shown in the figures. Values in the text, tables, and figures are means 6 SEMs. Tendencies were discussed only for cytokine expression.
Total and anti-ovalbumin plasma IgG concentrations were lower with quantitative feed restriction after 4 wk of treatment. The main objective of this study was to assess the effects of feed intake level and dietary energy concentration on the immune response. Therefore, the systemic immune response, assessed by plasma IgG concentrations, was studied. Plasma total IgG concentrations were not affected during feed restriction (Figure 4). However, after returning to ad libitum feed consumption, rabbits that previously were restricted had lower IgG concentrations than rabbits that previously consumed feed ad libitum (222%, P < 0.01). Concerning the effect of dietary energy concentration, after 1 wk of the study, IgG concentrations were higher in rabbits fed the HE diet than in those fed the LE diet (+23%, P < 0.05), but at all subsequent time points (50, 63, and 72 d), the diet energy concentration did not affect plasma IgG concentrations (Figure 4). The ovalbumin immunization protocol allowed us to investigate the effects of feed intake level and dietary energy concentration on
Results Body weight and PeyerÕs patch surface area was lower with quantitative feed restriction without affecting relative spleen and appendix weight. The feed intake of the rabbits consuming feed ad libitum averaged 135 g/d from age 35 to 63 d. The feed intake of the rabbits consuming the restricted diets was
FIGURE 2 Effect of feed intake level and dietary DE concentration on the growth of rabbits from age 35 to 72 d. Values are means 6 SEMs, n = 10. DE, digestible energy; HE100, high-energy ad libitum–feed group; HE75, high-energy restricted-feed group; LE100, low-energy ad libitum–feed group; LE75, low-energy restricted-feed group. Feed restriction modulates rabbit immunity
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Expression of mRNA encoding for cytokines by real-time PCR. Tissue RNA of 50- and 63-d-old rabbits was processed as previously described (17). Concentrations, integrity, and quality of RNA were determined spectrophotometrically (OD 260) through the use of the Nanodrop ND1000 (Labtech International). The sequences of primers used in the PCR for Il1b, Il2, Il8, Il10 and Tnfa are detailed in Supplemental Table 2 and were purchased from Sigma. Real-time qPCR was performed in 384 well plates with the use of SYBR Green as the reporter. qRT-PCR data were expressed as 2(2DDCt) and normalized to a housekeeping gene. b2 microglobulin (B2m) was chosen among 3 other candidate reference genes [Gapdh, hypoxanthine phosphoribosyltransferase 1 (Hprt1), and cyclophilin A (Ppia)] because its expression was not affected by our experimental treatments or age, and it was thus considered to be a valid reference. Amplification efficiency and initial fluorescence were determined by using the same method as for real-time PCR (18). Finally, gene expression was expressed relative to the LE100 group. The specificity of qRT-PCR products was assessed at the end of the reactions by analyzing dissociation curves.
consistent with the level of 75% initially planned (75% and 74% for the LE and the HE diets, respectively, from age 35 to 63 d). As expected, growth rate was lower with feed restriction, leading to lower weight in the rabbits for which feed was restricted starting at age 42 d, whereas diet energy concentration did not affect growth (Figure 2). The relative weights of each organ were not affected by the experimental treatments during the feed restriction period. However, the relative weight of the appendix was greater in rabbits fed the LE diet after 1 wk of ad libitum feed consumption at age 72 d (P < 0.05, 0.32% 6 0.01% compared with 0.28% 6 0.01%, data not shown). The surface area of the PeyerÕs patches was not affected by the experimental treatments at age 42 and 50 d but was lower with feed restriction at age 63 d (P < 0.01, 20.2 cm2). After 1 wk of ad libitum feed consumption, the surface area of the PeyerÕs patches of the rabbits for which feed previously was restricted was smaller than that of rabbits that consumed feed ad libitum throughout the study (P < 0.05, 20.2 cm2) (Figure 3). However, when the body weight of the rabbits was added as a covariable in our model, no effect from our treatments was detectable, regardless of age. In conclusion, body weight was lower with feed restriction, but the growth of secondary lymphoid organs (spleen and appendix) and the surface area of the PeyerÕs patches were not affected by feeding level, whereas dietary energy concentration only had a moderate effect on the growth of the appendix.
