1. Antinutritional factors, deterioration of feeds, mycotoxins
Dr. Éva Cenkvári Ph.D.
Szent István University
Faculty of Veterinary Sciences Institute of Animal Breeding, Animal Nutrition and
Laboratory Animal Sciences

2. Antinutitional factors and antimetabolites
Definition
Original function
Chemical groups
Functional groups!
Regulation

3. Antinutitional factors and antimetabolites
Antinutritional factors
Inhibit the forcasted production based upon the chemical composition
Decreased: - weight gain,
- feed utilization,
- milk and egg production.
But: they do not cause any symptom or toxicity
Classical test: Ostborn and Mendel, 1917
feeding raw soybean by rats
reduced/delayed growth/development
Significance: protein content is positively correlated with the level of antinutritional factors (excluded sunflower seed)

4. Classification of antinutritional factors
Original (in plant) functions:
a. Plant nutrient (storage) (e.g. carbohydrates are stored for own nourishment of plants (e.g. phytohemaglutinins and lectins);
b. Inhibits the decomposition of nutrients and inhibit the early germination of seeds;
c. Protection against the plant insects (trypsin inhibitors against pea-beetle, saponin against Trichoderma viridae).
In seeds of Leguminosae: protease inhibitors;
In legumes and potatoes: saponin, solanin, phytooestrogens,
In Brassicae: goitrogen substances (disturbing the functions of thyroid gland).

5. Classification of antinutritional factors (cont.)
2. Based on their chemical structure:
a. Protein type: protease inhibitors, biogenic amines, goitrogenic peptides, phytohemagglutinins, enzymes),
b. Alcaloids: lupinin and spartein (Vicia faba), vicin, convicin (Phaseolus vulgaris, bean),
c. Steroids: saponin (alfalfa), solanins (potatoe), phytooestrogens,
d. Sacharides: pentosans (barley), raffios, stachios (bean, peas, lentils, cow peas, chick peas),
e. Fitates: inosytol-hexa-phosphoric acid (cereals).

6. Classification of antinutritional factors (cont.)
2. Based upon chemical structure:
f. Antagonists of glutamic acid: N-oxalyl-diamino-propionate
(OADP), N-glutamil-amino-propionitril (GAPN),
g. Silicates: rice, rice bran, acid grasses,
h. Unsaturated fatty acid ethers: erucic acid (rape seed).
Binaftol compounds: gossypol (in pigment of cotton seed meal),
j. Compounds of phenolic structure: tannin (sorghum, chestnut).

7. Classification of antinutritional factors (cont.)
3. According to their functions in animal organisms
Digestion and utilization of proteins is decreased:
protease inhibitors in soybean in birds and in mammals (trypsin inhibitor!);
How to decrease: heat treatment
1.1. The most important proteinase inhibitors:
- Kunitz-factor (F1, F2, F3) → heat-labile;
- Bowmann-Birk-factor (A1, B1, B2, S) → heat-stabile.
Effects: inhibition of the activity of trypsin, chymotrypsin, binding of them → excretion
pancreozymin in blood, pancreas hypertrophy develops,
sulphur storages are depleted (supplementation!)
Measurement of activity: indirectly or by the measurement urease activity

8. Classification of antinutritional factors (cont.)
1.2. Lectins: (phytohemagglutinins, PHA)
Structure: contain proteins and sugars, immun-recognizers;
Functions: adhere to the mucous membran of intestine, damage the absorption (soybean, field bean).
Detection: based on their ability to agglutinate red blood cells by linking to the glycoprotein of the cell wall
→in vitro coagulation of red cells in blood;

9. Classification of antinutritional factors (cont.)
1.3. Saponins: in alfalfa against Trichoderma viridae
(test of saponin content)
Effects: based upon their physical structure,
decrease of tension on surface,
haemolysis of red cells (good for detection!),
absorb in the intestinal tract hardly,
colesterines bind them (no haemolysis in vivo);
decrease the absorption, diarrhea, enteritis.

