Serum antioxidants and trace minerals as influenced by vitamins, probiotics and proteins in broiler breeders

Abstract

One hundred and eighty birds at the age of 65 weeks were induced to moult by mixing zinc oxide in their feed at the rate 3000 mg/kg of feed. Upon completion of moulting, the birds were divided into six groups (five replicates) by a completely randomised design. One group was kept as control (16% crude protein [CP]) while the other five were supplemented with vitamin E (100 IU/kg feed), vitamin C (500 IU/kg feed), probiotics (50 mg/L), protein level (14% CP) and their combination. At the end of the experiment, the results revealed that serum total anti-oxidant response significantly increased in the vitamin E fed group compared to the other treated groups. Paraoxonase and arylesterase were significantly higher in vitamin E and C groups. The ceruloplasmin level decreased significantly in the vitamin C fed group as well as in the group having the combination of all the treatments. The homocysteine was significantly lower in the vitamin E fed group compared to the other treated groups. In trace elements, serum zinc was significantly high in vitamin E supplemented group. These findings suggested that compared to other treatments, vitamin E has pronounced influence on improving the anti-oxidant status in male broiler breeders.

Keywords:

• oxidative stress
• broiler breeders
• moulting
• vitamins
• probiotics
• protein

1. Introduction

The process of moulting and subsequent recovery from the moult is characterised by a complex physiological constellation, induced by nutritional cues that affect the endocrine, reproductive, immune and haematopoietic system (Khan et al. 2011a, 2012). Moulting can be induced through a variety of mechanisms; however, theconsiderable body of evidence has implicated that high dietary zinc (Zn) is an effective way of inducing moult and does not confront the normal physiological processes of birds (Khan et al. 2011a). Considerable studies are available on the improvement of productive performance and immune status in Zn-induced laying hens (Khan et al. 2011a). However, reports are rather scarce on the effect of Zn-induced moulting on the oxidative stress in poultry species, especially as a consequence of some nutrients’ supplementation.

The study of free radicals is immensely important as it results in the pathogenesis of lipid peroxidation of the plasma membrane (Khan 2011; Khan et al. 2011b). The blood plasma has been endowed with a considerable amount of enzymatic and non-enzymatic antioxidants, which are constantly combating against the free radicals to neutralise their deleterious impacts on the body (Khan 2011). Under normal physiological circumstances, there is an appropriate balance between the creation and destruction of free radicals (Khan 2011; Khan et al. 2011b). However, when the production of free radicals exceeds the neutralising capacity of the body, oxidative stress develops (Khan et al. 2011b). Antioxidants, in general, are compounds and reactants that dispose, scavenge and suppress the formation of free radicals or oppose their actions (Khan 2011). Physiological stresses like heat, cold, diseases or overcrowding may augment the chicken requirement for antioxidants (Nockels 1984).

Dietary vitamin E has received considerable importance as one of the antioxidants which alleviates the oxidative stress of the poultry birds (Khan 2011). Vitamin E is mainly found in the hydrocarbon part of the membrane lipid player towards the membrane interface and in close proximity to oxidising enzymes, which initiate the production of free radicals (McDowell 1989; Khan 2011). Vitamin E effectively scavenges the free radicals and reacts with them producing a stable ROOH group, thus, providing biological stability to the plasma membrane (Surai et al. 2000). Vitamin C or ascorbic acid is an anti-oxidant which is normally synthesised by the chicken (Khan 2011). It has been recommended in poultry feed as a supplement to alleviate stress on the assumption, that during stress the requirements may exceed the synthesising ability of the birds (McDowell 1989; Gous & Morris 2005; Khan 2011).

