DOI: 10.1111/jpn.12316

ORIGINAL ARTICLE

Effects of different sulphur amino acids and dietary electrolyte balance levels on performance, jejunal morphology, and immunocompetence of broiler chicks V. Nikoofard, A. H. Mahdavi, A.H. Samie and E. Jahanian Department of Animal Sciences, College of Agriculture, Isfahan University of Technology Isfahan, Iran

Summary As alterations of dietary electrolyte balance (DEB) can influence amino acid metabolism via changes the ions incur in their configurations, performance and immunological responses of broiler chicks might be affected. So, the current study was carried out to investigate the effects of different levels of sulphur amino acids (SAA) and DEB on performance, jejunal morphology and immunocompetence of broiler chicks. A total of 360 1-day-old male Ross 308 broiler chicks were randomly assigned to nine experimental treatments with four replicates of 10 birds each. Experimental treatments consisted of three levels of SAA (100, 110, and 120% of NRC recommendation, provided by methionine supplementation in diets with the same cysteine level) and three levels of DEB (150, 250, and 350 mEq/kg) that were fed during the entire of trial in a 3 9 3 factorial arrangement. Results showed that the relative weights of intestine and abdominal fat were decreased markedly (p < 0.001) with increasing levels of SAA and DEB respectively. Antibody titre against sheep red blood cell was neither individually nor in combination influenced by supplementation of SAA or DEB. Nevertheless, a decrease in DEB level led to a suppression in heterophile (p < 0.05) and an increase in lymphocyte counts (p = 0.06); consequently, heterophile to lymphocyte ratio was significantly decreased (p < 0.05) by decremental levels of DEB. Albumin to globulin ratio was increased after inclusion of at least 10% SAA (p < 0.001) and 150 mEq DEB/kg in the diet (p = 0.11). Although feeding high-DEB level led to a remarkable decrease in villus height (p < 0.01) and goblet cell numbers (p < 0.001), supplementing the highest level of SAA improved the height of jejunal villus. During the entire trial period, average daily feed intake (ADFI) was increased by incremental SAA levels (p < 0.05). However, inclusion of 150 mEq/kg led to not only a remarkable increase (p < 0.0001) in both ADFI and average daily weight gain (ADWG) but also to improved (p < 0.001) feed conversion ratio (FCR) both during the growing and over the entire trial periods. The present findings indicated that inclusion of low DEB decreased the heterophile to lymphocyte ratio and improved both the albumin to globulin ratio and intestinal health indices. The best growth performance was obtained with 150 mEq DEB/kg in the diet for each level of SAA. Keywords broiler chicks, dietary electrolyte balance, sulphur amino acids, immunological responses, performance Correspondence A. H. Mahdavi, Department of Animal Sciences, College of Agriculture, Isfahan University of Technology, Isfahan 84156-83111, Iran. Tel: +98-31-33913505; Fax: +98-31-33913471; E-mail: [email protected] Received: 31 August 2014; accepted: 11 February 2015

Birds’ sodium, potassium and chloride requirements are minimal because these monovalent electrolytes are provided by dietary ingredients and salts (Leeson and Summers, 2001). However, Na+, K+ and Cl- are referred to as ‘strong ions’ due to their greater effects on birds’ acid–base homeostasis than divalent ions (such as calcium, magnesium, phosphate or sulphate); therefore, the equation Na+K-Cl (mEq/kg) is usually used for evaluating dietary electrolyte balance (DEB) in poultry (Abbas et al., 2012; Ghasemi et al., 2014).

Acid–base balance in birds is affected by such factors as environmental conditions, diet and metabolism (Olanrewaju et al., 2007). Electrolyte balance plays important roles in tissue protein synthesis, maintenance of intracellular and extracellular homeostasis, maintenance of electric potential of cell membranes, creation of osmotic pressure (Borges et al., 2007), and minimization of deleterious effects of heat stress in addition to its roles in enzyme and nerve functioning (Ahmad et al., 2008). Although early reports indicated that maximum performance is usually observed in broiler chickens fed DEB 250 mEq/kg of diet

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Introduction

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SAA and dietary electrolyte balance in broiler chicks

