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Virginia Polytechnic Institute and State University, Blacksburg 24061
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: copper • intestinal morphology • performance • pig • sanitation • spray-dried plasma
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), and commercial application in the United States is substantial, particularly in weanling and starter pigs (NAHMS, 2002). However, the practice has become increasingly controversial (Phillips et al., 2004) and was recently banned in the European Union (Pradella, 2006). Of particular concern is the development of resistant microbial populations that could potentially have a negative impact on antibiotic treatment of disease in humans and animals.
Two dietary alterations that elicit growth performance responses in weanling pigs similar to those seen with antibiotic growth promoters include addition of elevated levels of Cu (Cromwell et al., 1989; Davis et al., 2002) and spray-dried plasma protein (SDPP; reviewed by van Dijk et al., 2001) Furthermore, poor sanitation conditions in the environment in which weanling pigs are reared can have a negative impact on performance (Bassaganya-Riera et al., 2001) and may influence the response to dietary addition of Cu or SDPP. For example, Coffey and Cromwell (1995) reported that the response of weanling pigs to SDPP was more pronounced in a conventional on-farm nursery than in an experimental nursery and suggested that differences in sanitation and subclinical pathogen exposure between locations were involved in the variable response. Effects of dietary Cu (Stahly et al., 1980) or SDPP (Coffey and Cromwell, 1995) are considered independent of and potentially additive with subtherapeutic antibiotics. The potential exists for dietary Cu and SDPP to have additive responses as well.
Considering these factors, the objective of this study was to determine the effects of including elevated Cu (200 ppm) and SDPP (6%) in nonmedicated diets, individually and in combination, on growth performance and intestinal morphology of weanling pigs housed under sanitary or nonsanitary conditions.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Before initiation of the experiment, the protocol was approved by the Virginia Tech Animal Care Committee. The study was conducted at the Virginia Tech Tidewater Agricultural Research and Extension Center Swine Unit in Suffolk, VA. The nursery facility at the unit included 2 adjacent, recently remodeled pig nursery rooms. Each room provided 24 identical pens (0.91 x 1.22 m) for the study with a nipple drinker and a stainless steel feeder, with 4 feeding spaces in each pen. Pen flooring was constructed of triangular metal bars over shallow manure collection pits. The heating and negative pressure ventilation equipment was also identical for each of the 2 rooms. Controls were set to maintain a temperature of 27 ± 1°C during the first week followed by a gradual decline to 24°C by the end of the second week and 22°C by the end of the 35-d nursery period. Throughout the study, the nursery rooms were continuously illuminated by fluorescent lighting.
Animals and Treatments
Crossbred weanling pigs (n = 192, 6.02 ± 0.2 kg of initial BW) produced from AI matings of terminal line Duroc boars and Yorkshire x Landrace sows were used in the study. The treatments were assigned according to a 2 x 2 x 2 factorial arrangement. This factorial arrangement included 2 levels of added dietary Cu (0 and 200 ppm), 2 levels of SDPP (0 and 6%), and 2 nursery sanitation conditions (manure application with no cleaning or disinfection of a previously populated room, or thorough cleaning and disinfection of an identical previously unpopulated room). There were 48 pens in the study, with 4 pigs housed in each pen. Therefore, there were a total of 6 replicate pens for each of the 8 Cu, SDPP, and sanitation environment treatment combinations. The sex ratio in each pen was 2 females and 2 castrated males.
The source of elevated supplemental Cu was a commercial Cu proteinate complex (Bioplex Cu, Alltech Inc., Nicholasville, KY), and the SDPP was a granular commercial product (Appetein, APC Inc., Ankeny, IA). To create the sanitary and nonsanitary pen conditions, the following procedures were performed. Two weeks before the experiment, healthy appearing pigs from another facility at the farm were moved into and housed temporarily in the 24 pens of the designated nonsanitary nursery room. Additionally, on d 11, 8, and 3 before the experiment, liquid manure slurry from healthy sows and finisher pigs was applied to the nonsanitary pen surfaces using a broom. In an identical adjacent nursery room providing 24 sanitary pens, no pigs had been housed and no manure was applied. Five days before initiation, pens in the sanitary room were washed with high-pressure water followed by application of a livestock premises disinfectant cleaner (Tek-Trol, BioTek, Atlanta, GA). The room and pens were then allowed to dry before the experiment. Pigs were blocked and assigned to treatment pens to minimize confounding effects, but the design was established with the recognition that the influence of individual nursery room and pen sanitation treatment could not be definitively separated because sanitary and nonsanitary conditions could not be maintained in the same nursery room.
