Effects of different levels of dietary crude protein and threonine on performance, humoral immune responses and intestinal morphology of broiler chicks

Effects of different levels of dietary crude protein and threonine on performance, humoral immune responses and intestinal morphology of broiler chicks

Abbasi MA^I; Mahdavi AH^I; Samie AH^I; Jahanian R^I

Department of Animal Sciences, College of Agriculture, Isfahan University of Technology, Isfahan 84156-83111, Iran

^{Corresponding author}

ABSTRACT

The present study aimed at investigating the effects of different dietary crude protein (CP) and threonine (Thr) levels on the performance, immune responses and jejunal morphology of broiler chicks. A total of 432 broiler chicks were randomly assigned to a 3×3 factorial arrangement of treatments including three different CP dietary levels (90, 95, and 100% of Ross 308 recommendations) and Thr (100, 110, and 120% of Ross specifications) dietary levels. Performance parameters were recorded for the starter (1-12 days), grower (13-24 days) and finisher (25-42 days) periods. Birds were subjected to different antigen inoculations to evaluate antibody responses. At day 42 of age, two randomly-selected birds per replicate were slaughtered to measure carcass traits. Although Thr dietary supplementation had no marked effect on Newcastle antibody titers, particularly the supplementation of Thr up to 110% of Ross specifications improved (p<0.05) antibody titers against sheep red blood cells during both primary and secondary responses. Reduction of dietary CP level resulted in significant decrease in villus height (p<0.05) and crypt depth (p<0.01) in jejunal epithelial cells, but the supplementation of low-CP diets with Thr up to 110 and 120% of the recommended values allowed overcoming these changes. Except for the starter period, reducing dietary CP level to 90% of Ross recommendations had no harmful effects on performance parameters; however, the best values were obtained with diets containing 110% Thr. The present results indicate that it is possible to reduce dietary CP level up to 10% after the starter period without any detrimental impact on growth performance, and dietary Thr supplementation up to 110% of Ross values may compensate for low CP-induced growth delay in broiler chicks.

Keywords: Body weight gain, broilers, immunocompetence, jejunal histological morphology, protein and threonine.

INTRODUCTION

Protein and amino acids have various biological functions, including the biosynthesis of tissues, and resulting in diverse economic products in all farm animals (Alleman et al., 2000). The importance of protein in feed formulation is more evident because it is the most costly nutrient in feeds is and dietary crude protein (CP) requirement is high in modern broiler strains as a result of their fast growth rates. In addition to CP, individual amino acids are important to supply nutritional requirements and to support optimal growth performance in modern broiler flocks (Han et al., 1992; Aletor et al., 2000; Kim et al., 2007). Han et al. (1992) showed that there are some amino acids that are crucially more important than others, and are known as critical or essential amino acids. During the last few years, attempts have been made to use low-CP diets supplemented with synthetic amino acids for broiler chicks. The reason is that such supplementation can provide significant opportunities for nutritionists to utilize the least-cost formulations in order to decrease feed cost and subsequently to reduce nitrogen excretion to the environment (Kidd et al., 1996; Aletor et al., 2000; Alleman et al., 2000).

Threonine (Thr) is usually known as the third limiting amino acid for broilers, after methionine and lysine, in corn-soybean meal-based diets (Han et al., 1992; Fernandez et al., 1994) and it has an important role in the structure and function of gastrointestinal tract (Law et al., 2000; Ball, 2001). The amount required depends on factors such as dietary CP content, main dietary feed ingredients, and broiler strain, age, and sex (Barkley & Wallis, 2001). The study carried out by Mack et al. (1999) showed that weight gain and breast meat percentage increased as a result of Thr supplementation of low-CP diets; consequently, feed conversion ratio (FCR) improved, but there was no noticeable effect on abdominal fat.

Many studies have been conducted to show varying roles of Thr in biological pathways of animals (Defa et al., 1999; Wang et al., 2006, 2007); however, there are few studies on the possible interaction between different dietary CP and Thr levels in broilers (Ciftci & Ceylan, 2004; Jahanian, 2010a,b). Although some first studies did not report any significant effect of dietary CP content on Thr requirements of broilers (Robbins, 1987; Holsheimer et al., 1994), Kidd et al. (2001) found a clear interaction between Thr needs and dietary CP levels for feed conversion ratio (FCR), but none for BWG or feed intake. Therefore, the exact relationship between dietary CP and Thr remains unclear. A study performed by Jahanian (2010a) showed a marked increase in weight gain caused by Thr supplementation of deficient diets, especially in high-CP diets. He also showed that, irrespective of dietary CP level, Thr supplementation of deficient diets enhances feed efficiency, particularly when high-CP diets are fed (Jahanian, 2010a). In addition of improving growth performance, Wang et al. (2006) and Jahanian (2010a,b) with studies with pigs and broilers, respectively, reported that Thr supplementation of diets enhanced immune functions as measured by antibody responses to different antigens. Furthermore, Jahanian (2010a,b) showed that reducing dietary CP content up to 3 percentage points impaired the immunocompetence of broiler chicks, and concluded dietary CP and individual amino acids have important roles in immune function.

