The invention relates to a feed additive for farm animals, in particular cattle, intended for use in improvement of animal well-being, suitable in particular for prevention of selenium deficiency and related diseases, as well as improving efficiency of rearing young farm animals, especially calves.

Selenium is essential for farm animals, and due to its deficiency in the environment, development of an effective animal feed supplement that supplements this element, taking into account its narrow therapeutic window and good bioavailability, remains a challenge for scientists dealing with this subject.

Vegetables for ages have played an essential role in feeding cattle; however, the amount of selenium provided in the diet based on plants grown in Central Europe is insufficient. In Poland and neighboring countries, this element is missing in the soil. In addition, due to the lack of selenium in the soil, plants and fodder obtained from them are not a good source of selenium and exacerbate the element deficiency problem. For this reason, the deficiency occurs in the organisms of herbivorous farm animals, and since selenium is absent in the meat of these animals, there are significant deficiencies of selenium in the human body (Molenda, Muszyhska, 2017).

The first country to use selenium supplementation was New Zealand (1967). In the same year, Finnish veterinarians used selenium to treat muscle diseases in pets, and in 1960 the use of selenium was approved as an animal feed additive (Koller and Exon, 1986). Selenium deficiency was diagnosed in 50% of tested cattle from 54% of farms in the Czech Republic, including dairy cows and calves (Pavlata et al., 2002; Pavlata etal., 2005). In the 1960s, white muscle disease caused by severe selenium deficiency was diagnosed in young ruminants for the first time (Muth etal., 1963).

Further results of animal studies have shown that selenium supplementation in cows with selenium has a positive effect on their fertility and growth, as well as strengthening of the immune system (Mehdi and Dufrasne, 2016). Selenium deficiency is especially critical for mothers during late pregnancy, when the fetus is provided with selenium even when the mothers are deficient in this element (Flefnawy etal., 2014). Adequate selenium supply for cows during late pregnancy and in newborn calves can be obtained through programmed dietary supplementation (Bayril etal., 2015). Ruminant selenium supplements are categorized into inorganic salts (such as sodium selenate and sodium selenite, currently being phased out due to scientifically proven toxic effects) and organic compounds (such as selenomethionine and selenocysteine) that are found in selenium-enriched yeast (selenium-yeast). The recommended amount of selenium in feed rations for cattle is 0.3 mg/kg dry matter (DM) (Nutrient Requirements of Dairy Cattle, 2001 ). The results of a study by Marounek et al. (2006) showed that selenium supplementation does not statistically increase the level of selenium in muscles, liver or kidneys, which is, respectively: 0.35 vs 0.69; 1.57 vs 1.87; 2.51 vs 2.65 mg/kg for the control group and with supplementation of the element.

Selenium in the rumen of cattle is metabolized by microorganisms that can integrate the element with their own proteins, and more precisely with amino acids, or reduce selenium compounds to non-digestible elemental selenium, which is excreted in the faeces (Galbraith etal., 2016).

Inorganic selenium has a lower bacterial uptake rate in the abdominal cavity than organic selenium sources (Panev et al., 2013).

Edible mushrooms, due to the ability to accumulate elements and numerous therapeutic effects (e.g. immunostimulating, anti-inflammatory, prebiotic), are an ideal model for this purpose. Particularly interesting in this respect is a species of edible mushroom: Lentinula edodes (Berk.) Pegler (shiitake) (Basidiomycota) - sawtooth oak mushroom. Polysaccharides and biologically active compounds contained in its fruiting bodies strengthen the immune system, eliminate the side effects of chemotherapy and radiotherapy, have anti-cancer, antioxidant, anti-atherosclerotic, as well as antiviral and antibacterial properties (Muszyhska etal., 2018).