Probiotics supplementation has been confirmed in numerous scientific investigations to modulate the composition of the gut microflora by eliminating the pathogenic bacteria (Panda et al. 2003). Previously, Lactobacillus and Bifadobacterium have been reported to improve the anti-oxidant status in birds (Sohail et al. 2011) by decreasing the production of free radicals (Fuller 1989). The lower levels of crude protein (CP) control body weight gain and decrease blood urea, which is translated into improved reproductive performance of the birds (Zhang et al. 1989; Romero-Sanchez et al. 2007; Laudadio et al. 2012). Some authors have indicated that different levels of dietary protein may affect the serum antioxidants in poultry (MachIn et al. 2004). However, studiesss are rather scarce on the effect of dietary levels of protein on oxidative stress indices.

To our knowledge, under the effect of certain feed supplements, the status of serum total anti-oxidant response (TAR), paraoxonase (PON1), arylesterase (Arys), ceruloplasmin (Cp), homocysteine (Hys) and trace elements have not been reported in avian species after Zn-induced moulting. Therefore, this research work was planned to investigate the effect of feeding different nutritional regimens on some of the indices of oxidative stress and trace minerals’ status of the moulted male broiler breeders.

2. Materials and methods

2.1. Feeding and management of birds

The present research work was conducted on male broiler breeders (hubbard) at the age of 65 weeks. One hundred and eighty birds were obtained from the commercial breeder's farm. The birds were acclimatised for one week, during which they were fed standard broiler breeder's ration containing 16% CP and water ad libitum. The lighting schedule was adjusted at 16 h per day. At the beginning of the second week, birds were subjected to moult with zinc oxide (ZnO) at the rate of 3000 mg/kg of feed with a moderate decrease in lighting schedule from 16 to 12 h, and offered 50 g/bird feed on a daily basis (Khan et al. 2012). The phase of moulting continued for two weeks. After completion of moult (Table 1), the birds were randomly assigned to six groups (five replicates per group) in a completely randomised design, and were reared on floor pans (3.96×3.96 m) in a window sided house with controlled temperature, ventilation and illumination.

Table 1. Composition of basal diet.

Ingredients (g/kg)	CP-16%	CP-14%
Yellow corn	430.0	450.0
Canola meal	170.0	180.0
Rice polishing	140.0	150.0
Rice tips	120.0	120.0
Soybean meal	60.0	20.0
Maize gluten (30% CP)	50.0	50.0
Dicalcium phosphate	10.0	10.0
Lime stone	10.0	10.0
Sodium chloride	0.1	0.1
Vitamin minerals premix*	4.9	4.9
Lysine	2.50	2.5
Methionine	2.50	2.5
Calculated composition
Crude protein (g/kg)	160.15	140.21
Crude fibre (g/kg)	50.50	50.50
Ash (g/kg)	70.00	50.00
ME (MJ/kg)	12.24	12.22

* Provided per kg of diet: Mn 80 mg; Zn 60 mg; Fe 60 mg; Cu 5 mg; Co 0.2 mg; I 1 mg; Se 0.15 mg; choline chloride 200 mg; vitamin A (retinol) 12000 IU; vitamin D3 (cholecalciferol) 2400 IU; vitamin E (dl-α-tocopherol) 50 IU; vitamin K (menadione) 4 mg; vitamin B1 (thiamine) 3 mg; vitamin B2 (riboflavin) 6 mg; vitamin B5 (pantothenic acid) 25 mg; vitamin B6 (pyridoxine) 5 mg; vitamin B12 (cyanocobalamin) 0.03 mg; folic acid 1 mg;

Note: Dietary treatments: Control =16% CP; Vitamin E=16% CP +100 IU/kg dl-α-tocopherol acetate; Vitamin C=16% CP +500 IU/kg L-ascorbic acid; Probiotics=16% CP+50 mg probiotics; Protein=14% CP only; Combination=14% CP+ 100 IU/kg dl-α-tocopherol acetate+500 IU/kg L-ascorbic acid+ 50 mg probiotics.

