Weaning is a critical period for piglets that involves stressful factors when the immune and digestive systems are still immature. This situation generally results in low voluntary feed intake and suboptimal growth rate, both of which are associated with effects on the intestinal mucosa integrity and with the occurrence of pathological disorders (Pluske et al., 1997; Spreeuwenberg, 2002). Antibiotic preventive medication has been an effective tool for improving the performance of piglets at weaning for years, but now concern about bacterial resistance to antibiotics and general food safety issues has led to legislation minimizing the use of these compounds. This situation has led to an increase in the investigation of the use of new feed additives (Kamel, 2001).
Spray-dried porcine plasma (SDPP) seems to be a good alternative to antibiotics (Coffey and Cromwell, 2001). Spray-dried porcine plasma benefits at weaning are mainly attributed to the maintenance of mucosal integrity and the reduction of inflammatory response in the intestine, helping the piglet to resist bacterial aggressions (Bosi et al., 2004).
Recently, the use of plant extracts has become an interesting alternative to antibiotics. Antimicrobial effects (Didry et al., 1994; Dorman and Deans, 2000), antioxidant (Aruoma et al., 1996), antitoxigenic activities (Sakagami et al., 2001), and immunomodulatory effects (Middleton and Kandaswami, 1992) have been reported for their use in human medicine and the food industry. Nonetheless, their modes of action are not yet fully understood.
An experiment was designed to evaluate the effect of dietary SDPP and a plant extract mixture (XT; containing carvacrol, cinnamaldehyde, and capsicum oleoresin) on intestinal morphology and on the leukocyte cell subset populations of peripheral blood and gut-associated lymphoid tissue (GALT) in conventionally weaned pigs during the postweaning mucosal adaptive phase. This experiment represents an expansion of the evaluation of plant extracts that are partially reported in Manzanilla et al. (2006) and Castillo et al. (2006).
MATERIALS AND METHODS
The experiment was performed at the Experimental Unit of the Universitat Autònoma de Barcelona and received prior approval from the Local Ethical Committee for Animal Experimentation of the institution. The treatment, housing, husbandry, and slaughtering conditions conformed to the European Union Guidelines (The Council of the European Communities, 1986).
Animals, Housing, and Dietary Treatments
A total of 24 piglets [(Landrace × Large White) × Pietrain; mixed males and females) from a commercial herd were used. No creep feeding was provided during the lactation period. The animals were weaned at 18 to 22 d of age, 6.0 ± 0.14 kg of BW, and were housed in a weaning room equipped with automatic heating and forced ventilation. The room temperature was gradually reduced from 29 to 25°C over a period of 3 wk. The piglets had free access to feed and water. During the study period, the piglets did not seroconvert against the most important swine infectious agents, including porcine circovirus type 2, porcine parvovirus, porcine respiratory and reproductive syndrome virus, swine in-fluenza virus, Aujeszky’s disease virus, Mycoplasma hyopneumoniae, and Lawsonia intracellularis.
The animals were allocated into 6 pens (2 pens per treatment) following a complete randomized design for 3 wk, taking the litter of origin into account. Details of ingredient composition and calculated nutrient content of the diets are given in Tables 1 and 2. A control (CT) diet was formulated containing soy protein concentrate and wheat gluten. In addition, 2 treatment diets were formulated. The first diet (SDPP diet) evaluated SDPP (Appetein, APC-Europe, Barcelona, Spain) as an alternate protein source to the soy protein concentrate (also replacing a portion of the wheat gluten and with adjustments to some AA). The second diet evaluated the addition of 0.03% of a plant extract combination to the CT diet (XT diet). The XT is a plant extract combination (Pancosma, S.A., Geneva, Switzerland) standardized in 5% (wt/wt) carvacrol (Origanum spp.), 3% cinnamaldehyde (Cinnamonum spp.), and 2% capsicum oleoresin (Capsicum annum), and included in an inert fatty carrier (that represented the remaining 90%).
Feeding Regimen and Sampling
Individual BW and pen feed consumption were registered weekly. On d 17 to 21 postweaning (PW), a controlled feed intake pattern was applied from 0800 to 2000 to standardize the digestive tract conditions upon sacrifice (Manzanilla et al., 2004). In particular, 30-min periods of feeding were alternated with 1-h fasting periods. The adequacy of timing and ad libitum conditions were confirmed when the pigs in the pen moved to the feeders to eat each feeding period and finished in a 0.5-h period. Pigs were fed ad libitum the remainder of the day (from 2000 to 0800 of the next day).
