Clinical signs and behavior

Clinical signs and behavior during aflatoxicoses has been investigated by many workers [2],[12],[13]. Increased water intake has also been reported in broiler chicks during aflatoxicosis [14]. A decrease in feed intake and increase in water intake may be due to avoid dehydration and replenish the water loss during watery and loose dropping Chohota et al.[15] described a general depression in broiler chicks suffering from aflatoxicosis. Robinson et al.[16] study demonstrated weakness, little response to feed and depression in wild birds suffering from aflatoxicosis. In our study group B having aflatoxin in feed exhibited abnormal signs and behavior throughout the course of experiment and was found sever at the end of 6^th week however groups D, E and F receiving aflatoxin and different levels of adsorbent in feed showed a very low cumulative score as compared to group B. The present study clearly indicated that the prepared adsorbent can play a vital role to ameliorate the ill effects of aflatoxin B1.

Blood serum biochemical parameters

Serum biochemical parameters such as Total protein, Serum globulin, Albumen contents [17], alkaline phosphatase and alanine transferase [13] blood level can be used to determine the hepatic malfunctioning and liver injury. Adsorbents can effectively reduce the negative effects of aflatoxin in chicks consuming aflatoxin contaminated feed [13],[18]. The effect of the prepared carbon nanostructures on different blood serum parameters are discussed below.

Alanine Transferase (ALT)

Weekly results of blood serum biochemistry analysis of broiler chicks given different levels of adsorbent revealed that the concentration of ALT of groups C, D, E, and F were significantly lower than group B throughout the course of experiment. The values of ALT concentration of groups C, D, E and F were noted non-significant at the end of 1^st and 2^nd weeks whereas a significant difference were observed at the end of 3^rd, 4^th, 5^th and 6^th weeks. ALT concentrations of groups D, E and F showed no significant difference, however lower values were observed in groups D and F (Figure 1).

Serum ALT and alkaline phosphatase activities, Total protein, serum albumens and globulin concentration have also been described as valuable parameters of liver injury and function [19]. The results of group B indicated high concentration of ALT fed on aflatoxin contaminated feed as compared to groups D, E and F offered feed containing aflatoxin and different levels of adsorbent. These results represented that addition of the prepared adsorbent in aflatoxin contaminated feed ameliorated the toxic effects of aflatoxin. Our findings are in close agreement to Pasha et al.[20], Gowda et al.[18], Ibrahim et al.[21], Shi et al.[22] and Watts et al.[23] who reported that different adsorbents have been found to diminish the toxic effects of aflatoxin in broiler chicks.

Alkaline Phosphatase (ALP)

The results of ALP concentration has been shown in Figure 2. The ALP concentration of chicks of group B were significantly higher than that of groups C, D, E and F, while the values of ALP concentration of groups C, D, E and F were observed non-significant throughout the experimental weeks. Concentration of group A showed no significant difference to that of groups C, D, E and F. However lower values of ALP concentration were noted in groups D and E.

Biochemical changes in blood serum represent a stress on liver function [24],[25]. In present study the activities of ALP increased, whereas the concentrations of total protein globulin and albumens in the serum were decreased in chicks of group B. All these are the prime indicators of aflatoxicosis [24],[25] whereas increases ALP level are associated with the hepatobiliary malfunction. It has been also reported that though ALP is not a liver specific enzyme but always associated with liver injuries, biliary stasis and other nonspecific injuries to the body tissues [24]. Our results also demonstrated that chicks of groups D, E and F fed on diet containing aflatoxin and different levels of adsorbent diminished the toxic effects of aflatoxicosis. Same results have been reported by Miazzo et al.[7], Kubena et al.[12], Gowda et al.[18], Ibrahim et al.[21], Jindal et al.[24] and Abdel-Wahhab et al.[25] whose investigations showed that different adsorbent can effectively reduce the negative effects of aflatoxin in chicks consuming aflatoxin contaminated feed.

