Dietary proteins in which lysine is converted to homoarginine through guanidination have been used to measure endogenous amino acid secretions in monogastric animals. In these studies, test diets containing the guanidinated protein were fed as a single meal following overnight food deprivation (Schmitz et al. 1991, Siriwan et al. 1994). However, a single meal does not represent normal feeding conditions, and this may influence the estimation of endogenous amino acid secretions. Nitsan et al. (1974), for example, reported that total secretions of digestive enzymes in chicks significantly increased with increased feed intake. Thus, ideally the homoarginine technique to determine endogenous secretions must be carried out with birds given free access to diets containing guanidinated proteins.

One assumption in using guanidinated proteins is that the homoarginine is transformed to lysine by arginase in the liver and kidneys (Ryan et al. 1968), thus preventing lysine deficiency. It is unclear whether this conversion occurs in chickens because previous experiments were conducted either in vitro (Ryan et al. 1968) or with mammals (Steven and Bush 1950). If this assumption is incorrect, then continuous feeding of guanidinated protein may cause lysine deficiency in chickens.

Table 1. Composition and calculated analysis of diets used in Experiment 11 [View Table]

In a preliminary study, in which broiler chickens were given free access to diets containing guanidinated casein (G-casein),⁶ we observed that feed intake and weight gains were markedly depressed compared to those fed casein diets. It has been reported that homoarginine may depress feed intake in rats (Tews and Harper 1986a). It is not known whether homoarginine will have a similar effect in chickens or whether homoarginine in guanidinated proteins will similarly influence feed intake. It was hypothesized that both homoarginine per se and lysine deficiency may have contributed to the adverse effects of the G-casein diet. This study was conducted to investigate the effects of homoarginine and lysine deficiency on the feed intake depression associated with G-casein diets. In the first experiment, the effects of supplementing graded levels of lysine to diets containing G-casein (20 g homoarginine/kg) on the feed intake and growth of broiler chickens were evaluated. Plasma and tissue concentrations of basic amino acids were also determined with the objective of examining possible in vivo transformation of homoarginine to lysine. Treatment effects on arginase activity were also determined. In the second experiment, the effect of administering homoarginine per se on the short-term feed intake of broiler chickens was investigated.

MATERIALS AND METHODS

Diets were fed from d 5 to d 12 post-hatching. Group feed intakes were recorded daily, and birds were weighed on d 2, 4, 6 and at the end of the experiment on d 7. At the end of the trial, two birds in each pen were randomly selected for blood sampling and then euthanized by an intracardial injection of pentobarbitone sodium. Plasma and tissue (liver, brain, breast muscle and kidneys) samples were obtained and kept frozen at 20°C until analyzed for free basic amino acids. Kidney arginase was analyzed as described by Kadowaki and Nesheim (1978).

Blood samples were centrifuged (1500 × g, 10 min), and the plasma was collected into plastic tubes and frozen at 20°C. Plasma (2 mL) was deproteinized by treating with 1 mL of 18% sulphosalicylic acid solution. After adding the internal standard, DL-norleucine (0.4 mmol/L, 2 mL), the solution was thoroughly mixed, centrifuged (1500 × g, 20 min) and the clear supernatant was stored at 20°C. The samples were subsequently thawed, and the pH was adjusted to 2.2 with NaOH before filtering through a 0.22 µm Millipore filter. Approximately 20 µL of filtrate was analyzed for basic amino acids.

Thirty-two, 5-wk-old broiler chickens (Inghams strain TM 70, Inghams Enterprises Pty Ltd., Casula, NSW, Australia) were used. The birds (average weight, 1.5 ± 0.06 kg) were housed in individual wire-mesh cages with 24-h lighting. Water was available at all times. Birds were given a 7-d adaptation period to the cages before measurements started. Prior to the experimental period, 16 birds were fed either a corn-soybean meal diet (CSBM) or a purified diet based on glucose and casein (Table 2) for 3 d, and feed intake was recorded. Birds with low feed intake were discarded, and the remainder were randomly divided into four groups of six birds based on feed intake during the pre-experimental period. Two groups were fed the CSBM diet, and the other two were fed the purified diet.