On d 19 and 21 PW, 24 pigs (4 pigs/dietary treatment on each day) were euthanized with an intravenous sodium pentobarbital overdose (200 mg/kg of BW; Dolethal, Vetoquinol S.A., Madrid, Spain). The pigs were opened immediately from sternum to pubis, and the whole gastrointestinal tract was removed, measured, weighed, and sampled. Sections of proximal jejunum, ileum, and proximal colon were removed, opened longitudinally, and fixed by immersion in 10% (vol/vol) buffered formalin for the histological study.
For hematological and immune cell subsets studies, blood samples from the anterior vena cava were collected in tubes containing EDTA and lithium heparin, respectively, on the day of weaning (d 0), on d 7 and 14 PW, and on the slaughter days. Also, immediately after slaughter, ileocolic lymph node (LN) and ileal Peyer’s patches (il-PP) were collected, cleaned with PBS, and the surrounding tissues were removed. The LN and il-PP were immersed in transport medium (2,000 IU of penicillin and 2 mg of streptomycin in 100 mL of PBS, Sigma, Madrid, Spain) at 4°C until their processing for flow cytometric analysis.
Tissue samples for the morphometric study were dehydrated and embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin. Morphometric measurements were performed with a light microscope (BHS, Olympus, Barcelona, Spain). Villus height and width, crypt depth, intraepithelial lymphocyte (IEL) number in the villi, the index of mitosis in the crypt, intravillus lamina propria cell density, and goblet cell number in villi and crypts were measured. Measurements were taken in 10 well-oriented villi and crypts from each intestinal section of each animal.
The villus height and crypt depth were measured using a linear ocular micrometer (Olympus, Microplanet). Villus:crypt ratio was calculated by dividing villus height by crypt depth. The same villus and crypt columns were used to determine the number of IEL, goblet cells, and mitosis; these variables were expressed per 100 enterocytes. On the basis of the cellular morphology, differences between the nuclei of enterocytes, mitotic figures, goblet cells, and lymphocytes were clearly distinguishable at 400× magnification. Intravillus lamina propria cell density was determined by counting total visibly stained nuclei and total lymphocytes in 10 fields (total area of 4,000 μm2) from each section using a grid ocular (Olympus, Microplanet). Cell density was expressed as the number of total stained cells and the number of lymphocytes per 1,000 μm2. The number of lymphocytes in relation to the number of total cells was also calculated. All morphometric analysis was done by the same person, who was blinded to the treatments.
Blood and Lymphoid Tissue Cell Subsets Analysis
A complete hemogram was performed on each pig blood sample collected in EDTA using a semiautomatic electric impedance blood cell counter (Sysmex F-800, Toa Medial Electronic Europa). The leukocyte differential count was performed by identification of 100 cells on a blood smear using light microscopy.
For flow cytometric analysis, mononuclear cells (MC) were isolated from blood, LN, and il-PP and processed based on a previously described protocol (Solano-Aguilar et al., 2000). In short, blood containing lithium heparin was mixed with Ficoll–Hypaque (specific density 1.077 g/mL, Sigma) and centrifuged (800 × g for 30 min) at room temperature. The MC were centrifuged twice (600 × g for 10 min), resuspended in RPMI-1640 (Sigma), and kept for flow cytometric analysis. Cells from LN were isolated after gentle trimming of the tissue in RPMI-1640. Cell suspensions were further filtered through a 100-μm nylon mesh (Sigma). After 2 consecutive centrifugations (600 × g for 10 min), the cells were resuspended and left to stand in RPMI-1640. Sections from il-PP (∼2 cm) were incubated in a water bath (20 min at 37°C) containing 2 mM HBSS-DTT (dithiothreitol; Sigma) and 5 mM HBSS–EDTA (Sigma). The remaining tissues were minced gently with collection media (2,000 IU of penicillin with 2 mg of streptomycin and 50 mg of Gentamycin in 100 mL of PBS, Sigma) and filtered through a metal sieve. After centrifugation of the supernatants (600 × g for 10 min), the pellet was purified (1,000 × g for 30 min) through a continuous 40 to 70% Percoll gradient (Sigma). The cells were centrifuged (600 × g for 10 min), resuspended, and left to stand in RPMI-1640. Then, contaminating erythrocytes of all cell suspensions were removed by lysis with ammonium chloride, and MC viability was tested with trypan blue dye.
