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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Department of Animal Sciences, University of Illinois, Urbana 61801
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: immune • nursery • photoperiod • piglet • weaning
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Weaning age affects immune responses of piglets due partly to postnatal development of the immune system. Lymphocyte proliferation and lymphocyte numbers increase as a piglet grows older (Blecha et al., 1983; McCauley and Hartmann, 1984a,b), but there is no age-related change in neutrophil phagocytosis (Hoskinson et al., 1990).
Photoperiod manipulation influences immune function in several species. In hamsters, 6 h of light stimulated lymphocyte proliferation and numbers (Bilbo et al., 2002; Zhou et al., 2002), whereas 8 h increased bovine neutrophil chemotaxis (Auchtung et al., 2004). However, in hamsters there was no effect of photoperiod on innate immunity (Zhou et al., 2002). Pigs exposed to 18 h of light had increased leukocyte numbers, phagocytosis, lymphocyte proliferation, and immunoglobulin production in response to an antigenic challenge (Yurkov, 1985). Also, piglet immune responses (e.g., immunoglobulin G) were found to be affected in utero by manipulation of the sow’s photoperiod, at least until the piglets were 21 d of age (Niekamp et al., 2006). Furthermore, piglets kept on 23 h of light per day had greater ADFI and ADG due to improved metabolism of dietary energy and a reduced energy requirement for maintenance (Bruininx et al., 2002).
There is limited scientific information on the combined effects of weaning age and photoperiod on performance and immune function of piglets. The objective of this study was to evaluate immune status and performance of pigs weaned at 14, 21, and 28 d of age and the impacts of photoperiod manipulation on these measures through the nursery phase.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The University of Illinois Institutional Animal Care and Use Committee approved all experimental procedures involving animals.
Sixty-eight piglets were used from 12 second-parity white crossbred sows from the University of Illinois research farm. Experiments were conducted during March through August. Sows were kept in standard gestation stalls in 2 environmentally controlled rooms (3 sows per room). Each stall was equipped with a feeding trough and nipple waterer. The gestation rooms were constructed of solid concrete floors and had mechanical ventilation. Ventilation-fan hoods were fitted with light-baffling boxes to eliminate the effects of the natural outdoor photoperiod. Farrowing rooms were constructed similarly to the gestation rooms. Sows were kept in standard farrowing stalls with woven-wire floors. The room temperature was 23 ± 2°C. Heated mats were used postfarrowing to provide supplemental heat to the neonatal pigs.
Gestating and lactating sows were individually fed their respective diets, which were formulated to meet or exceed established nutrient allowances (NRC, 1998). During gestation, each sow was fed 2.5 kg/d of corn-soy-based diet containing a calculated 12.5% CP and providing a ME density calculated to provide 3,300 kcal/kg, as-fed. Lactating sows had ad libitum access to a corn-soy-based diet containing a calculated 16.7% CP and providing a ME density calculated to provide 3,426 kcal/kg, as-fed. Cleaning, feeding, and other chores were on the same fixed schedule in all rooms.
At d 83 of gestation, all sows were exposed to a 12 h of light:12 h of dark photoperiod regimen for 1 wk. Following this 1-wk adjustment period, sows were placed on a short-day photoperiod of 8 h of light/d (lights on at 0700 h and off at 1500). Supplemental light was provided by fluorescent tubes to achieve an intensity of 250 lux at the eye level of the sows. A tendency has been shown for a short-day photoperiod during late gestation to increase litter size and survivability of the piglets through lactation (Niekamp et al., 2006). Crossfostering did not occur among litters, but litter size was adjusted based on the dam’s teat number; extra piglets were removed from the study within 24 h after birth. Litter processing procedures, including ear notching, teeth clipping, tail docking, castration, and an iron dextrose injection, were all completed within 24 h after birth.
