Effect of protein, probiotic, and symbiotic supplementation on serum biological health markers of molted layers

Effect of protein, probiotic, and symbiotic supplementation on serum biological health markers of molted layers H. Anwar,1 Z. U. Rahman, I. Javed, and F. Muhammad Department of Physiology and Pharmacology, Faculty of Veterinary Sciences, University of Agriculture, Faisalabad-38040, Pakistan ABSTRACT Dietary zinc was used to induce molt in 200 White Leghorn birds in caged housing at the age of 70 wk. The birds were equally and randomly allocated to 4 groups each of 50 birds as G1 (control; CP 16%, no supplement), G2 (CP 18%, no other supplement), G3 (CP 16%, symbiotic Perfectin: 85 mg·L−1 in drinking water daily), and G4 (CP 16%, probiotic Protexin: 85 mg·L−1 in drinking water daily) after the completion of molt. The sampling was conducted 3 times at 5% production, peak production, and end of production with 15 birds being killed at each sampling from each group to collect the blood for harvesting of serum. Serum health markers including total oxidant status, total antioxidant capacity, homocysteine concentration, and

paraoxonase, arylesterase, and ceruloplasmin activity were determined by the prescribed assays. The overall total antioxidant capacity was increased, whereas total oxidant status and homocysteine concentrations were reduced significantly (P ≤ 0.01) in all the supplemented groups compared with the control. The paraoxonase and ceruloplasmin activity were enhanced (P ≤ 0.01) in the supplemented groups compared with the control, and arylesterase activity was increased (P ≤ 0.01) in only G2 compared with the other groups. Although protein supplementation significantly reduced the oxidative stress, supplementation with symbiotic and probiotic also improved the health status by decreasing the oxidative stress in the birds.

Key words: molted layer, health biomarker, protein, probiotics 2012 Poultry Science 91:2606–2613 http://dx.doi.org/10.3382/ps.2012-02172

INTRODUCTION Zinc-induced molting resulted in an increased humoral immunity and more IgM and IgG production (Sandhu et al., 2007), as well as improved cell-mediated immunity (Sandhu et al., 2006). Zinc-induced molting was found to be an improved method regarding disease resistance along with production performance and the dynamics of pituitary-hormone-producing cells (Sandhu et al., 2010). Therefore, dietary zinc was used to induce molt in the birds in this experiment. Some studies reported about supplementation of the diet of molted layers to achieve early recovery from molting stress, better production performance, and improved health status. Togun et al. (2004) reported a positive effect of supplementation with protein and follicle-stimulating hormone in molted Nera black hens in terms of early recovery from postmolt stress and better performance. Likewise, Koelkebeck et al. (1991) showed that the birds on the comparatively high protein diet (13 to 16%) recovered earlier from molting stress. ©2012 Poultry Science Association Inc. Received January 19, 2012. Accepted June 19, 2012. 1 Corresponding author: [email protected]

Lactobacillus and Bifidobacterium are commonly used bacterium in probiotics. During stress-induced molting, these microorganisms were most commonly affected in birds (Patterson and Burkholder, 2003). The colonization of pathogenic Salmonella species was also increased after a period of stress (Craven, 2000). Supplementation with probiotics (Stern et al., 2001) and symbiotics was reported to increase the disease resistance and exclusion of pathogenic bacteria from the gastrointestinal tract. A study by Haghighi et al. (2006) suggested that probiotics have an immune-boosting role due to enhanced induction of natural antibodies in chickens. The accumulation of the reactive oxygen species (ROS) as a result of the increase of different physiological processes with age lead to the phenomenon of aging (Junqueira et al., 2004). The low generation of endogenous antioxidants and decreased intake in diet are contributing factors to oxidative stress (Maggio et al., 2003). Sahin et al. (2002) reported oxidative stress was increased in the stressed birds. The activity of antioxidant defense enzymes such as paraoxonase (PON) decreases because of environmental stress factors (Gumuslu et al., 2002). Sahin et al. (2004) reported the development of oxidative stress and diminished antioxidant status during the stressful condition in Japanese quail. They reported an

2606

2607

BIOLOGICAL MARKERS IN SUPPLEMENTED MOLTED LAYERS Table 1. Molting schedule of spent White Leghorn through high dietary zinc at 3 g/kg of feed Stage Premolt1 Start of molting Rest period2,3 Postmolt4