10. 1.4. Solanins: bitter poisonous alkaloids
in vegetative parts and in potatoe peel
To decrease: soaking, heat treatment  dissolving
1.5. Phenol-containing compounds: tannin (sorghum, amaranth grain, sunflower leaves)
Effects: denaturation of proteins (lysine, arginine, free amino acids!), decrease of protein digestion
1.6. Silica: rice bran, sedges, cane.
Effects: decrease the digestibility of organic matters (proteins!) by ca. 10%

11. Classification of antinutritional factors (cont.)
2. Disturbances of mineral metabolism:
2.1. Phytates: phosphorus supplier in cereal seeds,
monogastrics: P-availability 
Sensitivity: rabbit, swine (phytase activity)  poultry (daily P-requirement: in available P)
Effects: binding of minerals secondary Ca-, Zn- and Mn-deficiency; minerals in chelate-binding,
Solution: supplementation by phytase enzyme
2.2. Oxalates: spinach, garden sorrel, Amaranthus retroflexus.
Effects: Ca is not absorbed, Ca-oxalate crystals cause bleeding in the kidney damage or precipitation as uroliths

12. Classification of antinutritional factors (cont.)
2. Disturbances of mineral metabolism:
2.3. Goitrogenic peptides: glucosynolates, sinigrin (rapeseed, Brassica species, family Cruciferae)
Impacts: inhibition of I-intake, hyperthyroidism, (goitre)
To decrease: by heat treatment is difficult,
development of superior plant varieties
(double zero rape seed = canola rape seed  no erucic acid, goitrogen content; triple zero rape seed: no tannin)
Struminogenic (thyreostatic) compounds: field nut, raw soybean.
2.4. Cianogenic glycosides: linamarin, durrin (in linseed, sorghum plant)
Impacts: they bind Fe of citochrom oxidase, oxigen-deprivation respiratory, ataxy.

13. Classification of antinutritional factors (cont.)
2. Disturbances of mineral metabolism (cont.):
2.4. Phytooestrogens: isoflavonoids
(genistein, cumoestrol, daidzein, biocanin A, phormomonetin,);
Oestrol-type compunds: leguminose plants (soybean,
red clover, alfalfa, also green grasses)
Effects: induce sexual activities (spring pastures),
In high levels: reproductive failures,
oestrogen syndrome → oedema of vulva, cystal distrophy of ovaries, abortion

14. Occurance of protease inhibitors in plants Plants Occurance
Inhibited enzyme
Peanut
seed
trypsin, plasmin
Chickpea
trypsin
Soybean
trypsin, chymotrypsin, plasmin
Rice
Beans (Phaseolus)
Peas (Pisum)
non-specific
Potatoe
bulbs, leaves
papain (plant protease), trypsin, chymotrypsin
Wheat
Maize

15. Trypsin inhibitors
Reduction of activity of trypsin, pancreatin
Hypertrophy/hyperplasia
Lectins
Gut wall damage, immune response,
metabolic toxicity
Amylase inhibitors
Interference with starch digestion
Antigenic proteins
Gut wall damage, immune response
Polyphenols
Decrease of protein digestion
Tannins
Formation of complexes with proteins and carbohydrates, digestibility
Glycosides
Disturbance of thyroid functions

16. Vicine, convicine
Haemolytic anaemia, interference with fertility and hatchability of eggs
Saponins
Haemolysis, affect intestinal permeability
Flatulence factors
Gastro-intestinal discomfort
Glucosides
Glucosinolates
Impaired iodine utilization, affect intestinal permeability
Alkaloids
Liver damage, bitter taste
Quinolizidine
(lupin alkaloids)
Neural disturbances, affect liver function, bitter taste

17. Scopolamine and hyosciamine
(from datura)
Palatability, toxicity!
Other ANFs
Phytate
Forms complexes with minerals and protein, depresses absorption of minerals (availability of P and Zn)
Gossypol
Anaemia due to Fe-complexation, reduces egg weight, toxicosis!
Sinapins
Fishy egg (smell and taste)

18. Classification of antinutritional factors (cont.)
3. Natural antivitamines/antimetabolites
Functioning: by destruction, binding or competitive antagonism
3.1. Anti-vitamin A: lipoxygenase (rancidity in grain, raw soybean, bean)
Effects: spoiling of β-carotine, vitamin A
3.2. Anti-vitamin D: raw soybean
Effects: rachitis in growing chicks

19. Classification of antinutritional factors (cont.)
3.3. Anti- vitamin E: gossypol (field bean, cotton seed meal)
Effects: in cows low tocopherol levels, decreases level of β-carotine and fertility in cattle
3.4. Anti-vitamin K: dicumarol in sweet clover
(Melilotus officinalis), (rat poisons)
Effect: interferes with viamine K in blood clutting →
soft swellings beneath the skin
3.5. Anti-niacin impact: acetyl-pyridin, indol-acetate
(in maize and millet seeds; maize is poor of tryptophan, which is precursor of niacin!)