(Mongin and Sauveur, 1977), evidence shows that administrating low-DEB diets might have beneficial effects on birds’ performance due to their acidic characteristics (Borges et al., 2003). On the other hand, interactions have been shown to exist between DEB and dietary crude protein or some amino acids as evidenced by the effects of ion size or charge on other molecules, especially proteins, due to changes the ions incur in their configurations (Hampson et al., 1987). In this regard, Adekunmisi and Robbins (1987b) stated that diets with high levels of amino acids had adverse effects on birds’ performance by affecting the acid–base balance. In another study, the same authors reported that chickens fed low-CP diets exhibited reduced growth when DEB was changed by inclusion of Na and K, indicating that DEB alters according to dietary CP (Adekunmisi and Robbins, 1987a). On the other hand, manipulations in DEB might affect the metabolic pathway and destiny of many amino acids such as serine, glycine and branched-chain amino acids (Adekunmisi and Robbins, 1987a,b; Brake et al., 1998). Similarly, Patience et al. (1986) emphasized that the renal and hepatic metabolism of lysine and leucine could be influenced by the dietary acid–base balance. From these studies, it may be concluded that amino acid metabolism both influences and is influenced by acid–base homeostasis. Methionine (Met) as the first limiting amino acid in poultry diets (Leeson and Summers, 2001), especially those based on corn–soya bean meal, plays many vital roles; it is a donor of methyl groups for DNA and other molecules (Rubin et al., 2007), it is a precursor to glutathione and polyamine (Rubin et al., 2007), and it participates in the synthesis of proteins such as antibodies (Tsiagbe et al., 1987; Swain and Johri, 2000). Rubin et al. (2007) reported that the average weight gain and feed efficiency could be influenced by manipulating the level of Met in broiler diets. DEB (Borges, 2001; Borges et al., 2003; Ahmad et al., 2008) or Met (Weissman et al., 2008; Maroufyan et al., 2010) each has its own remarkable effects on the performance and immunological responses of birds. The interactions between these two factors could, therefore, be of great interest because of the possible reciprocal relationship(s) between DEB and amino acid metabolism (Patience et al., 1986; Brake et al., 1998). Although it has been claimed that there are interactions between DEB and dietary crude protein (Adekunmisi and Robbins, 1987a, 2009) or some amino acids such as lysine or leucine (Patience et al., 1986; Brake et al., 1998), there are no data concerning the

interactions between different levels of DEB and Met, as the first limiting amino acid in poultry, on the performance and immunological responses of broiler chicks. Therefore, this study was designed to evaluate the effects of different levels of DEB and sulphur amino acids (SAA, provided by Met addition in diets with the same cysteine level) on the performance, internal relative organ weights, jejunal histology and immunocompetence of broiler chicks.

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Material and methods Chicks, diets and experimental design

This study was performed at the Poultry Research Station of Isfahan University of Technology (Isfahan, Iran), and all the procedures used were approved by Isfahan University of Technology Animal Use and Care Committee. A total of 360 1-day-old male Ross 308 broiler chicks were used in this experiment during a 42-days feeding trial including 1–21 days of age (starting period) and 22–42 days of age (growing period). The chicks were assigned to different dietary treatments and housed in pens throughout the trial. Four replicate pens were assigned to each of the nine experimental diets. Dietary treatments included three levels of DEB (150, 250, and 350 mEq/kg) and three levels of SAA (100, 110, and 120% of NRC recommendation, provided by Met supplementation in diets with the same cysteine level), which were applied as a 3 9 3 factorial arrangement of treatments based on a completely randomized design (Table 1). Experimental diets were formulated using the analysed-feed ingredients to meet or exceed the nutritional requirements of broilers as provided by NRC (1994) during the starter and grower periods. DL-Met supplementation was used to meet SAA needs. The dietary SAA levels (100, 110, and 120% of NRC recommended values) provided for 0.820, 0.902 and 0.984% of the diet in the starter period respectively. The respective values for the grower period were 0.690, 0.759 and 0.828% of the diet. Ammonia chloride (NH4Cl) and sodium bicarbonate (NaHCO3) were used in this study to supply the required dietary electrolyte balance. Light was continuously on for the first 5 post-hatching days, after which a 23L:1D lighting schedule was maintained throughout the experiment. Feed and water were provided ad libitum. At 1 days of age, temperature was adjusted at 32–33 °C before it was reduced by 3 °C/ week. Body weight and feed consumption were measured periodically (1–21 and 22–42), and probable mortalities were recorded daily to adjust feed conversion ratio (FCR).

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SAA and dietary electrolyte balance in broiler chicks

Table 1 Feed ingredients and nutrient composition of basal diets Item Ingredients (%) Corn, yellow Soya bean meal Soya bean oil Corn starch Oyster shell Monocalcium phosphate Common salt DL-Methionine Bicarbonate sodium Mineral premix* Vitamin permix† Nutrient composition Calculated analysis MEn (kcal/kg) Methionine (%) Methionine + Cysteine (%) Lysine (%) Threonine (%) Measured analysis Crude protein (%) Calcium (%) Available Phosphorus (%) K (%) Cl (%) Na (%)

Starter (1–21)

Grower (22–42)

56.40 36.87 2.24 0.05 1.72 1.21 0.24 0.15 0.17 0.25 0.25

61.20 31.40 3.14 0.05 1.75 0.88 0.17 0.07 0.38 0.25 0.25

2930 0.49 0.82 1.13 0.86

3050 0.39 0.69 0.99 0.78

21.06 0.92 0.41 0.91 0.18 0.16

19.06 0.86 0.33 0.82 0.14 0.19

described by Lacroix et al. (1970). Finally, the dietary treatments were formulated based on the values thus obtained (Table 2). Measurement of the weights of internal organs

At the final day of the trial, two birds per replicate were randomly selected to measure the relative weights of some internal organs. Feed was removed 3 h before slaughtering; the birds were become anaesthetized, then sacrificed and allowed to bleed for approximately 2 min. Then, the thymus, bursa of Fabricius, spleen, intestine and abdominal fat were precisely removed and separately weighed using a sensitive digital scale. The relative weights of these organs were calculated as percentages of live body weight, and the mean data of two birds per cage were used for analysis of variance. Antibody responses to sheep red blood cells (SRBC)

