Vitamin E or clay in layer hens

A total number of 240 laying hens and 24 cocks of Silver Montazah strain at 40 weeks of age were randomly divided into 12 groups (20 hens and 2 cocks / each treatment group). Birds in all treatments were nearly similar in the average initial body weights and average daily egg production. A 4 x 3 factorial design experiment was performed including four levels of supplemented lead (0. 250,500 and 1000 mg/kg diet) and three levels of feed additives (without clay or vitamin E, 3 % Natural clay, tafla, and 200 mg /kg vitamin E). Slaughter traits, some biochemical blood component(serum total protein, albumin, globulin, serum transaminase enzymes, ALT and AST, creatinine, urea - N and uric acid)and lead residues in tissues and eggs were studied.

Results obtained revealed that lead contaminated feed decreased pre-slaughter body weight, carcass and heart weight and increased liver weight. Results of biochemical analysis revealed a significant decrease in serum total protein, albumin and globulin however, increased creatinine, urea - N, uric acid levels, AST and ALT activities. The addition of natural clay or vitamin E to the diet for laying hens caused a significant higher serum total protein and albumin, lower serum creatinine, urea - N, uric acid and lower amounts of lead residues in tissues and eggs. With respect to interaction between lead and feed additives, results showed that at each lead level natural clay or vitamin E supplementation increased the concentration of total protein and albumin, decreased creatinine, urea - N, uric acid levels and AST and ALT activities, reduced the lead residues in the tissues and  eggs of the hen.

On the basis of the results obtained in this study, it can be concluded that the consumption of diets polluted with heavy metal such as lead causes rious effects in the carcass, blood components and increases the level of lead residual in  tissues and eggs. However, the addition of zeolite or vitamin E to diets of laying hens caused beneficial effects on carcass characteristics, blood components and lead residual tissues and eggs. Moreover, adding clay or vitamin E as feed additives to lead polluted diets diminishes lead toxicosis and therefore improve all traits studied.

Key words: Lead, blood components, lead residues, slaughter traits.

Lead poisoning has been described in human, domestic livestock and poultry. High concentrations of lead are associated with highly industrialized areas, major cities and heavily traveled roads where it mainly comes from car emission deposits in soil and on grits (Carson et al., 1987). Lead is used, mainly, in the manufacture of storage batteries, cables and of various alloys in the chemical machine industry, also for the protection from radioactive radiation, in the protection of tetraethyl lead and lead pigment, boiled oils and in the rubber industry (Santiev et al., 1975).Lead is consider one of the major environmental pollutants. The effect of lead on chicken is  well documented (Hermayer et al. 1977 and Fathi et al. 1999). Furthermore consumption of lead by laying birds will result in an increase of lead concentration in eggs (Mazliah et ,1989 and Jeng et al., 1997), blood, kidney, liver and muscle (Finley et al., 1976). The lead pollution may also, cause rious effects on biochemical indices (Abou - Zeid et al. 2000).

Supplementation of 2.5, 5.0, 7.5 and 10% bentonite in diets decreases the toxicities effect of T-2 toxin, and  10 % level being the most effective one.  Residual of T-2 toxin in muscle is reduced, but in the liver and kidney is not affected by dietary bentonite supplementation, feeding reduces T-2 toxicities by reducing intestinal absorption and increasing feacal excretion of the toxin (Carson and Smith, 1983).

Vitamin E is a crucial lipid soluble antioxidant that protect unsaturated fatty acids in feeds and tissues against oxidation. Vitamin E also increases the immune response in the chicken (Franchin et al., 1995).

Therefore, the present study was carried out to investigate the efficacy of clay and vitamin E to alleviate severity of lead contaminated diets and its effect on the carcass characteristics, blood components and lead residues in the tissues and eggs of Silver Montazah hens and their egg component.

This work was carried out at Inshas Poultry Research Farm belonging to Animal Production Research Institute, Agricultural Research Center, Ministry of Agriculture, Cairo, Egypt.

A total number of 240 hens and 24 cocks of Silver Montazah strain at 40 weeks of age were randomly divided into 12 treatment groups (20 hens and 2 cocks /each treatment group). Birds in all treatment groups were nearly similar in the average initial body weights and average daily egg production A 4 x 3 factorial design experiment was performed including four levels of supplemented lead (0, 250, 500 and 1000 mg / kg diet) and three levels of feed additives (without clay or vitamin E, 3 % Natural clay, tafla, and 200 mg /kg vitamin E). Tafla is a desert clay and analyzed as soluble cations and anions (meq/ 100 g dry matter soil ) were Ca⁺⁺ 0.75, Mg⁺⁺ 0.25, Na⁺ 0.05, K⁺ 0.10, Cl^‑ 0.55, SO₄ 0.30 and HCO₃ 0.75. Exchangeable cations (meq/100 g DM soil) were 2.65 and available nutrients (mg/100 g dry matter soil) were P 5.0, K 1.2, Mn 2.4, Zn 0.74, Cu 0.30 and Fe 0.55 mg ( Maria et al 1996). The composition and calculated chemical analysis of the experimental ration are presented in Table l. The birds were fed on the contaminated diets from 40 to 52 weeks of age, while at the period from 52 to 56 weeks, birds were fed diets without lead addition. Birds of all experimental groups were reared in suitable pens during the experimental period and were kept in the same conditions. Feed and water were offered ad-libitum and 16 hours light per day were maintained.

