Effect of Different Levels of Egg Shell Powder on Gas Production, Fermentation Parameters and Digestibility of Aflatoxin B1 Contaminated Diet in vitro

Introduction Mycotoxins are secondary metabolites of fungi that are produced under stress condition. Aflatoxin is one of several extremely toxic, mutagenic and carcinogenic compounds produced by Aspergillus Flavus and Aspergillus Parasiticus. Numerous agricultural commodities such as Forages, cereal grains, oilseeds, and cotton seeds are potential sources of aflatoxins in ruminant diets. Many studies indicate that ruminants, like other animals, are affected by aflatoxins. Aflatoxin B1 reduces ruminal digestion, animal production, and in high doses causes liver damage and death in ruminants. Several chemical, biological, and physical strategies developed in order to, detoxification of aflatoxins or minimizing the production of aflatoxins and inhibiting the absorption of them in the gastrointestinal tract. Recently, many researchers are focused on aflatoxin adsorbents to reduce the bioavailability of aflatoxins in the diet. Eggshell has a porous structure and on the other hand has significant amounts of pure calcium carbonate, which has the ability to absorb toxins. Due to limit information on the ability of egg shell powder to absorb aflatoxin, the present study was designed to investigate the effect of adding egg shell powder as toxin binder in diets containing aflatoxin B1 on fermentation parameters and ruminal digestibility and gas production in vitro.  Materials and Methods To produce the aflatoxin required for the experiment, a standard strain of Aspergillus Parasiticus NRLL 2999 used and cultured on potato dextrose agar. In other to obtain proper amount of Aflatoxin, 2 ml of spore suspension with a concentration of 6.5× 106 grown fungi was prepared and added to a flask containing sterile culture medium. After 5 days, the culture medium was dried in an oven. Culture medium contained of 250.9 mg/kg aflatoxin B1. This experiment was performed in a completely randomized design with 5 treatments and 4 replications in each treatment. Experimental treatments consist of , control with methanol, control without methanol, Aflatoxin (800 ng/mg of rumen fluid), Aflatoxin+ level 1 egg shell powder (7 mg per 200 mg of diet), Aflatoxin +level 2 egg shell powder(75 mg per 200 mg of diet), Aflatoxin +level 3 egg shell powder(150 mg per 200 mg of diet). Rumen fluid was collected before the morning feed from three fistulated Moghani male sheep with 46 ± 3 kg live weight. Sheep fed with basal diet used in this experiment at a concentration of 50:50 forage to concentrate for 15 days before rumen fluid collection. In vitro gas production was measured in 4 replicate with 200 mg DM for each. The bottles were filled with 30 ml of incubation medium that consisted of 10 ml of rumen fluid plus 20 ml of buffer solution and placed in a water bath at 39 °C. Gas production was recorded at 2, 4, 8, 16, 24, 48, 72 and 96 h. Gas values corrected for blank incubation. The gas production and rate of gas production measured through 96 h incubation. A procedure similar to gas production with 4 replicate for each treatment was used for rumen batch culture system to measure NH3-N and in vitro digestibility after 24 h incubation. Contents of each glass bottle were filtered through three layers of cheesecloth and rumen fluid used to determination of NH3-N using the distillation method. Finally, all remaining contents oven dried at 60 °C for 48 h and analyzed for IVDMD and IVOMD. Metabolizable energy (ME), net energy for lactation (NEL), short chain fatty acids (SCFA), partitioning factor (PF), Microbial mass and Efficiency of microbial synthesis calculate throughout 24h incubation.  Results and Discussion Results indicate a significant decrease in GP in aflatoxin treatment compared to treatments without aflatoxin, so that the amount of gas production decreased from 295.05 and 294.38 in control with methanol and control without methanol to 228.48 ml/g of DM. This change in GP was associated with significant reduction in IVDMD and IVOMD, ME, NEL, SCFA, PF, microbial mass and Efficiency of microbial synthesis. Addition of different levels of egg shell powder as aflatoxin binder improved fermentation conditions which was significant in level 2 treatment compare to aflatoxin treatment. There was no significant difference in GP, IVDMD and IVOMD, ME, NEL, SCFA, PF, microbial mass and efficiency of microbial synthesis between control treatments and level 2 egg shell powder as toxin binder.  Conclusion Considering all the results of experiment, egg shell could be considered as an adsorbent of aflatoxin in ruminal conditions. Egg shell powder suggested as toxin binder to reduce the negative effects of aflatoxin B1 on microbial activity and degradability in ruminal conditions.