Because of the controversial results obtained relative to the interaction between dietary CP and Thr, the present study was conducted to investigate the effects of different dietary CP and Thr levels on the performance, carcass characteristics, immune responses and intestinal morphology of broilers.

MATERIAL AND METHODS

Birds, diets, and experimental design

The present study was performed at the Poultry Research Station of Isfahan University of Technology (Isfahan, Iran) and all procedures used were approved by Isfahan University of Technology Animal Use and Care Committee. A total of 432 one-day-old Ross 308 broiler chicks were randomly distributed in nine treatments with four replicate pens (12 birds per pen). Treatments consisted of diets containing three different dietary CP levels (90, 95 and 100% of Ross 308 recommendations [Ross Management Manual, 2009] for starter, grower and finisher periods) and three dietary Thr levels (100, 110 and 120% of Ross 308 specifications), and were distributed according to a completely randomized design in a 3×3 factorial arrangement. Of course, the 100% CP and Thr values were calculated after dilution relative to dietary metabolizable energy value, so that dietary CP levels of 23 and 21% for starter and grower, respectively (suggested by Ross manual, 2009) were reduced to 21.90 and 20.34% of diet, and respective 100% Thr values (0.93 and 0.82% of diet) were reduced to 0.89 and 0.79% of diet. Experimental diets were formulated using the analyzed composition of feed ingredients (crude protein, ether extract, crude fiber, total ash and gross energy values by standard protocols of AOAC, 2002) to meet or exceed the nutritional requirements of broilers as suggested by Ross Broiler Management Manual (2009) for the starter (1 to 12 d of age), grower (13 to 24 d of age) and finisher (25 to 42 d of age) phases (Table 1). Dietary CP levels to meet 90, 95 and 100% of Ross 308 specifications were equal to 19.71, 20.80 and 21.90% for starter phase, respectively. The respective values for grower phase were 18.31, 19.32 and 20.34%, and for finisher phase were 17.00, 17.95 and 18.89% of the diet. Three total Thr dietary levels (100, 110 and 120% of Ross 308 specifications) were equal to 0.89, 0.98 and 1.07% of the starter, 0.79, 0.86 and 0.94% of the grower, and 0.71, 0.77 and 0.84% of the finisher diets, respectively. The ratios between individual amino acids (except Thr) and dietary CP were the same for all dietary CP levels. Birds had ad libitum access to water and to the experimental diets at all times. Body weight gain (BWG) and feed intake (FI) were regularly measured (1 to 12, 13 to 24 and 25 to 42 d of age). Bird mortality was daily recorded to adjust feed conversion ratio.

Measurements of carcass traits

On the last day of trial (d 42 of age), two birds per pen nearest to the average weight of the same pen were selected to evaluate carcass traits. Feed was removed 3 h before slaughter. Each bird was exsanguinated by cutting the jugular vein and allowed to bled for approximately 2 min. Viscera and abdominal fat were immediately removed and the weights of breast, drumstick, pancreas, and abdominal fat were obtained. Carcass yield and abdominal fat, pancreas, breast and drumstick relative weights were calculated as percentages of live body weight and the average values of two birds per pen was used for analysis of variance (Jahanian et al., 2008).

Antibody responses to different antigens

On day 16, all birds were challenged with Newcastle disease virus (NDV) via cervical injection (Newcastle-Bronchitis vaccines, 2007). Twelve days after antigen inoculation (28 days of age), two birds per replicate were chosen at random and blood samples were collected from the brachial vein. Sera were separated by centrifugation (5000 rpm for 10 min) and antibody titers against NDV were measured by hemagglutination halter test using commercially available V-form ELISA plates according to manufacturer's instructions (Newcastle-Bronchitis vaccines, 2007).

Sheep red blood cells (SRBC) were washed three times in phosphate buffer saline (PBS) and diluted in PBS to a final dilution rate of 7% (vol/vol), as described by van der Zijpp & Leenstra (1980). At 23 and 33 days of age, two birds per replicate were injected with 0.5 mL of 7% SRBC suspension intramuscularly (thigh muscle). Heparinized blood samples were collected at 6 and 12 days after first and second immunizations, respectively. Plasma samples were stored at -20ºC until further analysis.