SAA was provided by Met addition in diets with the same cysteine level. Dietary SAA levels (100, 110, and 120% of NRC recommended values) provided 0.820, 0.902 and 0.984% for the starter period respectively. The respective values for the grower period were 0.690, 0.759 and 0.828% of diet. To provide the required dietary electrolyte balance in the present study, ammonia chloride (NH4Cl) and sodium bicarbonate (NaHCO3) were used. *Vitamin premix provided the following per kilogram of diet: vitamin A, 9000 IU; cholecalciferol, 2000 IU; vitamin E, 36 IU; vitamin K3, 2 mg; thiamine, 1.8 mg; riboflavin, 6.6 mg; pantothenic acid, 10 mg; niacin, 30 mg; choline chloride, 250 mg; biotin, 0.1 mg; folic acid, 1 mg; pyridoxine 3.0 mg; vitamin B12, 0.015 mg; BHT, 1 mg. †Trace mineral premix provided the following in milligrams per kilogram of diet: iron, 50 mg; zinc, 85 mg; manganese, 100 mg; iodine, 1 mg; copper, 10 mg; selenium, 0.2 mg.

Sheep red blood cells were washed three times in phosphate-buffered saline (PBS) and diluted in PBS to a final dilution rate of 7% (vol/vol). At 23 and 33 days of age, two chicks per replicate were intramuscularly injected with 1 ml of 7% SRBC suspension (0.5 ml per each thigh muscle). Heparinized blood samples were collected at d 6 and 12 after the first and second immunizations respectively. Plasma samples were stored at 20 °C until further analysis. Haemagglutination assay was performed as described in Leshchinsky and Klasing (2001). Briefly, each well of a 96-well plate received 0.05 ml of the diluent buffer containing PBS. The initial well received 0.05 ml of the plasma sample, which was doubly diluted serially by transferring 0.05 ml to the next wells. Then, 0.05 ml of 2% SRBC suspension in PBS was added to each well. The plates were shaken for 1 min, incubated for 1 h at room temperature and scored. The agglutination titre was expressed as log2 of the highest titre with 50% agglutination.

Chemical analysis and measurements

Circulating differential leucocyte count

Before starting the trial, the feed ingredients were analysed for basal chemical compositions (crude protein, ether extract, crude fibre, total ash and gross energy; AOAC, 2006) and Met content (Fontaine et al., 2001, 2002). Also, dietary anion–cation balance was evaluated based on Na++ K+- Cl- in terms of mEq/kg. Na+ and K+ were determined using the flame photometric method according to AOAC (2006), and Cl- was evaluated by volumetric analysis

At 42 days of age, two randomly selected birds from each pen were bled and total blood cells were counted by Automated Hematology Analyzer (KX21N; Sysmex, Kobe, Japan). Total and differential counts of leucocytes were performed by screening a Gimsastained slide. The different subpopulations of leucocytes were counted, and the heterophil to lymphocyte ratio was calculated as described by Stedman et al. (2001).

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Table 2 Effects of DEB and SAA levels on relative weights of organs (% of live BW)

SAA level (%) 100

110

120

DEB level

SAA level

DEB level (mEq/kg)

Intestine (%)

Spleen (%)

Bursa of Fabricius (%)

Abdominal fat (%)

Pancreas (%)

150 250 350 150 250 350 150 250 350 150 250 350 100 110 120

4.01 4.09 4.27 3.79 3.86 3.82 3.86 3.66 3.73 3.89 3.87 3.94 4.12a 3.82b 3.75b

0.14 0.15 0.16 0.18 0.16 0.14 0.13 0.17 0.14 0.15 0.16 0.15 0.15 0.16 0.15

0.08 0.08 0.05 0.05 0.06 0.06 0.07 0.07 0.06 0.07 0.07 0.06 0.07 0.06 0.07

2.01 2.23 1.43 1.67 1.85 1.12 1.72 1.71 1.29 1.80a 1.93a 1.28b 1.89 1.55 1.57

0.24 0.23 0.23 0.23 0.22 0.22 0.21 0.21 0.20 0.23 0.22 0.22 0.24a 0.22a 0.21b

p-value DEB level SAA level SAA 9 DEB level SEM

0.81 0.008 0.68 0.1

0.61 0.83 0.34 0.01

0.29 0.31 0.63 0.006

0.002 0.11 0.93 0.15

0.54 0.02 0.99 0.008

Means with no common superscript within each column are significantly (p < 0.05) different.

a-b

Measurement of serum protein fractions

Statistical analysis

At the end of the experiment, blood samples were taken from two randomly selected chicks per cage to determine the serum protein fractions. The share of individual serum protein fractions was determined by electrophoresis on tapes of gelled cellulose-acetate (Cellogelâ; MALTA Chemetron, Milan, Italy). Absolute and relative concentrations of protein fractions were determined using the relevant software as described in Savory et al. (1976).

