Chickens, diet and experimental design

A total of 60 unsexed chicks of native chickens’ breeds (Sinai) were used in this study. Chicks were randomly distributed into two feed restriction treatment groups keeping similar body weight at 7 d of age. Each treatment was replicated five times of six unsexed chicks each. During 0-6 d of age, chickens were fed ad libitum commercial diets containing adequate nutrients for growth, 22.92 % crude protein (CP) and 12.72 MJ ME/kg diet with adequate amino acids, minerals and vitamins. During d 7-14 of age, chicks were fed either 100 or 80 % of the daily amount of feed consumed by the control group during the previous day. From d 15 to 35, chickens were fed ad libitum diets containing 21.1 % CP and 12.98 MJ ME/kg until the end of the experiment.

Husbandry

Chicks were housed in battery brooders during d 1 to 35 of age and vaccinated with Hitchiner + IB + Gambaro, Influenza H5 N2, Colon30, Gambaro 123, Colon79 and Lasota at d 7, 9, 10, 16, 20 and 30 of age. During the experiment, the ambient temperature and the relative humidity were 31.4 ± 4 °C and 44.9 ± 6 %, respectively; with 24 h light in day one, and 23:1 light-dark cycle during d 2 to 35 of age.

Measurements

Chickens were weighed (g) on replicate basis at d 7, 14, 28, and 35 of age. Chicks were weighed in the morning before offering feed, while body weight gains were predicated by deducting body weight at end of each period from the initial body weight at the same period per replicate. Feed intake per replicate was recorded during the periods 7-14, 7-28 and 7-35 d of age. Feed conversion ratio (kg feed/kg gain) per replicate was calculated as the units (g) of feeds needed to yield one unit (g) of growth during the 7-14, 7-28 and 7-35 d of age. Survival rate was presented as number of lived chickens at the termination of the experiment in each group as relative to the initial number of chicks in each treatment.

At 35 d of age, six chickens comprising three males and three females representing all treatment replicates were selected randomly from each treatment, weighed after overnight fasting, slaughtered according to the Islamic method, bleed, and feather picked. The lymphoid organs thymus, bursa of Fabricius and spleen, were separated and individually weighed and expressed as relative to live body weight. At 14 and 35 d of age, blood samples (n= 6 chickens per treatment representing all treatment replicates at each age, which were selected randomly) were taken from the wing-vein of chickens before access to feed and water. The blood samples were collected in two clean dry centrifuge tubes with or without an anti-coagulant (heparin). The one with heparin was used for determination of blood hematology, phagocytic activity (PA) and phagocytic index (PI). Then the tubes were centrifuged at 3,000 rpm for 20 min to clearly separate serum and plasma. Serum and plasma were stored at -20 °C until analysis. Blood biochemical constituents were determined using commercial diagnostic kits purchased from Diamond Diagnostic company (23 EL-Montazah St. Heliopolis, Cairo, Egypt), while blood hematology were performed as cited by Attia et al^8,9. Plasma tri iodo threonine (T₃) and thyroxine (T₄) were determined according to the method of Young et al¹⁰. Phagocytic activity and phagocytic index were determined as cited by Kawahara et al¹¹. The serum antibody against New Castle disease virus (NDV) and avian influenza virus (AIV) was done via haemagglutination inhibition (HI) test^12,13. The antibody titer for infectious bursa disease virus (IBDV) was carried out according to Cosgrove¹⁴. Bactericidal activity (BacA) in serum was according to Rainger et al¹⁵.

Statistical analyses

Analysis of variance was done using one-way ANOVA of SAS®¹⁶ for growth performance and two-ways for traits containing two factors such as feed restriction and sex or age of chickens. All the percentages were transformed to log¹⁰ to normalize data distribution. Mean difference at P0.05 was tested using the Student Newman Keuls test¹⁶. Survival rate was analyzed using chi-square test.

Growth performance

Table 1 indicates the effect of FR on growth, feed intake and FCR and survival rate. FR significantly decreased growth during d 7-14, 7-28 and 7-35 of age. It was found that 27.2 % FR during the 2^nd wk of age had long term negative effect on growth of Sanai chickens (for example, 31.6, 14.6 and 11.7 % during 7-14, 7-28 and 7-35 d of age, respectively). These indicated a compensatory growth during 14-28 and 14-35 d of age without induction of complete recovery at the termination of the experiment (88.3 %). The decreased growth of FR group during the FR period coincided with lowered nutrients intake (72.8 %), which account for 86 % of the decrease in growth. Similar to the present results, growth of broilers was significantly higher for the control group than those of the FR groups^7,17,18. However, a compensatory growth in broilers following FR was reported^1,5,7. Moreover, broilers feed restricted grow faster and were heavier at market age than the control group, while group on the 4- and 6-d FR and the control chickens have similar weight at marketing⁶. The published results indicated that most weight loss during early FR in chickens could be compensated by 20-25 d of the re-feeding period¹⁹. Moreover, FR at 75 % or 50 % for 7 and 14 d period, respectively did not significantly influence the final body weight of broilers at 42 d of age²⁰. These contradictory results about the effect of FR on the growth could be due to the duration and intensity of FR and/or strain of chickens.