Age (wk) 70 71 to 74 74 to 75 76 77 onward

Feed offered (g/bird) 110 35 (ZnO 3 g/kg of spiked feed) 70 + 2% CaCO3 80 110

Water Ad Ad Ad Ad Ad

libitum libitum libitum libitum libitum

Light (h) 24 (d 1) then 16 12 12 14 16

1Deworming,

egg drop syndrome, and Newcastle disease vaccination. treatments of all the groups were started. 3Start of egg production. 4A 5% production level. 2Respective

increase in the malondialdehyde (MDA) and homocysteine levels in serum and decrease in PON and arylesterase activity in quail under heat-induced stress. The antioxidant system, its related enzymes, and health markers associated with the overall physiological health status have been extensively studied in humans under different physiological and pathological conditions. Such studies in poultry are lacking. Almost no reports are available regarding the overall status of these biological health markers in egg-laying hens, especially in the context of supplementation with proteins, probiotics, and symbiotics. The current experiment was planned to investigate various health markers in response to additional protein, probiotic, and symbiotic supplementations in postmolted White Leghorn hens.

MATERIALS AND METHODS The use of animals and protocols in this experimental trial was in compliance with the conduct established and approved by the University of Agriculture Institutional Animal Care and Use Committee.

Experiment The study was conducted on commercial White Leghorn hens, which are at the end of their first production cycle (70 wk of age). A total 200 caged housed birds from a commercial poultry farm were brought to the poultry research station of the Department of Physiology and Pharmacology, University of Agriculture, Faisalabad. The farm used cages, and birds were housed one bird per cage. The birds were given a week to acclimatize to the new environmental conditions. The birds were dewormed, and ration (CP 16%, energy 2,795 kcal) was offered at 100 g/bird per day with water ad libitum during the acclimatization period, and the birds were then randomly allotted into 4 groups (G1, G2, G3, and G4) having an equal number (50) of birds in each group. High dietary zinc (3 g/bird per day) was used to induce molt in all the birds after they were initially weighed. The molting schedule is given in Table 1. The birds were allowed to lose up to 35% of their initial BW until the completion of molting (Sandhu et al., 2007). Mortality was 3.78% during the process of molting. After molting, groups were allocated to

their respective treatments with G1 as control (diet CP 16%, no other supplement), G2 (diet CP 18% diet, no other treatment), G3 [diet CP 16%; symbiotic (Perfectin) in water at 85 mg∙L−1 of drinking water daily], and G4 [diet CP 16%; probiotic (Protexin) at 85 mg∙L−1 of drinking water daily]. The feed, Perfectin, and Protexin formulations are shown in Table 2. The viability for bacterial species of Perfectin (1 × 104 cfu∙mL−1) and Protexin (1 × 106 cfu∙mL−1) was determined in the research laboratory of the Department of Microbiology, University of Agriculture, Faisalabad, Pakistan.

Serum Biological Health Markers The biological health markers were determined in serum to assess the impact of supplementation on the dietary zinc induced molted birds throughout their 2nd production cycle. Fifteen birds from each group were killed at 5% production (5P), peak production (PP), and end (EP) of production. Blood was collected in sterilized chilled test tubes without anticoagulant. The test tubes with blood were kept at 4°C for 30 min and blood was allowed to clot. The clot was then dislodged from the walls of the test tube and centrifuged at 769 × g at 4°C (Beckman TJ-6 refrigerated centrifuge, Beckman Coulter Inc., Brea, CA) for 10 min to separate the serum. The serum was collected in triplicate for each sample in microfuge tubes and frozen at −20°C until further analysis. Fifteen serum samples from each group were repeated twice (total of 30 readings) at each sampling stage, and their means were used for statistical data analysis.

Chemicals and Instrumentation The analytical reagent grade chemicals used in assays were provided by Sigma (St. Louis, MO) and Merck (Lahore, Pakistan) excluding ABTS, which was provided by Fluka. The probiotic (Protexin) was provided by Probiotics UK International (Lopen Head, Somerset, UK) and the symbiotic (Perfectin) was supplied by Diasham, Diasham Resource Pte. Ltd., Jurong, Singapore. Bio System (BTS-330, Biosystems S.A., Costa Brava, Barcelona, Spain) was used for all the spectrophotometer analyses. Ceruloplasmin activity was measured

2608

Anwar et al.

