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PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION |
* Animal Science Department, California Polytechnic State University, San Luis Obispo, CA 93407; and Foster Poultry Farms, Modesto, CA, 95357
1 Corresponding author: ekoutsos{at}calpoly.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: turkey • breeder • intestinal histology • inflammation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immune challenge can also directly affect the physiology and performance of growing animals. When an animal faces an immune challenge, nutrients are diverted from anabolic to catabolic functions (Bistrian et al., 1992), and nutrient metabolism is altered for virtually every nutrient studied (Koutsos and Klasing, 2001). Immune challenges also directly affect the intestinal intrinsic primary afferent neurons that regulate villi movement, muscular contractions, luminal secretions, and blood flow (Furness et al., 2004). Therefore, an immune response can reduce gut motility, disrupt nutrient absorption and metabolism, allow for bacterial overgrowth, and increase morbidity and mortality (Woosley, 2004). Optimization of immune responses, in which responses occur when necessary to prevent disease but are minimized during subclinical challenge, will maximize performance in a production setting. However, characterization of the immune response of birds must take place before modulation of the immune system should be attempted.
The purpose of this trial was to examine the effects of turkey breeder hen age and immune challenge on performance, intestinal physiology, and systemic inflammatory responses of female turkey poults grown to market weight. Lipopolysaccharide (LPS) was used to induce the inflammatory immune response, which was then assessed with plasma haptoglobin and plasma mineral levels. Haptoglobin is an acute phase protein produced by the liver during an immune challenge, and it plays a role in hemolysis and binding of free hemoglobin for hepatic sequestration (Dobryszycka, 1997). Plasma minerals were also examined, because partitioning of some minerals is significantly altered during an inflammatory response. For example, inflammatory responses generally result in reduced plasma Fe, which is a limiting nutrient for invading organisms (Weinberg, 1978).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Inflammatory Immune Response and Intestinal Histology
On d 10, 24, and 65 posthatch, 1 bird/pen was vaccinated with LPS from Salmonella Typhimurium (0.5 mg/ kg of BW) intraabdominally. All vaccinations were administered by trained personnel under supervision, to ensure accuracy of administration. Saline injection does not induce an inflammatory response, thus the control group was not injected (Laurin and Klasing, 1987). At 24 h postadministration, the LPS-vaccinated bird and 1 unvaccinated (control) bird were sampled for blood via cardiac (d 11 and 25 posthatch) and wing (d 66 posthatch) puncture. Plasma was isolated by centrifugation, and then plasma haptoglobin levels (determined using a commercial kit) were used to assess inflammatory immune response. Additionally, plasma from d 11 and 66 posthatch were analyzed for P, K, Ca, Mg, S, Na, Fe, Zn, and Cu by infected-cell polypeptide analysis.
Following blood collection, all birds were euthanized by cervical dislocation, and intestine samples were taken. Duodenum, jejunum, and ileum samples were isolated, washed with cold PBS, rinsed with formalin, cut (2 cm), and placed in 50 mL of 10% formalin for embedding and hematoxylin and eosin staining. Intestinal segments were taken from the midpoint of the duodenum (duodenum), from the midpoint of the section cut to the Meckel’s diverticulum (jejunum), and the midpoint of the Meckel’s diverticulum to the ileocecal junction (ileum). Histological analyses were completed using a light microscope, and all tissues were examined by the same individual for villus length (distance from the top of the villus to the bottom of the villus), villus width (distance between 1 side of the brush border membrane to the other side of the brush border membrane), villus surface area (villus length x villus width), crypt depth (height of the invaginations at the bottom of the villus; Iji et al., 2001), lamina propria width (width of the vasculature in the middle of the villus; Sanderson, 2003), and number of lamina propria lymphocytes (LPL) and intraepithelial lymphocytes (IEL; Bjerregaard, 1975). For each parameter, 3 measurements were taken from each intestinal section then averaged for statistical analysis.
