The Journal of Nutrition Vol. 128 No. 10 October 1998, pp. 1760-1766

Lipopolysaccharide-Induced Reductions in Food Intake Do Not Decrease the Efficiency of Lysine and Threonine Utilization for Protein Accretion in Chickens¹

Douglas M. Webel², Rodney W. Johnson, and David H. Baker³

Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois, Urbana, Illinois 61801

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Exposure of animals to infectious agents induces immune responses that result in reductions in food consumption and weight gain. The effect of these changes on amino acid requirements and utilization remains unclear. Three assays were conducted with young chicks with Escherichia coli lipopolysaccharide (LPS) used to stimulate the immune system. An initial study was conducted to evaluate the effects of LPS on animal performance. In a daily or alternate day injection regimen for 9 d, chicks were given intraperitoneal injections of sterile saline containing 0, 100 or 400 µg LPS. Administration of 100 or 400 µg LPS daily, or every other day, decreased both weight gain and food consumption. In two subsequent growth assays, chicks were fed graded levels of lysine or threonine and injected with either 0 or 400 µg LPS every other day to evaluate the effect of LPS administration on the efficiency of amino acid utilization. At the three lowest amino acid doses, whole-body protein accretion was a linear function of supplemental lysine or threonine intake, and slopes of the accretion curves were not altered by LPS administration. The dietary lysine concentration required to maximize protein accretion was unaffected by LPS, but the absolute lysine intake required to maximize chick performance was lower in LPS-injected chicks than in saline-injected chicks. These results show that LPS administration reduces weight gain, food intake, efficiency of food utilization and the absolute quantity of lysine required to maximize these criteria. However, LPS administration does not affect the efficiency of amino acid utilization, nor does it affect the concentration of dietary lysine required to maximize performance.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Animals reared in unsanitary environments grow more slowly and consume less food than those reared under more sanitary conditions (Coates et al. 1963, Roura et al. 1992, Williams et al. 1997a and 1997b). The depressed growth performance of animals reared in unsanitary environments is thought to be a result of increased cytokine production (Dinarello 1996, Klasing and Johnstone 1991, Roura et al. 1992). Cytokines such as interleukin-1 (IL-1),⁴ IL-6 and tumor necrosis factor- (TNF-) are released from cells of the immune system and act to alter metabolic processes in animals, causing a generalized redistribution of nutrients. In general, nutrients are shunted away from growth and skeletal muscle accretion toward processes that support the immune system (Klasing and Johnstone 1991).

One of the hallmarks of sickness and disease is an increase in nitrogen excretion resulting from profound changes in whole-body protein metabolism (Garlick et al. 1980, Manary et al. 1997). Changes in protein metabolism include accelerated muscle protein breakdown, peripheral release of amino acids, and increased hepatic amino acid uptake and protein synthesis (Fong et al. 1989, Klasing and Johnstone 1991, Memon et al. 1994). The mechanisms regulating these processes are both hormonal (e.g., cortisol and somatotropin) and immunologic (e.g., TNF-, IL-1 and IL-6). These processes result in increased peripheral release of amino acids, with a large portion of the released amino acids being taken up by the liver for acute-phase protein synthesis and gluconeogenesis (Memon et al. 1994, Reeds et al. 1994, Takahashi et al. 1998).

The metabolic changes that follow an immune challenge may affect amino acid requirements and the efficiency of amino acid utilization for protein accretion. Klasing and Barnes (1988) suggested that immunologic stress in chicks induced by multiple immunogen injections may reduce the requirements for both methionine and lysine. However, the magnitude of the differences in requirements could not be evaluated because a limited number of amino acid levels were fed. More recently, Williams et al. (1997b) found that pigs reared in an unsanitary environment required a lower concentration of dietary lysine than pigs reared under more sanitary conditions. They also reported that the efficiency of lysine use for nitrogen accretion was not affected by the disease status of the environment (Williams et al. 1997a). Controversy exists in both poultry and swine production concerning whether (or how) daily amino acid requirements may change in response to anorexia caused by disease stress. To avoid over- or underfeeding protein and amino acids during acute or chronic disease stress, it is critical to know whether rations should be adjusted to maximize lean growth rate while at the same time minimizing nitrogen excretion.

