IN VIVO TRIALS ON RUMINANTS

IN VIVO TRIALS ON RUMINANTS

EFFICACY OF MINAZEL PLUS TO AMELIORATE THE TOXIC EFFECTS OF ZEARALENONE IN LAMBS Acta Veterinaria (Beograd), Vol. 54. No. 1, 1-62, 2004 STOJŠIĆ D*, STOJKOVIĆ M**, DAKOVIĆ ALEKSANDRA***, ADAMOVIĆ M*** and TOMAŠEVIĆ-ČANOVIĆ MAGDALENA*** *Militar Medical Academy, National Center of Poison Control, Department of Toxicological Chemistry, Belgrade **PKB, INI Agroekonomik. Belgrade ***Insitute for Technology of Nuclear and Other Mineral Raw Materials, Belgrade

In vitro and in vivo studies were conducted to evaluate the efficacy of Minazel Plus in reduction of Zearalenone (ZEN) toxic effects of in lambs.

Binding ability of Minazel Plus was tested as follows: 10 ml of the test solutions were added to test tubes containing 100 mg of Minazel Plus Controls were prepared by adding 10 ml of test solution without the addition of adsorbent. The tubes were mechanically shaken for 2 h and then centrifuged at 10 000 rpm. ZEN concentrations in electrolytes were determined by HPLC.

Group I Fed without zearalenone and without Minazel Plus. Group II Fed with 8,3 mg/kg zearalenone and without Minazel Plus. Group III Fed with 8,3 mg/kg zearalenone and with 0,2% of Minazel Plus. Group I Group II Group II 8,3 mg/kg of Zearalenone - + + Minazel Plus - - 0,2%

At the end of trial 6, randomly selected lambs from each group, were killed (4 males and 2 females) in order to test ZEA concentration in: Liver Kidneys Muscles

Adsorption index, % ph3 ph 7 ph 9 Minazel Plus 93 96 92

GROUP I II III Control +ZEN +ZEN and 0.2% Minazel Plus Start body weight, kg 17.12 17.41 17.41 Final body weight, kg 27.12 26.62 28.47 Daily gain, g 189 174 209 Kg of concentrate/kg of gain 3.12 3.39 2.82 Zearalenone, mg/100kg body weight 0 22.31 21.40

Zearalenone amount in liver, kidneys and muscles GROUP I II III Control + ZEN + ZEN and 0.2 %Minazel Plus Liver, g 0.00 9.45 0.17 Kidneys, g 0.00 8.53 0.09 Muscles, g/kg 0.00 7.64 0.02

IN VITRO trial has concluded that Minazel Plus successfully adsorbed over 90% of ZEA in the sample IN VIVO trial has confirmed positive effect of Minazel Plus on: Daily gain Decreasement of ZEA residues in: Liver Kidneys Muscules

APPLICATION OF MINAZEL PLUS IN PREVENTION OF CALF ZEARALENONE TOXICOSIS Srdan Nešic*, Goran Grubić**, Milan Adamović*** * Patent co. Belgrade ** Faculty pf Agriculture, University of Belgrade ***Insitute for Technology of Nuclear and Other Mineral Raw Materials, Belgrade

Trial were executed on 48 Holstein calves, divided into 4 groups, 12 calves in each group. Experiment duration - 90 days. Group A Feed was not contaminated with zearalenone and Minazel Plus was not added. Group B Feed was contaminated with zearalenone and Minazel Plus was not added. Group C Feed was contaminated with zearalenone and 0.2% of Minazel Plus was added. Group D Feed was contaminated with zearalenone and 0.5% of Minazel Plus was added.

Parameter Group A B C D Average age of calves, days 27.42 32.5 31.2 27.33 Minazel Plus, % - - 0.2 0.5 Body weight, kg Start of trial 63.92 63.79 63.96 63.87 30.Day 89.7 91.62 92.79 93.87 60.Day 121 121.95 121.55 121 90.Day 149 147.20 152.50 153 Daily gain, kg Milk feeding 0.758 0.818 0.848 0.882 Dry feeding 1.044 0.978 1.059 1.053 Average daily gain, kg 0.935 0.917 0.973 0.979 index 100 98.07 106.04 104.71 Average gain, kg - 0.917 0.973 0.979 index - 100 106.11 106.76

Amount of zearalenone in feces Time of sampling Group A B C D Start of trial 0 0 0 0 45.Day 0 0.96 0.48 0.32 90.Day 0 0.464 0.192 0.296 Average 0 0.475 0.224 0.196

EFFECT OF MINAZEL PLUS IN DAIRY COWS Srdan Nešic*, Goran Grubić**, Milan Adamović*** * Patent co. Belgrade ** Faculty pf Agriculture, University of Belgrade ***Insitute for Technology of Nuclear and Other Mineral Raw Materials, Belgrade

60 cows Holstein, divided into 5 groups, with 12 cows in each group. Duration of the experiment 120 days. Group A Feed without zearalenone contamination, without Minazel Plus. Group B Feed without zearalenone contamination, with 0.2% Minazel Plus. Group C Forage without zearalenone, concentrated feed with Zaralenone contamination, Without Minazel Plus. Group D - Forage without zearalenone, concentrated feed with Zaralenone contamination, with 0.2% Minazel Plus. Group E - Forage without zearalenone, concentrated feed with Zaralenone contamination, with 0.5% Minazel Plus.