The patent PL 225 547 B1 discloses a method of obtaining a preparation from the fungus edible mushroom Lentinula edodes, characterized by mycelium obtainted using a submerged culturing method in a liquid medium enriched with selenium at a concentration of 5 to 100 pg/mL. Selenium is introduced into the medium in the form of: sodium or potassium selenate(IV), selenic acid(IV), selentriglycerides or mixtures thereof. In a process known in the art, L. edodes are preferably grown on a culture medium containing 3 to 7% carbohydrate, 0.5 to 2% nitrogen source, 0.05 to 0.1% potassium dihydrogen orthophosphate, selenium source at a concentration corresponding to the concentration of the selenium element from 5 to 100 pg/mL. The sterilized medium is inoculated with Lentinula edodes’ s mycelium, preferably from strain ATCC 48085, at 5 to 10% of the culture medium volume. The culture is carried out for 7-14 days. The culture is preferably carried out with the following parameters: agitation 50 to 150 rpm, aeration from 10 to 30 liters per minute per liter of medium, temperature 20 to 30°C.

The object of the invention is to provide a preparation suitable for use in cattle rearing which improves both the weight gain and the degree of feed absorption by the farmed animals, especially calves.

Unexpectedly, the technical problem thus defined has been solved in the present invention.

The first subject of the invention is a feed additive for farm animals, characterized in that it contains L. edodes mycelium enriched in Se(IV), wherein the Se(IV) content in the mycelium is higher than 0.8 mg/100 g of mycelium DM.

Preferably, the Se(IV) content in daily dose of the feed supplement is about 0.005 mg Se(IV) per kg of animal body weight.

Preferably, the Se(IV) content in the mycelium is from 100 to 600 mg/100 g of mycelium DM.

Preferably, the additive of the invention is intended for use as a feed additive for cattle, especially calves.

Another subject of the invention is a method of improving the efficiency of rearing livestock, in particular cattle, characterized in that the feed additive of the invention as defined above is added to a feed provided to the animals, preferably to a young animal, especially a calf.

Another subject of the invention is the mycelium of L. edodes enriched in Se (IV) for use in the treatment or prevention of selenium deficiency in farm animals and diseases related to this deficiency, wherein the Se(IV) content in the mycelium is higher than 0.8 mg/100 g of mycelium DM.

Preferably, the Se(IV) content in the mycelium is from 100 to 600 mg/100 g of mycelium DM.

Preferably, the L. edodes mycelium enriched in Se(IV) for use in the present invention is intended to be used at a daily dose of about 0.005 mg Se (IV) per kg of animal body weight.

Preferably, the farm animal is cattle, especially a calf.

Preferably, the cattle disease related to selenium deficiency is: white muscle disease in young ruminants, impaired fertility, or immune disorders. The disclosed feed additive containing mycelium of L. edodes enriched with selenium unexpectedly improves both the weight gain and the degree of feed absorption by the farmed animals. In a study that led to the invention, the average daily body weight gain of calves was several times higher in the group fed with the feed supplement of the invention than in the control group. Moreover, the feed conversion rate of the calves in the experimental group was significantly lower (5.07) than in the control group (48.13).

Example 1. Obtaining L. edodes mycelium enriched in Se(IV)

Commercially-available strain Lentinula edodes ATCC 48085 was used in the exemplary cultures. L. edodes mycelium enriched in Se(IV) can be obtained by various methods, for example by a method described in patent PL 225 547 B1 or by a method modified by the inventors. a) the culture method known from PL 225 547 B1