We took advantage of a well-established model of disease stress by stimulating the immune system of chicks with lipopolysaccharide (LPS). This molecule, which is from the cell membrane of gram-negative bacteria, induces sickness, including anorexia, fever, lethargy and hypersomnia (Johnson et al. 1993). Several important metabolic responses have been reported in chicks after treatment with LPS (Johnson et al. 1993, Klasing et al. 1987, Roura et al. 1992, Takahashi et al. 1995 and 1998, Webel 1998). The behavioral and metabolic effects of LPS are attributed to IL-1, IL-6 and TNF-, which are released by stimulated macrophages and monocytes. Indeed, several recent studies demonstrate that if macrophages or monocytes do not secrete cytokines in response to LPS, animals will not become anorectic or lose body weight (Johnson et al. 1997, Segreti et al. 1997). By using repeated injections of a moderate dose of LPS, we were able to induce chronic anorexia as well as sickness behavior over assay periods of 9-11 d. As a result, it was possible to evaluate the effects of LPS-induced anorexia on the efficiency of lysine and threonine utilization for whole-body protein accretion in young rapidly growing chicks. The results indicate that LPS treatment does not decrease the efficiency of amino acid utilization above maintenance, but it does decrease the level of whole-body protein accretion that can be attained.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

General procedures.  All procedures were approved by the University of Illinois Laboratory Animal Care Advisory Committee. Female chicks derived from the mating of New Hampshire males and Columbian females were used in all studies. Chicks were housed in thermostatically controlled batteries with raised wire floors, and a 24-h constant light schedule was maintained. Water and experimental diets were freely available and diets were formulated to meet or exceed NRC (1994) requirements for all nutrients with the exception of the amino acid under study. Additions to experimental diets were made at the expense of cornstarch. The basal diets were analyzed for crude protein (CP) by the macro-Kjeldahl procedure (AOAC 1980), and amino acids were quantified by ion-exchange chromatography (Spackman et al. 1958) after 24-h acid hydrolysis under a nitrogen atmosphere. True digestibility of lysine and threonine in the two basal diets employed was determined with the use of adult cecectomized roosters as described by Chung and Baker (1992).

Immunologic stress was induced using Escherichia coli lipopolysaccharide (serotype 0127:B8), which was purchased from Sigma Chemical (St. Louis, MO) and reconstituted in sterile saline (7.5 g/L NaCl). Chicks were injected intraperitoneally with 2 mL of sterile saline containing the appropriate quantity of LPS.

Diets.  Basal diets for both experiments are shown in Table 1. In the lysine study (Experiment 2), a corn-feather meal basal diet was supplemented with graded levels of lysine to achieve digestible lysine levels ranging from 0.62 to 1.22 g/100 g diet. Previous work with this same diet and strain of chick indicated that these levels represented both the linear and plateau regions of the growth curve (Han and Baker 1993). In the threonine study (Experiment 3), a corn-peanut meal basal diet was used that had previously been shown to be severely deficient in threonine yet capable of supporting maximal growth when fully fortified with threonine (Webel et al. 1996).

Analytical procedures.  At the end of Experiments 2 and 3 and after 24 h of food deprivation, chicks were analyzed as described by Baker et al. (1996). In brief, frozen chicks were combined in replicate groups and then chopped into small pieces, after which the samples were ground thrice. The grinder was prechilled with solid carbon dioxide before grinding each group of birds to minimize thawing and moisture loss. After grinding, subsamples of ground carcasses from each replicate group were placed in freezer bags and frozen at 4°C overnight, after which they were lyophilized. After being freeze-dried, samples were weighed to determine weight on a dry matter basis. Freeze-dried samples were then further ground in a food processor and later analyzed in duplicate for total nitrogen by the macro-Kjeldahl procedure. These procedures were repeated for a group (12 birds) of 10-d-old chicks that represented initial chicks such that whole-body protein accretion could be calculated.

Experiment 1.  Chicks were fed a 24% CP corn-soybean meal diet from hatching throughout the experimental period. On d 10 posthatching, quadruplicate groups of three chicks were weighed and randomly assigned to pens and treatments. After a 3-d acclimation period, chicks were weighed and LPS treatments were initiated. Chicks were given daily intraperitoneal injections of sterile saline containing 0, 100 or 400 µg LPS, or they were given alternate daily injections of 100 or 400 µg LPS, with saline injections given on the intervening day. Food intake and body weight were monitored daily.