Nursing piglets were weighed on the afternoon before the beginning of the experiment, weaned, and moved to their assigned pens the following morning. The weaning age was 18 ± 2 d, and pigs were blocked to balance the initial BW, litter of origin, and sex and, within a block, were randomly assigned to treatment pens. Once in the nursery, the pigs were individually weighed in accordance with diet phase formulation changes on d 10 and 20 and at the conclusion of the feeding period at d 35. Pen feed consumption was measured as feed disappearance and was determined when the pigs were weighed.
Experimental Diets
Complex, phase I nursery diets (Table 1) were formulated to have similar nutrient and ME concentrations for all treatments and to meet or exceed nutritional requirements for weanling pigs (NRC, 1998). To assure consistent dietary formulation across treatments, the common ingredients for each experimental diet were prepared as a single basal diet, and each experimental diet was then prepared from this common basal diet. The phase I experimental diets were fed for the initial 10 d after weaning. Diets were reformulated as a phase II diet that was fed from d 11 to 20 and a phase III diet that was fed from d 21 to 35 (Table 2). The SDPP treatments were discontinued after phase I, similar to standard commercial practice. The elevated Cu treatments were continued through diet phases II and III to the conclusion of the 35-d nursery period. All diets were prepared and fed in meal form. Corn grain used in preparation of the phase I diets was ground to pass through a 3.2-mm hammer-mill screen; for phase II and III diets, corn grain was ground to pass through a 4.8-mm screen.
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On d 10, blood samples were taken from each pig for determination of plasma urea N (PUN) as an assessment of the efficiency of dietary protein utilization for protein accretion. Samples were collected by jugular venipuncture into vacuum tubes containing 0.117 mL of a 15% solution of EDTA (Becton Dickinson Co., Franklin Lakes, NJ). The plasma was harvested after centrifugation and stored at –20°C until assayed. An assay kit (Sterling Diagnostics Inc., Sterling Heights, MI) was used for PUN determination. Briefly, the procedure is based on hydrolyzation of urea by urease to produce ammonia and carbon dioxide. The ammmonia reacts with salicylate, nitroferricyanide, and an alkaline solution of hypochloride to yield a blue-green chromophore. The chromophore is measured photometrically and is proportional to the amount of urea in the plasma (Kaplan and Teng, 1982).
Also on d 10, a female pig identified as closest in BW to the average within each pen was selected for intestinal morphology assessment. The pig was humanely killed by i.v. injection of sodium pentobarbital (80 mg/kg of BW). The small intestine was dissected free of the mesentery and arranged in measured lengths on a chilled stainless steel tray. Three 10-cm cross-sectional segments were cut at points 25, 50, and 75% of the total intestinal length to represent samples for duodenum, jejunum, and ileum, respectively. The segments were gently rinsed in ice-cold phosphate buffered saline, cut into two 5-cm segments and fixed in 10% neutral buffered formalin. The tissue segments were transported to a commercial laboratory (HISTO Scientific Research Laboratories, Woodstock, VA) for embedding in paraffin, slide preparation, and staining. Two cross-sections of each intestinal segment were processed, embedded in paraffin, and stained with hematoxylin and eosin. Villous height and crypt depth were measured using a digitized board coupled to a video monitor receiving output from a video camera mounted on a binocular microscope (Olympus Polaroid DMC-IE camera, Polaroid Corp., Waltham, MA). Output from the digitizing board was collected using the program SigmaScan Pro 5. (SPSS Inc., Chicago, IL). Six long and straight villi and their associated crypts were chosen and measured in duplicate for each section. The villous height was measured from the tip to the base, and crypt depth was measured from the base of the villus to the base of the crypt. The villous height to crypt depth ratio (VCR) was also calculated. Mean villous height, crypt depth, and VCR for each pig (12 total measurements for each segment) were calculated for statistical analysis.