Leukocyte cell subsets were determined after staining with a panel of antiporcine monoclonal antibodies (mAb) and analyzed by flow cytometry. The cells were stained with: anti-CD45 (2A5, unconjugated, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria [INIA], Madrid, Spain) to identify all leukocytes, anti-SWC3 (BL1H7, unconjugated, INIA) to identify monocytes and granulocytes, anti-CD21 [BB6-11C9.6, fluorescein isothiocyanate-conjugated, Southern Biotechnology Associates (SBA), Birmingham, AL] to identify B lymphocytes, anti-CD3 [B23-8E6, phycoerythrin (PE)-conjugated, SBA] to identify T lymphocytes, anti-CD4 (74-2-11, unconjugated, INIA) to identify T helper lymphocytes, anti-CD8 (76-2-11, PE-conjugated, SBA) to identify cytotoxic T cells, and anti-γδTCR (PGBL22A, unconjugated, Veterinary Medical Research and Development, Pullman, WA) to identify γδ T lymphocytes.
Each sample was adjusted to 6 × 106 cells per mL of flow cytometry medium (FCM; PBS containing 0.1% BSA, Sigma). Fifty microliters of the resulting cell suspensions was introduced into the wells of a 96-well microtitre plate and incubated for 30 min with 50 μL of mAb (hybridoma supernatant). After 2 washes with FCM, the cells were incubated for 30 min with rabbit F(ab′)2 anti-mouse antibody (fluorescein isothiocyanate-conjugated; Dako, Glostrup, Denmark) diluted 1:40 in FCM (except when the mAb was CD3, CD21, or CD8 because they were already conjugated). Finally, the cells were washed twice more and fixed in FCM with 0.3% paraformaldehyde before being analyzed in an EPICS_XL MCL flow cytometer (Beckman-Coulter, Hialeah, FL). To check for the purity of MC, immunostaining was done against the leukocyte common antigen (CD45). Only blood samples with a percentage of CD45+ greater than 96%, and LN or il-PP samples with a percentage of CD45+ higher than 90%, were used. Irrelevant isotype-matched mAbs were used as negative controls.
Data on productive performance, intestinal morphology, and immune cell subsets analysis were subjected to 2-way ANOVA, with dietary treatment as the classification factor, using the GLM procedure (SAS Inst. Inc., Cary, NC). Additionally, the MIXED procedure of SAS was used for repeated blood-derived measures. In the productive performance analysis, pen was used as the experimental unit. In the intestinal morphology and leukocyte cell subsets analysis, pig was used as the experimental unit. The individual pig was used as the experimental unit because the treatments were qualitative and any potential differences in feed intake among pigs could not obscure those distinct treatment differences. Further, when pig was used as the experimental unit, the effects of sex, litter, pen (as a random effect), and day of sacrifice were initially studied in the model but were ultimately not included in the model because none were significant (all exceeded P = 0.30). Preplanned comparisons of each of the 2 treatment groups to the CT treatment were made using Tukey’s test. The alpha level used for determination of significance for all analyses was P < 0.05, with statistical tendencies reported when P < 0.10.
Productive Performance and Gross Findings
Data on growth performance are shown in Table 3. The ADG was 34.6 g/d (86%) and 33.1 g/d (82%) greater in SDPP and XT groups compared with CT group during the first week PW, respectively. Moreover, ADFI was also 53.6 g/d (39%) and 71.2 g/d (21%) greater in XT group than CT group during the first and second week PW, respectively. Animals fed SDPP had a greater G:F ratio (10 g/g; 18%) than those in CT group (P = 0.024).
No clinical signs during the study and no gross findings at necropsy were recorded in any animal. The weight and length of the whole gastrointestinal tract and the lengths of duodenum, jejunum, and ileum were not affected by dietary treatment.
Morphometric measurements are summarized in Table 4. There were no differences in villus height and crypt depth in the proximal jejunum, ileum, and colon. However, ileum villus width was decreased (P = 0.092) in the SDPP-fed pigs.
Counts of IEL/100 enterocytes were affected by diet. A reduction (P = 0.034) was observed in the proximal jejunum of animals fed with XT diet compared with those fed the CT diet. Moreover, a reduction (P = 0.026) in IEL was observed in the colon of animals that received the SDPP diet.
There were differences in intravillus lamina propria cell density associated with the treatment. We did not quantify the specific phenotype of the intravillus lamina propria cells. However, based on cellular morphology, we found that between 22 to 41% of the intravillus lamina propria cells were lymphocyte-like cells. Total cell density was reduced in the ileum (P = 0.002) and proximal colon (P = 0.01) of SDPP-fed pigs than CT animals. In contrast, lymphocyte-like cell density was increased (P = 0.003) in the proximal colon of XT-fed pigs. Number of goblet cells and number of mitosis were similar in pigs from different treatments.