20. 3. Natural antivitamins/antimetabolites
3.6. Anti-vitamin B1: similar to thiamine (antimetabolite); in horsetail (Equisetum)
Effects: it decreases vitamine B1 (neural degeneration: opisthotonus)
3.7. Anti-vitamin B6: linatinen (dipeptid), in linseed
Effects: depression of growth in chicks
3.8. Anti-vitamin B12: in raw soybean
Effect: anaemia especially in cat
3.9. Antibiotin-effect: avidin (in raw egg white)

21. Classification of antinutritional factors (cont.)
4. Effect of photosensibilation
4.1. Primary photosensitization
- Fagopyrum esculatum: fagopyrism;
Hypericum species: hypericosis.
Effects: sunshine activates the antinutrient in the skin.
4.2. Secondary photosensitization
- Swedish clover (Trifolium hybridum: trifoliosis), alfalfa,
Effects: skin inflammation (liver does not eliminate the chlorophyl metabolite, the phylloeritrine content),
UV-radiation → dermatitis, superficial necrosis

22. Classification of antinutritional factors (cont.)
4. Effect of photosensibilation (cont.)
Clinical findings for differential diagnosis:
head of sheep is swelled, thick ear →„big head” syndrome caused by Clostridium chauvayi bacteria
b. cattle- in aphthae epizooticae– salivation, (blisters) on the mucosa of mouth, lesions on muzzle,
mouth and feet disease (MFD) of cattle.

23. Classification of antinutritional factors (cont.)
5. Other effects
5.1. Lipoxygenase: self-rancidation of beans („beany taste”)
5.2. Polifenyloxidase: browning - apple, bananas
5.3. Raffinose, stachiose: legume seeds (non-starch carbohydrates in the nitrogen-free extracts)
5.4. Toxic amino acids: in Leguminosae
Effects: lathyrism (deformations of bones, neurological symptoms)

24. Classification of antinutritional factors (cont.)
5.5. Eruic acid: rape seed - bad taste, cancerogenic.
5.6. Lupinine, sparteine: bitter taste of lupins, liver damage
5.7. Sparteine, gossypol: O2-transporting capacity of blood is decreased

25. Acceptable limits (feed law)
Cianogenes: linseed mg/kg
linseed solvent mg/kg
concentrates 20 mg/kg
Gossypol: cottonseed meal mg/kg in concentrates
Mammals: mg/kg
Birds: mg/kg
Calves: mg/kg
Beef: mg/kg
Glycosinolates (goitrogenic peptides): total 4000 mg/kg
(in the double zero rapeseed meal)

26. Biogenic amins: Physiological role: neurotransmitters
(e.g. serotonin=5-OH-tryptamine from Trp,
histamine from His)
Produced by the decarboxilization of amino acids (mono-, poli-);
Monoamins: tyramin, octopamin, -phenyl-etil-amin
Poliamins, benzol-containing compounds:
- putrescin, cadaverin, spermine, spermidine (cell growth),
indol-copounds: serotonine, tryptamine,
imydasol-compounds: hystamin.
Effects on animals: local irritations, neural symptoms
Origin: foods, feedstuffs, gut microbes

27. Biogenic amines (cont.)
Positive effects: in newborns - renewing of mucosa of gut membrane, enzyme production;
exogenous - endogenous (mutual) effects;
putrescine, spermydin (low concentration!):
growth stimulation.
Negative effects: silages, haylages, high poliamine-content
Reactions: oxidation of amines, barrier-building in the gut, clinical symptoms are seldom
Symptoms: local erosions, vasodilatation, diarrhaea, urticaria
(hystamin toxicosis or „scombroid fish poisoning”)

28. Deterioration of feedstuffs. Warranty concerns
1. Heat damages of proteins
Rancidation of fats
Deterioration casused by bacteria and fungi
Rancidation:
Hydrolytical decomposition  glycerin, fatty acids
Activation: by fungi, lypase of bacteria, metal ions, heat, light
Impacts: organoleptic characteristics turn to be worse, but not toxic, slight nutrient losses
b. Oxidative decomposition  reacting free radical is produced
(ROO*, RO*, HOO*)  +O2  fatty acid peroxides + H2 
fatty acids
Activation: by oxigen
Impacts: damage the enzymes, membrane structure, lipoid substances (vitamines!)