All the data were subjected to ANOVA using the GLM procedures of SAS software (SAS Institute, 2001) as a 3 9 3 factorial arrangement of treatments that consisted of DEB and SAA levels as the main effects and their respective interactions. The treatment means were separated by LSD tests at p < 0.05 statistical level. Pen was the experimental unit. After testing normality with Shapiro–Wilk test, the lamina propria lymphatic follicles were analysed using the GLM procedure of SAS software.

Measurement of jejunal histological alterations

On day 42, two randomly selected chicks from each replicate were become anaesthetized and then sacrificed to determine the effects of dietary treatments on intestinal cell morphology as described in Boka et al. (2014). Tissue samples were collected, and a 2-cm segment of the jejunal region anterior to Meckel’s diverticulum was excised for light microscopic observations. The histological sections were immediately fixed in 10% formaldehyde solution, and the fixed samples were subsequently embedded in paraffin. Transverse and longitudinal sections 5 lm in thickness were prepared using a microtome and stained with haematoxylin–eosin for examination under a light microscope. 4

Results and discussion Different levels of DEB (Borges, 2001; Borges et al., 2003; Ahmad et al., 2008) as well as SAA (Weissman et al., 2008; Maroufyan et al., 2010) when administered individually have remarkable effects on performance and immunological parameters. Investigation of their interactions for additive or reductive effects with respect to the above-mentioned parameters should be interesting because manipulations in DEB have been shown to affect the metabolic pathway and the fate of some amino acids such as lysine and leucine (Adekunmisi and Robbins, 1987a,b; Brake et al., 1998). Journal of Animal Physiology and Animal Nutrition © 2015 Blackwell Verlag GmbH

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SAA and dietary electrolyte balance in broiler chicks

Relative weights of the internal organs

The relative weights of the small intestine, spleen, bursa of Fabricius and pancreas were not affected by supplementation of DEB, as also confirmed by Borgatti et al. (2004). Nevertheless, the relative weight of abdominal fat was depressed (p < 0.01) after addition of 350 mEq/kg DEB. This might have been the consequence of the lower average daily feed intake (ADFI) and the average daily weight gain (ADWG) in birds fed the highest level of DEB (Table 6), which may have, consequently, resulted in reduced fat deposition in the carcass. Incremental SAA level up to 120% led to a significant decline (p < 0.01) in the weight of the small intestine by approximately 9% in broilers receiving 120% compared to 100% SAA level. It has been shown that Met with antibacterial effects (Dahiya et al., 2007) results in decreased small intestine bacterial populations that lead to thickened intestine and increased intestinal secretion and weight (Ficken and Wages, 1997). The suppression of the relative weights of the pancreas (p < 0.05) and abdominal fat (p = 0.11) in birds fed Met supplement was in line with the findings of Zhan et al. (2006) who reported that dietary supplementation of Met significantly

decreased pancreas and abdominal fat weights. The effects of SAA on pancreas weight may be explained by the participation of SAA, especially Met, in the structure of proteins such as peptide hormones (Tsiagbe et al., 1987); hence, SAA helps the hormonal requirements, especially in the pancreas to be met. Circulating differential leucocyte counts

As shown in Table 3, the number of total leucocytes was not influenced by SAA or DEB supplementation either alone or in combination. Dietary inclusion of 350 mEq/kg of DEB led to an increase in the heterophile to lymphocyte ratio (p < 0.05), which may serve as a good index of stress (Teirlynck et al., 2009). This response was due to the increase (p < 0.05) in heterophils counts and the simultaneous decrease (p = 0.06) in lymphocytes enumeration; however, other subpopulations of leucocytes were not affected. Although Borges et al. (2004) and Ahmad et al. (2008) indicated no effect of DEB on the heterophil to lymphocyte ratio, the findings of Borges et al. (2003) showed that not only under heat stress but also in thermoneutral conditions were the lowest ratios of heterophil to lymphocyte obtained when diets were administrated with 140–240 mEq/kg of DEB, and this

Table 3 Effects of DEB and SAA levels on total leucocytes (TL), lymphocytes (Lymph), heterophils (Heter), monocytes (Mono), eosinophils (Eosin), basophile (Baso) and heterophils to lymphocytes ratio (H:L)

SAA level (%) 100

110

120

DEB level

SAA level

DEB level (mEq/kg)

TL (9103/ll)

Heter (9103/ll)

Lymph (9103/ll)

Mono (9103/ll)

Eosin (9103/ll)

Basophile (9103/ll)

