Epiphytic microflora and mycotoxin content in meadows-Is plant biodiversity affecting fungal contamination?

Ecosystem services are an important aspect of grasslands utilization; however, they are often contradictory to their main purpose, which is a production of good quality and safe feed. In this study, we evaluated the difference between grass monocultures and species-rich mixtures in terms of epiphytic microflora and mycotoxin contamination levels. We hypothesized that higher species diversity would lead to higher microbial counts, which could lead to higher mycotoxin contamination risk. Differences in epiphytic fungal, yeast and total amount of microorganisms (CFU g -1) depending on the species diversity in the field has been evaluated by cultivation method. Concentration of deoxynivalenol (DON), zearalenone (ZEN) and aflatoxin B1 (AFB1) was measured by ELISA. Results are suggesting that higher total amount of microorganisms were found in monocultures, however, fungal and yeast counts were higher in species-rich mixtures. Higher species diversity of grasses was related to higher total microbial count (TMC) and yeast colonization of phyllosphere. Our results suggest higher risk of fungal phyllosphere colonization of species-rich mixtures with higher biodiversity and therefore higher risk of mycotoxin contamination of such feed.

Ergosterol and polyphenol contents as rapid indicators of orchardgrass silage safety.

The ergosterol (ERG) has been proposed as a potential indicator of fungal contamination, along with polyphenol content analysis to predict silage safety. Despite efforts in controlling fungal growth in silage, mycotoxin co-contamination represents a possible risk for animal and human health. Modern analytical techniques determine a multitude of fungal metabolites contaminating feed. Nonetheless, these methods require sometimes arduous sample pre-treatment, long separation times, and expensive standard compounds to identified contaminants. Thus, the goal of this study was to suggest a rapid analysis of ERG and polyphenol contents to assess silage hygienic quality in ten orchardgrass varieties ensiled without and with biological and chemical additives. The determination of ERG on samples was performed by high-performance liquid chromatography using UV detection and UV/Vis spectrophotometry to determine the polyphenol content. Statistically significant differences (P < 0.05) between varieties, years and silage additives were found. Bepro was the unique variety that did not present ERG in the first cut in 2012. ERG content increased in the first cut in 2013 using biological additives as well as ERG and polyphenol contents in the first cut in 2013 using chemical additives compared with untreated silage. In addition, biological and chemical additives used in this study did not satisfactorily reduce the content of ERG and polyphenols in silage grass. Consequently, our results provide fast information about the progressive fungal contamination of grass silage. To our knowledge, it is the first time that the presence of ERG and polyphenols is determined in ten different orchardgrass varieties treated without and with additives. In general, ERG and polyphenol contents showed to be good indicators of orchardgrass silage safety.

Is a new generation of mycotoxin clay adsorbents safe in a pig's diet?

BACKGROUND: Bentonites, as a clay mineral, serve in pig farms as adsorbents of toxic substances. They are mainly used to reduce the negative impact of mycotoxins to maintain the performance and health status of animals. The new genotypes of pigs are highly sensitive to a range of antinutrients, including mycotoxins. Currently, attention is focused on more effective adsorbents of mycotoxins with a higher adsorption capacity. Such materials are in great demand among feed manufacturers. However, there is a concern that these new materials may also adsorb too many essential nutrients and decrease animal performance. The aim of the experiment was to evaluate the effect of the new generation of purified bentonites on the efficiency and health status of the pigs. RESULTS: Forty-eight slaughtered pigs with an average weight of 31.2 ± 2.6 kg were included in the experiment. The pigs were divided into two groups (2 × 24 pigs). Pigs were slaughtered at an average weight of 66.3 ± 5.2. The first group had a diet without clay (control-C). The second group (treatment-T) was fed a diet with a clay additive (purified bentonite) of 1.5 kg/t. Animals were fed the experimental diet for 35 days. In group T, a higher daily weight gain (by 4.8%) and feed intake (by 2.9%) was observed while the feed conversion decreased by 1.9%. There were no significant differences between the groups of pigs during observation in the evaluation of hematological, biochemical parameters of the blood. Morpho-pathological analysis of the jejunum showed similar signs of moderate lymphoplasmacytic infiltrate in the mucosa in the groups C and T, contained similar number of goblet cells. CONCLUSION: Taken together, the addition of the new generation of bentonite clays did not negatively influence the health status and the performance of pigs.

Importance of Zinc Nanoparticles for the Intestinal Microbiome of Weaned Piglets.