Table 2. Composition and calculated analysis of diets used in Experiment 21 [View Table]

Food was withheld for 12 h with free access to water before starting the first short-term feed intake study. One group of birds from each dietary treatment was orally administered a gelatin capsule containing 400 mg homoarginine-HCl. The other two groups were given placebo capsules containing 400 mg soluble corn starch. Because corn starch is well digested by chickens, it was chosen as the placebo. Diets were reintroduced immediately after the administration of capsules, and feed intake was accurately recorded every 30 min for 4 h, and then at 6, 9 and 12 h. After a rest period of two days, during which time the birds continued to consume their respective diets, the short-term feed intake study was repeated with the same birds. The only difference being that food was not withheld for 12 h prior to the administration of capsules. Feed intake was recorded every 30 min for 4 h.

At the termination of the second feed intake study blood samples were taken from all birds, and plasma was separated and subsequently analyzed for free basic amino acids including homoarginine.

All samples were analyzed using sodium buffers. For physiological samples, this was achieved by modifying the time program that controls different buffers and eluting the basic amino acids with a shorter run time. This was done because only the basic amino acids (lysine, arginine and homoarginine) were of interest in physiological samples. The analysis of a physiological standard (Sigma Aldrich Chemical, Milwaukee, WI) indicated that the concentrations of basic amino acids determined in this manner were reliable.

RESULTS

Table 3. Feed intake and intake of basic amino acids of broiler chickens fed diets containing casein or guanidinated casein (G-casein) from d 5 to d 12 post-hatching (Experiment 1)1 [View Table]

A similar trend was observed in relation to weight gains of chicks (Table 4). Birds fed Diet 2 lost weight and showed muscle atrophy, especially of breast muscles, but there were no deaths. The weight gains improved (P < 0.05) with additional lysine. The growth rate of birds fed Diet 4 was greater (P < 0.05) than those fed Diet 5. The birds fed Diet 1 (control diet) had a better (P < 0.05) overall feed conversion ratio than those fed other diets, with the exception of those fed Diet 4 which did not differ.

Table 4. Weight gain and feed conversion ratio (FCR) of broiler chickens fed diets containing casein or guanidinated casein (G-casein) from d 5 to d 12 post-hatching (Experiment 1)1 [View Table]

Table 5. Plasma concentrations of basic amino acids and mitochondrial arginase activity in broiler chickens fed diets containing casein or guanidinated casein (G-casein) from d 5 to d 12 post-hatching (Experiment 1)1 [View Table]

When plasma lysine:arginine ratios were calculated, birds fed Diet 5 had a significantly (P < 0.05) higher ratio compared to those fed Diet 1, although both diets had similar lysine and arginine concentrations (Table 1). Birds fed Diets 1 and 4 had similar lysine:arginine ratios, and this coincided with the similar performance (weight gain and intake) of these groups.

Arginase activity was almost 60 times higher in kidney than that in the liver of chickens (Table 5). Arginase activity was not affected by dietary supplementation with lysine and birds fed G-casein diets had significantly greater (P < 0.05) arginase activity than those fed the casein diet.

The trend observed in birds that had free access to food prior to the administration of homoarginine was similar, with the exception being that the effect of homoarginine was not evident until 90 min (Figure 6). The adverse effects of homoarginine were seen in birds fed the CSBM diet even after 240 min, whereas the difference in intake (g/h) only tended to differ (P < 0.10) after 180 min in groups fed the purified diet.

The influence of homoarginine on plasma concentrations of basic amino acids is summarized in Table 6. Significant (P < 0.01 to 0.05) diet type × treatment interactions were observed for plasma lysine and homoarginine concentrations. Administration of homoarginine lowered (P < 0.01) the plasma lysine and arginine concentrations in birds fed the CSBM diet. In contrast, lysine concentrations were greater (P < 0.05), and plasma arginine concentrations were lower (P < 0.01) in birds fed the purified diet. In both diet types, administration of homoarginine resulted in higher (P < 0.01) plasma lysine:arginine ratios.