The hemagglutination assay was performed as described by Leshchinsky & Klasing (2001). Briefly, each well of a 96-well plate received 50 µL of diluent buffer containing PBS. The initial well received 50 µL of plasma sample, which was serially doubly diluted by transferring 50 µL to the next wells. Then, 50 µL of 2% SRBC suspension in PBS was added to each well. The plates were shaken, incubated for 1 h at room temperature, and then scored. The agglutination titer was expressed as the log₂ of the highest titer with 50% agglutination.

Measurement of jejunal histological changes

At 42 days of age, two randomly-selected chicks from each replicate were slaughtered to determine the effects of dietary treatments on intestinal morphology as described by Mahdavi et al. (2010). Tissue samples were collected and a 2-cm segment of jejunal region anterior to Meckel's diverticulum was excised for light microscopic observations. The histological sections were immediately fixed in 10% formaldehyde solution, and then embedded in paraffin. Transverse and longitudinal 5-µm thick sections were prepared using microtome, stained with hematoxylin-eosin, and examined under a light microscope.

Statistical analyses

All data were analyzed using the General Linear Model procedure of SAS software (SAS Institute, 2001).

The following model was assumed in the analysis of all traits. Y_ijk = µ + A_i + B_j + ABij + e_ijk, where Y_ijk= observed value for a particular parameter, µ= overall mean, A_i= effect of the i^th level of dietary CP, B_j= effect of the j^th level of dietary Thr, AB_ij= the respective interaction between i^th and j^th levels of dietary CP and Thr, and e_ijk= random error associated with the ijk^th recording. Treatment means were separated using the Least Significant Difference test at p<0.05 significance level.

RESULTS AND DISCUSSION

Carcass traits and relative organ weights

As shown in Table 2, the lowest CP dietary level caused a significant (p<0.05) decrease in carcass yield; however, the dietary inclusion of different levels of Thr had no marked effect on this parameter. Although the decrease in dietary CP content to 90% of the recommended values had no considerable impact on drumstick and breast relative weights, irrespective of CP level, dietary Thr supplementation up to 110% of Ross recommendation resulted in significant (p<0.05) increase in the relative weights of both drumstick and breast. On the other hand, decrease in dietary CP content caused marked (p<0.05) decline in relative pancreas weight.

These results are in agreement with the findings of Dozier et al. (2000b, 2001), who indicated that total carcass yield was not affected by dietary Thr concentration. Alleman et al. (2000), however, reported that after reducing dietary CP concentration in broiler chicks, breast meat yield decreased. Similarly, several studies indicated that dietary Thr supplementation markedly increased breast and drumstick yields (Mack et al., 1999; Dozier et al., 2000a,b, 2001; Ciftci & Ceylan, 2004; Jahanian 2010b). Ciftci & Ceylan (2004) showed that increasing dietary Thr concentrations significantly increased breast yield when both high- and low-CP diets were fed, and drumstick yield only with high-CP diets. Some reports suggest that broiler diets containing more Thr than recommended levels improves edible meat percentage. Kidd & Kerr (1997) conducted an experiment to determine Thr dose responses in broilers and proposed that Thr needs for breast meat yield were higher than those reported for weight gain. Also, Chung et al. (1995) showed that breast meat yield and drumstick meat increased in response to dietary Thr supplementation. Consistently, the present findings suggest that although reducing dietary CP level up to 10% decreased (p<0.05) carcass yield, relative breast and drumstick weights were not influenced by dietary CP content. However, the dietary supplementation with L-Thr at levels at least 10% higher than recommended values markedly increased the relative weights of both carcass sections. It should be noted that Thr needs for breast and drumstick meat yields were higher than recommended values.

As described by earlier researchers (Aletor et al., 2000; Alleman et al., 2000), the relative weight of abdominal fat was numerically increased after feeding low-CP diets. However, Thr supplementation had no marked effect on abdominal fat pad. This finding is consistent with the observations of Mack et al. (1999), Dozier et al. (2001), Rosa et al. (2001) and Jahanian (2010b), who reported that Thr supplementation of diets deficient or sufficient in Thr had no reducing effect on abdominal fat.

As presented in Table 2, reduction of dietary CP content decreased (p<0.05) relative pancreas weight. Danicke et al. (2000) suggested that both synthesis and secretion of exocrine pancreas enzymes are up-regulated by feed intake and nutrient presence and density in the intestine. Consistent with the present findings, Swatson et al. (2000) indicated that increase in dietary CP level increased relative pancreas weight, in part due to the induction of high amount of enzymes and protein secretion.