H:L

150 250 350 150 250 350 150 250 350 150 250 350 100 110 120

17.10 17.50 17.20 18.20 18.00 19.1 18.7 19.2 18.7 18.2 18.0 18.8 18.67 18.6 18.8

7.33 9.20 9.81 9.56 9.64 10.90 9.72 9.54 11.16 8.94 9.34 10.62 8.77 9.45 9.78

9.45 7.53 6.57 7.92 7.63 7.43 8.04 8.91 7.82 8.60 7.91 7.27 9.22 8.38 8.23

0.66 0.65 0.69 0.59 0.50 0.64 0.75 0.66 0.65 0.67 0.59 0.68 0.72 0.58 0.68

0.06 0.07 0.12 0.09 0.09 0.12 0.05 0.08 0.06 0.07 0.08 0.10 0.09 0.10 0.06

0.02 0.05 0.00 0.05 0.05 0.00 0.00 0.00 0.00 0.02 0.03 0.00 0.02 0.03 0.00

0.77 1.22 1.49 1.21 1.26 1.47 1.21 1.07 1.42 1.04 1.18 1.46 0.95 1.13 1.19

0.15 0.17 0.66 0.07

0.04 0.10 0.31 0.06

p-value DEB level SAA level SAA 9 DEB level SEM

0.48 0.52 0.62 1.22

0.04 0.10 0.36 0.09

0.06 0.09 0.18 0.08

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0.58 0.18 0.95 0.10

0.59 0.48 0.91 0.14

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parameter was increased markedly with 40 or 340 mEq/kg DEB. It is interesting to note that phagocytic and bactericidal activities decreased although an increase was observed in the heterophil release by the bone marrow with the resulting increase in their numbers in circulation under stress (Swenson and Reece, 1996; Berne and Levy, 1998). These physiological responses of exposure to stress that are mediated by glucocorticoids would cause suppression in both humoral and cellular immune functions (Berne and Levy, 1998). An increase in SAA level resulted in both a slight (p = 0.10) increase in heterophil and a marginal (p = 0.09) decline in lymphocyte. It has been demonstrated that alterations in some dietary amino acids such as SAA (Met and cysteine) could affect the immune system functions because these nutrients or their products are implicated in the relationships within and between leucocytes in the systemic (Al-Maya, 2006) or mucosal immune systems (Swain and Johri, 2000). These findings are confirmed by Maroufyan et al. (2010) who claimed marginal responses of performance and white blood cell differentiation to Met and Threonine supplement (once, twice and thrice of NRC) in broiler chicks challenged by infectious bursal disease. Our data suggest that the lowest ratios of heterophil to lymphocyte were obtained when diets with 150

and 250 mEq DEB/kg of diet were supplemented with the highest SAA level. Humoral immune response

Antibody responses to SRBC were not influenced by supplementation of DEB and SAA either individually or in combination (Table 4). However, Santin et al. (2003) reported a significant linear increase in Newcastle disease virus antibody titres with increasing DEB (40, 140, 240, 340 mEq/kg) following vaccinations with LaSota strain of the vaccine at 7 and 21 days of age. Consistent with our results, Swain and Johri (2000) indicated that shortage or excess of Met did not alter the antibody response to SRBC or synthetic antigen in rats. Nevertheless, our observation might be explained by the fact that more Met might have been required for affecting the antibody titre in the present experiment, as some studies have also shown that Met plays a role in the immune system by improving both humoral and cellular responses (Payne et al., 1990; Rama Rao et al., 2003). Electrophoretic pattern of serum proteins

After inclusion of low level of DEB in the diets, the albumin to globulin ratio was marginally

Table 4 Effects of DEB and SAA levels on antibody production titres against sheep red blood cell (SRBC) and serum protein pattern of broiler chicks

SAA level (%) 100

110

120

DEB level

SAA level

DEB level SAA level SAA 9 DEB level SEM

DEB level (mEq/kg) 150 250 350 150 250 350 150 250 350 150 250 350 100 110 120

Sheep red blood cell (log2) Primary

Secondary

Albumin (%)

Globulin (%)

A:G*

2.37 2.12 3.00 1.62 2.12 3.25 2.62 2.62 2.37 2.20 2.29 2.87 2.50 2.33 2.54 0.31 0.89 0.53 0.41

2.25 2.62 2.57 2.50 2.50 2.50 2.50 2.50 1.62 2.41 2.37 2.06 2.48 2.33 2.04 0.67 0.58 0.83 0.37

32.45 33.14 31.40 39.56 38.93 37.61 42.93 35.69 36.36 38.31 35.92 35.13 32.33b 38.70a 38.33a 0.12 0.0003 0.31 1.47

67.54 66.86 68.59 60.44 61.06 62.38 57.06 64.30 63.63 61.68 64.07 64.86 67.66a 61.29b 61.66a 0.12 0.0003 0.31 1.47

0.48 0.49 0.46 0.67 0.65 0.60 0.75 0.57 0.57 0.63 0.57 0.54 0.48b 0.64a 0.63a 0.11 0.0005 0.35 0.037

a-b Means with no common superscript within each column are significantly (p < 0.05) different. *A: G = Albumin/Globulin ratio.

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(p = 0.11) increased (Table 4). Confirming the present findings, previous reports (Griminger and Scanes, 1986; Ots et al., 1998) have demonstrated that increasing albumins shifts metabolism nutrition towards weight gain; however, enhanced globulin content showed that amino acids tended to favour response to immunogenic antigens (Ots et al., 1998). The highest ADWG (Table 6) and albumin to globulin ratio were obtained when birds received 150 mEq/kg DEB in their diets containing 120% SAA. Although Weissman et al. (2008) showed that supplementation of Met in diets had no significant effects on total serum protein content and albumin to globulin ratio in fattened rats, these parameters were significantly (p < 0.001) increased as a result of dietary inclusion of Met in the present study as evidenced by the higher percentage of albumin (15.65%) in broilers receiving 120% SAA than those receiving 100% SAA. Nevertheless, globulin content was by 8.87% lower in chicks fed 120% of SAA compared with those on 100% of SAA (p < 0.001). It is therefore probable that the lack of any Met effects on antibody production observed here might have remarkably reduced globulin percentage.