Composition of probiotics mixture: Lactobacillus plantarum, L. bulgaris, L. acidophilus, L. rhamnosus, Bifadobacterum binfadum, Streptococus thermophilus, Enterococus faecium, Aspergillus oryzae, Candida pintolopesi.

The first groups was kept as control (CP–16%), the second group was fed vitamin E (CP–16%+100 IU vitamin E/kg feed as dl- α-tocopherol acetate; Neolait SA, France), third group vitamin C (CP–16%+vitamin C 500 IU/kg feed as l-acerbic acid; Neofarma, Italy; BA Traders, Lahore, Pakistan), fourth group probiotics (CP–16%+50 mg probiotics/kg feed; Protexin® HiltonPharma, Holland), fifth group lower protein (CP–14%) and the last group was fed the combination of vitamin E, C, protein and probiotics [CP–14%+100 IU/kg vitamin E+vitamin C 500 IU/kg+50 mg probiotics (per kg of feed)]. The detailed composition of the diet is given in Table 2. Sampling was conducted after 10 weeks of treatment.

Table 2. Moulting schedule during the experiment.

Stage of moulting	Weeks	Feed	Water	Light
Pre-moult	65	130 gm/day/bird (Normal feed)	Ad libitum	16 hrs
moult	66–67	50 gm/day/bird (Zinc added feed; 3 g/kg of feed)	Ad libitum	12 hrs
Rest period	68	75 gm/day/bird (Supplemented feed)	Ad libitum	12 hrs
	69–70	110 gm/day/bird (Supplemented feed)	Ad libitum	12 hrs
Post-moult	71 Onward	130 gm/day/bird (Supplemented feed)	Ad libitum	16 hrs

For the determination of serum oxidative stress parameters, about 5-ml blood samples from six birds was randomly taken by cervical dislocation. The blood samples were collected in sterile test tubes by decapitating the birds. Serum was separated after centrifugating the blood at 800×g for 15 min. Serum was collected in the small clean appendix for each sample and stored at –20°C until further analyses. All samples were analysed in duplicate.

2.2. Blood collection and sample analysis

All parameters of oxidative stress were measured spectrophotometrically (Biosystem, BTS-330; Biosystem, S.A. Costa Brava, Barcelona, Spain). TAR was measured in the serum samples by using a novel automated method developed by Erel (2004), using o-dianisidine dihydrochloride (Sigma Chemical Co. London, UK) as a substrate. PON1 was determined by the method described by Mackness et al. (1991), using paraoxon (Sigma Chemical Co. London, UK) as a substrate. Arylesterase was measured using phenyl acetate (Sigma Chemical Co. London, UK) as the substrate (Juretic et al. 2006). Cp was measured from its oxidising activity in serum using o-dianisidine dihydrochloride as the substrate (Schosinsky et al. 1974). Hys was measured by the commercially available assay kit (Diazyme Laboratories, Gregg Court Poway, USA). Hys concentration was calculated from the standards provided with the kit.

For minerals analyses, 5-ml blood samples from six birds per group were randomly taken by cervical dislocation and processed for serum collection as stated above. The preparation of serum samples for mineral analysis was carried out as described by Richard (1968). Briefly, the serum sample of 1 ml was taken into a digestion flask and added with 10-ml concentrated nitric acid. The mixture was placed on a hot plate until all the fumes evaporated. The flask was removed and let to cool for five min. After cooling, 5-ml perchloric acid was added into the flask, and the mixture was heated again on a hot plate. When the volume was reduced to 1–2 ml, the flask was removed and the content was cooled again. The contents were diluted with 50-ml deionized water, filtered and kept in a clean bottle until analysis. The analyses of Zn, manganese (Mn), iron (Fe) and copper (Cu) were performed using an atomic absorption spectrometer (Varian spectrAA 240). First standard solutions were run followed by samples. The concentration of minerals in the samples was obtained from the absorbance of standards and their corresponding concentrations (Richard 1968).