Table 2. Feed formulation (g/100 g) for molted White Leghorn hens1,2

Feed ingredient Corn Rice tips Rice polishing Maize gluten 30% Maize gluten 60% Canola meal Soybean meal Fish meal Dicalcium phosphate Limestone powder/0 chips Vitamin premix3 + amino acid4 Total

CP 16% energy = 2,795 kcal (g/100 g)

CP 18% energy = 2,800 kcal (g/100 g)

40 10 11 6 — 10 8 6 1.5 7 0.5 100

40 10 11 — 4 10 10 6 1.5 7 0.5 100

1Composition of Perfectin (Diasham, Diasham Resource Pte. Ltd., Jurong, Singapore; per kg): probiotic (viability: 1 × 104 cfu∙mL−1): Lactobacillus acidophilus, Bifidobacterium thermophilus, Bifidobacterium longum, Streptococcus faecium; prebiotics: vitamin A, 4,000,000 IU; vitamin D3, 800,000 IU; vitamin E, 500 IU; vitamin K, 200 mg; vitamin B1, 200 mg; vitamin B2, 2,000 mg; vitamin B6, 600 mg; vitamin B12, 2,000 mg; vitamin C, 2,000 mg; folic acid, 100 mg; niacin, 10,000 mg; l-lysine, 5,000 mg; dl-methionine, 15,000 mg; iron, 7,500 mg; copper, 1,000 mg; zinc 7,500 mg; manganese, 10,000 mg. 2Composition of Protexin (Probiotics UK International, Lopen Head, Somerset, UK) viability (1 × 106 cfu∙mL−1): Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacillus rhamosus, Bifidobacterium bifidum, Streptococcus thermophilus, Enterococcus faecium, Aspergillus oryzae, Candida pintolopesi. 3Composition per kilogram of diet: vitamin A, 8,300 IU; cholecalciferol, 2,200 ICU; vitamin E, 8 IU; vitamin B12, 0.02 mg; riboflavin, 5.5 mg; d-calcium pantothenic acid, 15 mg; niacin, 36 mg; choline, 500 mg; folic acid, 0.5 mg; vitamin B1, 1 mg; pyridoxine, 2.2 mg; biotin, 0.05 mg; vitamin K, 2 mg. 4Composition per kilogram of diet: methionine, 0.143 g; lysine, 0.72 g; threonine 0.35 g.

by using UV/Vis spectrophotometer, Hitachi model U-2000 (Hitachi Instruments, Schaumburg, IL).

Total Oxidant Status The method described by Erel (2005) was used to assess total oxidant status (TOS; µmol of H2O2equiv.∙L−1) in the serum samples. The TOS of samples were determined equivalent to H2O2 standards (12.50, 6.25, 3.12, and 1.56 µM/L) from the standard curve. The minimum detectable limit of the assay was 0.13 μmol of H2O2equiv.∙L−1, with <3% precision and linearity up to 200 μmol of H2O2equiv.∙L−1. The intraassay CV was kept under 10%.

Total Antioxidant Capacity To estimate the total antioxidant capacity (TAC; mmol of Troloxequiv.∙L−1), the method of Erel (2004) was used. The standards of 0.1, 0.3, 0.6, 0.9, 1.2, and 1.5 mmol/L were made from Trolox (vitamin E analog; 30 mM stock) to construct the standard curve. The minimum detectable range of this assay was 0.18 mmol∙L−1 and was linear up to 6 mmol of Trolox equiv.∙L−1 with intraassay CV below 3%.

Homocysteine The homocysteine (Hcy; µmol∙L−1) assay kit (enzymatic cycling), blank, and standards supplied by Diazyme Laboratories (San Diego, CA) was used to estimate the homocysteine in serum samples. The minimum detectability of the assay was 1.5 µmol/L and intraassay CV was 2.2 to 5%.

Paraoxonase Activity The method described by Juretić et al. (2006) was adopted with some modifications to estimate the paraoxonase (PON; U∙min−1∙L−1) activity in serum samples. Briefly, first absorbance reading was taken after the mixing of working reagent (350 µL) and serum sample (10 µL) at 37°C. After the first reading, an increase in the absorbance was recorded for 1 min at λ 405 nm. The prescribed formula was used to calculate the enzyme per minute activity. The minimum detection limit of this assay was 80 to 100 U∙min−1∙L−1. The sensitivity/initial rate of hydrolysis was stable up to 5 min, and intraassay CV was under 10%.

Arylesterase Activity (kU∙L−1) The enzymatic activity per minute was estimated and calculated by the formula as described by Juretić et al. (2006). The minimum level of detection was 40 to 55 kU∙min−1∙L−1. The intraassay CV was less than 7%, and the rate of hydrolysis was stable up to 5 min after initial hydrolysis.