29. Rancidation of fats (cont.)
b. Oxidative rancidation:
Multiple unsaturated fatty acids: autooxidative reactions
hydroperoxids  aldehyds  organic acids,
Activation: light, heat, metal ions (Cu, Fe, Mn)
Effects: hot, „screching” taste ( products of polimerization),
unpleasant smell (aldehydes; „keton-rancidity”)
Detection: number of peroxides, number of fatty acids, number of anizydines (earlier: number of aldehydes or benzydines)
High number of peroxides: at starting of peroxidation
Other parameters: secondary oxidation products
Warranty levels: values based upon lipid content!

30. Rancidation of fats (cont.)
Impacts:
In feedstuffs: bad taste, smell, feed refusal,
vitamines A and E are damaged,
toxic polimerization products.
In animals:
rats, 10-20% feeding rancid fat no appetite, losing hair, death;
slow growth of chiks, oedema (in abdominal and heart membranes)
(toxic fat syndrome of chicks)
c. laying hens: no egg production, aspecific mortality increases
d. testinal tissues in cocks are especially sensitive

31. Rancidation of fats (cont.)
Protection: by antioxidants (AHs)
ROO* AH ROOH A*
peroxide radical hydroperoxide
A*: reacts slowly with O2 (if at all), peroxidation stops
Plants: natural antioxidants
Lipids of animal origin: low levels of AHs
In feedtstuffs: mixing in vitamine E

32. Rancidation of fats (cont.)
Warranty: is that the only qualitative problem?
Basis of judgement: is it a detrimental process?
If: number of peroxides  20-30
number of acids  40-50
changes of nutrient contents, feed refusal
Protection
Antioxidants: butyl-hydroxi-anisol (BHA), butyl-hydroxi-toluol (BHT), cyclic amins (trimetyl-etoxi-dihydro-kvinolin, etoxikvin (EMQ), tocoferols (natural AHs)
Mixing concentration: max. 150 mg per kg BHA or BHT or EMQ

33. Data for the evaluation of the rancidity of feedstuffs
Peroxide number
Evaluation
Acid number
0-15
Fresh
0-35
16-25
Slightly rancid
36-45
26-40
Not fresh
46-55
41-50
Long storing
56-60
51-60
Early rancid
61-70
60<
Rancid
70<

34. Microbiological deterioration of feedstuffs
a. fungi  mycotoxin-production
b. proliferation of fungi or yeasts  mycosis
(e.g. Candida-enteritis in piglets, in calves)
c. pathological bacteria: Salmonellae, Clostridia, Staphylococci  production of toxins
d. non-pathological bacteria : non-specific toxins,
products of cell degradation subclinical inflammation, mucosa gets thick, absorption of nutrients decreases
Influenced by: temperature, water activity, pH, partial pressure of oxygen, chemical composition of feeds, conditions of harvest and of storage, kernel damage

35. Microbiological deterioration of feedstuffs
Water activity (aw): determines the growth opportunities of microbes
(expresses the available water in a substrate as a decimal fraction of
the amount present when the substrate is in equilibrium with a
saturated atmosphere, equilibrium relative humidity of 70% around
the substrate means _= 0.70)
aw = steam pressure of substrate / steam pressure of water
(chemically clean water aw-value: 1,0)
Expresses: how much „free” water is available
Water requirement of microbes is different :
Xerophil fungi (Aspergilli); aw = ;
Most of fungi (Penicillium genus) medium aw = ;
Hygrophyil Mucor-varieties: aw  0.95.