Statistical Analysis
Data were analyzed by GLM (JMP software, SAS Institute Inc., Cary, NC), using ANOVA. Dependent variables were examined for the main effect of breeder flock age and LPS administration (haptoglobin and intestinal histology only) and their interactions. When main effects or interactions were significant, differences among means were identified using Tukey’s LSMEANS comparisons. Differences among means were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). A similar response was seen in 7- and 21-d-old poults (P < 0.01 for each), and this trend continued through d 63 posthatch (P = 0.09). However, by d 87 or 103 posthatch, poult BW were not significantly different due to breeder flock age (P > 0.70). Feed offered was not significantly different during the trial (P > 0.20 for all dates; Table 1). However, feed offered was dramatically different than predicted, and these differences are likely due to feed wastage during the trial. Mortality was not affected by breeder flock age (P = 0.68; mean = 5.77%).
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). However, plasma Cu, Fe, Mg, and K were significantly increased post-LPS, and plasma S and Na were significantly reduced post-LPS (P < 0.01 for each; Table 2).
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, panel A), increased plasma Cu and Ca, and decreased plasma Zn, Fe, and K (P < 0.01 for each; Table 2
) compared with control birds. Similarly, in 66-d-old poults, LPS increased plasma haptoglobin compared with control birds (P < 0.01). However, a trend for a breeder flock age x LPS administration interaction was observed (P = 0.07), in which poults from the younger breeder flock had increased plasma haptoglobin post-LPS (P < 0.05), whereas poults from the older breeder flock did not (Figure 1, panel B). Plasma minerals were also affected by LPS at d 66 posthatch; plasma Cu was increased, and plasma Fe was reduced compared with control birds (P < 0.02 for each; Table 2). In contrast, there was no treatment effect on plasma Zn, Na, S, Mg, Ca, K, P, or Na (P > 0.10 for each).
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), wider villi in the ileum (d 11 post-hatch; P = 0.05; 146 vs. 129 µm ± 5.9), and wider lamina propria width in the jejunum (d 11 posthatch; P = 0.05; 46.3 vs. 40.5 µm ± 2.1, respectively). However, by d 66 posthatch, differences in villus length and width were not found (P > 0.10, data not shown), and lamina propria width was greater in the ileum of poults from the younger breeder flock compared with those from the older breeder flock (P = 0.03; 41.3 vs. 32.3 µm ± 2.9, respectively).
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Lipopolysaccharide administration also affected intestinal histology. Compared with control birds, LPS increased villus length and width in the ileum of 11-d-old poults (P < 0.01 for each; Figure 3, panel A) and increased lamina propria width in the duodenum and ileum of 11-d-old poults (P = 0.05, P < 0.01, respectively; Figure 3, panel B). A similar effect was seen on d 66 posthatch, in which LPS increased lamina propria width in the duodenum (P = 0.02; data not shown). Lipopolysaccha-ride increased crypt depth compared with control birds in 11-d-old poults (ileum, P = 0.04; 102.4 vs. 87.6 µm ± 4.9) but reduced crypt depth compared with control birds in 25-d-old poults (duodenum and ileum, P = 0.03, data not shown). Lamina propria lymphocytes were increased by LPS in the duodenum and ileum of 11-d-old poults (P < 0.05 for each; Figure 4, panel A) and in the duodenum of 25-d-old poults (P < 0.01; 1.85 vs. 1.09 LPL/villus ± 0.19). Additionally, there was a trend for increased duodenum LPL post-LPS in 66-d-old poults (P = 0.06; 2.70 vs. 1.81 LPL/villus ± 0.32).