antigen-specific immunity. Plasma anti-ovalbumin IgG concentrations were lower with feed restriction at age 63 d (241%, P < 0.05) (Figure 5), but after 1 wk of ad libitum feed consumption, previous feeding levels did not affect specific antibody concentrations. Diet energy concentration did not affect anti-ovalbumin IgG concentrations in any of the age groups. Taken together, these results demonstrate that feed restriction and dietary energy concentration had a limited effect on plasma total IgG concentrations, but feed restriction greatly affected treatment-specific anti-ovalbumin IgG production after 4 wk of treatment. Quantitative feed restriction reduced fecal and plasma IgA concentrations to a greater extent than did energy restriction. Another aim of this study was to assess the effect of feed intake level and dietary energy concentration on the local immune response in the digestive tract through measurements of fecal IgA concentrations. Fecal IgA concentrations were lower during feed restriction (240% at age 42 d, P < 0.05; 252% at age 50 d, P < 0.001; and 265% at age 63 d, P < 0.001) (Figure 6A). When rabbits returned to ad libitum feed consumption, no effect from the previous feeding levels was observed. Fecal IgA concentrations were higher in rabbits fed the HE diet than in those fed the LE diet from age 50 d onward (+56% at age 50 d, P < 0.01; +46% at age 63 d, P < 0.05; and +73% at age 72 d, P < 0.05).
FIGURE 5 Effect of feed intake level and dietary DE concentration on plasma anti-OVA IgG concentrations at ages 63 and 72 d in growing rabbits. Values are means 6 SEMs, n = 10. DE, digestible energy; HE100, high-energy ad libitum–feed group; HE75, high-energy restricted-feed group; LE100, low-energy ad libitum–feed group; LE75, low-energy restricted-feed group; OVA, ovalbumin.
When we plotted the mean fecal IgA concentrations against the mean DE intake per treatment, a linear relation between these variables was observed at ages 50 and 63 d but not at age 42 d (Figure 7). To compliment the fecal analyses, plasma measurements of IgA concentrations were performed. Plasma IgA concentrations were lower with feed restriction after 4 wk of treatment (age 63 d, 241%, P < 0.001) and after 1 wk of ad libitum feed consumption (age 72 d, 229%, P < 0.05). Diet energy concentration did not affect plasma IgA concentrations (Figure 6B). These results indicate that fecal IgA concentrations were lower with limited feed intake and reduced dietary energy concentration, and plasma IgA concentrations were also, to a lesser extent, lower with feed restriction. Quantitative feed restriction tended to modify cytokine expression in the ileum. Cytokines play a key role in regulating immunity, and more specifically, inflammation. Expressions of IL-1b and IL-2 were greater with feed restriction (+30% and +77%, respectively, 0.05 # P < 0.1), whereas those of TNF-a were lower with feed restriction (215%, 0.05#P < 0.1). Feed restriction did not affect the expression of IL-8 and IL-10 (Table 1). Immunologic values were affected by age. The relative weight of the spleen decreased with age (P < 0.01) for all groups, whereas the relative weight of the appendix increased with age until age 63 d and decreased afterwards for all groups except LE75, for which age did not affect the relative weight of the appendix (Supplemental Table 3). The surface area of the PeyerÕs patches moderately increased with age for all groups (P < 0.01) (Supplemental Table 3). Fecal IgA concentrations increased with age (P < 0.01) for the HE100 and HE75 groups but were unaffected by age for the LE100 and LE75 groups (Supplemental Figure 1A). Plasma total IgG and IgA concentrations increased with age regardless of study group (P < 0.001) (Supplemental Figure 1B and C).
FIGURE 4 Effect of feed intake level and dietary DE concentration on plasma total IgG concentrations from age 42 to 72 d in growing rabbits. Values are means 6 SEMs, n = 10 per treatment and per age. For an age, labeled means without a common letter differ, P , 0.05. DE, digestible energy; HE100, high-energy ad libitum–feed group; HE75, high-energy restricted-feed group; LE100, low-energy ad libitum–feed group; LE75, low-energy restricted-feed group. 486
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Discussion Dietary strategies influence animal health. The present study aimed to analyze the effects of quantitative feed restriction and dietary energy concentration on the immune response of
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FIGURE 3 Effect of feed intake level and dietary DE concentration on the development of PeyerÕs patches from age 42 to 72 d in growing rabbits. Values are means 6 SEMs, n = 10. For an age, labeled means without a common letter differ, P , 0.05. DE, digestible energy; HE100, high-energy ad libitum–feed group; HE75, high-energy restricted-feed group; LE100, low-energy ad libitum–feed group; LE75, low-energy restricted-feed group.