The scientific community is closely monitoring the replacement of antibiotics with doses of ZnO in weaned piglets. Since 2022, the use of zinc in medical doses has been banned in the European Union. Therefore, pig farmers are looking for other solutions. Some studies have suggested that zinc nanoparticles might replace ZnO for the prevention of diarrhea in weaning piglets. Like ZnO, zinc nanoparticles are effective against pathogenic microorganisms, e.g., Enterobacteriaceae family in vitro and in vivo. However, the effect on probiotic Lactobacillaceae appears to differ for ZnO and zinc nanoparticles. While ZnO increases their numbers, zinc nanoparticles act in the opposite way. These phenomena have been also confirmed by in vitro studies that reported a strong antimicrobial effect of zinc nanoparticles against Lactobacillales order. Contradictory evidence makes this topic still controversial, however. In addition, zinc nanoparticles vary in their morphology and properties based on the method of their synthesis. This makes it difficult to understand the effect of zinc nanoparticles on the intestinal microbiome. This review is aimed at clarifying many circumstances that may affect the action of nanoparticles on the weaning piglets' microbiome, including a comprehensive overview of the zinc nanoparticles in vitro effects on bacterial species occurring in the digestive tract of weaned piglets.

Effect of Lactic Fermentation and Cooking on Nutrient and Mineral Digestibility of Peas.

Peas are prospectively beneficial legumes in the human diet, and especially in a vegan and vegetarian diet, due to their high content of proteins and starch. Their frequent lack of appeal in human nutrition can be caused by their bloating effect and the content of some antinutritional compounds inhibiting the absorption of important nutrients. This study brings a comprehensive comparison of the nutrient content of pea flour after cooking and lactic fermentation before and after digestion in vitro. As a control sample, raw pea flour was used (sample 1). Raw pea flour was cooked for 10 min (sample 2) and 120 min (sample 3) at 100°C or it was fermented by Lactobacillus plantarum (sample 4) and cooked for 10 min at 100°C (sample 5). The samples were analyzed for protein and amino acids content, maltose, glucose, raffinose, total polyphenols, phytic acid, phytase, and mineral composition (P, Mg, Mn, Fe, Cu, Zn) before and after in vitro digestion. The results showed a significant (p < 0.05) increase in the protein digestibility of samples 3, 4 and 5. In the fermented samples were observed a higher concentration of Cys, Met, and Gln when compared to non-fermented samples. The fermentation of pea flour resulted in a significant (p < 0.05) decrease in glucose, maltose, and raffinose content. Cooking of pea flour for 10 and 120 min, but not fermenting, significantly (p < 0.05) decreased the polyphenols content. Cooking and fermentation together did not affect phytic acid concentration and phytase activity. Mg, Mn, Fe, Cu and, Zn concentration in pea flour was significantly (p < 0.05) decreased by cooking. On the other hand, fermentation significantly (p<0.05) improved the bioaccessibility of Mn and Fe. These findings suggest that lactic fermentation of pea flour is a promising culinary preparation that can improve the digestibility of peas.

Effect of Streptococcus uberis on Gamma Delta T Cell Phenotype in Bovine Mammary Gland.

In this study, we focused analyzing γδ T cells during bovine mammary gland inflammation induced by Streptococcus uberis. A mammary gland cell suspension was obtained using lavage 24, 48, 72, and 168 h after intramammary-induced infection. The proportion of lymphocytes increased during the entire week in which inflammation was present. The γδ T cells were also elevated during inflammation, reaching their peak at 72 h following induced inflammation. The percentage of apoptotic lymphocytes continually increased, with the highest proportion occurring 168 h after S. uberis infection. The results show that γδ T cells may be involved in the resolution of inflammation in bovine mammary glands, with the apoptosis of those cells potentially playing an important role.

Protective effect of a new generation of activated and purified bentonite in combination with yeast and phytogenic substances on mycotoxin challenge in pigs.

