Assessment of the level of contamination of feed with micromycetes and mycotoxins in the cattle industry of Ukraine in recent years

Goal. To study the degree of contamination, the species of micromycetes and the content of mycotoxins (aflatoxin B1, sterigmatocystin, zearalenone, patulin) in feed for cattle for 2018 and the first half of 2019 and to carry out a retrospective analysis of contamination of feed with micromycetes over the last 5 years. Methods. Mycological and micotoxicological study was conducted in accordance with generally accepted and developed in the laboratory of Toxicological monitoring methods of determination. Results. Level of contamination with micromycetes in 87 samples of feed for cattle in 2018–2019 exceeded the maximum permissible level (MPL) in 73.6% of samples. The major ones were: feed — 23,4%, mono feeds and silage — 18,7% each, straw — 10,9%, and hay — 7.8%. The main pollutants of the feed were micromycetes of the family Mucoraceaе — 25.6%, and of species: Aspergillus Mich. — 24,7, Penicillium Linc. 9.5, Fusarium Linc. — 3.3, other — 36.9%. 35 samples of feed were examined on the presence of mycotoxins. Only one sample of feed has zearalenone (0,88 mg/kg, which is 76% higher than MPL). Aflatoxin B1, sterigmatocystin, patulin and zearalenone were outside the method definition, and did not exceed MPL. Conclusions. The level of contamination with micromycetes of feed for cattle in 2018 and the first half of 2019 remained high, including poor quality (higher than MPL), and was 73.3% and 73.8% respectively. The main pollutants of the feed were moldy saprophytes of family Mucoraceaе — 25.6%, and species: Aspergillus Mich. — 24,7%, Penicillium Linc. — 9.5%, Fusarium Linc. — 3.3%, other — 36.9%. The presence of aflatoxin B1, sterigmatocystin, patulin and zearalenone in quantities higher than MPL in 34 samples of feed was fixed. Only in the sample of feed zearalenone was determined — 0,88 mg/kg of feed, which is 76% higher than MPL.

One of the many negative environmental factors that affect the safety of feed materials and feeds are micromycetes and their secondary metabolites -mycotoxins. Their special danger lies in the latent form of impact on animal health. The presence of microscopic fungi in feed leads to a decrease in their consumption due to the deterioration of organoleptic qualities and causes a decrease in the absorption of nutrients and metabolic disorders in the body.
As a result, the costs of treatment of animals, as well as due to shortages and reduced quality of livestock products cause great economic losses [1][2][3][4][5][6][7]. According to the Food and Agriculture Organization of the United Nations (FAO), due to the high prevalence of microscopic fungi in almost all habitats and their high adaptive properties, mold saprophytes affect 25 -40% of feeds each year [2]. Therefore, we believe that the systematic control of micromycetes and their secondary metabolites in feed at all stages of their manufacture and during storage is one of the main measures to prevent their negative impact on animal health and ensure the production of safe and quality products [4,6,7].
The purpose of the work is to investigate the degree of contamination, species affiliation of micromycetes and the content of mycotoxins (aflatoxin B1, sterigmatocystin, zearalenone, patulin) in the feeds used in the livestock industry of Ukraine in 2018 -the first half of 2019 and to compare the results with previous years. Materials and methods. The study was conducted on the basis of the laboratory for toxicological monitoring of the NSC "IECVM" in the period 2018 -I half of 2019. We analyzed 87 samples of feed for dairy cattle from different regions of Ukraine: Kharkiv, Sumy, Odesa, Donetsk, Kyiv, Poltava, Cherkasy, Kirovograd, Ivano-Frankivsk and Ternopil regions.
Mycological studies of the samples were performed according to generally accepted methods, in particular: the degree of contamination of feed with microscopic fungi was determined by the number of colony-forming units (CFU) per 1 g of feed under primary seeding in nutrient medium (agar wort and Chapek) [7,8]; species affiliation of isolates of microscopic fungi was determined by comparing cultural and morphological characteristics of the isolated mycobiota (features of crop growth on different nutrient media, their size, shape, width, structure of edges and center of colonies, growth intensity, surface characteristics, colony color, their reverse, mycelium, etc. .) with the descriptions given in the determinants of micromycetes, and with museum strains of test cultures (property of NSC "Institute of Experimental and Clinical Veterinary Medicine") [9][10][11][12].
Mycotoxicological studies were performed following the guidelines "Simultaneous determination of mycotoxins (aflatoxin B1, zearalenone, patulin, sterigmatocystin) in feed and cereals by thin-layer or liquid chromatography", which were developed in the laboratory and approved by the State Committee of Veterinary Medicine of Ukraine (protocol №1, December 24, 2009).
Research results. During mycological monitoring in 2018-2019, 87 samples of feed and feed materials for cattle were studied, in particular: roughage and succulent feed (silage, haylage, hay, straw, mono-feed, beet pulp, beer pellets) -56 samples, compound feed -17, cereals (grain mixture, corn, barley, wheat) -8 and other types of feed -6 ( Fig. 1). From the data shown in Fig. 1, it is seen that mostly coarse and succulent feeds, as well as compound feeds were subjected to mycological analysis.