Serum albumen

The values of groups A, C, D, E and F were significantly higher than group B, while there no significant difference in the values of groups A, C, D, E and F at the end of 1^st week. At the end of 2^nd week no significant difference were seen in all groups however the values of groups B and C were found significantly less than that of groups A, D, E and F throughout the experimental period. The values of group C showed significant difference than that of group B during 1^st, 3^rd, 4^th, 5^th and 6^th weeks (Figure 3).

Blood biochemical parameter alters in chicks consuming aflatoxin contaminated feed [18],[24],[26],[27]. The decreased levels of serum total protein, globulin and albumen contents are the prime indicators of aflatoxicosis which can be reduced by the addition of adsorbent in aflatoxin contaminated feed [7],[18],[21],[24],[25],[28]. Our findings showed that the level of albumen contents were found low in chicks of group B fed on diet containing aflatoxin alone whereas increased levels of albumen were noted in those groups offered feed containing aflatoxin along with different levels of adsorbent.

Serum total proteins

Weekly results of Serum blood biochemistry of chicks for total protein concentration have been given in Figure 4. The results indicated that the total protein concentration of group B was significantly different than that of groups C, D, E and F, while concentration of total protein in blood serum of chicks of groups C, D, E and F were not significantly different at the end of 1^st week. Significant lower values were found in group C than that of groups D, E and F in the 5^th week. The concentration of group D was significantly less than that of groups E and F at the end of 2^nd week. Concentration of total protein of groups E and F were significantly different at the end of 4^th week, whereas the values of groups D, E and F showed no significant difference at the end of 6^th week. High values of total protein concentration were observed in groups D and E. The levels of serum albumin and total protein are the sensitive indicators of aflatoxicosis. In the present study the level of serum total protein decreased in group B consuming aflatoxin contaminated feed when compared to Groups C, D, E, and F fed on diet containing aflatoxin and different levels of adsorbent.

Decrease concentration of serum total protein has been proposed as indicator of inhibition of protein synthesis [18]. Our results showed that addition of adsorbent to aflatoxin contaminated feed strongly ameliorated the hazardous effect of aflatoxin. Various reports are available which demonstrated that different adsorbents have the capability to reduce the toxic effect in chicks when offered aflatoxin contaminated feed [7],[18],[20].

Serum globulin

Weekly Serum globulins result of chicks given different levels of adsorbent has been presented in Figure 5. Serum globulins concentration in blood serum of chicks of group B were found significantly less than that of groups C, D, E and F. The concentration of serum globulins in blood serum of broiler chicken of groups C, D, E and F showed no significant difference throughout the course of study however the highest values for serum globulins were determined in groups D and E. Our results showed that the serum globulin levels decreased in group B fed on aflatoxin contaminated feed, while increased in groups C, D, E and F by the addition of the prepared adsorbent.

According to Yinghua et al.[28] the levels of serum globulin decreases when chick fed diets containing aflatoxin whereas no significant difference were seen between control and that of chicks fed on diets containing aflatoxin plus adsorbent.

Mortality

Weekly results of mortalities of broiler chickens at different level of adsorbent have been presented in Figure 6. There was no mortality in the groups A, C, D, E and F throughout the experimental period. Total mortality of broiler chickens in group B was 2 at the end of 6^th week which was recorded as 3.07%. These results are in accordance with the findings of Afzal et al.[29] according to which percent mortality increases in chicks fed on aflatoxin contaminated feed whereas percent mortality can be decreased when fed on diet containing aflatoxin plus adsorbents.

Mortality in chicks fed on aflatoxin contaminated feed has been also reported by many workers [29],[30]. Chicks of group B consuming aflatoxin contaminated feed without adsorbent showed high percent mortality as compared to groups C, D, E and F fed with aflatoxin plus different levels of adsorbent.

Body weights

Relative body weight of group B showed significant lesser body weight fed on toxin contaminated feed through the experimental period. Group D values were significantly higher than that of groups B, C, E and F fed on aflatoxin and different levels of adsorbent at the end of 2^nd week. During 3^rd week all the values of all groups were found non-significant except group B. The body weight of broiler chicks of groups D, E and F showed no significant difference, while the values of all these groups were significantly higher than that of groups B and C during 5^th week. No significant differences were noted in groups A and D, E and F whereas values of these groups were found significant higher than that of groups B and C at the end of 6^th week (Figure 7).