The study aimed to investigate the efficacy of new mycotoxin adsorbents based on purified and activated bentonites combined with yeast and phytogenic compounds in fattening pigs. The experiment involved 96 pigs (31.2±2.4 kg). Control (C) group was fed a diet naturally contaminated with mycotoxins (5 mg/kg deoxynivalenol, DON) without an adsorbent. Treated groups received the feed with mycotoxin adsorbents: purified and activated bentonite (T1), purified and activated bentonite, yeast derivatives, phytogenic substances (T2), and purified, activated, and sulphurated bentonite with phytogenic substances (T3). Evaluated parameters involved growth performance, organ weight, small intestine and liver histopathology, complete blood count, serum biochemistry, antioxidant status of the organism and total and free DON content in urine. In all treated groups, an significant increase in intestinal GSH and GSH/GSSG ratio was observed when compared to C. No significant effects on liver and kidney weight, complete blood count, serum or intestinal malondialdehyde concentration, or total/free DON content in urine were observed. All adsorbents improved histopathological findings in the liver when compared to C. Moreover, T1, and T2 groups showed no presence of inflammatory reaction or necrotic changes in the livers. Although, mycotoxin adsorbents investigated in this study had no significant impact on pig growth performance, they reduced the oxidative stress, and on the tissue level they protected the jejunal tissue and liver parenchyma under deoxynivalenol challenge.

Effects of Sub-Lethal Doses of Selenium Nanoparticles on the Health Status of Rats.

Selenium nanoparticles (SeNPs) are fast becoming a key instrument in several applications such as medicine or nutrition. Questions have been raised about the safety of their use. Male rats were fed for 28 days on a monodiet containing 0.5, 1.5, 3.0 and 5.0 mg Se/kg. Se content in blood and liver, liver panel tests, blood glucose, total antioxidant capacity (TAC), the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were analysed. Liver and duodenum were subjected to histopathology examination. The weight gain of rats showed no differences between tested groups. Se content in blood was higher in all treated groups compared to the control group. The liver concentration of Se in the treated groups varied in the range from 222 to 238 ng/g. No differences were observed in the activity of AST (aspartate aminotransferase), ALP (alkaline phosphatase) and TAS (total antioxidant status). A significant decrease in ALT activity compared to the control group was observed in the treated groups. GPx activity varied from 80 to 88 U/mL through tested groups. SOD activity in liver was decreased in the SeNP-treated group with 5 mg Se/kg (929 ± 103 U/mL). Histopathological examination showed damage to the liver parenchyma and intestinal epithelium in a dose-dependent manner. This study suggests that short-term SeNP supplementation can be safe and beneficial in Se deficiency or specific treatment.

Theranostic Approach for the Protein Corona of Polysaccharide Nanoparticles.

Polysaccharide nanoparticles are promising materials in the wide range of disciplines such as medicine, nutrition, food production, agriculture, material science and others. They excel not only in their non-toxicity and biodegradability but also in their easy preparation. As well as inorganic particles, a protein corona (PC) around polysaccharide nanoparticles is formed in biofluids. Moreover, it has been considered that the overall response of the organism to nanoparticles presence depends on the PC. This review summarises scientific publications about the structural chemistry of polysaccharide nanoparticles and their impact on theranostic applications. Three strategies of implementation of the PC in theranostics have been discussed: I) Utilisation of the PC in therapy; II) How the composition of the PC is analysed for specific disease markers; III) How the formed PC can interact with the immune system and enhances the immunomodulation or immunoelimination. Thus, the findings from this review can contribute to improve the design of drug delivery systems. However, it is still necessary to elucidate the mechanisms of nano-bio interactions and discover new connections in nanoscale research.

Usability of graphene oxide as a mycotoxin binder: In vitro study.

Mycotoxin management in agriculture is an essential challenge for maintaining the health of both animals and humans. Choosing the right adsorbent is still a question for many breeders and an important criterion for feed manufacturers. New adsorbents are still being sought. Graphene oxide is a promising material in the field of nanotechnology, which excels in its adsorption properties. Presented in vitro study investigates graphene oxide for the binding of mycotoxins from crushed wheat. The results show that graphene oxide has an adsorption capacity for aflatoxin 0.045 mg/g, zearalenone 0.53 mg/g and deoxynivalenol 1.69 mg/g at 37° C. In vitro simulation of crushed wheat digestion showed rapid adsorption during the gastric phase. Of the minerals, Mg, Cu and Zn were the most adsorbed. The applied dose of graphene oxide of 10 mg/g caused only a slight inhibition of the digestive enzymes α-amylase and trypsin compared to pepsin and gastric lipase. In vitro results indicated the suitability of graphene oxide in the adsorption of the aflatoxin, zearalenone and deoxynivalenol.

Zinc phosphate-based nanoparticles as alternatives to zinc oxide in diet of weaned piglets.