In this method, inoculum cultures were grown as an agitated culture in 500-mL flasks containing 150 mL of medium (5% w/v glucose, 1% yeast extract, 1% casein hydrolyzate, 0.1% KH2PO4, pH 6.5) at 26°C in a rotary shaker (New Brunswick Scientific) at 110 rpm for 7 days. Then, the obtained L. edodes inoculum was used to inoculate medium in the bioreactors. The culture was carried out in a BioTec bioreactor with a total capacity of 10 L, working capacity of 8 L. The fermentor was equipped with a paddle stirrer, aeration through a bubbler, heating system with a heating mantle. The following medium was used: beet molasses (5%), thick stillage (5%), corn steep extract (0.15%), KH2PO4 (0.3%). Selenium was introduced into the medium in the form of sodium selenate(IV) in an amount sufficient to achieve the target concentration of selenium in the culture medium of 50 ppm (pg/mL). Culture conditions: temperature 28°C, agitation 200 rpm, aeration 200 L/h. Appropriate components of the medium were weighed on laboratory scales, then dissolved in distilled water, poured into a bioreactor and sterilized. Selenium in the form of sodium selenate(IV) solution was prepared in a separate Erlenmayer flask by weighing out enough selenate to obtain a selenium concentration in the culture medium equal to 50 ppm and sterilized. The flask was closed with a cotton wool stopper and secured with aluminum foil. The fermentors with the medium and the selenate solution were sterilized for 20 min at 120°C. Once the sterilization was completed and the bioreactor cooled down, a sodium selenate solution was introduced into it under sterile conditions, and then the inoculum was inoculated in the amount of 10% v/v.

Once the culture was completed, L. edodes biomass was filtrated from the post cultivation medium. b) the modified method

L. edodes biomass was cultured in a liquid medium according to Oddoux enriched with organic selenium compounds - Selol 5% (50 mg Se (IV) per ml_ of preparation) in the amount of 1 mL/L of the medium.

In order to obtain an effective and significant increase in the L. edodes biomass, mycelium from cultures grown in Erlenmeyer flasks was transferred into a biofermenter with a mycelium mixing system, in which the volume of the medium was 9 L, and its mixing is provided by the inflow of sterile air and carbon dioxide removal. The cultures were grown at 25±2°C and with a natural photoperiod. After 10 days of cultivation in the biofermenter, the mycelium was separated from the medium.

The biomass obtained by the methods described above was frozen and subjected to lyophilization (Freezone 4.5 freeze dryer, Labconco; temperature: -40°C, property of the Department of Pharmaceutical Botany, Jagiellonian University Medical College).

The standardization of the obtained mycelium was carried out by determining the selenium content by the flame atomic absorption spectrometry (F-AAS) method, and selected organic compounds (lovastatin, a-linolenic acid, phenolic acids) by RP-FIPLC and GC methods.

According to the obtained results, improved growth of L. edodes biomass was achieved in case of aerated liquid cultures on a modified Oddoux medium and on the same medium with the addition of selenitetriglycerides (Selol 5%) at the temperature of 25±2°C with a natural photoperiod and during the 10-day growth cycle. Aeration and carbon dioxide removal have been found to be effective in optimizing the culture conditions. This resulted in the maximum mycelium growth which was obtained within 10 days of cultivation as compared to control cultures without aeration (which took about 21 days to grow). The yield of biomass grown on the modified Oddoux medium was in avarage 10.6 g of DM/L, but with the addition of selenetetriglyceride the yield was about 15.1 g DM/L.

Selenium content in the L. edodes mycelium grown on a medium with the addition of selenitriglycerides was evaluated. It was found that the selenium content in commercially-available mycelium grown without addition of selenium was 0.01 mg/100 g DM. A slightly higher selenium content was observed in the mycelium from in vitro culture without the addition of selenium, which was 0.79/100 g of DM (Table 1 ). Selenium was not detected in the medium after L. edodes cultivation. L. edodes cultivated in a medium supplemented with selenotriglycerides at concentrations of 25 mg/L and 50 mg/L showed a significant accumulation of selenium (from 192.6 to 678.6 mg/100 g DM) (Table 1 ). Higher selenium concentration in the medium increased the amount of selenium in L. edodes mycelium.