2.3. Statistical analysis

Data was statistically analysed with the help of the statistical software (SPSS, version 12.0). One-way analysis of variance was used to test the significance of the treatment (six diet treatments) on the studied traits (Steel et al. 1997). Means of the significantly affected traits were separated by Duncan's Multiple Range Test (Duncan 1955). A p-value less than 0.5 was considered to be statistically significant.

3. Results

Mean TAR, PON1, Aryl, Cp and Hys of the experimental and control groups are given in Table 3. Mean TAR was significantly higher in the vitamin E group as compared to the rest of the group at the end of the experiment. Mean serum PON1 and Aryl concentration was high (p<0.05) in vitamin E and C groups and low (p<0.05) in the control and combination groups. Overall, a significantly high serum Cp was recorded in the control group and a significantly low concentration was observed in the vitamin C and combination groups; an overall significantly high concentration of Hys was observed in the control, protein and probiotics groups in this study.

Table 3. Mean±SE serum total anti-oxidant response (TAR), paraoxonas, arylesteras, ceruloplasmin (Cp) and Hys of post-moult control and experimental male broiler breeders.

Groups	TAR (mmol Trolox equiv./L)	Paraoxonase (PON1; U/ml/min.)	Arylesterase (Aryl; U/ml/min.)	Cp (U/L)	Homocysteine (Hys; µmol/L)
Control	1.05±0.09^b	396.60±27.86^c	144.37±5.57^c	40.23±4.49^a	31.56±1.31^a
Vitamin E	1.61±0.14^a	513.74±18.31^a	179.93±9.33^a	31.51±2.57^b	25.95±1.94^b
Vitamin C	1.24±0.10^b	534.21±14.00^a	181.32±9.51^a	23.05±1.57^c	28.56±1.17^ab
Protein	1.27±0.09^b	476.83±35.68^b	172.06±8.63^ab	30.97±2.43^b	30.79±1.99^a
Probiotics	1.09±0.08^b	455.56±13.56^b	135.85±6.70^c	34.43±2.53^b	30.99±1.69^a
Combination*	1.18±0.07^b	412.62±30.94^c	161.24±5.97^b	23.76±1.50^c	28.65±1.82^ab

^a,b,c,d Mean values within a column, not bearing a common superscript differ significantly (p<0.05).

* 14% CP+100 IU/kg vitamin E+500 IU/Kg vitamin C+50 mg probiotics.

TAR, Total Antiixidant Response;

Mean serum Zn, Cu, Mn and Fe values of the experiment are given in Table 4. Overall serum Zn concentration was significantly high in vitamin E and C groups and significantly low in the control group. No significant difference was found in serum Cu, Mn and Fe concentration between the control and treated birds.

Table 4. Mean±SE serum zinc (Zn; mg/L), copper (Cu; mg/L), Manganese (Mn; mg/L) and Iron (Fe; mg/L) concentration of post-moult control and experimental male broiler breeders.

Groups	Zn (mg/L)	Cu (mg/L)	Mn (mg/L)	Fe (mg/L)
Control	5.81±0.18^c	1.19±0.10	0.25±0.01	22.69±1.31
Vitamin E	7.94±0.31^a	1.22±0.09	0.26±.01	22.00±0.94
Vitamin C	6.99±0.24^ab	1.31±0.07	0.27±0.01	22.09±1.44
Protein	6.89±0.44^bc	1.05±0.101	0.27±0.01	20.96±1.58
Probiotics	6.83±0.25^bc	1.36±0.09	0.27±0.01	20.04±1.05
Combination*	6.72±0.38^bc	1.31±0.14	0.26±0.00	22.07±1.40

^a,b,c Mean values within a column, not bearing a common superscript differ significantly (p<0.05).

* 14% CP+100 IU/kg vitaminE+500 IU/Kg vitamin C+50 mg probiotics.