Experiments 2 and 3.  In Experiments 2 and 3, a standard 24% CP corn-soybean meal diet was fed to chicks from hatching to d 10 posthatching. On d 8 posthatching, quadruplicate groups of four chicks were weighed and randomly assigned to pens and treatments in a manner that resulted in a similar average initial weight for each dietary treatment (Sasse and Baker 1974). After a 2-d acclimation period, chicks were weighed, and dietary and LPS treatments were initiated. Chicks received 2 mL of sterile saline containing 0 or 400 µg LPS every other day starting on d 0 of experimentation. Diets were removed after 11 d of feeding, and birds were then food deprived for 24 h after which all chicks were killed (CO₂ asphyxiation) and immediately frozen at 4°C for later processing.

Statistical analysis.  ANOVA was carried out on all data using the General Linear Model (GLM) procedure of SAS (SAS Institute 1985). In Experiment 1, differences between treatment means at each day of measurement were evaluated by the least significant difference pairwise multiple-comparison procedure (Carmer and Walker 1985). For the lysine requirement assay (Experiment 2) involving both control and LPS-treated chicks, the data were analyzed as a 7 × 2 factorial (seven levels of lysine and two levels of LPS). Digestible lysine requirements were also estimated using broken-line methodology (Robbins et al. 1979), and requirement estimates were determined to be different when asymptotic 95% confidence intervals did not overlap. The data from Experiment 3 were analyzed as a 6 × 2 factorial (six levels of threonine and two levels of LPS). Protein accretion data from the first three dose levels in Experiments 2 and 3 were also fitted to linear regression equations, with protein accretion regressed on supplemental lysine or threonine intake. In all cases, mean values for each pen of chicks were used to compute regression equations and fits (r²).

	RESULTS

Abstract Introduction Methods Results Discussion References

Experiment 1.  Administration of 100 or 400 µg LPS, daily or every other day, decreased (P < 0.05) weight gain, food consumption and gain:food over the 9-d assay period (Table 2). Weight gain, food intake and gain:food were reduced by 22, 14 and 8%, respectively, when chicks receiving 400 µg LPS were compared with chicks receiving saline. The LPS-induced reduction in food intake was greatest after the first injection, but chicks receiving 400 µg LPS every other day maintained a lower food intake throughout the experimental period (Fig. 1). In addition, chicks receiving LPS showed classic behavioral symptoms of sickness throughout the 9-d experimental feeding period. These included lethargy, hypersomnia and piloerection. Thus, chicks receiving repeated exposure to LPS did not develop complete tolerance to LPS as the assay progressed.

View larger version (13K): [in this window] [in a new window]   Fig 1. Daily food intake (g/d) of chicks treated with lipopolysaccharide (LPS) in Experiment 1. Chicks were injected intraperitoneally every other day with 2 mL of sterile saline containing either 0 (saline) or 400 µg LPS. Asterisks indicate that LPS-injected chicks at a given time point are different (P < 0.05) from saline-injected controls. Each data point represents the mean daily food intake value of four pens of four female chicks (pooled SEM = 1.2 g/d).

Experiment 2.  Lysine supplementation resulted in quadratic (P < 0.05) responses in gain, food efficiency and protein accretion in both control and LPS-treated chicks (Table 3). Chicks given LPS exhibited lower (P < 0.05) weight gains, food intakes, food efficiencies and whole-body protein accretions compared with saline-injected controls.

Regression analysis (Fig. 2) of protein accretion in chicks given the three lowest doses of lysine indicated that protein accretion was a linear (P < 0.01) function of supplemental lysine intake for chicks injected with either saline (r² = 0.96) or LPS (r² = 0.93). Slopes of the best-fit regression lines for the treatment groups were nearly equal, indicating that lysine utilization for protein accretion was not affected by LPS administration. Requirements estimated from the use of broken-line methodology suggested that LPS administration neither increased nor decreased the concentration of digestible lysine required to maximize weight gain, food efficiency or protein accretion. Chicks receiving LPS, however, required a lower (P < 0.05) absolute quantity of lysine to maximize both food efficiency (278 vs. 334 mg/d; not shown) and protein accretion ( 281 vs. 303 mg/d; Fig. 3).

View larger version (18K): [in this window] [in a new window]   Fig 2. Best-fit straight-line plots of whole-body protein accretion (g/d) as a function of supplemental lysine intake (mg/d) for chicks fed 0, 0.10 and 0.20 g/100 g supplemental lysine (Experiment 2). Chicks were injected intraperitoneally every other day with 2 mL of sterile saline containing either 0 (saline) or 400 µg lipopolysaccharide (LPS). Each data point represents the mean daily protein accretion value of four pens of four female chicks during an 11-d feeding period (pooled SEM = 0.05 g/d).