Table 1. Selenium content in the mycelium of Lentinula edodes cultivated with or without the addition of selenotriglycerides (Selol 5%)

L. edodes [mg/100 g DM ± standard deviation (SD)]

Fruit bodies 0.01 ±0.00 ^a

Mycelium 0.79±0.63 ^{a b}

Medium

Mycelium + Se (IV) 25 mg/L medium 192.65±5.12 ^{a b} Mycelium + Se (IV) 50 mg/L medium 532.31 ±35.53 ^{a b d e} Medium + Se (IV) 25 mg/L medium 95.68±2.18 ^{a b d e f} Medium + Se (IV) 50 mg/L medium 183.97±8.70 ^{a b d e f}

Mean ± standard deviation; n = 6, — not detected; Tukey's test was used to reveal differences between paired groups of items in rows; the same letters (a, b, d, e, f) denote differences that are statistically significant (P<0.05), (GraphPad InStat).

Example 2. Effect of the addition of Se (IV) enriched L. edodes mycelium to feed on the course of rearing cattle.

The group of experimental animals and the diet used

The animals were treated in accordance with the European Union's guidelines on the care of animals (European Union, Directive 2010/63 EU, 2010). The experiment design was previously approved by the Local Ethics Committee in Lublin (decision no. 68/2018). Six clinically healthy Holstein-Friesian calves, 4-8 weeks old, were used in the study. The experiment was carried out in the animal facility of the National Veterinary Institute - National Research Institute in Putawy. The animals were randomized into two equal groups: experimental (E) and control (C) and housed in two pens with free access to water. The calves were given 312.5 g of milk replacer suspended in 2.5 L of warm water twice a day from week 0 to week 3 of the experiment and 375 g of milk replacer suspended in 3 L of warm water twice a day and 500 g of compound feed for calves daily from week 4 to week 7 of the experiment. The calves of both groups were fed hay and water ad libitum. The feed composition is shown in Table 2.

Table 2. Feed composition.

Component Value

Milk replacer

Crude protein (%) 20.0

Crude fats and oils (%) 8.0

Ash (%) 6.0

Crude fiber (%) 1.3

Calcium (%) 0.7

Phosphorus (%) 0.45

Sodium (%) 0.1

Lysine (%) 1 .4

Vitamin A (lU/kg) 10,000

Vitamin D ₃ (lU/kg) 2000

Vitamin E (mg/kg) 80

Vitamin K ₃ (mg/kg) 1 .0

Vitamin Bi (mg/kg) 4.3

Vitamin B2 (mg/kg) 4.3

Vitamin Be (mg/kg) 4.3

Vitamin C (mg/kg) 100

Niacinamide (mg/kg) 6.6

Calcium D-pantothenate (mg/kg) 8.6

Folic acid (mg/kg) 0.35

Vitamin B12 (mg/kg) 0.05

Biotin (mg/kg) 0.07

Choline chloride (mg/kg) 300

Manganese (mg/kg) 64 Zinc (mg/kg) 56

Iron (mg/kg) 80

Copper (mg/kg) 8

Iodine (mg/kg) 0.96 Selenium (mg/kg) 0.2 Enterococcus faecium 1.2 c 10 ⁹ CFUs

Mixed calf feed

Crude protein (%) 18.5

Crude fats and oils (%) 3.3

Crude fiber max (%) 6.5

Crude ash max (%) 9.0

Phosphorus (%) 0.8

Calcium (%) 1 .3

Sodium (%) 0.23

Magnesium (%) 0.25

Vitamin A (lU/kg) 25,000

Vitamin D ₃ (lU/kg) 5000

Vitamin E (mg/kg) 25.0

In addition, calves from the experimental group were given the lyophilized L. edodes mycelium enriched in Se (IV) as a feed additive, which was added to the morning portion of the milk replacer at a dose of 0.005 mg Se (IV)/kg of calf body weight, i.e. 0.086 g of selenium-enriched mycelium lyophilisate/kg calf bw daily for 7 weeks. Body weight of the calves was recorded prior to administration of the feed additives (experiment week 0) and weekly from week 1 to week 7 of the experiment.