Immune Cell Subsets
Blood variables demonstrated differences associated with age and treatment (Table 5). Total blood leukocytes and total lymphocyte counts on d 14 and on slaughter days were greater compared with weaning day (P < 0.05). Percentages of T cells were also increased because of an increase of CD8+ and CD4-CD8+ cells on d 7 and on slaughter days (P < 0.05), whereas the percentage of B (CD21+) and γδTCR+ lymphocytes was reduced (P < 0.05) on d 7 and 21 PW, and on d 7 and 14 PW, respectively.
The SDPP-fed piglets had a greater percentage of monocytes on d 7 PW compared with CT-fed piglets (P = 0.004). However, they had a decrease in mean percentage of monocytes on slaughter days (P = 0.006). In contrast, on the slaughter days, XT-fed pigs showed increased percentages of SWC3+ cells (myeloid cells; P = 0.038) and lower percentages of CD21+ (P < 0.001) and CD8high+ cells (P = 0.07) compared with those in CT group. The other blood immune cell subsets studied did not differ in pigs fed with different diets.
Flow cytometry analysis was also performed in samples from GALT on slaughter days (Table 6). In il-PP, SDPP-fed pigs had lower mean percentages of macrophages (SWC3+) in comparison to CT (P = 0.04). Moreover, in LN, SDPP-fed pigs had low percentages of macrophages (P = 0.04), B lymphocytes (CD21+; P = 0.04), and γδTCR+ cells (P = 0.009). The XT-fed pigs also had reduced percentages of B lymphocytes in LN in comparison with CT-fed ones (P = 0.03). No other mono-nuclear cell subsets from LN or il-PP differed between diets.
The weaning transition involves complex social, environmental, and nutritional changes for piglets, and it is a stressful event (Pluske et al., 1997). Moreover, weaning is associated with intestinal inflammation and a systemic proinflammatory response (McCracken et al., 1999; Jiang et al., 2000). In the current study, an increase in blood leukocytes, lymphocytes, and several subsets of T cells at different times during the 21-d PW period was observed, suggesting a PW activation of the immune system.
The results reported here also indicate that SDPP can affect immune cell subsets of blood and GALT in weaned pigs and can modify the morphology of the small and large intestine. We demonstrated that SDPP diminishes several immune cell subsets in blood and GALT. The SDPP-treated piglets also show lower IEL numbers and lamina propria cell density in large intestine compared with the CT group. All these results indicate a role for SDPP in preventing GALT from possible activation, as has been demonstrated previously in laboratory animals (Pérez-Bosque et al., 2004). In addition, we found further evidence that the immune effect of SDPP is observable not only in piglets inoculated with bacteria or bacterial products (Touchette et al., 2002; Bosi et al., 2004) but also in nonchallenged weaned pigs.
The improved feed efficiency observed in SDPP pigs is in agreement with previous studies in which the replacement of soy protein concentrate by plasma protein products resulted in positive responses (Jiang et al., 2000; Coffey and Cromwell, 2001). Although the ADG response to SDPP was not significant in this study due to the low number of replicates, the 35 g/d increase is consistent with that observed in larger studies (Jiang et al., 2000; Coffey and Cromwell, 2001; Bosi et al., 2004) with greater numbers of pigs that were designed to evaluate the performance response and establishes that pigs in this study were responding to the SDPP in a normal manner. These benefits appear even in situations where SDPP is considered to be deficient in methionine (Kats et al., 1994) or to have lower apparent ileal digestibility of AA than dried skim milk, isolated soy protein or wheat gluten (Chae et al., 1999). This fact indicates that, as already mentioned by other authors (Torrallardona et al., 2003), the positive effects of SDPP cannot be explained solely from its nutrient composition. It has been recently proposed that the mechanism of action of dietary SDPP on growth performance involves the immune system because the degree of immune cell activation may limit the availability of energy for growth (Demas et al., 1997; Pérez-Bosque et al., 2004). In fact, SDPP may prevent stimulation of the immune system by preventing microbial growth or colonization in the small intestine or by an indirect effect helping the pig’s mucosal integrity (Touchette et al., 2002). Our results with immune cells of blood and GALT and data on intestinal morphology also suggest the hypothesis of lower activation of the immune system of the SDPP piglets.