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, panel B) and in the duodenum of 25-d-old poults (P = 0.04; 1.43 vs. 0.95 IEL/villus ± 0.17). Poults from the younger breeder flock had increases in IEL post-LPS at 11 d (duodenum and jejunum) and 66 d posthatch (jejunum), which was not observed in poults from the older breeder flock (breeder flock x LPS interaction, P < 0.05 for each tissue and age, data not shown). Finally, poults from the younger breeder flock had increased numbers of IEL in the duodenum (11 d posthatch; P = 0.02; 2.4 vs. 1.8 IEL/ villus ± 0.2) and jejunum (66 d posthatch; P = 0.05; 2.76 vs., 1.70 ± 0.35) compared with poults from the older breeder flock, regardless of LPS administration
Interactions between breeder flock age and LPS were notable (Table 3). In 11- and 25-d-old poults, poults from the older breeder flock had reduced villus width post-LPS compared with control birds (P < 0.05 for each), but this change was not seen for poults from the younger breeder flock. Similarly, 11-d-old poults from the younger breeder flock had increased crypt depth post-LPS (P < 0.05), whereas 66-d-old poults from the older breeder flock had increased crypt depth following LPS administration (P < 0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As expected based upon previously published research, breeder flock age affected intestinal histology, most noticeably in 11- and 25-d-old poults. At these ages, poults from the older breeder flock had greater villus surface area based upon increased villus length and width in the various regions of the small intestine. Previous research has shown that poults from older breeder hens have increased enterocyte migration rates at hatch compared with poults from younger breeder hens, although those differences do not persist through the first week of hatch (Applegate et al., 1999a). In that trial, however, breeder hen ages only differed by 14 wk (34 vs. 48 wk), thus the longer duration of observed changes in intestinal histology in the current trial may be a result of the larger difference in breeder hen age (33 vs. 55 wk). The observed increases in villus measurements would provide poults with a greater surface area for nutrient absorption and would provide a mechanism for increased BW observed in poults from the older breeder flock.
As the poults aged, the effects of breeder flock age on intestinal surface area were less noticeable; few differences were observed among breeder flock ages in 66-d-old poults. This also correlates with the performance data that were collected; by d 66 posthatch, there was no significant difference in BW between the poults from the older breeder flock and the younger breeder flock. These data suggest that differences in intestinal anatomy and subsequent growth performance are noticeable during the first 9 to 10 wk posthatch but are negligible thereafter. Thus, potential differences in nutrient requirements and optimal management may be more targeted for younger birds, rather than older birds.
Overall, the age of the poults affected the observed response to LPS administration. Plasma haptoglobin levels were numerically reduced over time in control birds and were increased over time in LPS-vaccinated birds. Previous research has shown that the mitogenic response of peripheral blood lymphocytes to LPS ex vivo was greater in 16-wk-old turkeys than in younger birds (Agrawal and Reynolds, 1999). Because there is still little research available concerning inflammatory immune responses in turkeys, these data provide information regarding the value of plasma haptoglobin as a marker of the inflammatory response. It is important to note that since this trial was conducted, new evidence has been published demonstrating that a haptoglobin analog (PIT54) exists in birds and that this protein has a similar function, but not sequence, to the mammalian haptoglo-bin molecule (Wicher and Fries, 2006). The haptoglobin assay in the current trial examined function, as opposed to antibody-specific binding of the haptoglobin molecule. Thus, our trials, and previous trials using haptoglo-bin as a marker of inflammation, are likely measuring the haptoglobin analog.
In general, young poults (d 11 posthatch) did not have a plasma haptoglobin response to LPS administration, although haptoglobin levels in control birds were elevated compared with control birds at later ages. Additionally, at d 11 posthatch, plasma Fe and Cu were increased by LPS administration. Increased plasma Cu during inflammation is a result of increased ceruloplas-min synthesis and secretion to facilitate Fe sequestration from invading organisms (Milland et al., 1990; Koh et al., 1996). Thus, plasma Fe was expected to be reduced after LPS administration, which was not observed in 11-d-old poults. These data suggest that the systemic inflammatory immune response was blunted or had an altered time course at d 11 posthatch, which may be a result of maternal protection, particularly because breeder hens were vaccinated against Pasteurella multicoda (fowl cholera), which is a gram-negative organism for which LPS is a primary antigen (Confer, 1993). Therefore, maternal antibodies were likely present against LPS, and because yolk-derived IgG is still present in 21-d-old poults (Dohms et al., 1978), immune responses may be blunted in younger vs. older birds (Lung et al., 1996). Therefore, a minimal response to LPS in young poults may have been a result of lingering passive immunity from the hen. Alternatively, it may be that the dose or source of LPS chosen for this experiment resulted in an altered time course of inflammatory immune response than has been previously observed. Previous research demonstrated a significant affect on BW (Piquer et al., 1995b), antibody responses, and in vitro blastogenesis (Piquer et al., 1995a) when 50 µg of LPS (Escherichia coli derived) was administered, whereas the current trial administered ~100 µg of LPS (S. Typhimurium derived).