Ceruloplasmin Activity Ceruloplasmin activity (U∙L−1) was determined by the method of Schosinsky et al. (1974) with some amendments. Delta absorbance was used to calculate the enzyme activity from the formula described in the reference method. The minimum detectable range of the assay was 7 to 10 U∙L−1 with CV ≤4.2%. The data were subjected to 2-way ANOVA technique using 2 factorial completely randomized designs (Steel et al., 1997). The Duncan multiple range test was applied (Duncan, 1955) to seek the significance of difference between means and to calculate their standard error. Pearson correlation coefficient was determined between different parameters at various production stages.

RESULTS At the 5% production level, TOS did not change between groups; however, it did decrease (P ≤ 0.01) in groups G2, G3, and G4 at PP and EP compared with the G1 at PP (Table 3). Overall mean TOS for all stages of the second production cycle decreased significantly in supplemented groups (G2, G3, and G4) compared with the control (G1) and shown in Figure

2609

BIOLOGICAL MARKERS IN SUPPLEMENTED MOLTED LAYERS Table 3. Serum level of biological health markers in zinc-induced molted White Leghorn hens at different production Item

G1 H2O2 equiv.∙L−1)

Total oxidant status (µmol of  5%  PP  EP Total antioxidant capacity (mmol of Trolox equiv.∙L−1)  5%  PP  EP Homocysteine (µmol∙L−1)  5%  PP  EP Paraoxonase (U∙L−1)  5%  PP  EP Arylesterase (kU∙L−1)  5%  PP  EP Ceruloplasmin (U∙L−1)  5%  PP  EP

G2

G3

stages1 G4

2.33 ± 0.18a 1.44 ± 0.08bc 1.23 ± 0.10cd

2.31 ± 0.21a 0.28 ± 0.04f 0.59 ± 0.07def

2.03 ± 0.18ab 0.23 ± 0.04f 0.99 ± 0.13cde

2.40 ± 0.19a 0.39 ± 0.07ef 0.74 ± 0.07cdef

0.21 ± 0.02f 0.98 ± 0.04bc 0.27 ± 0.02ef

0.33 ± 0.01ef 1.80 ± 0.08a 1.18 ± 0.09b

0.37 ± 0.01ef 0.83 ± 0.08cd 0.56 ± 0.07de

0.39 ± 0.04ef 0.76 ± 0.05cd 0.73 ± 0.04cd

82.11 ± 7.57a 75.34 ± 5.27ab 38.66 ± 4.93de

72.57 ± 5.07ab 21.15 ± 2.87e 27.66 ± 2.00de

79.73 ± 5.26ab 52.90 ± 6.65bcd 38.59 ± 4.16de

68.30 ± 7.09abc 41.25 ± 4.75cde 40.68 ± 4.70cde

730.88 ± 51.93d 209.56 ± 10.18e 210.66 ± 17.34e

1,245.49 ± 71.64c 281.62 ± 20.12e 207.56 ± 14.29e

2,445.19 ± 19.61a 181.02 ± 19.64e 261.40 ± 23.14e

2,033.73 ± 161.75b 250.26 ± 13.80e 292.65 ± 30.59e

237.93 ± 27.06d 537.87 ± 27.64ab 523.90 ± 51.30ab

305.66 ± 31.13cd 698.26 ± 35.55a 457.72 ± 39.46bc

371.61 ± 27.88bcd 324.27 ± 18.88cd 491.69 ± 59.79bc

362.77 ± 23.48bcd 356.75 ± 27.61bcd 420.35 ± 25.46bcd

63.91 ± 5.26bc 15.81 ± 2.34f 19.77 ± 2.16ef

117.29 ± 7.64a 74.11 ± 3.91b 41.57 ± 3.82cdef

60.31 ± 3.66bcd 44.76 ± 3.44cde 21.99 ± 2.42ef

57.54 ± 7.78bcd 67.42 ± 7.06bc 34.52 ± 3.39def

a–fSimilar

superscripts do not differ significantly for each parameter at P ≤ 0.01. = control CP 16%, energy = 2,795 kcal, no supplement; G2 = CP 18%, diet E = 2,800 kcal; G3 = CP 16%, energy = 2,795 kcal, symbiotic in water; G4 = CP 16%, probiotic in water, 5% = 5% egg production; PP = peak production; EP = end of production. 1G1

1. The TOS was positively correlated (r = 0.59) with homocysteine concentration and negatively correlated (r = −0.70) with ceruloplasmin activity. The TAC increased (P ≤ 0.01) in G2 at PP and in G2 and G4 (P ≤ 0.01) at EP compared with G1 at these production stages (Table 3). The overall mean treatment effect on TAC irrespective of production stage