Fig. 1. Types of feed for cattle, subjected to mycological research in 2018 and the first half of 2019: 1 -roughage and succulent feed; 2 -compound feeds; 3 -grain feed; 4 -other types of feed
The degree of contamination with microscopic fungi of feed for cattle was determined. It was found that in 2018 and the first half of 2019, only 26.7 and 26.2% of feeds had a permissible degree of contamination (less than 5 • 10 4 spores per 1 g of feed), and -73.3 and 73.8% of feeds exceeded the maximum allowable level (MAL),respectively [8]. Compound feeds -23.4% (15 samples), mono-feeds and silage 18.7% (12 samples each), roughage: straw -10.9% (7 samples), hay -7.8% (5 samples) were the most contaminated with micromycetes.
Among the studied, the smallest number of feed samples exceeding the MAL was found among cereals -3.1% (2 samples). To determine the composition of mycobiota of feed and feed raw materials in 2018 and the first half of 2019, 360 isolates of mold and yeast-like fungi were isolated and identified (in 2018 -163, in 2019 -197 isolates) (Fig. 2  Representatives of other genera -133 isolates (36.9%). It should be noted that the number of isolates of the genus Aspergillus Mich. was higher in 2018. This is due to heat and dryness in early summer of that year (temperatures in June reached 30-35°C), and such climatic conditions are optimal for the growth of many species of micromycetes of the genus Aspergillus Mich.
The number of isolates of the Mucoraceae family was also identified in a clear dependence on weather conditions (temperature and humidity). In particular, due to the cool, rainy weather in July 2019, the number of isolates increased 1.5 times compared to the previous year (in 2018 -37, in 2019 -55 isolates). The taxonomic affiliation of isolates of potentially toxin-forming microscopic fungi was determined (table).

Species taxonomy of toxin-forming micromycetes isolated from feed samples for cattle in 2018 and the first half of 2019
Type of toxin-forming micromycete, toxic metabolite  However, in the feed sample obtained from the dairy farm of Kharkiv region, the presence of zearalenone was determined at a concentration of 0.88 mg/kg, that is 76% higher than the MAL of this toxin [8]. Thus, it was established that during the mycological monitoring of 87 samples of feed and feed materials used for feeding cattle in 2018 and the first half of 2019, 73.6% of feeds were of poor quality, that is more than two thirds of the total.
According to our data obtained in previous years, this trend has been continuing for Also, due to the high content of zearalenone in the feed sample, special attention should be paid to the members of the family Fusarium Linc. Although a relatively small number of them was isolated -12 isolates (3.3%), but they led to toxin formation. Aflatoxin B1, sterigmatocystin, patulin and zearalenone in other test samples were outside the detection of the method and below the MAL. This trend persisted during 2014-2017, the degree of contamination with microscopic fungi of substandard feed was 51-88%. Prospects for further research are the systematic control of contaminants of biotic origin (micromycetes, mycotoxins) of feed materials and feeds used for feeding cattle, to prevent their negative impact on animal health and productivity and reduce economic losses in livestock.