Litters were randomly assigned to a weaning-age treatment of 14, 21, or 28 d. At weaning, 6 piglets (3 barrows and 3 gilts) were randomly selected from each litter and placed in nursery pens of 3 littermates per group. The controlled-environment nursery had a perforated plastic floor and mechanical ventilation. Ventilation-fan hoods were fitted with light-baffling boxes to eliminate the effects of the natural outdoor photoperiod. Piglets were assigned at weaning to a short- or a long-day (lights on at 0700 h and off at 2300) photoperiod regimen, on which they remained until 10 wk of age. The numbers of piglets from each weaning age treatment that were assigned to a short or long day were (a) 14 d of age, n = 12 long day and 12 short day; (b) 21 d of age, n = 12 long day and 11 short day; and (c) 28 d of age, n = 11 long day and 10 short day. Supplemental light was provided with fluorescent bulbs to achieve an intensity of 250 lux at the piglets’ eye level. All animals had ad libitum access to a diet formulated to meet or exceed nutrient requirements (NRC, 1998), and each pen was equipped with a cup waterer.
Blood and Plasma Analyses
Each piglet was weighed and bled at weaning and again at 6, 8, and 10 wk of age. Piglets were held in a supine position, and blood samples collected via jugular vein puncture (the procedure lasted ~1 min). Average daily gain was calculated over 42 d from 4 to 10 wk of age.
Total immunoglobulin G (IgG) was measured using an ELISA as previously described by Morrow-Tesch et al. (1994), with minor modification. Porcine plasma samples were diluted 1:3,000 in 0.05% Tween-PBS. In duplicate, 120 µL of diluted sample or standard was added to 96-well microtiter plates coated with porcine IgG (Jackson Immunoresearch, West Grove, PA). Rabbit anti-pig IgG (120 µL; Sigma, St. Louis, MO) was added, and plates incubated for 2 h at 25°C and then washed 3 times with 0.05% Tween-PBS. Enzyme-linked antirabbit IgG (200 µL; Jackson Immunoresearch) was added at a dilution of 1:7,500. Plates were incubated for 1 h, decanted, and then washed 3 times. Substrate solution (200 µL; 1 mg of p-nitrophenyl phosphate/mL; Sigma) was added, and after a 30-min incubation, the reaction was stopped with 100 µL of 2 M NaOH. Plates were read using a microplate reader (BioTek Instruments, Winooski, VT) at wavelength 405 nm. A standard curve (0, 0.1, 0.5, 1, 5, 10, 20, and 40 µg of IgG/mL) was used to estimate total plasma IgG. Intra- and interassay CV were 6.1 and 16.5%, respectively.
Plasma cortisol was measured using a commercially available RIA kit (Coat-A-Count, Diagnostic Products, Los Angeles, CA). Intra- and interassay CV were 7.0 and 16.5%, respectively, and the minimal detectable concentration was 2 ng/mL. Prolactin was also measured by RIA using a specific antiporcine prolactin antibody in a modification of the assay previously described by Miller et al. (1999). The primary antibody (pPRL AFP-0842255Rb, NHPP and NIDDK, Torrance, CA) was diluted to 1:50,000 for a working solution, with a final tube dilution of 1:200,000. Porcine serum depressed binding in parallel to the standard curve. Mean intra- and interassay CV were 8.5 and 16.4%, respectively, and the assay sensitivity averaged 0.54 ng/mL.
Cell Isolation and Counting
Whole blood (10 µL) was added to Isoflow (10 mL; Beckman Coulter, Miami, FL), and red blood cells were lysed. Total white blood cell counts were measured electronically using a Coulter Z1 Particle Counter (Beckman Coulter). Differential smears were made and manually counted using a light microscope to determine the percentages of differential cell populations. Whole blood was diluted with Roswell Park Memorial Institute (RPMI) medium (Gibco, Carlsbad, CA), layered over Histopaque-1077 (density of 1.077 g/mL; Sigma) and -1119 (density of 1.119 g/mL; Sigma) and centrifuged at 700 x g for 30 min at 25°C. Lymphocytes were collected from the Hisptopaque-1077 layer, washed twice in RPMI, resuspended, and counted. Neutrophils and red blood cells were removed from the Histopaque-1119 layer and washed once in RPMI. Red blood cells were lysed using cold endotoxin-free water, and isotonicity was restored using 10x PBS. Neutrophils were centrifuged for 10 min at 475 x g, the supernatant was decanted, and the pellet was washed twice and resuspended in RPMI. Cell concentrations were appropriately adjusted with RPMI based on immune-assay requirements.