Figure 1. Overall serum total oxidant status of different groups (G1–G4) for all stages of the second production cycle. A,BSimilar letters do not differ significantly (P < 0.01). TOS = total oxidant status.

shows a high TAC value (P ≤ 0.01) in G2 followed by G4 compared with G1 (Figure 2). The TAC was positively correlated with PON (r = 0.49), arylesterase (r = 0.72) activity, and ceruloplasmin activity (r = 0.36). The TAC was negatively correlated with homocysteine (r = −0.44) and TOS (r = −0.18). Total serum homocysteine (hcy) concentration did not differ significantly between groups at 5P; thereafter, it decreased significantly in G2 and G4 at PP com-

Figure 2. Overall serum total antioxidant capacity of different groups (G1–G4) for all stages of the second production cycle. A–CSimilar letters do not differ significantly (P < 0.01). TAC = total antioxidant capacity.

2610

Anwar et al.

Figure 3. Overall serum homocysteine concentration of different groups (G1–G4) for all stages of the second production cycle. A–CSimilar letters do not differ significantly (P < 0.01).

pared with G1 (Table 3). Irrespective of the production stages, mean hcy level was low (P ≤ 0.01) in G2 and G3 compared with G1 (Figure 3). The increase (P ≤ 0.01) in PON activity was seen in all the treatment groups at 5P compared with G1. The PON activity has a trend of decreasing (P ≤ 0.01) in all the groups at PP and EP compared with 5P; however, it did not differ statistically between the groups at PP and EP (Table 3). The overall mean PON activity was high (P ≤ 0.01) in all the treatment groups; however, in G3 and G4 it was even higher (P ≤ 0.01) than G2 being highest in G4 irrespective of production stages (Figure 4). The overall mean arylesterase activity was high (P ≤ 0.01) in G2 compared with G1 and G4 regardless of production stage (Figure 5). The PON was positively correlated with arylesterase (r = 0.41) and ceruloplasmin activity (r = 0.57), whereas PON was negatively correlated (r = −0.44) with homocysteine concentration. The arylesterase activity was significantly lower (P ≤ 0.01) in G3 compared with G1 and G2 at PP (Table 3). Ceruloplasmin activity increased significantly (P ≤ 0.01) in G2 at 5P compared with other groups. At the peak of production, enzyme activity increased (P ≤ 0.01) in G2, G3, and G4 compared with G1 (Table 3). The overall mean ceruloplasmin activity irrespective of production stage was significantly high in G2 and G4 compared with G1 (Figure 6). Ceruloplasmin activity was negatively correlated (r = −0.67) with homocysteine concentration. Pearson correlation of all the parameters is given in the Table 4.

Figure 4. Overall serum paraoxonase activity in all the groups (G1–G4) for all stages of the second production cycle. A–CSimilar letters do not differ significantly (P < 0.01).

(Sandhu et al., 2006, 2007). It was seen that immune response was enhanced and stress was alleviated in the zinc supplemented (80 ppm daily) broiler chickens up to 4 wk of age (Sunder et al., 2008). Thus high dietary zinc was selected as an induced molting method in the current study. There are many physiological and stressful conditions that cause the overproduction of oxidative molecules in the body and ultimately lead to oxidative stress due to an imbalance between oxidants and antioxidants (Sahin et al., 2001). In the current study, the decreased antioxidant status and increased total oxidant status of the body in all of the groups just after the molt at the

DISCUSSION The high dietary zinc-induced molting method has proved its competency over the conventional fast molting method based on the improved immune status and improved production performance of postmolted hens

Figure 5. Overall serum arylesterase activity of different groups (G1–G4) for all stages of the second production cycle. A,BSimilar letters do not differ significantly (P < 0.01).

2611

BIOLOGICAL MARKERS IN SUPPLEMENTED MOLTED LAYERS

Figure 6. Overall serum ceruloplasmin activity of different groups (G1–G4) for all stages of the second production cycle. A–CSimilar letters do not differ significantly (P < 0.01).