BACKGROUND: The high doses of zinc oxide (ZnO) administered orally to piglets for the prevention of diarrhea and increase of growth rate can contaminate pig farms and the surrounding environment. Therefore, there is a need to find a replacement of high doses of dietary ZnO with an equally effective alternative. In the present study, the effect of two formulations of zinc phosphate-based nanoparticles (ZnA and ZnC NPs) on growth performance, intestinal microbiota, antioxidant status, and intestinal and liver morphology was evaluated. A total of 100 weaned piglets were randomly divided into 10 equal groups with the base diet (control) or the base diet supplemented with ZnA, ZnC, or ZnO at concentrations 500, 1000, and 2000 mg Zn per kilogram of diet. Supplements were given to animals for 10 days. Fecal samples were collected on day 0, 5, 10 and 20. At the end of the treatment (day 10), three piglets from each group were sacrificed and analyzed. RESULTS: Comparing to that of control, the significantly higher piglet weight gain was observed in all piglet groups fed with ZnA (P < 0.05). Differences in the total aerobic bacteria and coliform counts in piglet feces after NPs supplementation compared to that of control and ZnO groups were also found (P < 0.05). The majority of aerobic culturable bacteria from the feces represented Escherichia (28.57-47.62%), Enterococcus (3.85-35.71%), and Streptococcus (3.70-42.31%) spp. A total of 542 Escherichia coli isolates were screened for the virulence genes STa, STb, Stx2, F4, and F18. The substantial occurrence of E. coli virulence factors was found on day 5, mainly in fimbrillary antigen and thermostable toxins, except for piglets fed by ZnC. Zn treatment decreased Zn blood levels in piglets fed with ZnO and ZnA (500 mg/kg) and increased in ZnC (2000 mg/kg) compared to that of control (P < 0.05). The antioxidant status of piglets was affected only by ZnA. While some changes in the liver and the intestinal morphology of piglets with NPs were observed, none were serious as reflected by the normal health status and increased weigh gain performance. CONCLUSIONS: Our results indicate that ZnA NPs have a positive effect on the piglet growth performance even at the lowest concentration. The prevalence of E. coli virulence factors was lowest in pigs supplemented with ZnC. Zinc phosphate-based nanoparticles may be an effective alternative to ZnO.

Development of pH-Responsive Biopolymeric Nanocapsule for Antibacterial Essential Oils.

It is generally believed that antibacterial essential oils have the potential to become one of the alternatives in preventing diarrheal diseases of monogastric animals. The disadvantage is their low efficiency per oral due to easy degradation during digestion in the stomach. This study compares the efficacy of chitosan, alginate-chitosan, guar gum-chitosan, xanthan gum-chitosan and pectin-chitosan nanocapsules to the synthesis of pH-responsive biopolymeric nanocapsule for Thymus vulgaris, Rosmarinus officinalis and Syzygium aromaticum essential oils. Using spectrophotometric approach and gas chromatography, release kinetics were determined in pH 3, 5.6 and 7.4. The growth rates of S. aureus and E. coli, as well as minimal inhibition concentration of essential oils were studied. The average encapsulation efficiency was 60%, and the loading efficiency was 70%. The size of the nanocapsules ranged from 100 nm to 500 nm. Results showed that chitosan-guar gum and chitosan-pectin nanocapsules released 30% of essential oils (EOs) at pH 3 and 80% at pH 7.4 during 3 h. Similar release kinetics were confirmed for thymol, eugenol and α-pinene. Minimal inhibition concentrations of Thymus vulgaris and Syzygium aromaticum essential oils ranged from 0.025 to 0.5%. Findings of this study suggest that the suitable pH-responsive nanocapsule for release, low toxicity and antibacterial activity is based on chitosan-guar gum structure.

Essential Oils as a Feed Additives: Pharmacokinetics and Potential Toxicity in Monogastric Animals.

Essential oils (EOs) are now a hot topic in finding modern substitutes for antibiotics. Many studies have shown positive results and confirmed their high antibacterial activity both in vitro and in vivo. Deservedly, there is an attempt to use EOs as a substitute for antibiotics, which are currently limited by legislation in animal breeding. Given the potential of EOs, studies on their fate in the body need to be summarized. The content of EO's active substances varies depending on growing conditions and consequently on processing and storage. Their content also changes dynamically during the passage through the gastrointestinal tract and their effective concentration can be noticeably diluted at their place of action (small intestine and colon). Based on the solubility of the individual EO's active substances, they are eliminated from the body at different rates. Despite a strong antimicrobial effect, some oils can be toxic to the body and cause damage to the liver, kidneys, or gastrointestinal tissues. Reproductive toxicity has been reported for Origanum vulgare and Mentha arvensis. Several publications also address the effect on the genome. It has been observed that EOs can show both genoprotective effects (Syzygium aromaticum) and genotoxicity, as is the case of Cinnamomum camphor. This review shows that although oils are mainly studied as promising antimicrobials, it is also important to assess animal safety.