Average daily milk yield, chemical parameters of milk and zearalenone residuals Parameter Group A B C D E Daily amount of zearalenone per cow, mg 0.00 0.00 10.20 9.60 9.32 Amount of added Minazel Plus in concetrate feed, % 0.0 0.2 0.0 0.2 0.5 Daily milk yield, kg 22.48 23.44 21.85 22.12 23.54 Index 100 104.27 97.2 98.4 104.72 Index - - 100 101.24 107.73 4% FCM, kg/dan 20.18 21.09 19.17 19.86 20.72 Index 100 104.51 94.99 98.41 102.67 Milk fat, % 3.32 3.33 3.18 3.32 3.2 Milk fat, kg 0.746 0.781 0.695 0.734 0.753 Proteins, % 3.09 3.08 3.01 3.11 2.97 Proteins, kg 0.695 0.722 0.658 0.688 0.699 Zearalenone concentration in milk, mg/kg 0.00 0.00 0.053 0.019 0.004 Zearalenone concentration in faeces, mg/kg 0.00 0.00 0.107 0.032 0.085

CASE STUDY: Effect of Minazel Plus on reduction of Aflatoxin M in milk

Aflatoxins are products, secondary metabolites, fungi of the genus Aspergillus (A. flavus, A. parasiticus Speare) Optimal temperature for Aflatoxin B1 production is 24-32 C (minimum is 13 C and 42 C maximum)

AFM1 (4-hydroxy aflatoxin B1) is metabolite of hydroxylated AFB1 by means of enzymes, primary of liver. 120 min AFB1 AFM1 15 min AFB1 4 hours AFM1 After accurеnсe, Aflatoxin M1 is excreted through urine and cow's milk.

Distribution AFM1 in dairy products made from contaminated milk: 40-60% in cheese 10% in cream less than 2% in butter

Biological effects: 1. Carcinogenicity 2. Embryotoxicity 3. Mutagenicity / genotoxicity 4. Immunomodulation 5. Teratogenicity

Aspergillus spp. is predominantly warehouse mold that requires high temperature and lower humidity of the substrate for growth. In spring/summer 2012. in Serbia and region, due to specific climate conditions there was an occurrence of Aspergillus spp. even in the period of vegetation of corn with consequential production of aflatoxins. There are assumptions that the damage of the corn during intensified presence of insects facilitated growth and development of molds.

In Serbia, traditional feed for diary cows is consisted of great amount of corn (corn stairways 20-25 kg per cow on day, grain corn 3-4 kg per cow on day) All above mentioned had for consequence occurrence of aflatoxin M1 in milk and milk products from Serbia!!!

Concentration of aflatoxin in milk collection from 3 Serbian local farms 1st farm - Mackovic Josip 130 cows Forage (kg per cow per day) - Corn plant silage 25kg -Corn grain silage 4kg -Alfaalfa hay 1,5kg - Alfaalfa haylage 3kg Concentrated feed sunflower meal, barley, oats, wheat, premix

Concentration of aflatoxin in milk collection from 3 Serbian local farms 2nd farm - Kecenovic Janos 160 cows Forage (kg per cow per day) - Corn plant silage 20kg - Alfaalfa hay 5kg Molases 1kg Concentrated feed soybean meal, sunflower meal, corn, barley, wheat, premix.

Concentration of aflatoxin in milk collection from 3 Serbian local farms 3rd farm - Bencik Petar-Gornji Tavankut 80 cows Forage (kg per cow per day) - Corn plant silage 20kg - Alfaalfa hay 4kg - Straw 2kg - Concentrated feed 6kg

Concentration of aflatoxin in milk collection from 3 Serbian local farms First farm - 130 cows Second farm - 160 cows Third farm 80 cows 6th of March -Mz 0,0334 0,240 0,670 Minazel Plus dosage (g/cow/day) 30 g 100 g 50 g 3rd April + Mz 0,008 0,068 0,340 18 th April + Mz 0,007 0,065 0,017 Referent concentration of aflatoxin in milk is max 0,5 μg/kg (Serbian Ordinance -Official Gazette No. 28/11 and 20/13 and Law- Official Gazette No. 41/09)