View larger version (13K): [in this window] [in a new window]   Fig 3. Best-fit broken-line plots of protein accretion (g/d) as a function of digestible lysine intake (mg/d) for chicks in Experiment 2. Chicks were injected intraperitoneally every other day with 2 mL of sterile saline containing either 0 (saline) or 400 µg lipopolysaccharide (LPS) and fed diets containing graded levels of digestible lysine ranging from 0.62 to 1.22 g/100 g. The minimal digestible lysine requirement was 281 mg/d for LPS-injected chicks and 303 mg/d for saline-injected chicks (P < 0.05). Each data point represents the mean daily protein accretion value of four pens of four female chicks during an 11-d feeding period (pooled SEM = 0.07 g/d).

Experiment 3.  Both control and LPS-treated chicks responded to supplemental threonine in a linear (P < 0.05) fashion (Table 4). Chicks injected with LPS grew more slowly, consumed less food, converted food into body mass less efficiently and accreted less protein than saline-injected controls (P < 0.05). The interaction of LPS by threonine level was significant (P < 0.05) for gain, food intake and food efficiency. Thus, at the lowest level of supplemental threonine, saline- and LPS-injected chicks consumed food and gained weight at the same rate, but at supplemental threonine levels of 0.05 g/100 g and above, saline-injected chicks gained body mass faster and consumed more food than LPS-injected chicks.

On the basis of the slopes of the linear regression equations relating whole-body protein deposition to supplemental threonine intake (Fig. 4), the efficiency of dietary threonine utilization for protein accretion was not different between saline-injected (r² = 0.92) and LPS-injected (r² = 0.93) chicks. The data were deemed inappropriate for requirement estimation because clear-cut plateaus in performance measures were not achieved. This suggests that the digestible threonine requirement of chicks between 10 and 21 d of age is 0.70 g/100 g diet or greater in diets containing 13.4 MJ metabolizable energy /kg.

View larger version (18K): [in this window] [in a new window]   Fig 4. Best-fit straight-line plots of whole-body protein accretion (g/d) as a function of supplemental threonine intake (mg/d) for chicks fed 0, 0.05 and 0.10 g/100 g supplemental threonine (Experiment 3). Chicks were injected intraperitonneally every other day with 2 mL of sterile saline containing either 0 (saline) or 400 µg lipopolysaccharide (LPS). Each data point represents the mean daily protein accretion value of four pens of four female chicks during an 11-d feeding period (pooled SEM = 0.08 g/d).

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Our experiments employed a model system of immunologic stress in which repeated injections of LPS resulted in significant reductions in food consumption and weight gain over an extended period of time. In agreement with previous reports (Klasing et al. 1987, Klasing and Barnes 1988), immunogen-injected chicks consumed less food, gained weight at a slower rate and converted food into body mass less efficiently than saline-injected chicks. Our model system was slightly different from that used by Klasing and Barnes (1988), however, in that we used only one immunogen (LPS) and our injections were given over a longer period of time.

The greater rates of weight gain and protein accretion in control chicks than in LPS -injected chicks in Experiment 2 were associated with a higher dietary lysine requirement when expressed on the basis of absolute intake (mg/d) (Fig. 3). However, due to the reduced food intake observed in LPS-treated chicks, there was no difference in the concentration of dietary lysine required to maximize performance when comparing saline- and LPS-injected chicks. Klasing and Barnes (1988) reported that immunogen-injected chicks may require a lower concentration of dietary lysine to maximize performance than untreated chicks. The design of their experiment, which consisted of only three levels of dietary lysine (0.70, 0.95 and 1.20 g/100 g), makes it difficult to determine the magnitude of the requirement differences between control and immunogen-injected chicks. In fact, based on the incremental increases in gain and food efficiency resulting from lysine supplementation, it appeared that the lysine requirement of control chicks was comparable to that of the immunogen-treated chicks. Recent work in growing pigs, however, does suggest that immunologic stress induced by rearing pigs in an unsanitary environment reduces both the absolute quantity and the concentration of dietary lysine required to maximize weight gain and food efficiency (Williams et al. 1997b). Thus, although it remains problematic whether immune-challenged animals require a lower concentration of lysine in the diet to achieve maximal performance, it seems clear that in cases in which voluntary food intake is reduced due to disease stress, it is not prudent to increase the dietary lysine concentration in an effort to bring lysine intake of the stressed animals up to the same levels as that of the nonstressed animals.