Zinc phosphate-based nanoparticles as a novel antibacterial agent: in vivo study on rats after dietary exposure.

BACKGROUND: Development of new nanomaterials that inhibit or kill bacteria is an important and timely research topic. For example, financial losses due to infectious diseases, such as diarrhea, are a major concern in livestock productions around the world. Antimicrobial nanoparticles (NPs) represent a promising alternative to antibiotics and may lower antibiotic use and consequently spread of antibiotic resistance traits among bacteria, including pathogens. RESULTS: Four formulations of zinc nanoparticles (ZnA, ZnB, ZnC, and ZnD) based on phosphates with spherical (ZnA, ZnB) or irregular (ZnC, ZnD) morphology were prepared. The highest in vitro inhibitory effect of our NPs was observed against Staphylococcus aureus (inhibitory concentration values, IC50, ranged from 0.5 to 1.6 mmol/L), followed by Escherichia coli (IC50 0.8-1.5 mmol/L). In contrast, methicillin resistant S. aureus (IC50 1.2-4.7 mmol/L) was least affected and this was similar to inhibitory patterns of commercial ZnO-based NPs and ZnO. After the successful in vitro testing, the in vivo study with rats based on dietary supplementation with zinc NPs was conducted. Four groups of rats were treated by 2,000 mg Zn/kg diet of ZnA, ZnB, ZnC, and ZnD, for comparison two groups were supplemented by 2,000 mg Zn/kg diet of ZnO-N and ZnO, and one group (control) was fed only by basal diet. The significantly higher (P < 0.05) Zn level in liver and kidney of all treated groups was found, nevertheless Zn NPs did not greatly influence antioxidant status of rats. However, the total aerobic and coliform bacterial population in rat feces significantly decreased (P < 0.05) in all zinc groups after 30 d of the treatment. Furthermore, when compared to the ZnO group, ZnA and ZnC nanoparticles reduced coliforms significantly more (P < 0.05). CONCLUSIONS: Our results demonstrate that phosphate-based zinc nanoparticles have the potential to act as antibiotic agents.

Nanoparticles as a Solution for Eliminating the Risk of Mycotoxins.

Mycotoxins are toxic secondary metabolites produced by certain filamentous fungi. The occurrence of mycotoxins in food and feed causes negative health impacts on both humans and animals. Clay binders, yeast cell walls, or antioxidant additives are the most widely used products for mycotoxin elimination to reduce their impact. Although conventional methods are constantly improving, current research trends are looking for innovative solutions. Nanotechnology approaches seem to be a promising, effective, and low-cost way to minimize the health effects of mycotoxins. This review aims to shed light on the critical knowledge gap in mycotoxin elimination by nanotechnology. There are three main strategies: mold inhibition, mycotoxin adsorption, and reducing the toxic effect via nanoparticles. One of the most promising methods is the use of carbon-based nanomaterials. Graphene has been shown to have a huge surface and high binding capacity for mycotoxins. Attention has also been drawn to polymeric nanoparticles; they could substitute adsorbents or enclose any substance, which would improve the health status of the organism. In light of these findings, this review gives new insights into possible future research that might overcome challenges associated with nanotechnology utilization for mycotoxin elimination from agricultural products.

Antioxidant status of rats' blood and liver affected by sodium selenite and selenium nanoparticles.

BACKGROUND: Selenium is an essential element; however, at higher doses, it can be toxic. Therefore, alternative nanotechnological solutions are required to overcome toxicological issues, rather than conventional alternatives. Nanoparticles show new and promising properties that may be able to suppress toxicity while maintaining the positive effects of selenium on an organism. The aim of the experiment was to determine the influence of sodium selenite and selenium nanoparticles (SeNPs) on the antioxidant status of rats. METHODS: The males of the outbreed rat strain Wistar albino were selected as a model organism. Animals were fed different forms of selenium. The control group was given a mixture without selenium addition, whereas other groups were fed a mixture containing sodium selenite, Se-49, and Se-100 SeNPs respectively. The duration of the trial was 30 days. RESULTS: Analysis of blood and liver was performed where the concentration of reduced (GSH) and oxidised (GSSG) glutathione, and total selenium content were measured. In the liver, a significant reduction in GSSG was found for all experiment groups. Blood samples showed a significant reduction in GSH and an increase in GSSG. DISCUSSION: These results show that SeNPs may be an alternative to dietary selenium for animal organisms.