36. Mikróbák-nedvesség, ábra
Storage molds
Growth
Field molds
Bacteria
Relative humidity/water activity
Correlations between microbial growth and environmental humidity

37. Microbiological deterioration of feedstuffs (cont.)
Field fungi: Fusarium alternaria, Cladosporium, Stachybotritis, ergosterum - before harvest,
Storage fungi: Aspergillus, Penicillium, Mucor, Fusarium (!)
In moderate climatic conditions:
In fields: Fusarium genus
In storages: Penicillium genus
Feedstuffs of animal origin: Salmonella, Coli
Silomaize silage: yeasts

38. Classification of secondary metabolites
No biological activity
(95%)
Biological activity
(5%)
Toxic
On animals/humans
On plants
On microbes
Antibiotics
Mycotoxins known: ca.1500
significant: 150
analyzed: 12-15

39. Life with mycotoxins, or else some mycotoxin history
Old Testament: toxicity by ergot (Claviceps purpurea),
Fusariotoxins (T-2, zearalenon): decline of etrusc empire, deaths in Athens (5th century B.C. ),
- in the Egyptian crypts: ochratoxins (deaths of archeologists)
in 1960, the first mycotoxin was invested (aflatoxin),
- in the last 15 years, identification of 10 mycotoxins

40. Some history of mycotoxins (cont.)
3rd book of Moses :
Protection against ‘lepra’
regulation of harvesting
and storage of cereals

41. Some more hystory of mycotoxins
Middle ages:
St. Antonius „Fire”
Ergot poisoning by rye bread
(ergotism)
Reason: Claviceps purpurea
Fiery pain of peripheral vasopasm

42. Characteristics of the mycotoxins:
1. Toxic fungal secondary metabolites.
2. They can be produced on the fields and during storage as result of the contamination of molds.
3. They can result no symptoms or clynical symptoms in the farm animals.
4. During processing of feedstuffs, they do not decompose.

43. Feed deterioration – production of mycotoxins
No mycotoxin production:
atoxic fungus species,
toxic geni in favourable conditions

44. Feed deterioration – production of mycotoxins
Effects: carbohydrates decrease, proteins and lipids decompose
Mouldy raw materials:
dry matter, nutrients ( minus 10-30%),
vitamin E, beta-carotine (minus 30-70%),
free nitrogen, pH increases,
starch content decreases, acid levels are higher,
unsaturated fatty acids: peroxide phase is short
(2-4 days), acid number is higher,
strong lypase activity (fungi).
mycotoxins (moulds), toxic for warm-blooded animals

45. Effects of mycotoxins (cont.)
Foods of animal origin → danger for human consumers
Examples:
toxins of fungi: meal, bread, coffee, tea, beer, meat and milk products,
fusarium toxin: disturbances of sexual development in puberty,
mycotoxin concentration is concentrated in bran (müzli)
Detectable: in human blood and milk (damage of phoetus!)

46. Molds that produce mycotoxins:
Fusarium spp.
DON, T2
zearalenone
fumonisin
Aspergillus spp.
Aflatoxin, ochratoxin
Penicillium spp.
Ochratoxin
Claviceps spp.
Ergot

47. Ingredients commonly affected by molds
- corn, corn by-products,
wheat, wheat by-products,
rice bran,
- sorghum,
peanuts,
soybean meal,
canola meal

48. Primary mechanism through which the mycotoxins affect animals
- Reduction of feed intake
Reduced nutrition of the animal
- nutrient content of feed
- nutrient absorption
- alter/block nutrient metabolism
Suppression of immunity
Hormonal effects- primarily estrogenic
Antibiotic effects on rumen fermentation
Cellular death – various target tissues
Increased stress- interactions with other stress

49. Mycotoxicosis 1. Primary acute form:
Symptoms: hepatitis, bleeding, nephrosis, necrosis of mucosa in mouth and in gut, death
2. Primary cronic form (more often!)
Symptoms: reproductional disturbances, growth delayed, quality of
end-products gets worse
3. Secondary mycotoxin diseases: immune capacity decreases
Symptoms: immun-deficiency, immun response at cellular level is lower,
aflatoxin: immun antibody production gets lower,
phagocytosis and complementary production is damaged,
 perception against infectious and parasitical deseases increases

50. Identifying a disease as a true mycotoxicosis
it must be feed-related
no pathogenic organism isolated
disease is not contagious or infectious
performance improves with feed withdrawal
mycotoxin identified at toxic concentration

51. Trichotecens Aflatoxin (B1, B2, G1, G2 ,, M1 ,M2):
primary liver damage/cancer
Ochratoxin A and B, citrinin:
damage of kidney
Stachyobotriotoxicosis:
bleeding, erosions, crusts
Zearalenon or F-2: genital organs;
oestrogenous syndromes
(pseudo-estrogen) of newborn piglets