The body weight of chicks decreased when fed on aflatoxin contaminated feed as previously reported by many workers [7],[26],[27],[31]. Our study showed that body weight of chicks of group B decreased throughout the course of study whereas those groups receiving aflatoxin contaminated feed along with different levels of adsorbent showed increase in body weight. Aflatoxins are prim concern to poultry industry because of their negative effects. The lower weight gains in chicks fed with aflatoxin alone as compared to other groups were consistent with previous studies on the toxic effects of aflatoxin [26],[27],[31]. They also investigated a trend in improvement in body weight gain when fed with adsorbent added to aflatoxin contaminated feed.

Liver weight

Figure 8 data revealed that weekly results of relative weight of liver of chicks given different levels of adsorbent were non-significant at the end of 1^st week among all groups (A, B, C, D, E and F). Significantly higher values were recorded in group B at the end of 2^nd week and continued till the end of 6^th week. The values of groups C, D, E and F were found non-significant, while low values of relative liver weight were determined in groups D and E. Liver is the main target organ of aflatoxin. In addition, the relative weight of liver in poultry has been reported to be increased by the presence of aflatoxin in the feeding diet.

Relative weight of liver in groups D, E and F fed on diet containing aflatoxin plus different levels of adsorbent showed no pathological signs which confirmed that addition of adsorbent to aflatoxin contaminated feed diminish the toxic effects of aflatoxin [32],[33]. The basic theory is that adsorbent detoxify aflatoxin contaminated feed by binding them strongly enough to prevent toxic interaction with the consuming animals and to prevent aflatoxin absorption from the gastrointestinal tract [32].

Kidney weight

The results of relative weight of kidney showed that values of all groups were non-significant at the end of 1^st week, while the values of group B were found significantly higher than other groups at the end of 2^nd to 6^th weeks. No significant difference were seen among groups A, C, D, E and F at the end of 5^th week, whereas significant difference was noted between group B and groups A, D, E and F at the end of 6^th week. Groups D, E and F values were found non-significant, however low values were recorded in groups D and E. The relative weights of kidney of group B were significantly increased in chicks fed on aflatoxin B1 alone (Figure 9).

The results abstained were in agreements with previous study reported by Huff et al.[34] in which he found that aflatoxin alone in feed increase the kidney weight whereas by addition of adsorbent to aflatoxin contaminated feed ameliorated the deleterious effects of aflatoxin in broiler chicks. The increased relative weight of kidney was probably due to slight renal damage [34].

Spleen weight

The results of relative weight of spleen of groups A and B were found significantly different, values of group B were noted significantly higher than that of group A and were recorded maximum at the end of 6^th week. Group C values were non-significant to group A, D, E, and F at the end of 2^nd week, whereas a significant higher values were noted at the end of 3^rd, 4^th, 5^th and 6^th weeks. The results also revealed that values of groups D, E and F were not significantly different, however lower values were recorded in groups D and E (Figure 10).

Our findings related to group B are in close agreement to Hesham et al.[27] in which the increase in relative spleen weight was due to the presences of aflatoxin in the diet. The results of groups D, E and F showed that addition of the prepared adsorbent in diet ameliorated the toxic effects of aflatoxin B1. These results were in accordance with the work of Miazzo et al.[7], Pasha et al.[20] in which they studied that adsorbents are effective in preventing or ameliorating the changes in organs weights of chicks fed on aflatoxin B1 contaminated feed.

Weight of Bursa of fabricious

Results of relative weight of Bursa of fabricious have been shown in Figure 11. The values of Bursa of fabricious weight of broiler chicks given different levels of adsorbent were non-significant among all the groups (A, B, C, D, E and F). No significant change were found throughout the course of study, however the values of groups D and E were closely resembled to that of group A. These results are in close agreement to Bailey et al.[35] in which they investigated that the relative weights of Bursa were not affected by any dietary treatments.