Comparison of Biogenic Amines and Mycotoxins in Alfalfa and Red Clover Fodder Depending on Additives.

In the production of fermented feed, each crop can be contaminated with a variety of microorganisms that may produce natural pollutants. Biogenic amines, mycotoxins, and undesirable organic acids can decrease health feed safety. The aim of this study was to compare the counts of microorganisms, levels of biogenic amines, and the mycotoxins in forage legumes, and also to compare the occurrence of microorganisms and levels of mycotoxins in green fodder and subsequently produced silage and the influence of additives on the content of natural harmful substances in silage. The experimental plot was located in Troubsko and Vatín, in the Czech Republic. Two varieties of Medicago sativa and one variety of Trifolium pratense were compared. Green fodder and subsequently produced silage reaching up to 23% of dry matter were evaluated and prepared using a bio-enzymatic additive and a chemical additive. Green fodder of Medicago sativa was more contaminated by Enterococci than Trifolium pratense fodder. The obvious difference was determined by the quality of silage leachate. The silage prepared from Medicago sativa fodder was more contaminated with butyric acid. Fungi were present in higher counts in the anaerobic environment of green fodder and contaminated it with zearalenone and deoxynivalenol. Lower counts of fungi were found in silage, although the zearalenone content did not change. Lower content of deoxynivalenol was detected in silage, compared with green fodder. Silages treated with a chemical additive were found not to contain butyric acid. Lower ethanol content was determined, and the tendency to reduce the risk of biogenic amines occurrence was evident. The additives proved to have no influence on the content of mycotoxins.

Determination of Histamine in Silages Using Nanomaghemite Core (γ-Fe2O3)-Titanium Dioxide Shell Nanoparticles Off-Line Coupled with Ion Exchange Chromatography.

The presence of biogenic amines is a hallmark of degraded food and its products. Herein, we focused on the utilization of magnetic nanoparticles off-line coupled with ion exchange chromatography with post-column ninhydrin derivatization and Vis detection for histamine (Him) separation and detection. Primarily, we described the synthesis of magnetic nanoparticles with nanomaghemite core (γ-Fe2O3) functionalized with titanium dioxide and, then, applied these particles to specific isolation of Him. To obtain further insight into interactions between paramagnetic particles' (PMP) surface and Him, a scanning electron microscope was employed. It was shown that binding of histamine causes an increase of relative current response of deprotonated PMPs, which confirmed formation of Him-PMPs clusters. The recovery of the isolation showed that titanium dioxide-based particles were able to bind and preconcentrate Him with recovery exceeding 90%. Finally, we successfully carried out the analyses of real samples obtained from silage. We can conclude that our modified particles are suitable for Him isolation, and thus may serve as the first isolation step of Him from biological samples, as it is demonstrated on alfalfa seed variety Tereza silage.

Determination of Phytoestrogen Content in Fresh-Cut Legume Forage.

The aim of the study was to determine phytoestrogen content in fresh-cut legume forage. This issue has been much discussed in recent years in connection with the health and safety of feedstuffs and thus livestock health. The experiments were carried out on two experimental plots at Troubsko and Vatín, Czech Republic during June and July in 2015. Samples were collected of the four forage legume species perennial red clover (variety "Amos"), alfalfa (variety "Holyne"), and annuals Persian clover and Alexandrian clover. Forage was sampled twice at regular three to four day intervals leading up to harvest and a third time on the day of harvest. Fresh and wilted material was analyzed using liquid chromatography-mass spectrometry (LC-MS). Higher levels ( p < 0.05) of isoflavones biochanin A (3.697 mg·g (-1) of dry weight) and formononetin (4.315 mg·g (-1) of dry weight) were found in red clover than in other species. The highest isoflavone content was detected in red clover, reaching 1.001% of dry matter ( p < 0.05), representing a risk for occurrence of reproduction problems and inhibited secretion of animal estrogen. The phytoestrogen content was particularly increased in wilted forage. Significant isoflavone reduction was observed over three to four day intervals leading up to harvest.