5% production stage showed that the effects of molting stress were not alleviated until 5% production. There are no reports regarding the impact of induced molting on the TAC and TOS in layer chickens; however, the impact of stressors such as environmental temperature was studied by Sahin et al. (2004). Sahin et al. (2004) reported that increases in ambient temperature in quail increased the MDA production and ultimately led to oxidative stress, which also supports higher TOS and lower TAC at the 5% production stage in our study. In the current experiment, supplementation with increased protein (18%), probiotics, and symbiotics resulted in overall improvement of antioxidant status with a decrease in the total oxidant status of the body. Khaled et al. (1995) studied protein energy supplementation on thiobarbituric acid reactive substances (oxidative marker) in malnourished children and concluded that supplementation significantly reduced oxidative stress. A recent study by Erdoğan et al. (2010) showed that the addition of symbiotics alone to the diet of broilers did not show any significant improvement in

oxidant/antioxidant status. In contrast to the results of Erdoğan et al. (2010), symbiotics and probiotics alone showed significant improvement in TAC and reduction of TOS in the current study. The difference in species and induced molting might explain these findings. The positive correlation of TAC with PON, arylesterase, and ceruloplasmin activity is according to expectation, as these are enzymes that contribute to the antioxidant system of the body. Homocysteine is an abnormal amino acid and normally converted back to methionine, which keeps its level low in the blood (Sahin et al., 2004). In the current study, the increased homocysteine level at the 5% production stage in all of the groups compared with later production stages is due to the impact of induced molting, which was sustained until 5% production. Sahin et al. (2004) reported the increasing effect of high and low ambient temperature stress on serum homocysteine levels in quail. It was reported that homocysteine levels were enhanced with the increased consumption of methionine (da Costa et al., 2005); however, in our experiment, protein supplementation did result in a significant decrease in homocysteine level. The increased availability of B vitamins, decrease in vitamin catalysis, and conversion of homocysteine into cysteine could be the reasons for decreased homocysteine levels after protein supplementation as suggested by Wulffelé et al. (2003). The probiotics supplementation has been reported to enhance the synthesis of vitamin B (Patterson and Burkholder, 2003) and attributed to the decreased homocysteine in the probiotic-supplemented group in the current study. Lipid peroxidation was probably decreased due to the enhanced TAC and increased activity of antioxidant enzymes, which results in the negative correlation between TAC, related enzymes, and homocysteine concentration. During stressful condition such as high environmental temperature (Gumuslu et al., 2002) and liver dysfunction (Kilic et al., 2005), PON activity decreases. In current experiment, increased PON activity at 5P and then decreased activity at PP in all the groups could be

Table 4. Pearson’s correlation of overall mean serum biological health markers irrespective of the group treatment1 Item2

TOS

TAC

Hcy

PON

Aryl

TOS TAC

— −0.18 (0.00) 0.59 (0.00)  

  —

   

   

   

   

−0.44 (0.01) 0.49 (0.00) 0.72 (0.00) 0.36 (0.04)

—

 

 

 

−0.44 (0.01)  

—

 

 

0.41 (0.01) 0.57 (0.00)

—

 

Hcy PON Aryl Cerul 1Significant

  −0.70 (0.00)

−0.67 (0.00)

 

Cerul

—

r values are given with P-value within parentheses. total oxidant status (µmol of H2O2equiv.∙L−1), TAC: total antioxidant capacity (mmol of Troloxequiv.∙L−1), Hcy: homocysteine (µmol∙L−1), PON: paraoxonase (U∙L−1), Aryl: arylesterase (kU∙L−1), Cerul: ceruloplasmin (U∙L−1). 2TOS:

2612

Anwar et al.

attributed to the increased mobilization as well as consumption of the cholesterol to meet up the increasing egg production with progress in the production span. In this aspect, a study by Thomàs-Moyà et al. (2007) using rats fed a high-fat diet showed a decreased response in PON activity, which was due to increased weight and higher energy provision in the high-fat diet group compared with the control. The decreased PON activity due to decreased serum protein levels in cold environments in rats (Gumuslu et al., 2002) supports the enhanced activity of PON after protein supplementation in the current experiment. The PON is a part of circulating high-density lipoprotein and present in the outer core (Thomàs-Moyà et al., 2007). A proposed increase in the high-density lipoprotein cholesterol with protein supplementation could be responsible for enhanced PON activity. In addition, improved TAC of protein-supplemented birds and decreased lipid peroxidation is also associated with increased PON activity in the current experiment. The overall improved antioxidant capability of the probiotic-supplemented birds could be attributed to the enhanced PON activity in this group (Shareef and Al-Dabbagh, 2009); however, the exact mechanism behind that is not clear yet. Likewise, Gursu et al. (2004) reported that the activity of PON and arylesterase enzymes were enhanced in vitamin C and folic acid-supplemented birds compared with heat stress birds. A study by Song et al. (2009) in lipopolysaccharideinjected broiler chickens showed that the supplementation with copper significantly increased ceruloplasmin activity, which is in line with increased ceruloplasmin activity in the symbiotic group in our experiment. There are no reports regarding the study of this enzyme in molted layer chickens. Boero et al. (2010) mentioned the prooxidant role of ceruloplasmin and its prime connection to the oxidation of low-density lipoproteins. Their prooxidant role was enhanced during the phase of high low-density lipoprotein oxidation. The decrease in oxidative stress in the G2 and G4 could be attributed to the enhanced ceruloplasmin activity in these groups.