At the end of the experimental period (at 52 and 56 weeks of age) three hens were selected from each group and were sacrificed alter being fasted for 12 hours. After slaughter and complete bleeding, the birds were dressed. The carcass and some non - carcass components were weighed. At the same time liver, kidney and muscle samples were taken, dried and stored at - 20°C to determine the lead residues. At the time of slaughter (52 and 56 weeks of age) blood samples were collected from the birds and were centrifuged at 3000 rpm for 20 minutes to separate the serum. The collected serum samples were stored at -20 °C until assy. Serum total protein, albumin, serum transaminase enzymes (ALT and AST), creatinine, urea - N and Uric acid were determined by calorimetric methods using commercial kits (Biocony, D-57299 Burbach, Germany).      

At 52 and 56 weeks of age ten eggs from each treatment group were randomly taken to determine lead residues, broken and albumin, yolk and shell were separated. Egg shell was washed, dried and stored at room temperature to determine the lead residues. Separated yolk and albumin were dried and stored at -200C to determine the lead content. The lead contents in the tissues (muscle, liver and kidney) and egg  components, (shell, albumin and yolk) were determined at the age of 52 & 56 weeks by using the atomic absorption spectrohotometric technique according to Nation and Robinson (1971).

The obtained data were statistically analyzed by using 4x3 factorial design according to Snedecor and Cochran (1982) by the following model:   Yijk = μ+ Li + Aj + LAij + eijk 

where, μ = the overall mean, Li= the fixed effect of i^th lead level in diets (i = 1,.. 4), Aj = the fixed effect of j^th feed additives (j = 1,.. 3), LAij = the interaction between the i^th lead level and feed additives and eijk = random error. Significant differences were determined by Duncan´s Multiple Range test (Duncan, 1955).

 Pre-slaughter body weight, carcass and non-carcass component weights (heart and liver) were significantly decreased with increasing lead level in the diets at 52 weeks of age (Table 2).

Such decrease in the heart weight of treated birds could be due to a direct effect of lead  on heart, muscles and the accumulation of it in the heart could prevent the normal growth of heart muscles. Although the decrease in the  liver weight of treated birds may be due to the damage in the liver cells in portal areas accompanied by dilation of central viens and hepatic sinusoids, some of the hepatic cells showed hyper trophy and vacillation (Youssef et al 1995 and Abou -Zeid et al., 2000). This result is in agreement with the previous report of Fathi et al., (1999) who found that dressed carcass yield decreased in response to increasing dietary lead.

At 56 weeks of age, carcass  and heart weights were not significantly affected by the lead addition to the diets of hens (Tables 2). While  significant decrease was shown in liver weight in hens fed diet contained 250 mg lead/ kg  diet when compared with other experimental groups .

Pre-slaughter body weight, carcass and non-carcass component weights were not affected significantly by  the feed. additives at 52 or 56 weeks of age (Table 2).

Pre-slaughter body weight, carcass and non-carcass component weights were not affected significantly by the interaction between feed additives and lead levels at 52 or 56 weeks of age (Table 2).

Serum total protein and albumin were significantly (P<0.001) decreased by lead contamination in diet at 52 or 56 weeks of age, while globulin levels were not affected significantly (Table 3).The reduction in the concentrations of blood total protein and albumin indicates the impaired protein synthesis in the liver (Khan et al 1993). The depression in the concentrations of total protein and albumin in the hen blood may be the reason for the reduction in the growth rate, egg production and the fertility. Previous results are in agreement with  those report of Abou-Zeid et al. (2000) who indicated a sharp decrease in total protein, albumin and globulin in ducks fed 400 mg lead.

