Poultry farming has metamorphosed into an organized industry in recent past and registered a rapid growth over last three decades in India. Many problems are continuously challenging against successful poultry farming operations and the common one is threat of mycotoxicosis in poultry. Aflatoxins (AF) have been demonstrated to be carcinogenic, mutagenic, teratogenic and toxic. Surveillance of AFB1 content of poultry feedstuffs in U.P., India by Johri et al. (1986) revealed that 71 per cent of groundnut cake, 47 per cent of maize, 25 per cent of fish meal and 16.7 per cent of rice bran samples were contaminated by AFB1, and the authors also found AF in starter feed mixture at 0-2 ppm and in grower feed mixture at 0-1 ppm levels. Surveillance in Southern part of India also showed that more than 40 per cent of the poultry and animal feed samples contained moderate to high level of AFB1, ranged from 0.01 to 12 ppm (Selvasubramanian et al., 1987). Dietary manipulations have received considerable attention and increased dietary concentration of protein (Beura, et al., 1993), αtocopherol, ascorbic acid (Hoehler and Marquardt, 1996), choline, folic acid, pyridoxine, riboflavin were extensively studied for counteracting aflatoxicosis. However, studies on effect of trace minerals supplementation and its interaction with AF in vivo in poultry are very few. Increased dietary concentration of copper (Johri et al., 1986), have been tried to counteract AF with moderate to significant response and in all the studies the trace elements were supplemented as inorganic form, not as organic chelates.
It is now recognized that the organic chelates of minerals are different from its inorganic counterpart in that the trace minerals are held with metal binding agents (ligand) by high energy bond known as co-ordinate covalent bond (Leeson and Summers, 2002). Several studies reported that the chelated minerals are more bioavailable to animals and poultry (Wedekind et al., 1992) than its inorganic counterpart and hence one can expect more counteracting effect of this form of minerals against AF than inorganic form. But, studies regarding the effect of chelated minerals on AF and their interaction in vivo in poultry received very little attention. Hence, a study was designed to evaluate the interaction of chelated and inorganic copper on growth performances i.e. body weight gain, feed intake, feed conversion ratio, livability of broiler chicken.
MATERIALS AND METHODS
Production of aflatoxin
AF was produced from Aspergillus parasiticus NRRL 2999 in rice as per method of Shotwell et al. (1966) and in Yeast Extract Sucrose (YES) broth as per Tsai et al. (1984). The AF B1 content was measured by preliminary extraction of AF (Pons et al., 1966) and subsequent analysis by TLC method. The AF B1 in contaminated rice ranged from 25 to 35 ppm and in YES broth the AFB1 ranged from 250 to 300 ppm.
The feeding experiment was conducted in broiler chicken from day one to 42 day of age. Two hundred and seventy day-old synthetic dam line broiler chicks were obtained from Experimental Broiler Farm, CARI and were wing banded, weighed and randomly allotted into nine treatment groups. Each treatment had three replicates and containing ten chicks per replicate.
All the chicks were reared on wire floor, electrically heated, battery brooder with provision of feeder and waterers under uniform and standard management practices. Feed and water were offered ad libitum. All the chicks were apparently healthy and were free from Marek’s Disease, Newcastle Disease and Infectious Bursal Disease. All birds were immunized against Newcastle Disease and Marek’s Disease at day old by occulonasal route and against Infectious Bursal Disease at 14th day of age by occulonasal route. The experimental protocol had the agreement of Institute of Animal Ethics Committee. The feed ingredients of broiler starter and finisher diets were checked for presence of AF before preparation of experimental feed to ensure that the feed was free from natural AF.
Preparation of experimental diets
Two standard control diets were formulated (Table 1) separately for starter (0-21 days) and finisher phase (22- 42 days) of growth to meet the requirement of all the essential nutrients for broilers.