Jejunal histological changes

Table 5 summarizes the effects of different levels of DEB and SAA on jejunal morphology. Increasing DEB up to 350 mEq/kg led to a remarkable decrease in villus height (p < 0.05) as well as in crypt depth (p = 0.11). This DEB level led to diarrhoea in birds (data not shown). Borges et al. (2004) showed that feeding higher DEB levels led to an increase in excess Na excretion so that the absorptive surface area was declined due to the failure to provide for villi growth requirements. This is because the considerable proportion of nutrients absorbed in the small intestine is used by the mucosal layer (Xu et al., 2003). The highest villus height belonged to the birds fed diets with 150 mEq of DEB/kg; this might have been related to the beneficial acidic characteristics (Hassan et al., 2009) as the ionized forms of minerals might have been increased leading to better absorption for villus growth (Borges et al., 2007) and/or reduced proliferation of intestinal pathogenic bacteria (Roe et al., 2002; Xiao et al., 2008; Hassan et al., 2009). An increase in SAA levels led to an increase in villi height (p = 0.09) and a slight decrease (p = 0.10) in crypt depth (Table 5). It has been indicated that Met is an important amino acid for protein synthesis; hence,

Table 5 Effects of DEB and SAA levels on histological alterations of jejunal epithelial cells

SAA level (%) 100

110

120

DEB level

SAA level

DEB level (mEq/kg)

Villi height (lm)

Crypt depth (lm)

Villi height to Crypt depth ratio

Goblet cell number

Lamina propria follicle number

150 250 350 150 250 350 150 250 350 150 250 350 100 110 120

1440.87 1313.17 1191.25 1428.11 1461.22 1223.32 1414.57 1396.62 1352.32 1427.85a 1390.34a 1255.63b 1315.09 1370.88 1387.84

189.03 186.72 175.21 194.26 186.08 176.51 194.97 157.03 152.97 192.75 176.61 168.23 183.65 185.61 168.32

7.72 7.02 6.77 7.40 7.88 6.98 7.18 8.89 7.41 7.43 7.87 7.05 7.17 7.42 7.78

1921.60 1850.00 1637.20 1741.80 1633.20 1547.10 1620.20 1566.80 1310.35 1761.20a 1683.33b 1498.21c 1802.93a 1640.70b 1499.12c

2+* 1+ 3+ 1+ 1+ 2+ 3+ 2+ 4+ 2+ 1.3+ 3+ 2+ 1.3+ 3+

p-value DEB level SAA level SAA 9 DEB level SEM

0.011 0.09 0.48 10.25

0.11 0.10 0.46 3.01

0.44 0.51 0.56 4.38

0.0009 0.0004 0.5012 44.83

0.07 0.1 0.19 0.23

a-c Means with no common superscript within each column are significantly (p < 0.05) different. *Number of +’s indicates severity of the histological changes.

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SAA and dietary electrolyte balance in broiler chicks

it might induce villi growth. Met reportedly depresses intestine microbial populations (Dahiya et al., 2007), which decreases damages to jejunum and ileum villi, the two of which are not only recognized as important sites for invading enteric pathogens (Edelman et al., 2003), but also have slight nutrient requirements for cell turnover. On the other hand, supplementation of SAA declined crypt depth. The crypts of Lieberkuhn can be assumed as manufacturers of villus. New epithelial cells are produced by the stem cells that reside at the bottom of the crypts and migrate along with the villi to the top (Schat and Myers, 1991). A lower crypt depth may indicate a slower tissue turnover, suggesting that fewer attempts are required to compensate for normal sloughing or atrophy of the villus due to undesirable local factors and, thus, less nutrient supply is required to support the slower tissue turnover (Boka et al., 2014). An increase in DEB and SAA levels caused the jejunal goblet cell numbers to be suppressed, probably due to the reduced crypt depth. The crypt contains numerous specialist cells including germinal and goblet cells. A smaller crypt may reflect a decrease in goblet cell counts. Goblet cells produce mucine to protect intestinal villus from adverse luminal environmental

agents such as bacteria (Shirkey et al., 2006). Higher lamina propria lymphatic follicle populations may form in 120% SAA level due to the enhanced availability of Met for induction of local immunological responses to protect the intestinal villus from microbial injuries. Moreover, an increase (p = 0.07) in lamina propria lymphatic follicle numbers, after supplementation of 350 mEq DEB/kg, might be related to the adverse intestinal conditions that appear as diarrhoea. These may explain our findings that the highest jejunal villus height and goblet cell numbers in each SAA level were observed in birds fed ionic diets (150 mEq/kg). Growth performance

Table 6 summarizes the effects of different levels of DEB and SAA on the performance of broiler chicks. Dietary inclusion of different levels of DEB had no effect on ADFI during the starting period; however, reductions (p < 0.0001) in ADFI observed during the growing stage were about 9.8% and 10.6% and those for the entire experimental period were 11.92% and 11.66% in chicks receiving 350 mEq/kg compared with those fed 150 and 250 mEq/kg of DEB. These