Hemorrhages in organs

Results showed that tendency of hemorrhages on different organs except heart of group B chicks were found maximum, while that of groups D, E and F consuming aflatoxin and different levels of adsorbent in feed remained normal as compared to group B score. Hemorrhages on different organs in broiler chicks during aflatoxicosis have also been reported by Ortatatli and Oguz [2]. Impairment of coagulation mechanism and hematopoieses increased indothelial cells fragility which might be responsible for tendency of hemorrhages in the tissues [2].

Enlargement of organs

Liver enlargements of intoxicated broiler chicks followed by moderate kidney enlargement during aflatoxicosis have also been documented by Ortatatli and Oguz [2] Enlarged kidney and liver in group B were also evident by the significant increase in relative organs weight of chicks of group B consuming aflatoxin contaminated feed. Enlarged livers and kidneys due to aflatoxicosis have also been reported in other birds like Japanese quil, ducks and water fowls [2],[12],[36]. Our results also demonstrated that by adding different levels of adsorbent in aflatoxin contaminated feed ameliorated the hazardous effects of aflatoxin as previously, reported by [12],[36] in which they studied the ameliorating effects of different adsorbents in checks fed on aflatoxin contaminated feed.

Preparation of adsorbent

Bagasse was used as biomass to produce magnetic carbon nanocomposite. The biomass was shredded and transferred to ethanolic FeCl₃.6H₂O (10% w/v). After 30 minutes biomass was separated and air dried at room temperature for 24 hours. Thereafter, biomass was charred in a specially designed chamber (designed by the author) consisting of a stainless steel container equipped with an electric heater, wire gauze, nitrogen gas (to provide a nitrogen rich atmosphere) inlet and exhaust outlet. The prepared adsorbent was characterized by surface area analyzer, SEM, XRD, FTIR, TG/DTA and EDX.

Production of aflatoxin B1

Aflatoxin B1 was produced from A. flavous by culturing on rice following the method of Shot Well [2]. The Aflatoxin B1 residues produced were suspended into poly ethylene glycol and the required quantities were evenly mixed with basal feeds to get different level of aflatoxin B1contaminated feed samples. The preparation of basal feed without toxin binder were ordered and purchased from feed mill and checked for Aflatoxin B1 ≤ 1 μg/kg.

Induction of aflatoxicoses in broiler checks

All animal experiments were carried out according to the Scientific Procedures Issue-1 of Animal Bylaws-2008 approved by the legal bodies of the University of Malakand Khyber Pakhtoonkhwa, Pakistan. The ethical committee of the department of Biochemistry granted approval for conducting this study under the said protocols (ProceduresIssue-1 of Animal Bylaws-2008). A total of 480 one day old broiler chicks were purchased from local hatchery (Rawalpendi, Pakistan) and were kept in poultry house under standard environmental conditions. All these chicks were then equally divided into six different groups (A-F) and given access to fresh water ad libitum.

Experimental protocol

Broiler chickens of different groups were given the following experimental diets.

Group A: Chicks were kept on normal feed throughout study duration.

Group B: Chicks were kept on Aflatoxin B1 (200 μg/kg feed) contaminated feed for 6 weeks.

Group C: Chicks were given Aflatoxin B1 (200 μg/kg feed) contaminated feed mixed with 0.2% of the prepared adsorbent throughout study duration.

Group D: Chicks were given Aflatoxin B1 (200 μg/kg feed) contaminated feed mixed with 0.3% of the prepared adsorbent throughout 6 weeks.

Group E: Chicks were given Aflatoxin B1 (200 μg/kg feed) contaminated feed mixed with 0.4% of the prepared adsorbent throughout study duration.

Group F: Chicks were given Aflatoxin B1 (200 μg/kg feed) contaminated feed mixed with 0.5% of the prepared adsorbent throughout study period.

Observations and analysis

The body weights and mortality of the checks were recorded at the end of every week, while blood serum biochemistry for alanine transferase, alkaline phosphatase, albumen, globulin and total protein were carried out by collecting blood of 3 chicks individually from each group at the end of 1^st to 6^th weeks using commercially available kits (Diasys Diagnostic system Germany) [37].