Immune Assays
The mitogen-induced lymphocyte proliferation assay was performed using a CellTiter 96 nonradioactive cell proliferation assay (Promega, Madison, WI) following the manufacturer’s protocol, with minor modifications. Briefly, porcine lymphocytes were adjusted to 5 x 106 cells/mL in RPMI plus 10% FBS (Sigma) and placed in triplicate into a sterile 96-well flat-bottom plate. The mitogens concanavalin A (Sigma) and lipopolysaccharide (Sigma) were added at 0, 25, and 50 µg/mL to stimulate T- and B-cells, respectively. The plates were incubated for 72 h at 37°C under 5% CO2 in a humidified incubator. Twenty microliters of MTT [3-(4, 5-dimethyl-thiazol-2yl)-2, 5-diphenyl tetrazolium bromide; Sigma] were added to each well, and the plates were incubated for 4 h. Acidified isopropanol (100 µL of 0.1 N HCl in anhydrous isopropanol) was added, and the plates were incubated overnight at 37°C and then read using a microplate reader (BioTek Instruments) at a wavelength of 550 nm with a reference wavelength of 690 nm. The results were expressed as a proliferation index (PI):
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The ability of neutrophils to migrate toward the assay medium (control; random migration), recombinant human complement-5a (hC5a; Sigma), or recombinant porcine (p) IL-8 (Sigma; chemotaxis; directed migration) was measured using an assay previously described by Salak et al. (1993). Briefly, medium, hC5a (10–7 M), and pIL-8 (100 µg/mL) were added to the bottom wells of a 48-well microchemotaxis chamber (Neuro Probe, Gaithersburg, MD), in duplicate, and neutrophils that were adjusted to 3 x 106 cells/mL in RPMI were added to the top wells of the chamber and incubated for 45 min at 37°C. The polyvinylpyrrolidone-free filter (pore size, 5 µm; Neuro Probe) was fixed and stained using the Hema-3 system (Fisher Scientific, Houston, TX). Four fields per well were counted, using a light microscope and 100x magnification, by an individual unaware of treatment groups.
Neutrophil phagocytosis was measured using a flow cytometry-based assay as previously described by Jolie et al. (1997), with a minor modification. Briefly, neutrophils were adjusted in RPMI to a cell concentration of 2 x 106 cells/mL. Fluorescent beads (yellow-green, 1.0 µm; Molecular Probes, Eugene, OR) were preincubated for 30 min with nonheat-inactivated porcine serum and then added to the samples at a 10:1 ratio (beads:cells). Cells and beads were incubated together for 45 min at 37°C on a rotator, then centrifuged for 5 min at 1,000 x g. Samples were washed once in 1 mL of RPMI, decanted, resuspended in 1 mL of RPMI, and fixed in 4% paraformaldehyde. Samples were protected from light and held at 4°C until analyzed. The engulfment of beads by the cells was determined using an XL flow cytometer (Beckman Coulter). Data were transformed logarithmically, and the results were expressed as the total percentage of neutrophils engulfing 1 or more beads.
Natural killer cytotoxicity (NK) was measured using a commercially available nonradioactive cytotoxicity detection kit (Roche Diagnostics, Indianapolis, IN), as previously described by Sutherland et al. (2005). Briefly, porcine lymphocytes were used as effector cells, and K-562 chronic human myelogenous leukemia cells (American Tissue Type Culture Collection, Manassas, VA) were used as target cells. Lymphocytes were adjusted to 1 x 107 cells/mL and K562 cells adjusted to a constant 10,000 cells per well. Samples were run in triplicate at effector- (lymphocytes) to target- (K-562) cell ratios of 12.5:1, 25:1, 50:1, and 100:1. Plates were read using a microplate reader (BioTek Instruments) at a wavelength of 490 nm and a reference wavelength of 690 nm. Percent cytotoxicity was calculated as described by Lumpkin and McGlone (1992), and the assay was considered valid if maximal release divided by spontaneous release was 
20%.