Table 6. Plasma concentrations of basic amino acids to broiler chickens fed a corn-soybean meal (CSBM) or a glucose-casein (GC) based purified diet 4 h after oral administration of capsules containing either 400 mg homoarginine or corn starch (placebo control)1 [View Table]

DISCUSSION

While a lysine deficiency is the most likely reason for the depressions in feed intake associated with G-casein diets, it is possible that other factors may also have contributed in part to the observed depression. Firstly, the possibility that the initial reductions in body weight may have caused the lowered feed intake cannot overlooked. It is also possible that the homoarginine content of G-casein diet may have caused negative postingestive stimuli in chicks to form a conditioned aversion such that they subsequently reduce their intake of G-casein diets. As shown in Experiment 2, homoarginine reduced feed intake within the first hour after administration. The effect was more pronounced in birds fed purified diets than in those fed CSBM diets. Several mechanisms have been proposed to explain the effect of homoarginine on feed intake. Studies with rats suggest that homoarginine affects feed intake and growth by causing a lysine imbalance, since both lysine and homoarginine compete for the same membrane transport mechanisms at the blood-brain barrier (Tews and Harper 1983). Homoarginine has been shown to reduce the entry of ¹⁴C-lysine into the brain of the rat (Tews and Harper 1986b). Thus, high plasma homoarginine concentrations will substantially reduce the entry of lysine into the brain and this situation will be exacerbated when lysine-deficient diets are fed to birds (for example, Diet 2). Another possible explanation for homoarginine-induced intake depression is that release of homoarginine from dietary protein in the gut may stimulate chemoreceptors that influence the central nervous system directly via the vagus nerve, or indirectly by the release of gastrointestinal hormones (Li and Anderson 1983). However, the lower brain lysine concentrations observed in chicks are more likely to have been caused by lysine deficiency, and to a lesser extent by homoarginine per se, because lysine supplementation of G-casein diets increased brain lysine concentrations and largely overcame the reductions in feed intake and weight gain.

Certain concerns that have been raised in relation to the guanidination process deserve some discussion at this point. The first limitation relates to the potential racemization of amino acid residues to D-forms during guanidination of proteins under alkaline conditions (Liardon and Hurrel 1983). However, as shown recently by de Vrese et al. (1994), the problem of protein racemization at pH values above 10.5 can be avoided by guanidinating at 4°C instead of at room temperature. Recent observations in our laboratory that the guanidination process has no influence on the in vivo digestibility of amino acids (except lysine) by broiler chickens (L. I. Hew, V. Ravindran and W. L. Bryden, unpublished data) also suggest that protein racemization is not a practical problem. Another concern relates to the possibility of some destruction of other essential amino acids during guanidination, but this does not appear to be a limitation in the use of guanidinated proteins, since previous work from this laboratory (V. Ravindran, K. Angkanaporn and W. L. Bryden, unpublished data) and others (Moughan and Rutherfurd 1996) have shown that guanidination does not cause significant modification in the concentrations of any of the acid-stable amino acids, except lysine, when O-methylisourea is used as the guanidinating agent. In most feed proteins, the recoveries of amino acids were close to 100%.

Experiments with rats (Steven and Bush 1950) and isolated perfused liver (Ryan et al. 1968) have shown that homoarginine can be hydrolyzed by arginase to lysine and urea. It was shown in rats (Tews et al. 1988) that the addition of homoarginine to a lysine-deficient basal diet increased plasma lysine concentrations, perhaps following conversion of homoarginine to lysine by arginase. High kidney arginase activity in birds fed G-casein diets may indicate a greater activity for conversion of homoarginine to lysine. The induction of renal arginase activity in birds by basic amino acids is well known (Austic and Nesheim 1970). Thus high plasma concentrations of both lysine and homoarginine may be responsible for the increased renal arginase activity, though this may not correspond with in vivo transformation of homoarginine. Steven and Bush (1950) observed a small growth response in rats given homoarginine, but Aoyagi and Baker (1994) are of the opinion that the growth response was likely to have resulted from coprophagy. The latter workers fed broiler chickens a lysine-deficient diet and found that the addition of 2 g homoarginine/kg diet significantly reduced feed intake and weight gain, indicating that homoarginine had no lysine bioactivity.