Statistical Analysis
Data were analyzed using the GLM procedure (SAS Inst. Inc., Cary, NC). The experiment was a completely randomized design with a 2 x 2 x 2 factorial arrangement of treatments. The model included the main effects of dietary Cu, SDPP, and pen sanitation condition and all appropriate 2-way interactions. The 3-way interaction was removed from the model once it was determined to be nonsignificant (P > 0.10). Pen served as the experimental unit. Response criteria included ADG, ADFI, G:F, and PUN. Intestinal morphology variables included villous height, crypt depth, and VCR. The intestinal morphology measures were analyzed separately for each intestinal segment.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Subjective observations indicated that pigs in certain pens displayed mild diarrhea during the second week after weaning. This was noted in 9 of 24 pens in the nonsanitary room and in 1 of 24 pens in the sanitary room, with no apparent pattern across diet treatments. However, throughout the study there were no mortalities, and no pigs needed to be removed for poor performance or health reasons. Growth performance data are presented as 2-way treatment means in Table 3. During the initial 10-d postweaning (phase I), there were pronounced improvements (P < 0.003) in ADG and ADFI as a result of SDPP or Cu addition to the diet. Because gain and feed intake increased concomitantly, there were no effects of SDPP (P = 0.22) or Cu (P = 0.14) on G:F. Conditional on the potential for effects of separate rooms, there was a trend for pigs reared in the unsanitary pens to grow at a slower rate than those in sanitary pens (P < 0.10). A tendency for interaction (P = 0.07) was observed between SDPP and pen sanitation for G:F during phase I in which the response to SDPP was numerically greater in unsanitary pens than in sanitary pens. However, the interaction of elevated dietary Cu and pen sanitation condition for G:F during phase I was not significant (P = 0.11).
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; P > 0.13). Likewise there were no significant (P > 0.45) 2-way interactions during phase II. During phase III (d 21 to 35) pigs fed diets with elevated Cu had slightly reduced (P = 0.04) ADFI. Because ADG was not reduced by the same magnitude, Cu-supplemented pigs tended to have a better G:F (P = 0.08). Pigs fed SDPP-supplemented diets during phase I tended (P = 0.09) to have greater ADFI during phase III. There were no interactions between Cu and SDPP supplementation for all growth performance traits during any discreet diet phase or throughout the total experiment (P > 0.30). Furthermore, during the overall study period, there were generally no main effects of diet treatment on growth performance. Including SDPP in the diet during phase I tended to result in greater ADFI (P < 0.10) for the overall study.
Plasma Urea Nitrogen
Simple 2-way means of PUN concentrations determined from samples taken on d 10 ranged from 11.5 to 13.0 mg/dL (Table 4). There were no main or treatment interaction effects of SDPP, elevated dietary Cu, or pen sanitation condition on PUN (P > 0.36).
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All intestinal morphology measurements were collected and analyzed separately for each segment (duodenum, jejunum, ileum); no major segment effect or interaction was observed. The method of selecting the female pig within each pen that was closest to the average pig BW in the pen was chosen to reduce variation in morphology associated with BW variation. No regression relationships (P = 0.14) between BW and villous height or BW and crypt depth were detected (data not presented). Therefore, BW was not considered as a covariate when analyzing the histology data.
Conditional on the influence of separate rooms, pen sanitation affected intestinal morphology (Table 4). More pronounced effects were observed in the duodenum, but differences in the jejunum and ileum were also observed. Pigs housed in nonsanitary pens had shorter villous height and shallower crypts (P < 0.05) than those in sanitized pens. Because villous height and crypt depth were decreased to a similar extent in the pigs raised in the nonsanitary environment, the VCR was not affected (P > 0.58) by sanitation treatment. Supplementing the diet with SDPP tended to increase (P = 0.09) villous height in the duodenum (Table 4), but not in the jejunum and ileum. Pigs fed elevated dietary Cu tended (P = 0.10) to have longer villi than the control group in the jejunum. Copper supplementation decreased the duodenal crypt depth and increased (P < 0.05) VCR in the duodenum and jejunum as a result of increased villous height and decreased crypt depth.