The following parameters were calculated:

ADG = end of period body weight - start of period body weight c duration of period ^-1 ,

ADFI = amount of feed consumed in grams per animal per period c duration of period-

1

FCR = feed consumption over period c mean body weight gain over period ^-1 the above-mentioned parameters were calculated for the entire duration of the experiment, i.e. from week 0 to week 7.

The results of the performed experiment are presented in Table 3. Table 3. The effect of the addition of Se (IV) enriched L. edodes mycelium to the feed on the production parameters of calves.

Description Control group Experimental group (n=3) ± SD (n=3) ± SD

Average body weight of calves (kg):

The beginning of the experiment 61.019.85 65.83±9.75 1 ^st week of the experiment 60.67110.4 67.83±11 .3 2 ^nd week of the experiment 61.0111.27 68.17111.56 3 ^rd week of the experiment 60.83112.27 69.17114.51 4 ^th week of the experiment 62.33111.93 68.0112.32 5 ^th week of the experiment 61.33114.43 70.0114.53 6 ^th week of the experiment 61.33115.7 73.17113.79 7 ^th week of the experiment 62.0115.87 75.33115.01 ADG of calves (g):

Weeks 0-7 of the experiment 20.41 ±167.05 193.88±107.51 Calf ADFI (g):

Weeks 0-7 of the experiment 982.1410.0 982.1410.0

FCR of calves (gxg ¹ )

Weeks 0-7 of the experiment 48.13 5.07

Abbreviations: ADG, Average Daily Gain; ADFI, Average Daily Feed Intake; FCR, Feed Conversion Ratio; SD, Standard Deviation.

Results and conclusions

In this study, it was found that the feed consumption was 100%. The calves from the experimental group ate all portions of the tested selenium-enriched L. edodes mycelium feed additive. In the study, weight gain in calves fed with the addition of selenium-enriched L. edodes mycelium was faster compared to the control calves. The average daily gain of calf weight for the entire experimental period was higher in the experimental group than in the control group. Moreover, throughout the experiment, the feed conversion ratio in calves of the experimental group was significantly lower (5.07) than in the control group (48.13). Example 3. Medicinal and pro-health effects of Se (IV)-enriched L. edodes mycelium feed supplement

The group of experimental animals described in Example 2 was also tested in terms of selenium content in the blood and the effect of supplementation on the calf immune system.

The effect of supplementation with Se (IV)-enriched L. edodes mycelium on selenium concentration in calf serum

Selenium concentration in serum samples was analyzed by inductively coupled plasma mass spectrometry using Varian 820 MS, Bruker M 90 and Plasma Quant MS (Analytik Jena) and ICP-OES (Optical Emission Spectrometry) Varian Vista Pro, Thermo-Fisher 1-Cap Duo 7000.

The effect of supplementation with Se (IV)-enriched L. edodes mycelium on calf immune parameters

The total white blood cell counts with leukocyte differentiation: LYM, MON and GRA were tested in peripheral blood using an automated veterinary blood analyzer (Exigo, Boule Medical AB, Sweden).