Nevertheless, the present results suggest that some transformation of homoarginine to lysine does occur at a low rate. The similar plasma lysine concentrations and lysine:arginine ratios of birds fed Diet 4 (G-casein + 11.6 g lysine/kg diet) and those fed the control diet (Table 5), despite the significantly higher lysine intake of birds fed the control diet (Table 3), suggest that part of the homoarginine in Diet 4 may have been converted to lysine by these birds. Other evidence for this conversion was the discrepancy in plasma lysine concentrations of birds fed the control diet and those fed Diet 5 (G-casein + 17.0 g lysine/kg diet), though both diets had similar lysine concentrations. Plasma lysine concentrations of bird fed Diet 5 were twofold higher and caused a high lysine:arginine ratio or lysine-arginine imbalance which may have been detrimental to growth of chickens. The imbalance may explain the higher intake and weight gain of birds fed Diet 4 compared to those fed Diet 5, despite the higher lysine levels in the latter diet. These results also suggest that a level between 11.4 and 17.0 g/kg diet of added lysine may have given the most rapid rate of gain.

This study also showed that homoarginine is distributed throughout body tissues following absorption. Plasma concentrations of homoarginine in chickens were marginally higher than those reported in rats (1000 ± 60 µmol/L) receiving similar dietary levels of homoarginine (Tews and Harper 1986a), but the tissue concentrations in birds were lower than in rats. Of the organs analyzed, kidney had the highest homoarginine concentrations followed by liver, muscle and brain. In experiment with rats, Tews and Harper (1986a) showed that muscle had the highest homoarginine concentrations, which were about three times higher than those of plasma. From a comparison of the present results with those from rats (Tews and Harper 1986a), it appears that chickens accumulate less homoarginine in tissues, perhaps because they excrete homoarginine efficiently via urine (Angkanaporn et al. 1994).

Despite the differing plasma concentrations, homoarginine levels in the kidneys and brain of birds fed G-casein diets were similar in all treatment groups. This may indicate that the transport mechanism for homoarginine into these tissues is saturated (Banos et al. 1975) or that cellular uptake is proportionately lowered by increased concentrations of circulating lysine. The concentrations of arginine in the kidneys of chickens fed the G-casein diet (Diet 2) were significantly lower compared to those fed the normal casein diet, probably due to the high levels of homoarginine in the kidneys, which may have inhibited the resorption of arginine or increased arginine catabolism due to increased arginase activity (Austic and Nesheim 1972).

Interestingly, muscle lysine concentrations were similar for all treatment groups (Figure 1) despite different dietary lysine levels and lysine intakes. This may reflect conservation of this essential amino acid within cells for protein synthesis (Riis 1983). In addition, birds fed Diet 2 (G-casein diet) had the lowest intakes of lysine and arginine, and yet had the higher hepatic concentrations of these amino acids, suggesting reduced transport from the liver or accumulation following breakdown of skeletal muscle. Muscle atrophy was observed in this group and is consistent with the observation of Tesseraud et al. (1996) that lysine deficiency reduces breast muscle weight in chickens.

These experiments showed that feeding guanidinated casein under a continuous regimen depressed feed intake and weight gain of chickens, and that this can be largely ameliorated by dietary supplementation with lysine. Both lysine deficiency and homoarginine per se were responsible for these adverse effects. These results also suggest that the transformation of homoarginine to lysine in chickens is limited and that the amount of lysine produced by such conversion is too low to support growth in poultry.