Trends (P = 0.08) for interaction between pen sanitation and SDPP were observed for crypt depth and VCR. This resulted from a numerical increase in crypt depth with SDPP supplementation only under the unsanitary pen condition and a numerical increase in VCR with SDPP only under the sanitary pen condition (Table 4). Significant interactions were observed (P < 0.05) between SDPP and Cu supplementation for VCR in the duodenum and ileum. For example, adding Cu to the diet without SDPP resulted in a slight numerical decrease in ileum VCR, whereas adding Cu to the diet with SDPP resulted in a numerical increase in ileum VCR (Table 4).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The purpose of this study was to assess 2 readily available dietary modifications to nonmedicated diets and the response of weanling pigs to these dietary supplements under variable sanitation conditions in the initial rearing environment. With the model used, the potential effects of different rooms cannot be completely separated from pen sanitation treatment. However, the identical design of the newly constructed adjacent nursery rooms and the blocking procedure that placed diet treatments within replications across rooms were employed to minimize potential confounding effects. A similar experimental model with weanling pigs was employed by Bassaganya-Riera et al. (2001), but as in this experiment, the potential impact of having sanitation treatments separated by individual rooms must be considered in interpretation of the data. Nevertheless, our results are in general agreement with that study in that during the initial 10 d postweaning, pigs reared in the nonsanitary pens grew at a slower rate than pigs in the sanitized pens, due in part to numerically reduced feed consumption. The principle that pigs perform better in more sanitary all-in-all-out production systems than in facilities managed on a less sanitary or continuous flow basis has practical and research support (Williams et al., 1997; Ice et al., 1999; de Grau et al., 2005). The mechanism with the most support for this response is that immune system stimulation in growing pigs, through environmental exposure or direct challenge, results in temporal release of proinflammatory cytokines and a concomitant reduction in feed consumption and growth (Johnson, 1997; Webel et al., 1997; Williams et al., 1997). In our study the reduction in growth performance was small and occurred only during the initial 10 d after weaning; whereas in the study by Bassaganya-Riera et al. (2001), pigs reared in the nonsanitary environment had reduced growth performance throughout a cumulative 7-wk period. Variable levels of response of weaned pigs to an unclean environment would not be unusual considering that microbial populations, lack of or degree of pathogen exposure, pig genetics, health status, and other factors are inherently variable.
Plasma urea N has been used as a measure of dietary protein utilization (Eggum, 1970), but in this study neither pen sanitation nor dietary treatments had any influence on PUN concentration. Bassaganya-Riera et al. (2001) reported lower postweaning PUN concentration for pigs housed in a nonsanitary environment and attributed the response to reduced feed intake associated with the nonsanitary treatment. In the current study, sanitation effects on feed intake may not have been severe enough to alter PUN.
Negative alterations in intestinal morphology of pigs occur during the initial postweaning period including reduced villous height and, to a lesser extent, reduced crypt depth. Reduced food intake after weaning is considered a major contributing factor to this condition (Cera et al., 1988; Pluske et al., 1996). In this study, rearing pigs in nonsanitary rather than sanitary pens resulted in profound reductions in villous height and crypt depth 10 d after weaning in each segment of the intestinal epithelium. Numerical reductions in feed intake were observed during this period in the nonsanitary pens and perhaps were severe enough to contribute to alterations in morphology. However, other factors associated with the nonsanitary environment or the influence of separate rooms that cannot be determined from these data may also have been involved.
Dietary inclusion of SDPP improves postweaning growth performance of pigs (Kats et al., 1994; Angulo and Cubilo, 1998; Grinstead et al., 2000), although the response appears greater for pigs weaned at younger ages. The improvement in growth rate is associated primarily with improved feed intake during the initial postweaning period (Hansen et al., 1993; de Rodas et al., 1995), but improvement in G:F has been reported (Gatnau and Zimmerman, 1991). Multiple regression analysis of 68 experiments indicated that dietary addition of up to 6% SDPP induced improvements in ADG, ADFI, and G:F during the initial 2 wk after weaning of 26.8, 24.5, and 3.2 %, respectively (van Dijk et al., 2001).
In the current study, dietary inclusion of SDPP during the initial 10 d after weaning produced improvements in ADG and in ADFI with no significant effect on G:F. Coffey and Cromwell (1995) reported that positive growth and feed intake responses in weanling pigs to SDPP were more pronounced in a conventional on-farm nursery than in an experimental nursery and suggested that differences in sanitation and microbial exposure accounted for this observation. Based on this and the potential immunological properties of SDPP, we hypothesized that postweaning response would vary in identical on-site nursery rooms with altered initial sanitation conditions. Our results are not contrary to those of Coffey and Cromwell (1995) in that growth and feed intake responses to SDPP were numerically greater in the unsanitary environment. However, potential interactions of pen sanitation with dietary treatment for growth performance traits were limited to a trend in which SDPP supplementation numerically improved G:F under the nonsanitary pen condition but produced no improvement under the sanitary condition.