The percentage of leukocyte subpopulations containing CD2 ⁺ (T-cell), CD4 ⁺ (T- helper), CD8 ⁺ (suppressor/cytotoxic) and WC4 ⁺ (B-cell) surface antigens were analyzed with a flow cytometer (Coulter Epics XL 4C, Beckman Coulter Company, USA). The expression of CD markers was determined using the following primary mouse anti-bovine monoclonal antibodies (mAbs): anti-bovine CD2-FITC, anti-bovine CD4-FITC and anti-bovine CD8-FITC. WC4 ⁺ expression was determined using: anti- bovine WC4 monoclonal antibody and rabbit polyclonal F(ab')2 anti-mouse IgG with FITC secondary polyclonal antibody. Monoclonal antibodies: anti-bovine CD45-FITC and cross-reacting anti-human CD14RPE-Cy5 (Bio-Rad Company, USA) were used to standardize the analysis. Whole blood samples (100 pL) were incubated at room temperature (18-25°C) with the respective monoclonal antibodies for 15 minutes. Erythrocytes were lysed with lysis solution for 20 minutes. For WC4 ⁺ analysis, samples were incubated for 30 minutes with the respective primary mAb, washed with PBS solution containing 5% inactivated fetal calf serum, and incubated for 15 minutes with the secondary antibody. After washing the samples with PBS, the mixture was resuspended in 500 mI_ of the same PBS. Data were obtained in the form of histograms using SYSTEM II 3.0 software.

Table 4. The effect of supplementation with Se (IV)-enriched L. edodes mycelium on selenium concentration in the calf blood serum.

Serum selenium Serum selenium SEM P concentration in the concentration in the control group experimental group

[MQ/L] [pg/L]

I 19.33 23.00 9.82 0.37

II 23.00 ^a 177.67 ^b 81.17 0.01

III 23.33 ^a 175.00 ^b 78.09 0.01

IV 27.00 ^a 236.00 ^b 106.43 0.00

V 23.67 ^a 177.67 ^b 79.39 0.01

VI 33.67 ^a 138.33 ^b 53.06 0.00

VII 33.00 ^a 178.67 ^b 73.24 0.00

Abbreviation: SEM, Standard Error of the Mean.

Note: n = 3; a, b values with different letters differ significantly (P<0.05).

Fig. 1 shows the results of total count of white blood cells (WBC) (3A) with leukocyte differentiation (lymphocytes (LYM), 3B; monocytes (MON), 3C; and granulocytes (GRA), 3D) in the peripheral blood of calves (± SD) (10 ⁹ cells/L).

Fig. 2 shows the results of analysis of leukocyte subpopulations percentage in the peripheral blood of calves (%). CD2 ⁺ (4A), CD4 ⁺ (4B), CD8 ⁺ (4C), WC4 ⁺ (4D) (± SD). E, experimental calves; C, control calves. The letter a indicates differences that are statistically significant (P<0.05).

Example 4: Shaping the immune response in calves with the Se (IV)-enriched Lentinula edodes mycelium. The initial period of rearing calves is characterized by numerous threats to their health and life. Despite the use of many zootechnical and veterinary treatments, losses are high and vary depending on the farm from 4 to 30%.

The main aim of the research was to evaluate effects of the preparation of the invention on the health status and production parameters of calves.

A feeding experiment was carried out on 18 calves, hybrids of breeds cb x Hf (50- 75% of Hf genes). As the animals were born, they were assigned to 2 groups (control and receiving the additive of the invention). Each group consisted of 6 calves. The preparation was administered from 10 ^th to 70 ^th day of life in a form dissolved in water, milk or milk replacer. At the beginning of the experiment (age of 10 days) and at the age of 35, 70 and 120 days, blood was taken from the external jugular vein in order to determine the haematological parameters indicators (hematocrit, hemoglobin concentration, red and white blood cell count, trombocytes, RDW, MPV, MCV, MCVC, MHC, ), blood biochemistry and selenium concentration. The following data was collected during the experiment:

- zootechnical indicators (body weight, body weight gain, feed intake, milk and feed conversion ratio, )

On the 70 ^th day of life, 3 individuals from each group were euthanized to collect intestinal and rumensamples. Liver and muscles were also collected to determine the selenium level.

The mean body weights of calves on the day of birth did not differ between the groups and amounted to: control group - 45.2; formulation of the invention - 46.2 (Fig. 3). Later in life, until day 70, no statistical differences were noted. In the individual rearing periods, no differences were also observed in the average daily gain (Fig. 4). In the rearing period of 1 -53 days of age there were no differences in the average daily feed intake and feed conversion ratio in any of the groups. In the group receiving the additive of the invention, selenium concentration in blood serum (Fig. 5), muscles and liver increased significantly (P O.0001 ) (Fig. 6). In the control group on the 35 ^th day of life it was 44.33 (pg/L), while in the group receiving the additiveof the invention - 132.00 WL).