Table 6 Effects of DEB and SAA Met and levels on performance parameters of broiler chicks during 1–42 days of age

SAA level (%) 100

110

120

DEB level

SAA level

DEB level (mEq/kg) 150 250 350 150 250 350 150 250 350 150 250 350 100 110 120

ADFI (g/day per bird)*

ADWG (g/day per bird)*

FCR (g of feed: g of gain)*

1–21

22–42

1–42

1–21

22–42

1–42

1–21

22–42

1–42

49.15 48.75 48.29 48.38 48.33 48.32 49.24 49.44 48.54 48.92 48.84 48.38 48.73 48.34 49.07

155.32 155.78 134.85 153.83 152.56 135.45 151.94 156.76 145.51 153.70a 155.03a 138.60b 148.65 147.28 151.40

102.23 102.16 84.22 101.16 100.28 88.17 102.00 102.11 96.62 101.80a 101.51a 89.67b 96.20b 96.53b 100.24a

32.07 32.70 30.67 31.79 31.07 31.81 33.09 32.98 31.86 32.32 32.25 31.45 31.81 31.56 32.64

74.32 66.23 52.57 72.66 71.78 50.37 74.72 69.87 58.48 73.90a 69.29b 53.81c 64.37 64.94 67.69

53.19 50.27 41.48 52.55 51.36 40.92 53.68 51.05 45.07 53.14a 50.89ab 49.42b 48.32 48.27 49.93

1.52 1.49 1.56 1.51 1.55 1.52 1.48 1.49 1.52 1.51 1.51 1.53 1.52 1.53 1.50

2.05f 2.35cd 2.58ab 2.15ef 2.12ef 2.68a 2.03f 2.25ed 2.48bc 2.08c 2.24b 2.58a 2.32 2.32 2.25

1.92 2.03 2.03 1.92 1.95 2.16 1.89 1.99 2.14 1.91b 1.99b 2.11a 1.99 2.01 2.01

p-value DEB level SAA level SAA 9 DEB level SEM

0.36 0.21 0.85 0.35

0.0001 0.18 0.36 2.24

0.0001 0.04 0.03 1.47

0.42 0.31 0.63 0.64

0.0001 0.26 0.30 1.87

0.0001 0.11 0.60 1.00

0.64 0.54 0.63 0.02

0.0001 0.28 0.034 0.04

0.0001 0.95 0.60 0.04

a-f Means with no common superscript within each column are significantly (p < 0.05) different. *ADWG: Average daily weight gain; ADFI: Average daily feed intake; FCR: Feed conversion ratio.

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SAA and dietary electrolyte balance in broiler chicks

depressions in ADFI were occurred in birds given the highest level of DEB as a result of higher dietary inclusion of Na leading to higher water intake and diarrhoea, which consequently affected feed consumption (Borges et al., 2004). Contrary to these findings, Borgatti et al. (2004) reported that different levels of DEB had no effect on feed intake during the rearing period. The lack of ADFI response to dietary inclusion of SAA during starter and grower periods observed in this study was similar to the results reported by Rubin et al. (2007), indicating that higher SAA levels of NRC recommendation did not influence ADFI during these phases. However, an increase in SAA level up to 120% enhanced (p < 0.05) ADFI during the entire experimental period by up to about 4% compared with other SAA levels. Previous studies have reported various effects of SAA supplementation on feed consumption. Feed intake in birds has been reported to increase (Ohta and Ishibashi, 1995), to decrease (Xie et al., 2007), or to remain unaffected (Rubin et al., 2007) by SAA administration. Although ADFI was not significantly affected by the interaction of DEB and SAA, it was decreased when 350 mEq/kg of DEB was administered. Consistent with the present findings, Adekunmisi and Robbins (2009) showed that different levels of DEB had no effect on ADFI in broilers fed different levels of CP. Our findings also indicate that the best ADWG was observed when birds received diets with the lowest level of DEB (150 mEq/kg) during both the growing and the entire experimental periods (p < 0.0001). Some mechanisms have been proposed to explain the beneficial effects of low-DEB diets on growth performance of broiler chicks. One such mechanism proposed for the antibacterial action of inorganic supplements (Hassan et al., 2009) such as NH4Cl (Xiao et al., 2008) used in anionic diets involves the changes in cytoplasmic pH and the energetic status of the microbial cells due to the anion accumulation and cytoplasmic membrane disturbance (Roe et al., 2002). Another mechanism is the beneficial effect of intestinal acidized content in low-DEB diets on the intestinal ionization, which leads to improved bioavailability of ions (Borges et al., 2007). On the other hand, increase in DEB content up to 350 mEq/kg decreased ADWG markedly (p < 0.0001). This depression could be explained by the reduced feed intake as well as the significant suppression in villus height in the small intestine, especially in the jejunum (p < 0.05, Table 5). Similar results have been reported by Johnson and Karunajeewa (1985) who demonstrated that a dietary electrolyte balance higher than 300 mEq/kg caused the weight of broiler chicks to decline. Con-