Significant (P<0.01 or P< 0.001)increase was shown in serum total protein and albumin with the supplementation of  natural clay or vitamin E to the diets at 52 or 56 weeks of age, while globulin levels were not affected significantly (Table 3).The increase in the concentrations of blood total protein and albumin was an indicator of the improvement in protein synthesis in the liver and may be the reason for the increase  growth rate, egg production and fertility. Andronikashvih el al. (1994) found that zeolite addition to the diet delayed transit time of digesta through the digestive tract by 2 to 2.5 h and promoted absorption of nutrients, which may reflect the higher values of serum protein including albumin. Abd El-Latif (1999) reported that dietary vitamin E improved plasma total protein, albumin and globulin .Gore and Qureshi (1997) noticed that dietary vitamin E enchanced the cellular immunity which was partially dependent on plasma globulins.

There was no significant interaction between lead level and feed additives in serum total protein, albumin and globulins  at 52 and 56 weeks of age (Table 3). At each dietary lead level, natural clay or vitamin E supplementations increased the concentrations of total protein and albumin. These indicated that the supplementation of clay or vitamin E reduced the toxic effect of the lead. The positive effect of vitamin E on the total protein and albumin content of the blood plasma possibly caused by its stabilizing effect of liver cells during lead contamination. The liver as the main source of blood plasma albumen damaged by lead contamination and the vitamin E supply possibly decrease that effect. Natural clay can adsorb toxic products of digestion and decreases accumulation of toxic substances in tissues, thus decreasing the incidence of internal disorders (Mumpton and Fishman, 1977).

Serum creatinine, urea-N and uric acid concentration  were significantly (P<0.001) affected by the lead additions to the diets at 52 or 56 weeks of age (Table 3).This refers to occurrence of renaltoxicity.The higher concentrations of creatinine, urea-N and uric acid in the blood of hens fed diets containing lead may be due to the over function of the kidney to extract the lead from the body of the hens, also may be due to the damage in kidney nephrons (Fathi et al, 1999).Our results are in agreement with those obtained by Abou Zeid et al. (2000) who found that a highly significantly increase in plasma uric acid and creatinine in ducks fed 200 or 400 mg/kg lead in the diet.

Results in Table 4 showed that creatinine, urea-N and uric acid  levels were significantly (P<0.001) decreased in the blood of hens fed diets supplemented with clay or vitamin E at 52 and 56 weeks of age when compared with those fed diets without these supplementation. These results indicated that the supplementation of natural clay or vitamin E improved the function of kidney to the normal case.

Serum creatinine, urea-N and uric acid  were significantly (P<0.001) affected by the interaction between lead level and feed additives in  the  diet at 52 and 56 weeks of age (Tablet 4).At each dietary lead level, supplementation of clay or vitamin E reduced the concentrations of creatinine, urea-N and uric acid in the blood of hens. These results indicated that the natural clay or vitamin E supplementation reduced the toxicity effect of lead.

Serum transaminase enzymes (AST and ALT) were significantly (P<0.001) affected by the lead addition to the diet at 52 and 56 weeks of age (Table 5).This results refers to occurrence of hepatotoxicity. The increase in the concentrations of AST or ALT may indicate the over function in liver to reduce the level of lead in the blood of hens. The activites of ALT and AST as indicators of hepatic dysfunction were  shown to be elevated at 400 mg lead acetate (Abou-Zeid et al., 2000).Our results are in agreement with those obtained by Fathi et al.(1999) who reported that the plasma ALT increased in broiler chicks fed on a diet containing 500 mg of lead, but plasma AST did not affected significantly by the lead in the diet.

Data in Table 5 showed that AST and ALT were significantly (P<0.001) decreased in the blood of hens fed diets supplemented with clay or vitamin E at 52 and 56 weeks of age when compared with those fed diets without supplementation. These results indicated that the supplementation of natural clay or vitamin E improved the function of liver to the normal case. Also, clay may be decreased the lead absorption from the intestine, so the load on the liver may have been decreased.                      

Serum transaminase enzymes (AST and ALT) were significantly (P<0.001 or P< 0.05) affected by the interaction between lead level and feed additives in the diet at 52 and 56 weeks of age (Table 5).Within each dietary lead level, supplementation of clay or vitamin E reduced the concentrations of AST or ALT in the blood of hens. These results indicated that the natural clay or vitamin E supplementation reduced the toxic effect of lead by reducing the over function of the liver.

Lead residues in muscles, kidney and liver were significantly (P0.001) increased with increasing dietary levels of lead at 52 or 56 weeks of age (Table 6).. The lead retention in the tissues was lower at 56 weeks than at 52 weeks of age, this finding may be due to the ability of birds to recover them self when the lead content reduced in the diet. Higher lead residues were obtained in kidney than in liver and lower value was obtained in muscles. There are not many papers discussing the effect of lead on muscle in birds . Elevation of lead in blood and tissues by lead exposure is well-decumented (Cibulka et al., 1989; Mazlia et al., 1989). Rabinowitz et al (1976) suggested a three - compartment model for lead metabolism, including blood, soft tissues and skeleton. Among the tissues tested in this experiment, the kidney accumulated the highest amount of lead after lead exposure. This result is in agreement with Di Giulio and Scanlon (1984) who found three fold higher kidney to liver ratio of lead in mallard ducks. Jeng et al., (1997) found that the average concentration of lead in kidney of laying Tsaiy ducks was 2.7 times greater that lead in liver tissues. The concentration of lead in liver, kidneys, femur and gluteus muscle increased with increasing dietary lead (Stanchev et al., 1989). Foteva et al. (1997) also, found that lead content in chickens increased with increasing dietary lead during the experimental period.