The experimental design followed was 3 × 3 factorial and the experiment consisted of nine treatments as follows:
T1 – Control diet
T2 – Control diet + 0.5 ppm AF B1
T3 – Control diet + 1 ppm AF B1
T4 – Control diet + 40 ppm Cu from organic source
T5 – Control diet + 0.5 ppm AF B1 + 40 ppm Cu from organic source
T6 – Control diet + 1 ppm AF B1 + 40 ppm Cu from organic source
T7 – Control diet + 40 ppm Cu from inorganic source
T8 – Control diet + 0.5 ppm AF B1 + 40 ppm Cu from inorganic source
T9 – Control diet + 1 ppm AF B1 + 40 ppm Cu from inorganic source
Copper sulphate and copper propionate with 30 per cent Cu (supplied by M/s. Kemin Nutritional Technologies India Pvt. Ltd., Gummidipundi, Chennai) were used as the source of inorganic and organic copper, respectively.
The data on weekly body weight gain of broiler chicks during 0-6 weeks of age were collected and analyzed for starting (0-21days), finishing (22-42 days) and overall (0- 42 days) experimental period. The feed intake of the experimental birds was measured at weekly intervals up to 6 weeks of age. The weekly and cumulative feed conversion ratio (FCR) was calculated by dividing the amount of feed consumed with corresponding per unit gain in body weight by the chicks. The mortality as and when occurred under each experimental treatment was duly recorded and accordingly calculated.
The experimental design followed was 3 × 3 factorial design. The data obtained from the above experiments were subjected to statistical analysis as per standard procedure of Snedecor and Cochran, (1989) and Duncan’s multiple range test (Duncan, 1955) for verifying significance of treatment means.
RESULTS AND DISCUSSION
The result revealed that the BWG was significantly (P<0.01) depressed in a dose dependent fashion due to dietary AF levels and it was lowest in 1 ppm AF level and highest in basal AF level during 0-3 weeks, 4-6 weeks and also during 0-6 weeks, due to the effect of AF levels (Table 2). The mean BWG showed a significant difference due to Cu supplementation and it was higher in Cu chelate and Cu supplemented group than Cu inorganic group during 0-3 weeks. The mean BWG during 4-6 weeks and 0-6 weeks were highest in Cu chelate group followed by control and it was lowest in Cu inorganic group. Similar findings was made by several authors (Gopi, 2006; Vasan et al., 1998; Verma, 1990; Doerr et al., 1983) who reported reduced weight gain, feed intake and feed efficiency in a dose dependent fashion due to dietary AF in poultry. However, these authors reported reduced performance of broiler at various dietary concentration of AF, ranging from 0.5 ppm AF to 2 ppm AF level. In contrary to this finding, Sinha and Arora (1987) could not observe any adverse effect on the performance of broilers up to 1 ppm level of dietary AF. The reduced BWG might be due to reduced palatability of feed leading to reduced feed intake and feed utilization of nutrients.
The BWG during 0-3 weeks did not differ significantly due to interaction between Cu supplementation and AF levels whereas, the mean BWG during 4-6 weeks and 0-6 weeks differed significantly (P<0.01) due to the interaction. The BWG during 4-6 weeks was highest in all Cu groups at basal AF level and lowest in Cu inorganic group at 1 ppm AF level, while the mean BWG of other groups were found to be intermediary. The Cu chelate increased the BWG at 0.5 ppm AF level, although it was not significant. But the Cu chelate significantly increased the BWG at 1ppm AF. In agreement with the result, Llewellyn et al. (1981) indicated that Cu acetate caused higher body weight in Syrian hamster. However, Seffner et al. (1997) found more severe liver injury in guinea pigs administered AFB1 and Cu as CuSO4 than animals with AFB1 alone.