growing rabbits. We demonstrated that feed restriction rather than dietary energy concentration is responsible. The spleen, the appendix, and PeyerÕs patches are major secondary lymphoid organs. As previously demonstrated in mice (20), feed restriction did not affect the relative weight of the spleen or the appendix. Dietary energy concentration had only a very moderate effect on the relative weight of the appendix at the
FIGURE 7 Fecal IgA concentrations per treatment according to the mean digestible energy intake at ages 50 and 63 d in growing rabbits. Values are means 6 SEMs, n = 10. HE100, high-energy ad libitum– feed group; HE75, high-energy restricted-feed group; LE100, lowenergy ad libitum–feed group; LE75, low-energy restricted-feed group.
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FIGURE 6 Effect of feed intake level and dietary DE concentration on fecal (A) and plasma (B) IgA concentrations from age 42 to 72 d in growing rabbits. Values are means 6 SEMs, n = 10. For an age, labeled means without a common letter differ, P , 0.05. DE, digestible energy; HE100, high-energy ad libitum–feed group; HE75, high-energy restricted-feed group; LE100, low-energy ad libitum–feed group; LE75, low-energy restricted-feed group.
end of this study. Similarly, the smaller surface area of the PeyerÕs patches in the rabbits consuming a restricted diet may be correlated to the lower weight of these rabbits. Feed restriction and energy restriction therefore did not impair growth of the secondary lymphoid organs. Similar results were reported by Rogers et al. (20), who showed that a 30% reduction in feed intake did not affect the number of PeyerÕs patches or their number of cells. However, Kubo et al. (21) demonstrated that 50% feed restriction reduced the number of cells in the spleens of growing mice. Thus, immune activity might be compromised by feed restriction despite the lack of effect on the physiologic variables of the secondary lymphoid organs measured. Concerning the adaptive systemic immune response, feed restriction affected total IgG concentrations only after its application. A similar delayed dietary effect was reported by Zhu et al. (10) in growing rabbits fed diets differing in starch and fiber concentrations. As for the effects of feed restriction on the immune response to a specific antigen, the response was much more prompt: specific anti-ovalbumin concentrations were considerably lower after 4 wk of feed restriction. Similar results were obtained by Martin et al. (22) in 30% feed–restricted deer mice submitted to a keyhole limpet hemocyanin challenge. These findings could indicate that immunologic memory is an energetically costly process, which is negatively affected by feed restriction. Another hypothesis could be that antibody production is indirectly lowered through variations in nutrient or metabolite signals caused by reduced feed intake. Surprisingly, previous feeding level had no effect on the specific response to ovalbumin after 1 wk of ad libitum feed consumption. This could indicate that the specific immune response is readjusted very quickly after a change in the amount consumed. The present study evaluated the effect of feed intake level and dietary energy concentration on the local immune response through fecal and plasma IgA concentrations and ileal cytokine expressions. Fecal IgA concentrations were lower with feed restriction in growing rabbits, confirming the results obtained in intestinal fluids and submandibular gland cultures in mice subjected to long-term feed restriction (8, 23). However, to our knowledge, this is the first study to find effects of short-term feed restriction on fecal IgA concentrations. As we published previously (19), DE intake was greater with the HE diet and ranked in the following order: LE75 < HE75 < LE100 < HE100. In this study a linear relation between DE intake and fecal IgA concentrations was found. We could therefore hypothesize that fecal IgA concentration might be regulated by DE intake rather than the quantity of feed ingested. However, the regression analyses were based on the mean energy intake of a sample of rabbits and not on individual measurements. Thus, additional studies would be necessary to confirm our hypothesis. Muthukumar et al. (23) showed that a 40% reduction in feed intake resulted in lower submandibular gland IgA concentrations and attenuated the onset of diseases in autoimmune-prone mice, indicating a possible protective effect from a reduction in IgA concentrations. The mycotoxin deoxynivalenol also induces an increase in IgA levels in mice (24) and is associated with intestinal lesions in pigs (25). Thus, the lower fecal IgA concentrations could be related to the lower incidence of digestive disorders observed in rabbits consuming the restricted diet and detailed in our companion paper (19). In addition, our results showed that plasma IgA concentrations were lower with feed restriction after 4 wk of treatment. The change in plasma IgA concentrations lagged behind the one occurring at the fecal level (age 63 compared with 42 d), suggesting that the immune response in peripheral blood occurs subsequent to the response in the digestive tract. This hypothesis is consistent with the results obtained in plasma IgG concentrations in our study and the study by Zhu et al. (10).