Statistical Analysis
Statistical analyses were performed using SAS (SAS Inst. Inc., Cary, NC). All traits were tested for departures from a normal distribution. Natural logarithmic transformation was applied to all traits deviating from a normal distribution. A linear, mixed-effects model was used to analyze piglet variables across weaning ages, piglet variables repeated through the nursery phase, and the effects of light treatment and weaning age on piglet BW. Pig was the experimental unit. Main fixed effects were treatment, age, sex, and their interactions. Values from the previous experimental time point were used as respective covariates. The model had a repeated structure on age, allowing accommodation of heterogeneity of variances across ages. Significance was set at P 0.05, and trends were discussed at P 0.10.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The age of the pig at weaning significantly influenced leukocyte populations and various immune measures (Tables 1 and 2). Piglets weaned at 28 d had lower (P < 0.001) neutrophil counts than did pigs weaned at 14 or 21 d of age (Table 1). Likewise, percentages of neutrophils were lowest (P = 0.008; Table 1) in piglets weaned at 28 d of age; however, these same pigs had greatest percentage of lymphocytes (P = 0.002), resulting in a reduced (P = 0.005) N:L ratio compared with piglets weaned at 14 d (Table 1). Piglets weaned at 14 and 21 d also had greater (P = 0.006) percentage of monocytes relative to those weaned at 28 d. Total plasma IgG concentration and lipopolysaccharide-induced (50 µg/mL) proliferation were greater (P < 0.05) in piglets weaned at 14 d relative to those weaned at 21 or 28 d of age (Table 2). In contrast, piglets weaned at 21 or 28 d had greater (P < 0.001) NK activity than piglets weaned at 14 d (Table 2). Weaning age did not affect neutrophils chemotaxis or phagocytosis, cortisol, or prolactin concentrations (P > 0.20).
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Age affected all variables measured during the nursery phase. Weaning age and photoperiod treatments affected cortisol, neutrophil phagocytosis, and lymphocyte counts (Tables 3 and 4). Plasma cortisol concentration was greater (P = 0.003) and neutrophil phagocytosis was less (P = 0.009) in piglets weaned at 28 d and kept on long day than those in any other treatment group (Table 3). Phagocytosis was greatest (P < 0.05) in those short-day piglets that had been weaned at 14 d compared with pigs in all other treatment groups (Table 3). Total lymphocyte count was greater (P = 0.007) in piglets weaned at 28 d and kept on long day but less in long-day piglets that had been weaned at 21 d (Table 4). Photoperiod had no effect on chemotaxis, lymphocyte proliferation, NK cytotoxicity, IgG concentration, or prolactin concentration.
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). By 8 wk of age, the piglets weaned at 28 d had a lower (P < 0.05) percentage of neutrophils regardless of photoperiod treatment (Table 5). Similarly, piglets weaned at 28 d and regardless of light treatment had reduced (P = 0.02) N:L ratio at 8 wk of age than did those in all other treatment combinations (Table 4
). During the nursery phase, total plasma IgG concentration was less (P = 0.05) in piglets weaned at 28 d and kept on long-day photoperiod regimen (Table 3). Neutrophil chemotaxis, NK cytotoxicity, and prolactin concentration were similar regardless of photoperiod treatment and weaning age.
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Both photoperiod and weaning age affected pig ADG in the nursery phase (Figure 1). Piglets kept on long day regimen had greater (P = 0.001) ADG than did those on short day (Figure 1A). Piglets weaned at 28 d had greater (P < 0.001) BW gain per day compared with pigs weaned at 14 or 21 d (Figure 1B). Overall, both photoperiod and weaning age influenced pig BW during the nursery phase (P = 0.05; Figure 2). At 6 wk of age, piglets weaned at 28 d and kept on long day had greatest BW relative to those in other treatment groups. By 8 wk of age, BW was similar among treatment groups, except that piglets weaned at 14 d and kept on short-day photoperiod continued to have the lowest BW of all treatment groups. Finally, at 10 wk of age, 28-d-weaned, long-day piglets remained heaviest. Those weaned at 14 d and kept on short-day photoperiod were the lightest.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Complete development usually occurs at approximately 1 mo of age (McCauley and Hartmann, 1984a,b), as confirmed in our study. As the piglet aged, total lymphocyte counts increased, whereas neutrophil counts, total IgG concentrations, and mitogen-induced B-cell proliferation all decreased. These results are similar to previously reported findings (Blecha et al. 1983; McCauley and Hartmann, 1984a; Hoskinson et al., 1998). Additionally, NK cytotoxicity increased as the pigs got older. Cytolytic activity of NK cells toward K562 cells is absent in pigs until approximately 1 wk of age (Huh et al., 1981; Yang and Schultz, 1986) and may take 2 wk or more to fully develop (Charley et al., 1985). This may account for the results found here; piglets weaned at 14 d had less NK cytotoxicity than those weaned at 21 or 28 d. The shift in leukocyte cell populations and reduction in total IgG and B-cell proliferation as weaning age increased reflects the stages of development of the innate immune responses in piglets ranging in age from 14 to 28 d. More specifically, it is possible that this increase in total IgG among pigs weaned at 14 d of age is reflective of passive immunity obtained from the sow. Because piglets weaned at an older age apparently have a more developed immune system, they may be more efficient at mounting an adequate immune response when challenged with an antigen from the environment at weaning.