4. Discussion

Birds are unique animals that exhibit many physiological processes that render them vulnerable to degenerative processes. These factors include almost little more than a double metabolic rate, two to six times higher sugar level and 3°C higher body temperature compared to other mammals (Nockels 1984). Each of these factors increases the production of free radicals. Without a unique protection system, birds would be short-lived and would age more rapidly than other mammals (Klandorf et al. 2001). With the advancing of age, the progressive deterioration of oxidative stress develops because of the increased production of free radicals (Khan 2011). According to the free radical theory of ageing, the defence system of the body is weakening with advancing age, which results in damage of the cell structure and function (Klandorf et al. 2001). Living organisms have evolved a sophisticated anti-oxidant system to cope with deleterious compounds of oxygen reduction. This system is composed of chemical substances capable of scavenging reactive oxygen species, which include vitamins C and E, as the most important in defending the body against the free radicals (Khan 2011).

In the present study, at the end of the experiment, TAR was significantly high in vitamin E supplemented group. It is conceived that exogenous supplementation of antioxidants can ameliorate the injury of free radicals in the body (Khan 2011). Vitamin E directly scavenges the free radicals and regulates the activities of the anti-oxidant system (Khan 2011). Sahin et al. (2004) reported that the supplementation of melatonin, which is considered a potent anti-oxidant, improved the anti-oxidant status in the plasma of Japanese quails, exposed to heat stress (Khan et al. in press).

In the present study, supplementation of dietary vitamins E and C significantly increased PON1 and Aryl, which are two members of the anti-oxidant defence system (Sahin et al. 2004). PON1, an anti-oxidant enzyme, circulates in the blood and is associated with HDL particle, and can destroy lipids in the form of oxidised low-density lipoproteins (Sahin et al. 2004). Jayakumari and Gopalan (2009) observed that low serum PON1 activity is correlated with low serum vitamin C, suggesting that PON1 level is positively correlated with the anti-oxidant level. Jarvik et al. (2002) reported that vitamin C is a positive predictor of PON1, and its level increases with anti-oxidant vitamins. Gursu et al. (2004) concluded that PON1 and Aryl activities increased by the supplementation of vitamin C. Several studies have shown that plasma antioxidants are effective in protecting the birds from the negative effect of stress (Khan 2011). Sahin et al. (2004) also reported the restoration of PON1 and Aryl activities in Japanese quail, exposed to heat stress, after treating them with melatonin, which acts as an anti-oxidant.

In the current work, Cp was significantly lower in vitamin C and in the combined group of treatments. Cp has a variety of functions. It is essential for Fe homeostasis, Cu transportation, and acts as an anti-oxidant substance (Sohail et al. 2011). In serum, Cp level is positively correlated with thiobarbituric acid reactive substances (TBARS) and negatively correlated with vitamin C (Jacobs et al. 1987). In literature, an apparent antagonism has been documented between ascorbic acid and Cu. Ascorbic acid decreases absorption of Cu which results in a decreased serum and tissue Cp (Jacobs et al. 1987). Finly and Cerklewskfi (1983) found decreased Cp at the supplementation of 1500 mg/day ascorbic acid and concluded that a high level of ascorbic acid is antagonistic to Cu level, which results in a lower Cp in serum. An alternative explanation of reduced Cp due to high intake of ascorbic acid is that ascorbic acid dissociates Cu from Cp metalloenzyme (Jacobs et al. 1987). In this research work too, the overall decrease in the serum concentration of Cp, either in vitamin C fed birds or in the combined group of treatments, seems to be in agreement with the above reports. Furthermore, in our study, we did not find a significant reduction in the serum Cu concentration due to vitamin C supplementation. The exact reason could not be traced from the published literature; however, we could infer that the dose of vitamin C is vital in determining this relationship.