The result on feed intake revealed a significant (P<0.01) difference due to AF levels during 0-3 weeks, 4- 6 weeks and 0-6 weeks, and it was significantly lower at 1 ppm AF level, followed by 0.5 ppm AF than basal AF, due to the effect of AF levels. The feed intake during 4-6 weeks of age differed significantly (P<0.05) due to different Cu supplementation. Significantly higher feed intake was observed in Cu unsupplemented group and Cu chelate group than Cu inorganic group. However, the Cu unsupplemented group did not differ significantly with either Cu chelate group or Cu inorganic group. The feed intake during 0-3 and 0-6 weeks of age did not differ significantly due to different Cu supplementations. The feed intake during 0-3 weeks did not differ significantly due to interaction between Cu supplementations and AF levels. But the feed intake during 4-6 weeks and 0-6 weeks differed significantly (P<0.01) due to the interaction. The mean feed intake during 4-6 weeks and 0- 6 weeks was significantly highest in Cu groups at basal AF level followed by Cu group at 0.5 ppm AF level than Cu chelate or Cu unsupplemented groups and 1 ppm AF, while the feed intake of other treatments were found to be intermediary.
The FCR during 0-3 weeks, 4-6 weeks and 0-6 weeks differed significantly (P<0.01) among treatments due to AF levels. The FCR during 0-3 weeks was lower in 0 and 0.5 ppm AF level than 1 ppm AF level. The FCR during 4-6 weeks and 0-6 weeks was significantly increased in a dose dependent fashion with lowest FCR in basal AF level and highest at 1 ppm AF level. The FCR during 0-3 weeks was significantly (P<0.01) lower in Cu unsupplemented and Cu chelate group than Cu inorganic group, due to the effect of Cu supplementation. Non significant effect was observed on FCR during 4-6 weeks and during 0-6 weeks due to Cu supplementation. The interaction effect of Cu supplementation with AF level on FCR was significant (P<0.05) at 4-6 weeks and non-significant at 0-3 weeks and at 0-6 weeks. The FCR during 4-6 weeks was lower in Cu groups at basal AF level than Cu inorganic group at 1 ppm AF level, whereas the FCR of other groups were found to be intermediary.
The result of the study is also supported by earlier finding of many authors (Sharma et al., 1989; Bartov, 1983) who observed that CuSO4 did not counteract aflatoxicosis in chicken. In contrary, Johri et al. (1986) found that the growth depression due to mouldy corn was overcome by addition of 150 ppm of CuSO4.
The result suggests that there is a difference between chelated and inorganic Cu in their effect under AF challenge. The role of propionic acid in Cu-propionate against aflatoxicosis is also doubtful because of the trace amount of propionic acid, whereas the minimum effective level of propionic acid in feed was found to be 0.15 per cent (Johri et al., 1986). Arias and Koutsos (2006), in one experiment found that broilers with supplemented Cu as tribasic copper chloride had higher body weight and not with Cu in the form of CuSO4 and concluded that the broiler performance could be influenced by dietary Cu source and level. However, there is a little study comparing the chelated and inorganic Cu under AF challenge and more detailed study and comprehensive information appears necessary on the form of Cu and its influence during aflatoxicosis.
The present study indicates that the livability percentage ranges from 96.67 to 84.44 with higher livability in basal AF group and lower livability in 1 ppm AF group. These findings are well in agreement with Gopi (2006), Reddy et al. (1982) who reported significant reduction in livability at 0.5 to 1.25 ppm level of dietary AF in broilers.
The supplemental Cu in either form at 40 ppm was found to have no effect on the decrease in livability due to dietary AF. On the contrary, Llewellyn et al. (1981) observed higher livability in Syrian hamster undergoing AF and Cu treatment, than AF alone.
From the results, it was observed that there was a difference between organic and inorganic copper in their effect in counteracting aflatoxicosis in broilers. The organic form of copper was able to counteract the aflatoxicosis in broiler, while inorganic copper was found to aggravate the aflatoxicosis.
The authors thank Tamil Nadu Veterinary and Animal Sciences University, Chennai for providing external deputation at IVRI for the first author. The authors also thank M/s. Kemin Nutritional Technologies India Pvt. Ltd., Gummidipundi, Chennai for supplying copper propionate for research for the cause of poultry nutritional science.
This article was originally published in International Journal of Veterinary Science, 2(3): 106-110. www.ijvets.com.