CONCLUSIONS The supplementations of molted layers did show a significant impact in lowering oxidative stress; however, among different supplementations in this study, protein supplementation has shown remarkable improvement in antioxidant capacity. This improved antioxidant status in protein supplementation could also be related to the enhanced activity of antioxidant enzymes and thus proved to be an adaptable feed supplement for better performance of molted hens.

ACKNOWLEDGMENTS The authors are thankful to the Higher Education Commission, Islamabad, Pakistan for extending

the grant under PhD Indigenous scholarship scheme, Batch-III.

REFERENCES Boero, L., L. Cuniberti, N. Magnani, M. Manavela, V. Yapur, M. Bustos, L. G. Rosso, T. Meroño, L. Marziali, L. Viale, P. Evelson, G. Negri, and F. Brites. 2010. Increased oxidized low density lipoprotein associated with high ceruloplasmin activity in patients with active acromegaly. Clin. Endocrinol. (Oxf.) 72:654–660. Craven, S. E. 2000. Colonization of the intestinal tract by Clostridium perfringens and fecal shedding in diet-stressed and unstressed broiler chickens. Poult. Sci. 79:843–849. da Costa, K. A., C. E. Gaffney, L. M. Fischer, and S. H. Zeisel. 2005. Choline deficiency in mice and humans is associated with increased plasma homocysteine concentration after a methionine load. Am. J. Clin. Nutr. 81:440–444. Duncan, D. B. 1955. Multiple range and multiple F-test. Biometrics 11:1–42. Erdoğan, Z., S. Erdoğan, Ö. Aslantaş, and S. Çelik. 2010. Effects of dietary supplementation of synbiotics and phytobiotics on performance, caecal coliform population and some oxidant/antioxidant parameters of broilers. J. Anim. Physiol. Anim. Nutr. (Berl.) 94:e40–e48. Erel, O. 2004. A novel automated direct measurement method for total antioxidant capacity using a new generation, more stable ABTS radical cation. Clin. Biochem. 37:277–285. Erel, O. 2005. A new automated colorimetric method for measuring total oxidant status. Clin. Biochem. 38:1103–1111. Gumuslu, S., A. Y. Gocmen, and S. B. Sarikcioglu. 2002. Paraoxonase (PON1) activity in serum rats stressed by cold: Effects of vitamin E supplementation. 17. Page 74 (Abstr.) in Ulusal Biyokimya Kongresi, Ankara, Turkey. Gursu, M. F., M. Onderci, F. Gulcu, and K. Sahin. 2004. Effects of vitamin C and folic acid supplementation on serum paraoxonase activity and metabolites induced by heat stress in vivo. Nutr. Res. 24:157–164. Haghighi, H. R., J. Gong, C. L. Gyles, M. A. Hayes, H. Zhou, B. Sanei, J. R. Chambers, and S. Sharif. 2006. Probiotics stimulate production of natural antibodies in chickens. Clin. Vaccine Immunol. 13:975–980. Junqueira, V. B. C., S. B. M. Barros, S. S. Chan, L. Rodrigues, L. Giavarotti, R. L. Abud, and G. P. Deucher. 2004. Aging and oxidative stress. Mol. Aspects Med. 25:5–16. Juretić, D., A. Motejlkova, B. Kunovic, B. Rekic, Z. F. Mestric, L. Vujic, R. Mesic, J. L. Bajalo, and V. S. Rudolf. 2006. Paraoxonase/arylesterase in serum of patients with type II diabetes mellitus. Acta Pharm. 56:59–68. Khaled, M. A., I. Kabir, and D. Mahalanabis. 1995. Effect of protein energy supplementation on oxidative stress in malnourished children. Nutr. Res. 15:1099–1104. Kilic, S. S., S. Aydin, N. Kilic, F. Erman, S. Aydin, and İ. Celik. 2005. Serum arylesterase and paraoxonase activity in patients with chronic hepatitis. World J. Gastroenterol. 11:7351–7354. Koelkebeck, K. W., C. W. Parsons, R. W. Leeper, and J. Moshtaghian. 1991. Effect of protein and methionine levels in molt diets on post molt performance of laying hens. Poult. Sci. 70:2063–2073. Maggio, D., M. Barabani, M. Pierandrei, C. Polidori, M. Catani, P. Mecocci, U. Senin, R. Pacifici, and A. Cherubini. 2003. Marked decrease in plasma antioxidant in aged osteoporotic women: Result of a crosssectional study. J. Clin. Endocrinol. Metab. 88:1523–1527. Patterson, J. A., and K. M. Burkholder. 2003. Application of prebiotics and probiotics in poultry production. Poult. Sci. 82:627– 631. Sahin, K., O. Kucuk, N. Sahin, and M. Sari. 2002. Effects of vitamin C and vitamin E on lipid peroxidation status, some serum hormone, melite, and mineral concentrations of Japanese quails reared under heat stress (34°C). Int. J. Vitam. Nutr. Res. 72:91–100. Sahin, K., M. Onderci, M. F. Gursu, O. Kucuk, and N. Sahin. 2004. Effect of melatonin supplementation on biomarkers of oxidative