52. 1. Aflatoxin (B1, B2, G1, G2, M1, M2)
(Aspergillus flavus, parasiticus, nomius)
Occurance: groundnut meal, maize, cottonseed meal
(at high temperature → tropical, subtropical areas)
Effects: feed intake↓
enterithis,
ascites
liver damage, cancer
Limit: (≤ 0,5-1 mg/kg feedstuff)

53. Aflatoxin (cont.) Effects: appetite ↓,vomiting,
damage of intestinal musosa;
fagocitosis of macrophages ↓;
delayed hyposensitivity of skin;
IgG- és IgA- concentration in serum ↓,
activity of a complementary serum (produced in liver) ↓,
bactericid activity of serum ↓

54. Trichotecens (cont.) Toxin T-2 and its metabolites:
Toxin HT-2, deoxinivaleol (DON), nivaleol, fusareon-X
Effects: protein synthesis is inhibited at cellular level,
less progeteron is produced in corpus luteum,
lymphocytes T and B are less
immunosupressive effect
immune cells (depletion)
corpora lutea (decreased progesteron synthesis)
Detection: cultivation, chemical analysis,
ELISA test, neutralization,detoxication

55. 2. Trichotecens
F-2 (zearalenon):
Effects: oestrogenous effects,
foetus distrophy,
phoetus-absorption, pseudo-ovulation in femal animals,
in swines: perinatal oestrogen syndrome,
sperm quality gets worse.

56. Zearalenon: Symptoms in pigs (1-5 ppm in the ration):
- swollen and enlarged vulva
- vaginal and rectal prolapsus
- precocious enlargement of the mammary glands
- reduced testis size in young males

57. Zearalenon: Sypmtoms in pigs (1-5 ppm in the ration):
- shrunken ovaries in young females
- enlarged, oedomatous and tortous uterus
- infertility and abortion
- reduced litter size
- stillborn, weak and spayleg piglets

58. Deoxinivaleol (DON)
Altered feed consumption
Digestive disorders
Reduced milk production
Weight loss
Reduced immunity
Increased incidence of disease

59. Trichotecens (cont.)
Ochratoxin A and B, citrinin:
Produced by:
Penicillium sp., Aspergillus sp.
Occurance: cereals
Effects: damage of kidney, diuresis

60. 2. Trichotecens (cont.)
Patuline: antibiotic impact (also in false-fermented silages)
Satratoxin: bleedings (on straw, stachyobotiritis; seldom novadays)
Effects is increased (synergism):
polichloro-bifenils – aflatoxin B1 (cancerogenity);
Fusarium acid - DON (low development/growth in swine)

61. Fumonizin B1 (cont.) Occurance: maize Effects: ataxia, blindness,
leuco-encephalomalatia (mouldy corn disease)
porcine exudative lung syndrome,
oesophagal cancer in human

62. Alcaloids of ergot Claviceps purpurea, C. paspali Occurance:cereals
Effects: bleeding,
disturbances of central nervous system
fertility↓
milk production↓

63. Regulation of quantity of mycotoxins
Warranty problems: aflatoxin B1 is concered
Recommendation: to considere also ochratoxin A,
two limit values are given for fusariotoxins
Interpretation of critical values:
Under the lower depressive value: tolerable for a long period,
Over the toxicity limit: serious value decrease, direct risk of animal health,
Medium value: decreased value of animal product, application/utilization is risky.

64. Tolerable mycotoxin values in the European Union

65. Only aflatoxins are neutralized,
Decrease of mycotoxins in feeduffs: binding, absorption
Contaminated cereals:
- treatment by 61% hydrogen-peroxide + Na-hydroxide → significant decreased of aflatoxin,
- Detoxication of cereals with Na-carbonate, Na-dihydrogen-sulfide
Adsorbents: silicats have selective binding effects
(bentonite, hydrated sodium-calcium-aluminium-silicate)
But: high mixing concentrations are required,
Only aflatoxins are neutralized,
(vitamins, trace elements, medicals can also be bound)
Novelty: esterified glucomannane-based toxin-binders
(toxins get from feeds into milk less by 60%)