firming our findings, Ahmad et al. (2008) showed that in chicks fed DEB 50, 150 and 250 mEq/kg, compared with those receiving DEB 0 and 350 mEq/kg, the 42-day body weight gain and FCR were improved and the mortality rate declined. Supplementation of the highest level of SAA slightly (p = 0.11) improved ADWG. This partial improvement might be related to an increase in feed intake as well as the improved intestinal health indices such as villi height or lamina propria lymphatic follicle numbers (Table 5). Thinner intestinal epitheliums reportedly promote nutrient absorption and reduce the metabolic demands of the gastrointestinal system (Visek, 1987; Hampson, 1986; Sakamoto et al., 2000; Boka et al., 2014). Also, taller villi raises the enzyme activities secreted from the tips of the villi, leading to improved digestibility and absorption (Hampson, 1986). Swain and Johri (2000) found no remarkable effects on weight gain when different levels of Met were supplemented at 42 days of age; however, Weissman et al. (2008) claimed that SAA inclusion was able to improve ADWG during the rearing period. The best FCR in both the growing and the entire experimental periods were obtained when birds fed diets with the lowest DEB level (p < 0.0001). These improvements were mainly due to the remarkable increase in ADWG (p = 0.0001). At the end of the experiment, the poorest FCR was found to belong to birds fed diets containing 350 mEq DEB/kg. In agreement with our findings, Borges et al. (2007) showed that the worst FCR was observed when chicks were fed diets containing high-DEB level (340 mEq/kg). The lack of FCR response to different SAA levels may be due to the concomitant increase in ADFI and ADWG in chicks (Table 6). Meirelles et al. (2003) reported that SAA levels had no significant effects on FCR in chickens. However, this finding was in contrast to Garlich’s (1985) who found remarkable improvement in the feed conversion ratio in broilers fed 6.3 g/kg Met supplement. Although some previous reports (Adekunmisi and Robbins, 1987a, 2009; Murakami et al., 2003) claimed that there were interactions between different levels of DEB and dietary crude protein on growth performance of broilers, our findings indicate that the interaction of DEB and SAA are not significant with respect to ADWG and FCR over the entire trial period so that inclusion of 150 mEq/kg in each SAA level led to an improvement in growth performance.

Journal of Animal Physiology and Animal Nutrition © 2015 Blackwell Verlag GmbH

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Conclusions The findings of the present study indicated that inclusion of low-DEB in diets decreased the ratio of hetero-

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phile to lymphocyte but improved that of albumin to globulin and the intestinal health indices. Moreover, the best growth performance was obtained with diets containing 150 mEq DEB/kg for each level of SAA. References Abbas, A.; Jamshed Khan, M.; Naeem, M.; Ayaz, M.; Sufyan, A.; Hussain Somro, M., 2012: Cation anion balance in avian diet: a review. Agriculture Science Research Journal 2, 302–307. Adekunmisi, A. A.; Robbins, K. R., 1987a: Effects of dietary electrolyte balance on growth and metabolic acid-base status of chicks. Nutrition Research 7, 519–528. Adekunmisi, A. A.; Robbins, K. R., 1987b: Effect of dietary crude protein, electrolyte balance and photoperiod on growth of broiler chicks. Poultry Science 66, 299– 305. Adekunmisi, A. A.; Robbins, K. R., 2009: Dietary electrolyte requirement of broiler chicks as affected by dietary protein content. Pakistan Journal of Nutrition 8, 1613–1616. Ahmad, T.; Mushtaq, T.; Khan, M. A.; Babar, M. E.; Yousaf, M.; Hasan, Z. U.; Kamran, Z., 2008: Influence of varying dietary electrolyte balance on broiler performance under tropical summer conditions. Journal of Animal Physiology and Animal Nutrition 93, 613–621. Al-Maya, A. A. S., 2006: Immune response of broiler chicks to DL-methionine supplementation at different ages. International Journal of Poultry Science 5, 169–172. AOAC, 2006: Official Methods of Analysis. AOAC International, Arlington, VA. Berne, M. R.; Levy, M. N., 1998: Physiology, 4th edn. Guanabara Koogan, Rio de Janeiro. Boka, J.; Mahdavi, A. H.; Samie, A. H.; Jahanian, R., 2014: Effect of different levels of black cumin (Nigella sativa L.) on performance, intestinal Escherichia coli colonization and jejunal morphology in laying hens. Animal Physiology and Animal Nutrition 98, 373–383. Borgatti, L. M. O.; Albuquerque, R.; Meister, N. C.; Souza, L. W. O.; Lima, F. R., 2004: Performance of broilers fed diets with different dietary electrolyte balance under summer conditions. Brazilian Journal of Poultry Science 3, 153–157. Borges, S. A., 2001: Balanco eletrolitico e sua interrelacao com o equilibrio acidobase em frangos de corte submetidos a

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Effects of different sulphur amino acids and dietary electrolyte balance levels on performance, jejunal morphology, and immunocompetence of broiler chicks.

As alterations of dietary electrolyte balance (DEB) can influence amino acid metabolism via changes the ions incur in their configurations, performanc...
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