In addition to effects of LPS on systemic inflammatory responses in 11-d-old poults, LPS administration affected intestinal physiology at this age. Lamina propria width was increased by LPS in the duodenum and ileum, and the number of IEL and LPL in the duodenum and ileum were increased compared with control birds. Previous research has demonstrated correlations between lamina propria width and intestinal leukocyte numbers (Coates et al., 1955). This correlation is likely related to the induction of intercellular adhesion molecule-1 expression in intestinal epithelial cells by LPS and provides a mechanism by which leukocytes can be recruited to the intestine following a bacterial challenge (Kim and Jobin, 2005). Lipopolysaccharide administration also increased crypt depth in the duodenum of poults from the older, but not younger, breeder flock at d 11 posthatch. Interpretation of these data is difficult, particularly because similar changes were not observed in other regions of the intestine, or at other poult ages.
In contrast to very young poults, 25-d-old poults did have increased plasma haptoglobin following LPS, and the response was similar across breeder flocks. At d 66 posthatch, LPS again induced a haptoglobin response, although this response was only observed in poults hatched from the younger breeder flock. This observed difference among breeder flock progeny may be a result of altered nutrient uptake (related to surface area differences) during the first few weeks posthatch and subsequent long-term effects on immunity, because delayed access to feed and nutrients reduces development of the gastrointestinal associated lymphoid tissue and particularly that of the bursa and cecum (Shira et al., 2005) and affects the type of bacteria found in the gastrointestinal tract (Potturi et al., 2005). However, in contrast to differences in plasma haptoglobin due to breeder flock age in 66-d-old poults, LPS administration reduced plasma Fe and plasma Cu compared with control birds, demonstrating that some magnitude of inflammatory response was induced, regardless of breeder flock age. Again, these data provide insight as to the value of different measures of the inflammatory response in turkeys.
There were several significant interactions between LPS and breeder flock age on the turkey poults. Poults from the younger breeder flock generally had a higher lymphocyte infiltration in the intestine in response to LPS, and this was observed throughout the experimental period. Crypt depth in the ileum at d 66 decreased post-LPS in poults from the older breeder flock, whereas the opposite effect was observed in poults from the younger breeder flock. Additionally, as noted above, at 66 d post-hatch, poults from the older breeder flock had a lower plasma haptoglobin response compared with poults from the younger breeder flock. Different responses to LPS based upon breeder flock age may be related to nutrient availability for the developing immune system, although this hypothesis remains to be tested.
Overall, poults from the older breeder flock weighed more than their counterparts from the younger breeder flock, but only during the early stages of the growout (<63 d posthatch). However, in the later stages of the growout, differences in BW were no longer observed. Poults from the older breeder flock also had increased intestinal villus length and width, which is most likely responsible for their improved BW when compared with poults from the younger breeder flock. Thus, effects of breeder flock age on progeny performance and intestinal histology were noticed early in growth, which is in contrast to effects on the systemic inflammatory response. At d 11 posthatch, neither poult group responded to LPS, which is likely a result of passive immunity. At 25 d posthatch, poults had similar plasma haptoglobin responses to LPS administration, but at 66 d posthatch, poults from the older breeder flock had a lower plasma haptoglobin response compared with poults from the younger breeder flock. These data demonstrate that breeder flock age can affect performance and intestinal physiology of young birds at later ages than previously examined. However, as birds approach market weights, breeder flock age did not have significant effects on intestinal physiology or BW.
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ACKNOWLEDGMENTS |
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Received for publication February 3, 2006. Accepted for publication May 11, 2006.
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REFERENCES |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