Table 1. Effect of feed restriction regimen on growth performance of native strain during 7-35 d of age (g) 

¹=Number of observation were 5 replicates of 6 unsexed chicks each/treatment; SEM= standard error of mean, FR= Feed restriction.

^{a, b} Differences among means within a column within each factor not sharing similar superscripts are significant (P<0.05).

Feed intake of FR group was decreased during 7-14 d of age and there was a significant difference (P=0.046) compared to the control. The re-alimented chickens consumed more feed by 6.6 % during d 14-35 of age; this indicates an attempt to compensate for the decrease (27.2 %) in feed intake that occurred during FR period. Similarly, feed intake at 21 d of age was significantly decreased for feed-restricted chicks². In addition, the feed-restricted chicks and the control groups at 63 d of age consumed similar amount of feed. In addition, FR did not affect feed intake for the whole period²¹.

Feed conversion was not affected by FR treatments during the period 7-14 and 7-35 of age, but significantly impaired by 19.7 % compared to the control group, during d 7-28 of age. In concert with the current results, there was no significant effect of FR regimen on the body weight, feed intake, and FCR in the whole period^21,22. However, an improvement in FCR due to increased growth and decreased feed intake via FR was observed⁷. Others, observed that feed efficiency for maintaining body weight may be affected by the plan of nutrition, but this was confounded by nutritional regimen and duration, growth, strain and age of chickens²³.

Survival rate was similar among the experimental groups, showing that FR had no harmful effect and correlate to the findings of other experiments^1,5,20. However, published results indicated that FR increased survival rate due to less sudden death, ascites and skeletal disorders^24,25.

Lymphoid organs, antioxidant status and immune markers

FR regimen did not have significant influence on lymphoid organs (Table 2), total antioxidant capacity (TAC), malondialdehyde (MDA), Immunoglobulin M (IgM), Immunoglobulin G (IgG), HIIBD, HIAI, Lysosome activity (LyzA), BacA, PA and PI, but immunoglobulin A (IgA) significantly increased by 3.9 % compared to the control (Table 3). On the other hand, humeral immunity HINDV decreased by 24.6 % and non-specific immune components such as monocyte (macrophages; Table 4) decreased by 13.6 %. The increase in IgA of the FR groups indicated the stressful condition of chickens exposed to FR regimen undergo as proved by the low antibody titter to NDV and low growth at the end of the experiment. IgA involves in mucosal surface protection²⁶, and prevents immunoglobulin degradation by proteolytic enzymes, thus improve its survival in the harsh gut environment and protection against the multiplication of microbes in body secretions²⁷. IgA can also prevent inflammatory influences of other immunoglobulin²⁸.

Table 2. Effect of feed restriction regimen and/or sex of chickens on the absolute and relative weight of thymus, bursa and spleen at 35 d of age 

¹= number of observation were 6 chickens/treatment. ²= number of observation were 6 chickens/sex, ³= number of observation were 3 chickens/treatment; SEM=standard error of mean.

Table 3. Effect of feed restriction regimen and/or age of chickens on antioxidant status and immune markers 

TAC=Total antioxidant capacity; MAD= Malondialdehyde; IgA= Immunoglobulin A; IgM=Immunoglobulin MM IgG=Immunoglobulin G; HINDV= Newcastle disease; HIIBD= Infection bursa disease; HIAI=Avian influenza; lyzA=Lysosome activity; BacatA= Bactrio activity; SEM= standard error of mean.

¹= Number of observation were 12 chicks/treatment, ²= Number of observation were 12 chicks/age, ³= Number of observation were 6 chicks/treatment/age.

^a,b,c Differences among means within a column within each factor not sharing similar superscripts are significant (P<0.05).

Table 4. Effect of feed restriction regimen and/or age of chickens on characteristics of white and red blood cells 

WBC= white blood cells; HLR= Heterophile/Lymphocyte ratio; Hgb= Hemoglobin; PCV= Packed cell volume; MCV= Mean corpuscular values; SEM= Standard error of mean.