BIOLOGICAL MARKERS IN SUPPLEMENTED MOLTED LAYERS stress and serum vitamin and mineral concentrations in heatstressed Japanese Quail. J. Appl. Poult. Res. 13:342–348. Sahin, N., K. Sahin, and O. Küçük. 2001. Effects of vitamin E and vitamin A supplementation on performance, thyroid status and serum concentrations of some metabolites and minerals in broilers reared under heat stress (32°C). Vet. Med. (Czech) 46:11– 12,286–299. Sandhu, M. A., Z. U. Rahman, and S. U. Rahman. 2006. Dynamics of macrophages during second and third production cycles after zinc induced molting. Jpn. Poult. Sci. 43:286–295. Sandhu, M. A., Z. U. Rahman, and S. U. Rahman. 2007. Effects of induced molting on some immunological parameters in laying hens (Gallus domesticus). Arch. Geflügelk. 71:110–116. Sandhu, M. A., Z. U. Rahman, A. Riaz, S. U. Rahman, I. Javed, and N. Ullah. 2010. Somatotrophs and lactotrophs: An immunohistochemical study of Gallus domesticus pituitary gland at different stages of induced moult. Eur. J. Histochem. 54:e25. Schosinsky, K. H., H. P. Lehmann, and M. F. Beeler. 1974. Measurement of ceruloplasmin from its oxidase activity in serum by use of o-dianisidine dihydrochloride. Clin. Chem. 20:1556–1563. Shareef, A. M., and A. S. A. Al-Dabbagh. 2009. Effect of probiotic (Saccharomyces cerevisiae) on performance of broiler chicks. Iraqi J. Vet. Sci. 23:23–29. Song, Z., L. Zhu, T. Zhao, H. Jiao, and H. Lin. 2009. Effect of copper on plasma ceruloplasmin and antioxidant ability in broiler chickens challenged by lipopolysaccharide. Asian-australas. J. Anim. Sci. 22:1400–1406.

2613

Steel, R. G. D., J. H. Torrie, and D. A. Dieky. 1997. Principles and Procedures of Statistics. 3rd ed. McGraw Hill Book Co. Inc., New York, NY. Stern, N. J., N. A. Cox, J. S. Bailey, M. E. Berrang, and M. T. Musgrove. 2001. Comparison of mucosal competitive exclusion and competitive exclusion treatment to reduce Salmonella and Campylobacter spp. colonization in broiler chickens. Poult. Sci. 80:156–160. Sunder, G. S., A. K. Panda, N. C. S. Gopinath, S. V. Rama Rao, M. V. L. N. Raju, M. R. Reddy, and V. Kumar. 2008. Effects of higher levels of zinc supplementation on performance, mineral availability, and immune competence in broiler chickens. J. Appl. Poult. Res. 17:79–86. Thomàs-Moyà, E., M. Gianotti, A. M. Proenza, and I. Lladó. 2007. Paraoxonase 1 response to a high-fat diet: Gender differences in the factors involved. Mol. Med. 13:203–209. Togun, V. A., J. I. Okwusidi, O. A. Amao, and S. U. Onyiaoha. 2004. Effect of crude protein levels and follicle stimulation on egg production of aged hens. Niger. J. Physiol. Sci. 19:77–81. Wulffelé, H. G., A. Kooy, P. Lehert, D. Bets, J. C. Ogterop, and B. B. van der Burg. 2003. Effects of short-term treatment with metformin on serum concentrations of homocysteine, folate and vitamin B12 in type 2 diabetes mellitus: A randomized, placebocontrolled trial. J. Intern. Med. 254:455–463.