Antibody responses to NDV and SRBC

The effect of different levels of dietary CP and Thr on antibody titers against NDV and SRBC are shown in Table 3. Diets with different CP and Thr levels had no marked effect on antibody titers against NDV. The reduction of dietary CP content tended to decrease primary (p=0.16) and secondary (p=0.13) immune responses against SRBC; however, incremental levels of Thr up to 110% of the recommended values enhanced (p<0.05) antibody responses against SRBC inoculation. Kidd et al. (1997) evaluated cellular and humoral immunity in broilers fed diets supplemented with Thr and did not observe any improvement in immunocompetence. On the other hand, there are some evidences that Thr modulates immune functions (Bhargava et al., 1971; Wang et al., 2006; Li et al., 2007) and that the immune system is sensitive to dietary Thr intake (Li et al., 1999).

Reduced dietary CP level diminished (p<0.05) anti-SRBC antibody titers, but the dietary supplementation of Thr up to 110% of recommended values resulted in the highest primary and secondary antibody responses. It was reported that Thr is a major component of plasma γ-globulins (Kim et al., 2007) and Thr requirements for antibody production in broilers are higher than those for growth performance (Bhargava et al., 1971; Jahanian, 2010a,b). Wang et al. (2006) and Li et al. (2007) demonstrated that dietary administration of Thr influences immune system components by increasing serum IgG levels as well as jejunal mucosal concentrations of IgA and IgG. Our findings support previous reports that, irrespective of dietary CP level, Thr supplementation of diets enhances primary immune responses and could preserve memory immune responses for a long time.

Jejunal morphology

The effects of different dietary CP and Thr levels on jejunal villus height (VH), crypt depth (CD), VH to CD ratio, and goblet cell numbers are presented in Table 4. Decrease in dietary CP content caused significant decrease in VH (p<0.05) and CD (p<0.01); however, VH to CD ratio was not influenced by dietary treatments. Dietary Thr supplementation, especially 110% level, had a significant effect (p<0.05) on VH to CD ratio. These changes were mainly due to the marked increase in VH (p<0.05) and significant decrease in CD (p<0.05). Proteins maintain intestinal viability and mass, in addition to providing energy for normal intestinal functions. The gastrointestinal tissues have relatively high protein turnover rate and high-protein diets provide adequate nutrient (especially CP) for basal metabolism and development of intestinal structure. On the other hand, Thr has special importance as an essential nutrient, because, compared with other amino acids, it has the highest metabolism in the portal-drained viscera (Schaart et al., 2005). Relative to jejunal histological changes, it can be concluded that increasing dietary Thr levels promote an increase intestinal absorptive surface area. These findings confirm the reports by Law et al. (2000) and Ball (2001) with piglets and Zaefarian et al. (2008) with broilers, who showed that VH in animals fed on Thr-deficient diets decreased in comparison with groups receiving adequate Thr. As described by Wu (1998), 30 to 50% of Thr as well as some other amino acids (Arg, Pro, Ile, Val, Leu, Met, Lys, Phe, Gly, Ser) are directly used by the small intestine and are not available for extra-intestinal tissues. Therefore, it is probable that increases in dietary Thr levels provide adequate concentrations of this critical amino acid for the high turnover of mucosal tissues.

On the other hand, CD was influenced (p<0.05) by dietary CP and Thr interaction. Increasing Thr level up to 120% of Ross 308 specifications caused considerable increase in CD when the diets had low CP concentration (90% of recommended values). Our findings showed that the longest villi belonged to the birds fed on diets containing high-CP level and 110% Thr. Also, the lowest CD was observed in the group fed on low-CP, highest-Thr diets.

As shown in Table 4, reducing dietary CP decreased the number of goblet cells; whereas increasing dietary Thr levels increased the number of these cells in the jejunal epithelium. Goblet cells are mucin-producing and secreting sites in digestive system. As described in previous studies (Schaart et al., 2005; Kim et al., 2007), one of the primary fates of absorbed Thr is the synthesis of intestinal proteins, which are mainly secreted into the lumen as mucus, whereby protecting the gut from pathogens and anti-nutritional factors. Mucins are glycosylated proteins secreted along the intestinal epithelium and involved in the diffusion and absorption of the nutrients along the digestive tract. Mucins are particularly rich in threonine, proline and serine, with Thr representing as much as 28 to 40% of total amino acid profile of mucins. The jejunal goblet cell numbers was reduced in low-CP diets. Reduced goblet cell numbers may be related to lower endogenous protein losses associated with lower CP levels. Interestingly, increasing dietary Thr level caused a marked increase in goblet cell numbers. Nichols & Bertolo (2008) described that the de novo synthesis of mucosal and mucin proteins appeared to be highly sensitive to luminal Thr concentration, which demonstrates the importance of dietary Thr supply for gut metabolism. Our results suggest that although after reduction of dietary CP level the jejunal VH, CD and goblet cell numbers were decreased, but Thr dietary supplementation of diets at at least 110% of recommended values could compensate for these changes.