¹= Number of observation were 12 chicks/treatment, ²= Number of observation were 12 chicks/age, ³= Number of observation were 6 chicks/treatment/age.

^a,b,c Differences among means within a column within each factor not sharing similar superscripts are significant (P<0.05).

Age of chickens had a significant effect on TAC and IgA showing a significant decrease at 35 d of age compared to 14 d of age; but, IgG and HIIBD significantly increased. FR increased TAC and IgA of FR group at 14 d of age, but IgG and LysA were decreased. In addition, 35-d-old FR group increased IgG and LysA, revealing the positive impact of re-alimentation on immune response of chickens^29,30. Nonetheless, IgG of broiler chickens at 14 and 35 d of age were not significantly influenced by either quantitative FR regimen³¹, qualitative FR regimen or one-third reduction in amino acids, protein and energy³². However, there were no significant effects of FR on immune structure and function of the gut, including bursa mass, spleen mass, and total IgA content of intestinal flush samples³³.

There was significant interaction between FR regimen and age of chicks on only TAC, IgA, IgG and LysA. The results indicate that TAC and IgA of FR group at 14 d of age was significantly greater than those of the other groups, but IgG and LysA were lower than those of the control group at d 14 and FR group at d 35 of age. On the other hand, IgG and LysA of 35-d-old FR group were higher than the control at the same age. In accordance with the current results, immune response against NDV and IBD at 30^th d of age was found to be lower in the FR group than the control group³⁴. In addition, FR at 75 and 50 % of daily feed intake during 7-14 or 7-21 d of age did not affect absolute and relative weight of spleen and thymus, but groups on FR at 75 and 50 % for 7 d significantly increased Fabricius bursa compared to their counterparts groups²⁰. Similarly, nutritional status had a crucial role in immunity and under-nutrition can negatively affect immune function^29,31.

Blood components

Table 4 shows the effect of FR regimen and age of chicks on the parameters of white and red blood cells. Obviously, FR regimen had no significant effect on most of white blood cell parameters except for monocyte, showing that lower value of FR group than that of the control group, while there was a trend for heterophile to increase (P≤0.068).

Age of chickens had a significant influence on white blood cells (WBC) count and monocyte, showing higher WBC of 35-d old than those of 14 d-old, but monocyte was lower. There were significant interactions between FR regimen and chickens age in the lymphocyte, herterophile and heterophile/ lymphocyte ratio. The results show that 14 d-old chickens on FR regimen showed lower WBC count (P≤0.092) than the other groups, but higher lymphocyte (P≤0.004) than the 14 d-old control group and 35 d-old FR group. Chickens (35 d-old) on the control treatment exhibited significantly lower heterophile than those of the other groups, and lower heterophile/lymphocyte ratio than those of the control group at 14 d of age and FR group at 35 d of age. At the end of FR period (14 d of age), WBC count decreased, but lymphocyte (cell-mediated immunity) increased compared to 35 d-old FR group. These results indicate that though WBC count decreased at the end of FR period, chickens are compensated with increasing cell mediated immunity and had similar non-specific immune component (heterophile) and stress index heterophile/lymphocyte ratio. However, at 35 d of age, FR groups exhibited greater stress than the control group at the same age; this can be attributed to the significant decrease in cell-mediated immunity (lymphocyte). These changes in WBC count, lymphocyte and heterophile/lymphocyte ratio indicate that slow-growing chickens adapted to FR regimen by modifying different immune components. The decrease in IgG, HINDV and HIAI at the end of FR period and at the end of the re-alimentation period confirm this hypothesis. During the first 3 wk of age, a transition from maternal immunity to humoral immunity and development and maturation of immune system occurred³⁵. These changes in immunoglobulins IgG and antibody against NDV, IBDV and AI proved the improvement in the defence system with increasing age of chickens. However, the decrease of TAC with advanced age of chickens may indicate the use of antioxidants for antibody production during the transient period. Similarly, antibody to NDV of ad libitum fed chickens declined with duration of stress³⁶. H-L ratio and tonic immobility duration were higher at 41 d of age in broilers fed ad libitum than those FR for 4 h per day from 7 to 21 d and fed ad libitum thereafter²³, and FR induced stress on WBC during only the FR period^37,38.