Improved diet palatability (Ermer et al., 1994) and protein utilization (Jiang et al., 2000a) have been identified as contributing factors for improved postweaning performance with SDPP, but some studies indicate that immunological properties of SDPP is the principle mechanism of action. Immunoglobulins make up 15 to 20% of SDPP (Thomson et al., 1994), and earlier reports provide strong evidence that a component or components of this fraction stimulated growth as effectively as the intact product (Gatnau et al., 1995; Owen et al., 1995; Weaver et al., 1995). More recently Pierce et al. (2005) published results of a series of experiments with early weaned pigs in which the immunoglobulin-G fraction of plasma protein from porcine and bovine sources was identified as the component responsible for improved postweaning performance. Providing a source of immunoglobulin-G to weanling pigs via SDPP under nonpathogenic or subclinical conditions appears to modulate innate immune system activation because supplementation with SDPP reduces expression of pro-inflammatory cytokines such as tumor necrosis factor-
, interleukin-1-ß, and interleukin-6 (Touchette et al., 2002). In the current study, poor sanitation was associated with negative effects on intestinal morphology; however, there were no effects of SDPP on intestinal morphology (P > 0.05). Nevertheless duodenal villous height (P = 0.09) and VCR (P = 0.10) tended to be greater with SDPP supplementation. Other studies with weanling pigs demonstrated increased intestinal villous height and VCR with SDPP supplementation (Spencer et al., 1997; Owusu-Asiedu et al., 2003), but this response has not been consistent in all cases (Jiang et al., 2000b; Touchette et al., 2002).
Adding pharmacological levels (100 to 250 ppm) of Cu to the diet has a growth-stimulating effect in weanling pigs (Stahly et al., 1980; Cromwell et al., 1989; Davis et al., 2002). The most common source of added Cu in earlier studies was Cu sulfate. For this study, a commercial complex of Cu with amino acids and short-chain peptides (Waldroup et al., 2003) was used because some data indicate that organic Cu complexes may be more bioavailable and efficacious than inorganic Cu sources (Coffey et al., 1994; Harper et al., 2001). During the initial 10 d after weaning, the main effect of elevated dietary Cu on growth performance was similar to the main effects observed with SDPP, with significant increases in ADG and ADFI.
When a growth promoting level of Cu is used under commercial conditions, supplementation is typically continued throughout the starter period. This is in contrast to strategic use of SDPP, which is typically included only in phase I postweaning diets due to the contribution of this ingredient to overall diet cost and reduced responses in later phases. The response to elevated dietary Cu is typically maintained for the entire 4 to 5 wk starter period (Stahly et al., 1980; Coffey et al., 1994; Davis et al., 2002), but in this study, the Cu effect was statistically significant only during phase I (initial 10 d). Because there was no significant interaction of SDPP and Cu for performance variables measured, the supplements appeared to act independently; and during phase I, inclusion of both supplements produced numerically additive responses in growth (Table 3). During this period pigs fed diets supplemented with SDPP and elevated Cu consumed more feed and grew faster and more efficiently than those fed diets without either supplement.
It was originally proposed that the mode of action of Cu is through an antibacterial-like action in the gastrointestinal tract (Fuller et al., 1960). More recent evidence suggests that Cu may also act systemically because i.v. treatment of Cu in weanling pigs had similar effects as elevated dietary Cu (Zhou et al., 1994). The intestinal morphology results of this study suggest that growth stimulation with elevated dietary Cu may, in part, be related to its impact on the intestine. Pigs fed high Cu levels had less crypt depth (P = 0.005 in duodenum) and numerically greater villous length resulting in greater VCR in the duodenum and jejunum. Crypt depth gives a general indication of the rate of crypt cell production (Hampson, 1986). When crypt cells migrate, they become increasingly differentiated and their absorptive capacity develops. Therefore, measurement of crypt depth gives a general indication of the maturity and functional capacity of enterocytes. Increased VCR generally equates to more absorptive area, and fewer epithelial cells are recruited for cell renewal. The energy required to maintain the gastrointestinal tract has been estimated to be approximately 24% of the pig’s daily maintenance energy requirements (Yen et al., 1988). Observations of this study provide additional support to the hypothesis that high dietary Cu may decrease the energy requirements of the gastrointestinal tract and consequently decrease the maintenance energy requirement of the pigs, making more energy and nutrients available for growth (Radechi et al., 1992).
In summary these data are supportive of the concept that cleaning and sanitation between pig groups is beneficial to postweaning performance, but this conclusion must be considered conditional on possible influences of the separate nursery rooms used to create the sanitation treatments in our protocol. Furthermore, under production conditions, which may require use of diets free of antimicrobial growth promoters, strategic use of SDPP, and elevated dietary Cu, alone or in combination, can ameliorate reduction in performance typically seen during the early postweaning period. In terms of growth performance, these dietary alterations act independently and therefore offer potential for additive responses in weanling pigs.
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Footnotes |
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2 Corresponding author: alharper{at}vt.edu
Received for publication July 5, 2006. Accepted for publication January 20, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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