The applied preparation did not increase the body weight of the calves, however, in the last two weeks there was a tendency for higherbody weight gain in the group of calves receiving the additiveof the invention. Additionally, the additiveof the invention significantly increased the level of selenium in blood serum, muscles and liver.

Literature:

Galbraith M.L., Vorachek W.R., Estill C.T., Whanger P.D., Bobe G., Davis T.Z., Hall J.A. (2016). Rumen Microorganisms Decrease Bioavailability of Inorganic Selenium Supplements. Biol Trace Elem Res., 171 (2): 338-343.

Hefnawy A.E., Youssef S., Aguilera P. V., Rodriguez C. V., Perez J.L.T. (2014). The relationship between selenium and T3 in selenium supplemented and nonsupplemented ewes and their Lambs. Veterinary Medicine International Volume, Article ID 105236.

KollerL.D., and Exon J.H. (1986)The two faces of selenium-deficiency and toxicity-are similar in animals and man. Can J Vet Res., 50:2971986-306. Moienda A., Muszyhska B. (2017). Selenium - meaning in the prevention and therapy of cancer diseases. Medicina Internacia Revuo, 28 (109): 272-279.

Mehdi Y., Dufrasne I. (2016). Selenium in Cattle: A Review. Molecules., 21: 545. Moienda A., Muszyhska B. (2017). Selenium - meaning in the prevention and therapy of cancer diseases. Medicina Internacia Revuo, 28 (109): 272-279.

Muth R., Muth M.F. (1963). White muscle disease, a selenium-responsive myopathy. J. Am. Vet. Med. Assoc . ₄ 1 (142): 272-277.

Muszyhska B, Grzywacz-Kisielewska A, Kala K, Gdula-Argasihska J.(2018). Anti inflammatory properties of edible mushrooms: a review. Food Chemistry,; 243(3): 373- 381.

Muszyhska B., Kala K., Wiodarczyk A., Krakowska A., Ostachowicz B., Gdula- Argasihska J., Suchocki P. (2019). Lentinula edodes as a source of bioelements released into artificial digestive juices and potential anti-inflammatory material. Biological Trace Elements Research, DOI: 10. 1007/s 12011 -019-01782-8.

Nutrient Requirements of Dairy Cattle. Seventh Revised Edition. Subbcommittee on Dairy Cattle Nutrition.2001. National Academy Press. 142 pp.

PanevA., Hauptmanova K., Pavlata L., PechovaA., Filfpe, J., Dvorak R. (2013). Effect of supplementation of various selenium forms and doses on selected parameters of ruminal fluid and blood in sheep. Czech J. Anim. Sci., 58 (1): 37-46.

Pavlata L., Illek J., Pechova A., Matejicek M. (2002). Selenium Status of Cattle in the Czech Republic. Acta Vet., 71: 3-8. Pavlata L, Misurova L, Pechova A., Husakova T., Dvorak R. (2012). Direct and indirect assessment of selenium status in sheep - a comparison. Veteriniarni Medicina, 57 (5): 219-223.

Pavlata L, Slosarkova S., Fleischer P., Pechova A. (2005). Effects of increased iodine supply on the selenium status of kids. Vet Med -Czech, 50: 186-194.

Stewart I N.C., Bobe, G., Vorachek W.R., Pirelli G.J., Mosher W.D., Nichols T., Van Saun R.J., Forsberg N.E., Hall J.A. (2014). Organic and inorganic selenium: IV. Passive transfer of immunoglobulin from ewe to lamb. Journal of Animal Science, 91: 1791-1800.