It was demonstrated that FR regimen and age of chickens did not significantly affect Hgb, PCV and MCV. However, there were significant interactions between FR regimen and chickens‘ age on the Hgb, PCV and MCV. It was found that 14 d-old chickens on FR showed significantly higher Hgb and PCV than its counterpart group on the control regimen. However, MCV of the control group at 35 d of age was significantly higher than that of the FR group at the same age. Moderate FR regimen (27.2 %) during the 2^nd wk of age had no deleterious effect on blood haematology, as difference within FR groups was not significant between 14 and 35 d old chickens. Results on the effects of FR regimen on blood haematology are rare in the literature. In concert with the current results, haematological values were not affected by FR or sex (P>0.05)¹⁷. In addition, feed removal for 3 or 6 h a day during 5-37 d of age did not significantly affected the PCV and RBC, but feed removal for 6 h significantly increased Hgb compared to the control and 3 h FR groups³⁹.

Metabolic profiles

Table 5 reveals the influences of FR regimen and age of chicks on blood plasma biochemical constituents. It was found that FR had a significant effect on only plasma albumin and total cholesterol, but the effect on plasma total protein, globulin, T₃, T₄, T₃/T₄, glucose and cortisone was not significant. FR significantly increased plasma albumin compared to the control group, but decreased plasma cholesterol. On the other hand, increasing age of chickens significantly increased plasma albumin, but decreased plasma total protein, globulin, T₃ and glucose. There were significant interactions between FR regimen and age of chicks on plasma total protein, albumin and globulin. The control group at d 14 of age showed higher plasma total protein and globulin than the other groups except for total protein of the FR group at d 35 of age. However, the contrary trend was observed in the plasma albumin. FR significantly increased plasma albumin (non-specific immune protein) by 5.3 % compared to the control group, but decreased plasma cholesterol by 3.2 %. Serum glucose (P<0.05) and triglyceride (P<0.001) levels decreased at 41 d of age in broilers fed ad libitum compared to those restricted for 4 h per day from 7 to 21 d and fed ad libitum thereafter²⁰. FR at 12.5 and 25 % of the predicted requirement did not significantly influence Hgb, PCV, serum glucose, total protein, globulin, albumin/globulin ratio goats⁴⁰, but decreased serum albumin and urea and improved digestibility of DM, OM, CP, EE and ADF and decreased excretion of N and P. Hepatic leakage (AST and ALT enzymes), muscle catabolism (creatinine) and welfare of chickens (cortisone) at 14 d (end of restriction period) and 35 d of age (end of re-alimentation period) were not affected by FR regimen.

Table 5. Effect of feed restriction regimen and/or age of chickens on blood plasma biochemical traits 

C= control group; FR= Feed restriction; T₃=Tri-iodotherionine; T₄= Tetra-iodotherionine; T₃/T₄=Tri-iodotherionine/Tetra-iodotherionine ratio; SEM= Standard error of mean.

¹= Number of observation were 12 chicks/treatment, ²= Number of observation were 12 chicks/age, ³= Number of observation were 6 chicks/treatment/age.

^a,b,c Differences among means within a column within each factor not sharing similar superscripts are significant (P<0.05).

FR group decreased total protein and globulin (specific immune protein) at the termination of the FR period compared to the control group, but increased plasma albumin (nonspecific immune protein and the most favourable source of amino acids for protein synthesis). At the end of the experiment, total protein, albumin and globulin were completely restored. This is connected with lower IgG, HINDV and HIIBD at the termination of the FR period than those at d 35 of age after 21 d of ad libitum feeding, which indicates that FR decreased nutrient availability for building up specific immunity and that re-alimentation improved immune system³⁴. Similarly, plasma total protein and albumin are significantly decreased in either quantitative (fed 85 and 70 % of feed intake by the control group) or qualitative FR (fed 85 and 70 % of the energy control diet). However, at 35 d of age these blood traits were higher in the qualitative FR groups³⁶.

FR regimen did not negatively affect liver leakage (AST and ALT enzymes), protein metabolism and renal function (creatinine) and welfare of chickens (cortisone) at 14 (end of restriction period) and 35 d of age (end of re-alimentation period) and markers of liver leakage⁴⁰. However, a significant changes of corticosterone, T_4, T₃, protein, alkaline phosphatase, creatinine kinase in meat type female chickens caused by moderate and severe quantitative FR, illustrating that FR induces an intensive stress during the rapid growth period of fast-growing chicks⁴¹. In addition, plasma levels of both T₃ and T₄ were reduced in the FR chicks, and up to 40 % diet dilution decreased cholesterol, triglycerides, plasma T₃⁴², but uric acid and plasma T₄ increased at 21 d of age⁴³. Differences in metabolic profiles and thyroid hormones could be attributed to strain and growth of chickens, duration and intensity of FR.