TABLE 1 Effect of feed intake level and dietary DE concentration on cytokine relative expressions in the ilea of growing rabbits between ages 50 and 63 d1 50 d
IL-1b TNF-a IL-8 IL-2 IL-10
P values2
63 d
LE100
LE75
HE100
HE75
LE100
LE75
HE100
HE75
Pooled SEM
Age
Diet
Intake level
1.05 1.09 1.18 1.26 0.89
1.77 0.73 0.88 3.79 1.57
1.18 0.95 1.01 1.67 0.95
1.47 0.84 1.06 2.41 0.93
1.02 1.03 1.04 1.11 1.24
1.21 0.86 1.24 1.96 1.31
1.10 0.93 1.13 1.56 1.35
1.24 0.96 1.56 1.80 1.29
0.08 0.04 0.07 0.30 0.10
0.38 0.59 0.16 0.87 0.30
0.97 0.91 0.49 0.57 0.10
0.053 0.07 0.52 0.07 0.65
Values are means and pooled SEMs, n = 10. DE, digestible energy; HE100, high-energy ad libitum–feed group; HE75, high-energy restricted-feed group; LE100, low-energy ad libitum–feed group; LE75, low-energy restricted-feed group. 2 All interactions between factors were nonsignificant. 1
Acknowledgments We thank P Aymard, E Balmisse, and J-M Bonnemere from the INRA (Institut National de la Recherche Agronomique) UE (Unit´e Exp´erimentale) PECTOUL (Poˆle Exp´erimental Cunicole de Toulouse) for rabbit handling and care during the study. We also thank J Laffitte, AM Cossalter, and A Pierron from the TOXALIM (Toxicologie Alimentaire) unit and Y Lippi from the GeT-TRIX (G´enome et Transcriptome-Impact Transcriptomique des X´enobiotiques) platform for their technical support in ELISA protocols and qRT-PCR analyses. CK, CB, JD, GR, J-MS, AT, DW, and TG designed the research. CK, SC, and TG conducted the research. IPO provided the immunologic reagents and materials. CK performed the statistical analysis. CK, SC, TG, and IPO wrote the paper. TG had primary responsibility for final content. All authors read and approved the final manuscript. 488
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The results for cytokine expressions suggested that feed restriction tended to regulate the proinflammatory response (IL-1b and TNF-a) and increase the expression of IL-2. In contrast, LaraPadilla et al. (8) found greater expressions of TNF-a and IL-10 and lower expression of IL-2 in the duodenum of mice feeddeprived on alternate days for 17 wk, whereas Kurki et al. (26) recently found that long-term feed restriction at 70% of ad libitum intake in lean mice increased the protein expression of IL-2, IL-1b, IL-10, and TNF-a in adipose tissues. This suggests that either the cytokines are differentially expressed in the ileum, or the immune response is dependent on the duration and type of feed restriction. Finally, IL-2 and TNF-a regulate the synthesis of IgA (27); thus, the potential differential expression in these 2 cytokines could be related to the reduced IgA concentrations observed with feed restriction. However, this hypothesis remains to be confirmed, because the mechanisms by which cytokines regulate the production of IgA are complex. In conclusion, the present study demonstrated the impact of short-term feed restriction on the systemic and local digestive immunity of growing rabbits. Fecal IgA concentrations rapidly decreased with both feed restriction and energy restriction, whereas the effects on the systemic immune response (plasma IgG and IgA concentrations) lagged behind. Thus, the local immune response was modulated at an earlier stage than the systemic immune response. The innate immune response seemed less affected by our treatments because cytokine expressions in the ileum only tended to be modulated by quantitative feed restriction. Taken together, our results demonstrated that feed restriction and, to a lesser extent, the dietary energy concentration of the feed modulated gut immunity. However, it is not known whether these variables are associated with the beneficial effects of feed restriction on postweaning digestive health in rabbits.
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