To model amino acid requirements of growing animals accurately, it is critical to have precise information on the efficiency with which amino acids are utilized for whole-body protein accretion (Baker et al. 1996, Edwards et al. 1997). However, there is little available information on the efficiency with which amino acids are utilized for protein accretion and almost no information on how immunologic stress may affect this efficiency. We hypothesized that the profound changes in protein metabolism that follow an immune challenge might alter the efficiency with which amino acids are utilized for whole-body protein accretion. However, we found that the efficiency of lysine and threonine utilization for accretion of whole-body protein was not influenced by immunologic stress. Our finding that the efficiency of utilizing digestible lysine was unaltered by chronic immunologic stress agrees with recent work conducted in pigs in which nitrogen balance was used to determine the efficiency of lysine retention (Williams et al. 1997a). We evaluated the effect of immunologic stress on lysine and threonine utilization because these two amino acids are among those most limiting in practical diets fed to growing chicks and pigs. However, it is possible that the efficiency of utilizing other amino acids, especially those that have more prominent roles in the host's response to infection (e.g., arginine), might be altered as a result of stimulation of the immune system. Preliminary results in our laboratory, however, have indicated that LPS treatment does not affect the efficiency of arginine utilization for protein accretion in chicks (Webel 1998).

In agreement with previous reports on utilization of a limiting amino acid, the efficiency of utilizing both lysine and threonine for protein accretion was constant when given below required levels (Baker et al. 1996, Batterham et al. 1990, Beech et al. 1991, Edwards et al. 1996 and 1997). That lysine was utilized with a lower efficiency than threonine is in agreement with previous reports in growing chicks (Edwards et al. 1996 and 1997). Previous studies in pigs (Adeola 1995, Batterham et al. 1990, Batterham 1994, Beech et al. 1991) and rats (Gahl et al. 1996) indicated that lysine is utilized more efficiently than threonine for protein accretion. An explanation for the species differences in the efficiency with which lysine and threonine are utilized is not obvious and requires further study.

Administration of LPS has been reported to induce a transient refractory period to subsequent LPS challenges, resulting in reduced cytokine production and the associated metabolic changes (Cavaillon et al. 1994). In fact, previous research has shown that cytokine responses after repeated injections of LPS in chicks are reduced compared with that after a single injection (Takahashi et al. 1995), although Takahashi et al. (1998) recently reported elevations in plasma 1-acid glycoprotein 10 d after initiating repeated injections of LPS. It is uncertain whether the LPS-related reductions in performance seen in our experiments were primarily a function of the initial LPS injection or whether the animals were actually recovering during the later stages of the experiment. However, although the LPS-related reductions in food intake were most pronounced after the first LPS injection (Fig. 1), LPS-injected chicks also consumed significantly less food than saline-injected chicks during the later portions of the experiments.

An interesting observation was that LPS tended to have minimal effects on chick performance when diets severely deficient in threonine or lysine were fed. This was not totally unexpected because previous investigators have noted similar results when chicks were fed diets deficient in methionine or lysine and were subjected to either immunogen injection (Klasing and Barnes 1988) or coccidial infection (Willis and Baker 1981). Klasing and Barnes (1988) suggested that amino acid-deficient chicks may have a decreased capacity to synthesize and release cytokines. They found that plasma IL-1-like activity was increased by immunogen injection when chicks were fed amino acid adequate diets, but a similar increase in plasma IL-1-like activity was not observed in chicks fed methionine- or lysine-deficient diets. Therefore, it seems possible that the lowest levels of lysine and threonine in our experiments were sufficiently deficient to inhibit the synthesis or release of cytokines, and this in turn would not allow the occurrence of catabolic events typically associated with cytokine release.

In summary, these studies demonstrate that stimulation of the immune system by multiple injections of LPS reduces performance and decreases the absolute quantity of amino acids required for maximal performance of chicks. The decreased requirement for amino acids is apparently due to a reduction in the chicks' capacity to accrete protein and not to a change in the efficiency of amino acid utilization. Our data, however, indicate that the concentration of dietary lysine required to maximize performance is generally unaffected by immunologic stress because the reductions in food and amino acid intake associated with LPS administration are proportional to the decreased need for amino acids in LPS-injected chicks.

	FOOTNOTES

Manuscript received 14 January 1998. Initial reviews completed 13 April 1998. Revision accepted 3 June 1998.

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