In this study, Hys concentration significantly decreased in vitamin E fed birds. Hys is an intermediate compound formed during the metabolism of methionine into cysteine (Stangl et al. 2000). Stangl et al. (2000) reported that Hys has some association with the anti-oxidant system of the body, and its level is increased with the depletion of antioxidants. The influence of vitamin E on the trans-sulfuration or remethylation pathway is unclear. However, it was suggested that vitamin E may indirectly affect the pathway by oxidative destruction of folate, which is involved in the remethylation of Hys to methionine (Can et al. 2002). In support of this concept, Can et al. (2002) found that administration of vitamin E at the rate of 100 mg/kg/day in rats significantly decreased serum Hys level with respect to non-treated rats. Similar to this conclusion, Brude et al. (1999) reported negative correlation with dietary vitamin E supplementation and Hys in smokers.

Among the trace elements, no significant change was observed in serum Fe, Mn and Cu. However, overall serum Zn concentration was significantly high in the vitamin E fed group compared to control. In support to our study, Sahin et al. (2002) found that three levels of vitamin E (125, 250 and 500 mg/kg) significantly increased egg yolk Zn concentration in laying Japanese quails. In another study, Sahin et al. (2003) documented that vitamin E increases the retention of Zn, and decreased its secretion from the body of Japanese quails, when they were supplemented 250 mg/kg vitamin E. Zn plays a very important role in the anti-oxidant defence system of the body, and its deficiency has been linked with oxidative damage of the cell membrane leading to oxidative stress (Salgueiro et al. 2000; Prasad & Kucuk 2002). The exact mechanism of the Zn anti-oxidative role is still vague; however, it is speculated that Zn scavenges free radicals by increased synthesis of metallothionein (Oteiza et al. 1996). One way of the mechanism of action of this mineral is its role as an anti-oxidant through its interaction with vitamin E, the synthesis of which is impaired in Zn deficiency (Kim et al. 1998; Prasad & Kucuk 2002). Zn may play a role in suppressing the creation of free radicals, as it is a substantial part of the anti-oxidative enzyme copper–zinc superoxide dismustase (Khan 2011). Vitamins E and Zn have some common functions like membrane stabilisation and anti-oxidant function, and the previous studies show some of the interaction between the two nutrients (Goode et al. 1991).

In the current study, probiotics supplementation increased PON1 and Cp concentrations compared to control and did not affect other parameters. In contrast to our study, Sohail et al. (2011) reported that the addition of probiotics mixture decreased serum PON1 concentration in heat-stressed broilers compared to control (thermo-neutral temperature). The difference could be attributed to the experimental condition, probiotics species, dose and duration of the experiment. Studies regarding the role of probiotics on the status of Cp are not available. It is speculated that the probiotics bacteria may have some role in the absorption of Cu from the gut, the level of which is positively correlated with this enzyme.

In this study, significant differences were also found in serum anti-oxidative status in terms of PON1, Aryl and Cp between lower protein level (14%) and the control. To the best of our knowledge, we could not trace the effect of lower dietary protein level on the anti-oxidant status in the literature. Previously, some of the authors have reported improved reproductive performance by feeding lower protein levels in male broiler breeders (Zhang et al. 1999; Romero-Sanchez et al. 2007). Similarly, Laudadio et al. (2012) recently found that a lower protein level (20.5%) is a better choice for optimum level of growth in broilers than a higher protein level (22.5%). In all these reports, the antioxidants’ status of the birds has not been measured; however, it is inferred that the improved performance achieved with the lower level of protein may have some association with enhanced anti-oxidant status, which needs to be further explored.

In conclusion, in the present study, it was found that serum statuses of TAR better improved by vitamin E supplementation than other groups. However, both vitamins were equally effective in elevating the statuses of PON1 and Aryl. Moreover, vitamin C suppressed Cp more than vitamin E, and vitamin E was more effective than vitamin C on reduction of Hys concentration. Serum Zn was also improved in vitamin E supplemented group. Additionally, the interaction of vitamins E and C had no positive effect on enhancing the serum anti-oxidants and trace minerals in broiler breeders.