The γδ+ T cell subset plays a role in modulating the inflammatory response by promoting the influx of lymphocytes and monocytes to mucosal surfaces (Soltys and Quinn, 1999). Interestingly, SDPP-fed piglets showed reduced numbers of γδ+ T cells in LN and a decreased leukocytic infiltration in intestinal mucosa (IEL and lamina propria cell density), supporting the theory that there is a relationship between LN and the intestinal leukocytic population. Another possible explanation for the reduction in the numbers of IEL is the probable antimicrobial properties of this protein source because proliferation of IEL has been associated with the exposure to bacterial antigens (Rothkotter et al., 1999).
Jiang et al. (2000) speculated that local intestinal proinflammatory response associated with weaning could be prevented by dietary plasma and thereby could reduce leukocytic infiltration into the mucosal lamina propria in conventional early-weaned pigs. In accordance with these results, we observed a decrease in intravillus lamina propria cell density not only in the ileum, but also in the proximal colon. In contrast, dietary SDPP had no significant effect on villus length, crypt depth, and number of mitosis, indicating that it has no specific trophic effects in the small and large intestine. This is in accordance with most of the published studies on the effect of SDPP in nonchallenged pigs (Jiang et al., 2000; Van Dijk, et al., 2002).
Walter and Bilkei (2004) observed nonspecific immunostimulatory effects on several leukocyte populations (CD4+, CD8+, CD4+CD8+, MHCII+, and nonT/nonB cells) in the blood of finishing pigs fed diets including 0.3% oregano (which contains 6% carvacrol). The differences with our results, where no immunostimulatory effects were detected in blood, could be due to the real amount of carvacrol added to the diets (180 mg/kg in Walter and Bilkei diet vs. 15 mg/kg in our diet). However, this effect could be produced not by carvacrol but by other compounds present in oregano, such as thymol. On the other hand, capsaicin and cinnamaldehyde have demonstrated antiinflammatory properties. These compounds inhibit activation or proliferation of T cells and can modulate their differentiation (Koh et al., 1998; Sancho et al., 2002). Thereby, these 2 XT compounds could explain the reduced numbers of IEL observed in the jejunum, which are mainly T cells, and the decreased percentages of blood cytotoxic T cells.
Infiltration of lymphocytes in the lamina propria of ileum and colon was increased in XT-fed animals. This could be related to an increase in the number of lactobacilli observed in the large intestine of the same animals (Castillo et al., 2006) because lactic acid bacteria, in general, can stimulate the immune system (Schley and Field, 2002).
In summary, our results indicate that SDPP in weaning pig diets enhances feed efficiency, and reduces percentages of immune cell subsets in blood and GALT, numbers of IEL and the density of lamina propria cells, suggesting lower activation of the immune system. Conversely, dietary XT caused a reduction of IEL numbers and percentages of lymphocyte subsets in blood and LN and an increase of blood monocytes and of the density of lamina propria lymphocyte-like cells.
The results of the current study show that spray-dried porcine plasma was able to modify intestinal morphology and intestinal-related immune cells in weaned pigs. These data provide further support that the immune system seems to be less activated in those piglets fed with spray-dried porcine plasma. Further, the plant extract mixture was also capable of modifying intestinal and systemic leukocyte cells, albeit in an inconsistent manner. Although the observed findings could be related to microbial modulating or antiinflammatory properties of these products, more specific studies are required to clarify the mechanisms whereby the products modify the pig intestinal morphology and immune system. In addition, this study suggests that the use of these products can be considered a valid alternative for some functions of antibiotic growth promoters being removed from use in some areas of the world.
|Soybean meal, 44% CP||40.0||40.0|
|Full-fat extruded soybeans||40.0||40.0|
|Low temperature fish meal||50.0||50.0|
|Sepiolite (a clay)||10.0||10.0|
|Choline chloride, 50%||2.0||2.0|
|Vitamin and mineral premix3||3.0||3.0|
|0 to 7 d postweaning|
|7 to 14 d postweaning|
|0 to 14 d postweaning|
|Goblet cells/100 enterocytes||3.1||2.2||3.5||0.86|
|Goblet cells/100 enterocytes||10.1||10.7||11.2||1.07|
|Goblet cells/100 enterocytes||5.9||4.5||6.1||0.85|
|Goblet cells/100 enterocytes||16.0||17.0||18.6||1.39|
|Goblet cells/100 enterocytes||9.1||10.5||10.1||0.96|
|Leukocyte count, thousands·μL−1||14.9||12.3||13.4||15.9||1||19.2||19.9||16.4||1||20.1||19.4||17.3||1.3|