Additionally, some aspects of the immune system of the pig continued to differ postweaning through their residence in the nursery. This finding is similar to that reported by Sutherland et al. (2005), in which baseline immune responses varied with age, implying continued differential development of several aspects of the immune system. Here, not only did weaning age affect immune function during the nursery phase, but photoperiodic treatment also altered this response. Specifically, N:L ratio, neutrophil phagocytosis, and total plasma IgG concentrations were elevated in piglets weaned at 14 d and kept on a long day. Piglets weaned at 14 d of age appeared to have a more active innate immune response than did those weaned at 28 d, and therefore they would have been required to divert biological reserves (e.g., energy) toward metabolism and away from other biological functions, such as growth, to maintain that immune response (Moberg, 2000). Conversely, the greatest cortisol concentration was observed in piglets weaned at 28 d and kept on long-day photoperiod. Moreover, piglets weaned at 28 d and kept on a long day had lower percent neutrophils as well as lower neutrophil phagocytosis, perhaps implying that 28-d weaned piglets had a less activated innate immune system than did 14-d weaned pigs. It is possible that the lower immune responses observed in these 28-d weaned pigs compared with those weaned at 14 d resulted from greater basal concentration of cortisol, which could have had an immunosuppressive effect on phagocytosis. If so, then piglets weaned at 14 d have a more stimulated innate immune response and may divert their energy resources to maintain their activated immune status, whereas piglets weaned at 28 d and kept on the long-day photoperiod regimen might have had more energy available to divert for growth.
Although piglets weaned at 28 d and kept on a long day may have less stimulated innate immune responses than those in other treatment groups, they did have the greatest ADG from 6 to 10 wk of age. Perhaps these piglets were able to use more ME for growth rather than having to divert it for use in maintaining baseline immune function and regulation. Another possibility is that ADG was enhanced by increasing daily photoperiod to 16 h. Bruininx et al. (2002) reported that 23 h of light per day increased ADFI and decreased the energy requirement for maintenance. In our study, we neither measured ADFI nor observed feeding behavior. It is possible that longer days increased number of feeding bouts. Moreover, piglets weaned at 14 d and kept on 8 h of light daily lagged in BW and ADG in the nursery, suggesting that these piglets consumed less feed or diverted less ME away from growth in order to maintain their immune response and thus continued to be at a disadvantage. Photoperiod and weaning age can affect both immune status and piglet BW and BW gain through at least 10 wk of age. The piglets that were most immunologically developed at weaning and had lower neutrophil phagocytosis, total IgG concentrations, and leukocyte numbers throughout the nursery were at an advantage for increased growth, presumably resulting in healthier, heavier pigs into the grow phase. Conversely, piglets weaned at 14 d and kept on the short-day photoperiodic regimen from birth through the nursery phase had greater phagocytosis, total IgG concentrations, and leukocyte numbers, which may have been reflected in diversion of energy from growth to immune system support. Support of continued immune development postweaning may be more costly in terms of a biological cost to the piglet that is weaned at 14 d of age compared with a piglet weaned at 28 d. Moreover, a piglet weaned at 14 d and kept on a short day apparently is at a greater disadvantage relative to its long day counterpart because at the end of the 10-wk period it still lagged in BW. More importantly, it is unknown if the results we observed here were further influenced by the direction of change from a short-day photoperiod preweaning to long-day photoperiod that some piglets experienced postweaning. More research would be required to evaluate this as well as identify the potential mechanism(s) responsible for any photoperiodic effects on growth and energy partitioning that may have caused the piglet responses found here.
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Footnotes |
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2 Corresponding author: johnso17{at}uiuc.edu
Received for publication March 17, 2006. Accepted for publication August 2, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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