In our experiments, lead residues in egg shell, yolk and albumin increased significantly (P<0.001) with increasing dietary lead level at 52 and 56 weeks of age (Table 7). These results are in agreement with those obtained in laying hens and mallard ducks (Finley et al.,1979 and Mazliah et al., 1989). Burger and Gochfeld (1991, 1993) reported that female birds can excrete metals in the egg so they measured lead concentrations in whole eggs of birds to evaluate geographic contamination by heavy metals. It is worth noting that higher lead residues were obtained in egg yolk than in egg shell, the lowest value was obtained in egg albumin. The lead retention was lower in the eggs of hens at 56 weeks of age than those at 52 weeks. Albumin contained lower lead than yolk or egg shell as demonstrated by Mazliah et al., (1989). According to Jeng et al. ( 1997) a daily dose of 4 mg lead / kg weight caused significant increase in lead concentration in the egg yolk of laying ducks. These phenomena should receive more attention from, the point of public health, because yolk is food constituent for humans.

Lead residues in tissues ( muscles ,kidney and liver ) and egg (shell, yolk and albumin ) were significantly (P < 0.01 or P< 0.001) decreased by clay or vitamin E supplementations in the diet at 52 and 56 weeks of age (Tables 6 and  7). Lower lead retention was obtained in the tissues and eggs of hens fed diets supplemented with clay, than without supplementation. Natural clay addition in lead contaminated diets clearly reduced the level of lead residues in the viscera and muscles. Natural clay prevents the lead toxicity by reducing lead absorption in the intestinal tract and increasing fecal excretion.

Lead residues in tissues and eggs were significantly (P < 0.01 or P<0.001) decreased by the interaction between feed additives and lead levels in the diet at 52 weeks of age, except the egg albumin which was not significantly affected at 56 weeks (Tables 6 and 7). At each lead level, the clay supplementation in the diet reduced the lead residues in the tissues and eggs below the values measured in other groups.

On the basis of the results obtained in this study, it can be concluded that the consumption of diets polluted with heavy metal such as lead causes rious effects in the carcass, blood components and increases the level of lead residual in  tissues and eggs. However, the addition of zeolite or vitamin E to diets of laying hens caused beneficial effects on carcass characteristics, blood components and lead residual tissues and eggs. Moreover, adding clay or vitamin E as feed additives to lead polluted diets diminishes lead toxicosis and therefore improve all traits studied.

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Table 1. Composition and calculated analysis of the experimental laying ration:

* Each kg of premix contain : 5, 000, 000 I. U. Vit. A; 1, 250, 000 I. U. Vit.D₃;  2 g Vit. K; 3g Vit B₂ ; 15 g nicotinic acid; 4 g calcium D-Pantothenate; 8 g Vit. B₁₂ ; 150 choline chloride ; 80 g D. O. T. (35 Dinitro ortho toluamide); 40 g manganese; 20 g iron; 20 g zinc; 1 g copper; 1 g iodine and 1g cobalt.

Table 2. Carcass traits of Silver Montazah laying hens as affected by lead, feed additives and their interaction at 52 and 56 weeks of age.

Means in the same column within each classification with different letters, differ significantly (P<0.05).
NS = not significant, * P<0.05, ** P<0.01 and *** P<0.001. 

Table 3.Total protein, albumin and globulin (mean±SE)  of Silver Montazah layer as affected by lead, feed additives and their interaction  at 52 and 56 weeks of age.

Means in the same column within each classification with different letters, differ significantly (P<0.05).

NS = not significant, ** P<0.01 and *** P<0.001. 

Table 4. Serum creatinine, urea - N and uric acid (mean±SE ) of Silver Montazah layer as affected by lead, feed additives and their interaction  at 52 and 56 weeks of age.

Means in the same column within each classification with different letters, differ significantly (P<0.05).

*** P<0.001. 

Table 5. Serum transaminase enzymes ,AST and AL ,mean±SE,  of Silver Montazah layer as affected by lead, feed additives and their interaction  at 52 and 56 weeks of age.

Means in the same column within each classification with different letters, differ significantly (P<0.05).

* P<0.05 and *** P<0.001.