Performance parameters

Data on performance measurements are presented in Table 5. The present results showed that average daily feed intake (ADFI) was influenced (p<0.01) by dietary CP level in grower phase; however, different levels of Thr had no pronounced effect on ADFI. Reduction of dietary CP level caused significant (p<0.01) increase in FCR values in starter phase. This change was largely due to the decrease (p<0.01) in BWG during this period. Dietary supplementation of Thr (at least 110% of Ross recommended levels) improved FCR in the grower phase (p<0.05) and also tended to improve it during entire trial period. These improvements were largely due to the marked increase (p<0.05) in BWG. Although the interaction between dietary CP and Thr was not significant for FCR during the starter period, the best FCR values were obtained when the birds fed on high-CP, high-Thr diets. Interestingly, in the grower period, the best FCR and BWG values were obtained in birds fed on diets containing 95% CP level supplemented with 120% Thr.

It seems that the response of broiler performance to different dietary CP and Thr levels was time-dependent, because our findings showed that reducing dietary CP (to 90% of recommended values) decreased BWG only during the starter phase, with no suppressive effect during remaining experimental period. Also, dietary Thr addition at least up to 110% of recommended levels immediately after the starter phase caused a significant improvement (p<0.05) in BWG.

These findings are consistent with those of previous studies that reported improvements in broiler growth performance during the grower (Smith & Waldroup, 1988; Chung et al., 1995; Ciftci & Ceylan, 2004; Jahanian, 2010b) and finisher (Chung et al., 1995) periods in response to incremental levels of dietary Thr. Also, Kidd et al. (1996) observed significant improvements in FCR values as a result of dietary Thr supplementation in broilers.

The higher crystalline amino acids content of diet may result in better amino acid availability and better performance because it is assumed that the availability of free crystalline amino acid is higher than that of amino acids in intact proteins. Therefore, our data suggest that after the starter period, reducing dietary CP to 90% of the recommended values had no detrimental impact on broiler performance, with the best result obtained when diets supplemented with Thr at the level of 110% of recommended values. Furthermore, it is probable that Thr supplementation enhances growth performance by improving intestinal health. As previously explained, using at least 110% Thr in experimental diets increased VH to CD ratio. Higher VH to CD ratio may indicate slower tissue turnover, suggesting lower demands to compensate for villus atrophy. Therefore, lower energy would be required to support slower tissue turnover. Taller villi indicate more mature epithelia and enhanced absorptive efficiency due to the increased absorptive surface area. Greater villi heights increase the activity of the enzymes secreted from the tips of the villi (Hampson, 1986), resulting in improved digestibility coefficients. Moreover, it was shown that increasing dietary Thr levels caused a significant increase in goblet cell numbers, improving the protection of intestinal absorptive area due to the production of mucin (Nichols & Bertolo, 2008). The mentioned results suggest that dietary Thr supplementation with at least 110% of recommended values could improve the health status of the gastrointestinal tract and consequent growth performance mainly due to the development of intestinal structures and functions, such as increase in absorptive surface area and goblet cell numbers.

Some of previous findings (Kreukniet et al., 1996; Koenen et al., 2002) suggest that there is a negative relationship between growth performance and stimulation of immune system, but the present data indicate that dietary Thr supplementation not only improved FCR and BWG, but also induced and preserved high antibody levels for a long time. The present findings suggest that Thr broiler needs for antibody production were higher than those for optimum growth performance.

From the present findings, it seems that Ross nutritional specifications for dietary CP content in starter period is precise; after starting period, however, one could reduce dietary CP content up to 10% without any adverse effect on broiler performance. Of course, the best results obtained when diets were supplemented with Thr at the level of 110% of Ross 308 recommendations. It seems that the beneficial impacts of Thr on immune responses, as well as intestinal health indices, may improve broiler performance.

Submitted: April/2013

Approved: July/2013

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Publication Dates

• Publication in this collection
  16 May 2014
• Date of issue
  Mar 2014

History

• Accepted
  July 2013
• Received
  Apr 2013